EA046388B1 - ANTI-CD40 ANTIBODIES FOR USE IN THE TREATMENT OF AUTOIMMUNE DISEASE - Google Patents

Description

Field of technology to which the invention relates

The present invention generally relates to humanized anti-CD40 antibodies for diagnostic and therapeutic use. More specifically, humanized anti-CD40 antibodies and methods of use for the treatment of various diseases or disorders characterized by the presence of cells expressing CD40 are described. Also disclosed are pharmaceutical compositions and kits containing a humanized anti-CD40 antibody.

Prerequisites for creating the invention

CD40 is a 48 kDa type I integral membrane glycoprotein with a molecular mass of 48 kDa and is a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is expressed on a variety of cell types, including normal and neoplastic B cells, interdigital cells, carcinomas, epithelial cells (eg, keratinocytes), fibroblasts (eg, synoviocytes), and platelets. It is also present on monocytes, macrophages, some endothelial cells and follicular dendritic cells. CD40 is expressed early in B cell ontogeny, appearing on B cell precursors after the appearance of CD10 and CD19, but before the expression of CD21, CD23, CD24 and the appearance of surface immunoglobulin M (sIgM) (Uckun et al., 1990, Blood, 15: 2449). CD40 has also been detected on tonsils and bone marrow plasma cells (Pellat-Decounynck et al., 1994, Blood 84:2597).

The ligand for CD40 is CD40L (also referred to as CD154, gp39, and TRAP), a member of the TNF superfamily. CD40L is a transmembrane protein that is expressed primarily on activated CD4+ T cells and a small subpopulation of CD8+ T cells (Reviewed by (Van Kooten C. and Banchereau, 2000).

The interaction of CD40 with CD40L induces both humoral and cell-mediated responses. CD40 regulates this ligand-receptor pair to activate B cells and other antigen presenting cells (APCs), including dendritic cells (DCs) (for review, see (Toubi and Shoenfeld, 2004); (Kiener, et al., 1995). Function CD40 on B cells has been extensively studied. Activation of CD40 on B cells induces proliferation, differentiation into antibody-secreting cells, and isotype switching in germinal centers of secondary lymphoid organs. In vitro studies have demonstrated direct effects of CD40 activation on cytokine production (IL-6, IL- 10, TNF-α, LT-α), expression of adhesion molecules and co-stimulatory receptors (ICAM, CD23, CD80 and CD86) and increased expression of MHC class I, MHC class II and TAB transporter molecules by B lymphocytes (Liu, et al., 1996) In most of these processes, CD40 acts in concert with either cytokines or other receptor-ligand interactions.

CD40 signaling on monocytes and DCs leads to increased survival as well as cytokine secretion (IL-1, IL-6, IL-8, IL-10, IL-12, TNF-α and MIP-1a). CD40 ligation on these APCs also results in upregulation of co-stimulatory molecules such as (ICAM-1, LFA-3, CD80 and CD86). Activation of CD40 receptors is one of the critical signals that lead to the full maturation of DCs into effective APCs that mediate T cell activation (Banchereau and Steinman, 1998) (Van Kooten C. and Banchereau, 2000).

Recent studies in mouse models have shown that CD40 signaling on dendritic cells also plays an important role in the generation of TH17 cells, which are implicated as mediators of autoimmunity 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, has made it possible to study the role of CD40-CD40L interactions in several disease models. Administration of blocking hhtu-CD40L has been demonstrated to be beneficial in several models of autoimmunity, including spontaneous diseases such as lupus nephritis in SNF1 mice or diabetes in NOD mice, or in experimentally induced forms of disease such as collagen-induced arthritis (CIA) or experimental autoimmune encephalomyelitis (EAE) (Toubi and Shoenfeld, 2004). CIA in mice was inhibited by an anti-CD40L mAb, which blocked the development of joint inflammation, serum anti-collagen antibody titers, and inflammatory cell infiltration into subsynovial tissue in addition to cartilage and bone erosion (Durie, et al., 1993). For both lupus nephritis and EAE, it has been demonstrated that anti-CD40L can also improve ongoing disease, supporting a role for CD40-CD40L in the effector phase of the disease (Kalled, et al., 1998); (Howard, et al., 1999).

The role of CD40-CD40L interactions in the development of EAE has also been studied in CD40L-deficient mice carrying a transgenic T-cell receptor specific for myelin basic protein. These mice did not develop EAE after antigen priming, and CD4+ T cells remained quiescent and did not produce INF-γ (Grewal, et al., 1996).

In addition, inhibitory antibodies directed against CD40 have shown beneficial effects in models of inflammatory diseases such as EAE. Laman and colleagues demonstrated that the antagonistic mouse mAb mu5D12 against human CD40 and a chimeric version of this mAb efficaciously

- 1046388 effectively prevent clinical expression of chronic demyelinating EAE in outbred marmosets (Laman, et al., 2002); (Boon, et al., 2001). A subsequent study showed that therapeutic treatment with chimeric antibodies against human CD40 reduced inflammation detected on MRI and delayed the expansion of pre-existing brain lesions in a marmoset model of EAE (Hart, et al., 2005).

Anti-CD40 antibodies with agonist activity have been tested in mouse models of arthritis with some conflicting results. As expected for an immunostimulatory agent, the agonistic mouse anti-CD40 mAb FGK45 has been shown to exacerbate disease in the CIA DBA/1 mouse model (Tellander, et al., 2000). However, in another chronic model of CIA, FGK45 and another agonistic mouse anti-CD40 mAb, 3/23, both exhibited beneficial therapeutic effects (Mauri, et al., 2000). This group suggested that agonistic antibodies in this therapeutic regimen have a beneficial effect by inducing an immune bias toward a Th2 response with decreased levels of INF-γ and increased levels of IL-4 and IL-10 (Mauri, et al., 2000 ).

Prevention of graft rejection by blocking CD40/CD154 interactions has also been documented. The use of ch5D12, a chimeric anti-CD40 antagonist, in renal allograft studies in rhesus monkeys shows that CD40 antagonism is sufficient to modify the disease and increase the median survival time beyond 100 days. When ch5D12 was combined with anti-CD86 antibody and given only at the start of allograft studies followed by long-term treatment with cyclosporine, a median survival time of over 4 years was achieved, indicating that this combination has the potential to induce tolerance (Haanstra, et al., 2005 ).

Thus, there are more than enough preclinical studies that provide evidence for the critical role of the CD40-CD40L dyad in driving an effective T-cell-dependent immune response. Thus, blocking CD40 signaling is considered a suitable and necessary therapeutic strategy to suppress the pathogenic autoimmune response in diseases such as RA, multiple sclerosis or psoriasis. However, to date, there are no anti-CD40 antibodies that have been approved for therapeutic intervention in such disorders, as previously developed anti-CD40 antibodies have been found to have significant side effects. Thus, there remains a significant need for therapeutic agents that can be used to interfere with the action of CD40-CD40L and block CD40 signaling. This need may be met by novel humanized anti-CD40 antibodies that specifically bind CD40 and exhibit antigen-binding specificity, affinity, and pharmacokinetic and pharmacodynamic properties that enable their use in therapeutic intervention for CD40-related disorders.

Summary of the invention

The present invention provides a humanized monoclonal antibody, wherein said antibody specifically binds to human CD40 having an IC ₅₀ antagonistic activity of less than 1 nM and has no agonism up to 100 μg/ml against B cell proliferation, wherein said antibody is further characterized in that the antibody has an in vivo half-life of at least 10 days in non-human primates.

The humanized monoclonal antibody may further be characterized in that the antibody has a half-life in cynomolgus monkeys of more than 8 days at a dose of less than 30 mg/kg.

In exemplary embodiments, an antibody of the invention comprises a heavy chain sequence selected from the group consisting of any of SEQ ID NO: 1 through SEQ ID NO: 4, and a light chain sequence selected from the group consisting of any of SEQ ID NO: 5 SEQ ID NO: 8.

In other embodiments, the antibody is a humanized antibody or an antigen binding fragment of an antibody having the heavy chain variable region amino acid sequence of any of SEQ ID NO: 1-4, 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 an antigen binding fragment of an antibody that contains the light chain variable domain amino acid sequence of SEQ ID NO: 5 - 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.

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In certain embodiments, a monoclonal antibody described herein is characterized in that it contains a heavy chain and a light chain, wherein the heavy chain CDR1 sequence is selected from the group consisting of SEQ ID NO: 9 - SEQ ID NO: 11, the heavy chain CDR2 sequence the chain is selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 15, and the heavy chain CDR3 sequence is selected from the group consisting of SEQ ID NO: 16 SEQ ID NO: 17; and wherein the light chain CDR1 sequence has a sequence selected from the group consisting of SEQ ID NO: 18 - SEQ ID NO: 21, the light chain CDR2 sequence selected from the group consisting of SEQ ID NO: 22 - SEQ ID NO: 23, and a light chain CDR3 sequence selected from the group consisting of SEQ ID NO: 24 to SEQ ID NO: 25.

In certain embodiments, a monoclonal antibody described herein is characterized in that it comprises a heavy chain CDR1 sequence of SEQ ID NO: 10, a heavy chain CDR2 sequence of SEQ ID NO: 13, and a heavy chain CDR3 sequence of SEQ ID NO: 16; wherein said antibody comprises a light chain CDR1 sequence of SEQ ID NO: 19, a light chain CDR2 sequence of SEQ ID NO: 22, and a light chain CDR3 sequence of SEQ ID NO: 24.

In other specific embodiments, a monoclonal antibody described herein is characterized in that it comprises a heavy chain CDR1 sequence of SEQ ID NO: 9, a heavy chain CDR2 sequence of SEQ ID NO: 14, and a heavy chain CDR3 sequence of SEQ ID NO: 16; wherein said antibody comprises a light chain CDR1 sequence of SEQ ID NO: 20, a light chain CDR2 sequence of SEQ ID NO: 22, and a light chain CDR3 sequence of SEQ ID NO: 24.

In other specific embodiments, a monoclonal antibody described herein is characterized in that it comprises a heavy chain CDR1 sequence of SEQ ID NO: 9, a heavy chain CDR2 sequence of SEQ ID NO: 14, and a heavy chain CDR3 sequence of SEQ ID NO: 16; wherein said antibody comprises a light chain CDR1 sequence of SEQ ID NO: 20, a light chain CDR2 sequence of SEQ ID NO: 22, and a light chain CDR3 sequence of SEQ ID NO: 24.

In other specific embodiments, a monoclonal antibody described herein is characterized in that it comprises a heavy chain CDR1 sequence of SEQ ID NO: 11, a heavy chain CDR2 sequence of SEQ ID NO: 15, and a heavy chain CDR3 sequence of SEQ ID NO: 17; wherein said antibody comprises a light chain CDR1 sequence of SEQ ID NO: 21, a light chain CDR2 sequence of SEQ ID NO: 23, and a light chain CDR3 sequence of SEQ ID NO: 25.

Also described herein are selected sequences for the heavy chains of preferred antibodies in accordance with the invention. For example, the invention relates to an anti-CD40 antibody comprising a heavy chain variable domain sequence of any one of SEQ ID NOs: 1-4. In addition, the anti-CD40 antibody is characterized in that it contains a light chain variable domain sequence from any one of SEQ ID NO: 5 through SEQ ID NO: 8.

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

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

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

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

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

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

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

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

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

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

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

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

In another embodiment, the invention provides a humanized antibody or antibody fragment having a heavy chain variable domain and a light chain variable region comprising the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 36, respectively,

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

Another embodiment provides an isolated antibody or antigen binding fragment that binds to human CD40 comprising a humanized heavy chain variable domain comprising a framework region having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework region of a human heavy chain variable domain 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 that is at least 90% identical to the corresponding light chain variable domain of SEQ ID NO: 31.

In another aspect, the invention provides an isolated antibody or antigen binding fragment described in the embodiment immediately above, 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; and in another embodiment, the heavy chain amino acid sequence is SEQ ID NO: 35,

Also provided is an isolated antibody or antigen binding fragment that specifically binds to human CD40, comprising a humanized heavy chain variable domain comprising a framework region having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework region of the amino acid sequence of the human heavy chain variable domain SEQ ID NO: 37, SEQ ID NO: 38; SEQ ID NO: 39 or SEQ ID NO: 40, and containing 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 provides an isolated antibody or antigen binding fragment described in the embodiment immediately above, 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; and in another embodiment, the heavy chain amino acid sequence is SEQ ID NO: 40,

Antibodies in accordance with the present invention can be further characterized by the fact that these antibodies are unable to stimulate the production of cytokines by B cells in the absence of CD40L.

Antibodies in accordance with the present invention can be further characterized

- 4 046388 in that these antibodies bind to human CD40 in the presence of 50% human serum at a rate of less than twofold.

Antibodies in accordance with the present invention can be further characterized by the fact that the specified antibody causes inhibition of IgM and IgG production in mammals at a concentration of 1 mg/kg.

Antibodies in accordance with the present invention can be used in a variety of therapeutic, prophylactic, diagnostic and other methods. For example, the present invention describes a method of blocking the function of human CD40 in a mammal, comprising administering to said mammal a composition containing an antibody according to the invention in an amount sufficient to block a CD40-mediated immune response in said mammal.

The present invention also provides a method of treating or ameliorating graft-versus-host disease in a mammal, comprising administering to said 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 said animal.

By way of example, an autoimmune or inflammatory disease may include, but is not limited to, rheumatoid arthritis, lupus nephritis, 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 myxedema, thyrotoxicosis/Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune carditis, Addison's disease, premature menopause, type 1 diabetes mellitus, Goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis (HBsAg negative), cryptogenic cirrhosis, Sjogren's syndrome, dermatomyositis, scleroderma, mixed connective tissue diseases, discoid lupus erythematosus and systemic vasculitis. In exemplary embodiments, the mammal has rheumatoid arthritis.

The methods of the invention may further include administering a second therapeutic agent selected from the group consisting of a TNF antagonist, a disease-modifying antirheumatic drug, a CTLA4 antagonist, an anti-IL-6 receptor mAb, and an anti-CD20 mAb.

In certain embodiments, the inflammatory disease or autoimmune disease is an inflammatory disease or autoimmune disease that is associated with cells expressing both CD40 and CD20.

In specific methods, treatment involves administering the antibody composition parenterally.

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

Additional methods in accordance with the invention include inhibiting the production of antibodies by B cells in a human patient, comprising administering to said human patient an effective amount of an anti-CD40 antibody in accordance with the invention.

More specifically, the human patient has an inflammatory disease or autoimmune disease that is associated with cells expressing CD40.

In exemplary embodiments, the human patient suffers from 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 thrombocytopenia purpura (ITP), Crohn's disease and systemic lupus erythematosus (SLE), Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis/Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune carditis, Addison's disease, premature menopause, type 1 diabetes mellitus, Goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis (HBsAg negative), cryptogenic cirrhosis, Sjogren's syndrome, dermatomyositis, scleroderma, mixed connective tissue diseases, discoid lupus erythematosus and systemic vasculitis.

Another method according to the invention relates to inhibiting the growth of cells expressing human CD40 antigen, comprising introducing an antibody or an antigen-binding fragment thereof into the cells, wherein said antibody or antigen-binding fragment thereof specifically binds to a CD40 antigen on the surface of human cells, wherein binding of the antibody or its antigen-binding fragment with the CD40 antigen inhibits cell growth or differentiation.

Also provided is a method of treating a subject having a CD40-related disease, comprising administering to the subject an antibody or antigen-binding fragment of the invention, said antibody or antigen-binding fragment specifically binding to human CD40, wherein binding of the antibody or antigen-binding fragment to CD40 inhibits the growth or differentiation of CD40-related cells. diseases. Cells may be, but are not limited to,

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B lymphoblastoid 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.

A treatment method for inhibiting cell growth or differentiation may be useful in the treatment of rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, chronic lymphocytic leukemia, Burkitt's lymphoma, multiple myeloma, T-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Waldenström's macroglobulinemia, or Kaposi's sarcoma.

Also provided is a method for inducing peripheral B cell depletion, comprising introducing into the cells an antibody or antigen binding fragment in accordance with the invention, wherein said antibody or antigen binding fragment specifically binds to a CD40 antigen on the surface of a human cell, wherein binding of the antibody or antigen binding fragment to the CD40 antigen causes cell depletion.

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

Also contemplated is a method of treating rheumatoid arthritis in a subject, comprising administering to said subject an antibody in accordance with the invention, wherein said antibody is an antagonist antibody that blocks CD40 function in said subject.

Also contemplated is a method of treating systemic lupus erythematosus, or lupus nephritis, in a subject, comprising administering to said subject an antibody in accordance with the invention, wherein said antibody is an antagonist antibody that blocks CD40 function in said subject.

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

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

In other embodiments, the method is further characterized in that the antibody is administered in an amount effective to inhibit the production of proinflammatory cytokines, chemokines, matrix metalloproteinases, prostaglandins, and downregulation of adhesion molecules in non-immune cells of the subject.

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

The subject receiving therapy is a patient who has rheumatoid arthritis and is refractory to treatment with methotrexate alone.

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

The method according to the invention may be further characterized in that treatment of said subject with said antagonistic anti-CD40 antibody has higher efficacy than treatment with methotrexate alone, Enbrel alone, or the Enbrel+methotrexate combination.

The method according to the invention may be further characterized in that treatment of said subject with said antagonistic anti-CD40 antibody has higher efficacy than treatment with Enbrel+MTX in patients who have had an inadequate response to methotrexate.

In certain embodiments, the antibody is administered in combination with a regimen that includes an anti-TNF agent.

In certain embodiments, the subject is characterized as having rheumatoid arthritis and not responding to treatment with an anti-TNF agent alone. In such embodiments, the method may include treating said subject with a regimen comprising treatment with an anti-TNF agent in combination with said anti-CD40 antagonist antibody.

In certain embodiments, treatment of said subject with said antagonistic anti-CD40 antibody has greater efficacy compared to treatment with an anti-TNF agent.

In still other embodiments, the method is characterized in that treatment of said subject with said antagonistic anti-CD40 antibody has greater efficacy than treatment with Orencia or Rituxan in patients who have had an inadequate response to treatment with anti-TNF agent alone.

In addition, the present invention provides a pharmaceutical composition containing (i) an antibody or antigen binding fragment described herein; and (ii) a pharmaceutically acceptable excipient. In such compositions, the antibody or antigen-binding fragment thereof may advantageously be conjugated to a second agent, such as, for example, a cytotoxic agent.

- 6 046388 Chinese agent, PEG carrier, enzyme or marker.

The present invention also provides an isolated polynucleotide encoding the amino acid sequence of the heavy chain variable region of any of SEQ ID NO: 1-4, 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 present invention also provides an isolated polynucleotide encoding the amino acid sequence of the light chain variable region of any of SEQ ID NO: 5-8, 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 also relates to the use of antibodies described herein for the manufacture of a medicament for blocking the function of human CD40 in a mammal, wherein the medicament blocks a CD40-mediated immune response in said mammal.

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

In exemplary embodiments, the medicament is formulated for the treatment of an autoimmune or inflammatory disease selected from the group that includes rheumatoid arthritis, lupus nephritis, 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 myxedema, thyrotoxicosis/Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune carditis, Addison's disease, premature menopause, type 1 diabetes mellitus, Goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis (HBsAg negative), cryptogenic cirrhosis, Sjogren's syndrome, dermatomyositis, scleroderma, mixed connective tissue diseases, discoid lupus erythematosus and systemic vasculitis.

In some embodiments, the drug may further comprise a second therapeutic agent selected from the group that includes a TNF antagonist, a disease-modifying antirheumatic drug, a CTLA4 antagonist, an anti-IL-6 receptor mAb, and an anti-CD20 mAb.

The medicinal product may be formulated for parenteral administration. The drug may be formulated for intravenous or subcutaneous administration.

Another embodiment involves the use of antibodies described herein for the manufacture of a medicament for inhibiting the production of antibodies by B cells in a human patient.

Another embodiment involves the use of antibodies described herein for the manufacture of a medicament for inhibiting the growth and/or differentiation of cells expressing a human antigen.

Another embodiment involves the use of antibodies described herein for the manufacture of a medicament for treating a subject having a CD40-related disorder, wherein binding of the antibody or antigen-binding moiety in the medicament to CD40 CD40 inhibits the growth or differentiation of cells of the CD40-related disorder.

The drug may be formulated for use in the treatment of CD40-related disorder cells selected from B lymphoblastoid cells, pancreatic cells, lung cells, breast cells, ovarian cells, colon cells, prostate cells, skin cells, scalp cells and neck, bladder cells, bone cells or kidney cells.

The drug may be formulated for use in the treatment of chronic lymphocytic leukemia, Burkitt's lymphoma, multiple myeloma, T-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's disease, Waldenström's macroglobulinemia, or Kaposi's sarcoma.

Another embodiment involves the use of antibodies in accordance with the invention in the manufacture of a drug for inducing depletion of peripheral B cells, wherein the antibody or antigen binding fragment of the drug specifically binds to a CD40 antigen on the surface of a human cell, wherein binding of the antibody or antigen binding fragment to the CD40 antigen causes depletion cells.

The drug may be formulated for use in treating a subject having an immune disorder.

The drug may be manufactured for use in the treatment of rheumatoid arthritis

- 7 046388 or systemic lupus erythematosus.

Another embodiment involves the use of antibodies in accordance with the invention in the manufacture of a medicament for treating rheumatoid arthritis in a subject.

Another embodiment involves the use of antibodies in accordance with the invention in the manufacture of a medicament for the treatment of systemic lupus erythematosus or lupus nephritis in a subject.

The drug may be formulated for use in inhibiting B cell differentiation and antibody isotype switching in a subject.

The drug may be formulated for use in inhibiting the production of cytokines and chemokines and upregulating adhesion molecules in T cells and macrophages in a subject.

The drug may be formulated for use in inhibiting dendritic cell activation in a subject.

The drug may be formulated for use in inhibiting the production of proinflammatory cytokines, chemokines, matrix metalloproteinases, prostaglandins, and downregulation of adhesion molecules in non-immune cells in a subject.

In some embodiments, the drug is formulated as a combination drug for administration in combination with a regimen comprising administration of methotrexate and/or administration of Enbrel/Humira.

In other embodiments, the drug is formulated as a combination drug and the drug, in addition to the antibodies of the invention, further comprises an anti-TNF agent.

Brief description of several types of drawings

In fig. Figure 1 shows a simple dose escalation design. S-FU represents safety surveillance over time. *Refers to two subjects randomized to the placebo group and eight subjects randomized to the antibody group of the present invention. t Refers to S-FU, which was longer for Cohort 4 (56 days instead of 42 days).

In fig. 2 shows the blood concentrations before taking the next dose of the antibody in accordance with the present invention on days 8, 15 and 22 (Cpre) and the minimum concentration on day 29.

In fig. 3A shows the arithmetic average percentage of CD40 receptor occupancy across different dosage treatments.

In fig. 3B shows the percentage of inhibition of CD54 upregulation by different doses.

Detailed Description of the Invention

CD40-mediated signaling is now known to be involved in a variety of target disorders. Despite the availability of a variety of preclinical data indicating that intervention in these disorders will be therapeutically beneficial, there remains a need for antagonistic CD40 antibodies that can be used in the treatment of autoimmune diseases. In preferred embodiments, the present invention relates to humanized antibodies that recognize CD40. These antibodies are also disclosed in US Patent No. 8,591,900 and WO 2011/123489, the contents of each of which are included herein. In specific embodiments, the sequence of these humanized antibodies was identified based on the sequences of certain lead murine antibodies.

Despite therapeutic progress in recent years, there is still an unmet need for new treatments for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and lupus nephritis. The interaction of the cell surface CD40 receptor and its ligand CD40L (CD154) is known to play a major role in the regulation of humoral and cellular immunity, and in the pathogenesis of these autoimmune diseases. Therefore, the CD40-CD40L interaction is an attractive target for the modulation of autoimmune diseases.

CD40 is a cell surface receptor that belongs to the tumor necrosis factor receptor family and is expressed on B cells, dendritic cells, monocytes, macrophages, kidney cells and other non-immune cells. CD40 is a key co-stimulatory molecule involved in the development of antigen-driven acquired immunity by activating B cells and other antigen presenting cells (APCs), including dendritic cells and macrophages, but is also involved in the activation of non-immune resident cells. 4 CD40L is a member of the tumor necrosis factor superfamily that is expressed primarily by activated T cells, but also by activated B cells and platelets. Binding of CD40 to CD40L results in upregulation of E-selectin (CD62E), vascular cell adhesion molecule-1 (CD106), and cell-cell adhesion molecule-1 (CD54), thereby increasing leukocyte margination and diapedesis.

CD40-CD40L interaction appears to be required for optimal activation of APC T cells. The CD40-CD40L pathway is thought to be particularly important for enhancing T cell responses and is involved in several autoimmune diseases. It has been shown that blocking the signaling pathway

- 8 046388

CD40 inhibits T helper 1 (Th1) cell differentiation and maintenance of the immune response. Increased expression of CD40 and CD40L is associated with active disease in patients with rheumatoid arthritis. Elevated levels of CD40L on B and T cells are associated with disease activity in systemic lupus erythematosus, and renal expression of CD40 on mesangial cells is increased in patients with lupus nephritis grades III and IV.

Previous clinical development of anti-CD40L monoclonal antibodies failed due to cases of thromboembolism that were initiated by platelet activation and aggregation, possibly due to the Fc region of anti-CD40L antibodies activating the platelet receptor FcYRIIa (CD32a). Recent studies show that antibodies lacking a functional Fc region do not cause thromboembolic events, fail to activate platelets, and retain both pharmacological activity and clinical activity.

In one embodiment, the antibody of the present invention is a humanized anti-CD40 antagonistic monoclonal antibody that selectively binds CD40 and blocks the CD40-CD40L interaction; it was designed to have no agonist activity and to prevent stimulation of cytokine production. Two replacement mutations in the Fc region (Leu234Ala and Leu235Ala) were included to prevent Fc-mediated antibody-dependent or complement-mediated cellular cytotoxicity and platelet activation. The antibody of the present invention demonstrated strong and comparable binding properties in both human (EC ₉₀ =6.85±0.74 nM) B cells and cynomolgus monkey cells and potent inhibition of CD40L-induced proliferation of peripheral blood mononuclear cells without agonism. When bound to platelets, the antibody of the present invention does not alter platelet activation, aggregation or function. In preclinical studies in cynomolgus monkeys with repeated doses of up to 50 mg/kg of the antibody of the present invention over 26 weeks, a reversible decrease in B-cell levels, a reversible decrease in germinal center lymphoid organs, and good overall tolerability without thromboembolic events or corresponding cytokine release were demonstrated. (and unpublished data). The freedom from observed side effects rate in these studies was 50 mg/kg, the highest dose administered (unpublished data).

As shown in Example 9, in a single dose escalation study in healthy volunteers, increasing single intravenous (IV) and subcutaneous (SC) doses to 120 mg of the antibody of the present invention was well tolerated and showed high potential for blocking the CD40-CD40L pathway. A dose-related increase in CD40 receptor occupancy (RO) and inhibition of B cell activation (measured by inhibition of CD54 up-regulation) were observed following both intravenous and subcutaneous dosing of an antibody in accordance with the present invention.

Definitions

CD40 and surface antigen CD40 refers to an approximately 48 kDa glycoprotein expressed on the surface of healthy and neoplastic B cells that acts as a receptor for signals involved in cell proliferation and differentiation (Ledbetter et al., 1987, J. Immunol 138:788–785). The cDNA molecule encoding CD40 was isolated from a library generated from the Burkett's lymphoma cell line Raji (Stamenkovic et al., 1989, EMBO J. 8:1403).

As used herein, a cell that endogenously expresses CD40 is any cell that exhibits surface expression of CD40, including, but not limited to, healthy and neoplastic B cells, interdigital cells, basal epithelial cells, carcinoma cells, macrophages, endothelial cells, follicular dendritic cells, tonsil cells and bone marrow plasma cells. In some embodiments, the CD40 molecule is a human CD40 molecule.

Antibodies in accordance with the invention specifically bind to human recombinant and native CD40. A humanized monoclonal antibody, wherein said antibody specifically binds to human CD40, has an IC ₅₀ antagonistic activity of less than 1 nM, and has no agonism up to 100 μg/ml in B-cell proliferation, and wherein said antibody is further characterized in that the antibody has a time half-life in vivo in non-human primates, which is at least 10 days.

Preferably, the antibody specifically binds to CD40 in a CD40-Fc conjugate with an EC ₅₀ of less than 1 nM and CD40 in CD40-expressing cells with an EC ₅₀ of less than 2.5 nM. The antagonistic properties of an antibody are determined by the fact that it has an antagonistic activity against B cells or dendritic cells with an IC ₅₀ of less than 1 nM. In addition, the antibody has superior pharmacokinetic properties, having an increased in vivo half-life compared to other anti-CD40 antibodies (eg, anti-CD40 antibody 4D11).

As used herein, a cell that expresses CD40 is any cell characterized by surface expression of CD40, including, but not limited to, normal and

- 9 046388 neoplastic B cells, interdigital cells, basal epithelial cells, carcinoma cells, macrophages, endothelial cells, follicular dendritic cells, tonsil cells and bone marrow plasma cells. In some embodiments, the CD40 molecule is a human CD40 molecule.

Antibodies in accordance with the present invention recognize a specific epitope of the CD40 antigen and a CD40 epitope. As used herein, these terms refer to a molecule (eg, peptide) or fragment of a molecule capable of immunoreactivity with an anti-CD40 antibody, and, for example, includes a CD40 antigenic determinant recognized by any of the antibodies having a heavy chain/light chain sequence combination such as as a light chain SEQ ID NO: 26 with any heavy chain SEQ ID NO: 27, 28, 29 or 30; or light chain SEQ ID NO: 31 with any heavy chain SEQ ID NO: 32, 33, 34 or 35; or the light chain of SEQ ID NO: 36 with any heavy chain of SEQ ID NO: 37, 38, 39 or 40. CD40 antigen epitopes may be included in proteins, protein fragments, peptides, and the like. Epitopes are most often proteins, short oligopeptides, oligopeptide mimics (ie, 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; these molecules are heterotetrameric glycoproteins, 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 a single disulfide bond to form a heterodimer, and a heterotramer molecule is formed by a covalent disulfide bond between two identical heterodimer heavy chains. Although the light and heavy chains are linked together by a single disulfide bond, the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also has evenly distributed intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the amino terminus, followed by three or four constant domains ( _CH1 , CH2, CH3 and CH4), and a hinge region between CH1 and CH2. Each light chain has two domains, an amino-terminal variable domain ( _VL ) and a carboxy-terminal constant domain ( _CL ). The V _L domain binds non-covalently to the V _H domain, whereas the C _L domain is typically covalently linked to the _CH 1 domain via a disulfide bond. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains (Chothia et al., 1985, J. Mol. Biol. 186:651-663).

Some regions in the variable domains vary greatly between different antibodies, e.g. are hypervariable. These hypervariable regions contain residues that are directly involved in the binding and specificity of each particular antibody for its specific antigenic determinant. Hypervariability in both the light chain variable domains and the heavy chain variable domains is concentrated in three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). CDRs are determined by sequence comparison in Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., while HVLs are structurally defined according to the three-dimensional variable domain structure as described by Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. If the two methods result in slightly different CDR identifications, the structural identification is preferred. As defined by Kabat, CDR-L1 is located at approximately residues 24-34, CDR-L2 at approximately residues 50-56, and CDR-L3 at approximately residues 89-97 in the light chain variable domain; CDR-H1 is located at approximately residues 31-35, CDR-H2 at approximately residues 50-65, and CDR-H3 at approximately residues 95-102 in the heavy chain variable domain.

Thus, CDR1, CDR2, CDR3 of the heavy and light chains determine unique and functional properties specific to a given antibody.

The three CDRs in each of the heavy and light chains are separated by framework regions (FRs) that contain sequences that tend to be less variable. From the amino to 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 FR4. Mainly, the β-sheet configuration of FR brings the CDRs in each strand closer to each other, as well as to the CDRs from the other strand. The resulting conformation contributes to the antigen binding site (see Kabat et al., 1991, NIH Pub. No. 91-3242, Vol. I, pp. 647-669), although not all CDR residues are necessarily directly involved in antigen binding.

FR residues and Ig constant domains are not directly involved in antigen binding, but promote antigen binding and/or mediate antibody effector function. Some FR residues are believed to have a significant effect on antigen binding in at least three ways: by noncovalent binding directly to the epitope, by interaction with one or more CDR residues, and by influencing the interface between the heavy and light chains. Constant domains are not directly involved in antigen binding but mediate various Ig effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

The light chains of vertebrate immunoglobulins belong to one of two distinct

- 10 046388 classes, kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. In comparison, mammalian immunoglobulin heavy chains are classified into one of five major classes according to the sequence of constant domains: IgA, IgD, IgE, IgG, and IgM. IgG and IgA are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1 and IgA2. The heavy chain constant domains that correspond to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of native immunoglobulin classes are well known.

The terms antibody, anti-CD40 antibody, humanized anti-CD40 antibody, and humanized anti-CD40 antibody variant are used herein in the broadest sense and specifically include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and fragments antibodies, such as variable domains and other parts of antibodies that exhibit the desired biological activity, for example, binding to CD40.

The term monoclonal antibody (mAb) refers to an antibody from a population of essentially homogeneous antibodies; those. the individual antibodies in this population are identical except for naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific; they are directed against one antigenic determinant, an epitope. Therefore, the modifier monoclonal indicates an essentially homogeneous population of antibodies directed to an identical epitope and should not be interpreted as requiring the antibody to be produced by any particular method. It should be understood that monoclonal antibodies can be obtained by any method or method known in the art; including, for example, the hybridoma method (Kohler et al., 1975, Nature, 256:495), or recombinant DNA methods known in the art (see, for example, US Pat. No. 4,816,567), or methods for isolating recombinantly produced monoclonals using phage antibody libraries using techniques described in Clackson et al., 1991, Nature, 352:624-628, and Marks et al., 1991, J. Mol. Biol. 222:581-597.

Chimeric antibodies consist of the heavy and light chain variable regions of an antibody from one species (e.g., a non-human mammal such as a mouse) and the heavy and light chain constant regions of an antibody from another species (e.g., a human) and can be made by linking DNA sequences encoding the variable regions of an antibody of a first type (eg, mouse), with DNA sequences for the constant regions of an antibody of a second type (eg, human) and transforming the host with an expression vector containing related sequences allowing the production of a chimeric antibody.

Alternatively, a chimeric antibody may also be an antibody in which one or more heavy and/or light chain regions or domains are identical, homologous, or a variant of the corresponding sequence in a monoclonal antibody from another class or isotype of immunoglobulins or from a consensus or germline sequence. Chimeric antibodies can include fragments of such antibodies, provided that the antibody fragment exhibits the desired biological activity of its parent antibody, such as binding to the same epitope (see, for example, US Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 6851-6855).

The terms antibody fragment, anti-CD40 antibody fragment, humanized anti-CD40 antibody fragment, variant humanized anti-CD40 antibody fragment refer to a portion of a full-length anti-CD40 antibody that retains a variable region or functional ability, such as specific binding of a CD40 epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fd, Fv, scFv and scFv-Fc fragment, diabody, linear antibody, single chain antibody, minibody, diabody formed from fragments antibodies, and multispecific antibodies formed from antibody fragments.

Full length antibodies can be treated with enzymes such as papain or pepsin to produce useful antibody fragments. Papain digestion is used to produce two identical antigen-binding antibody fragments, called Fab fragments, each with one antigen-binding site and a residual Fc fragment. The Fab fragment also contains a light chain constant domain and a heavy chain CH1 domain. Treatment with pepsin produces an F(ab') ₂ fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

Fab' fragments differ from Fab fragments by the presence of additional residues, including one or more cysteines from the antibody hinge region at the C-terminus of the C _H1 domain. F(ab') ₂ antibody fragments are pairs of Fab' fragments linked by cysteine residues in the hinge region. Other chemical compounds of antibody fragments are also known.

The Fv fragment contains a complete antigen recognition and binding site, consisting of a dimer of one heavy chain variable domain and one light chain domain in a tight non-covalent bond. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs impart antigen-binding properties to the antibody.

- 11 046388 calling specificity.

A single chain Fv or scFv antibody fragment is a single chain variant of Fv containing the VH and VL domains of the antibody, where the domains are present on the same polypeptide chain. Single-chain Fv is able to recognize and bind antigen. The scFv polypeptide may also optionally contain a polypeptide linker located between the VH and VL domains to facilitate formation of the desired three-dimensional structure for antigen binding by the scFv (see, for example, Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

Other recognized antibody fragments include those that contain a pair of tandem Fd segments (V _H -CH1-VH-C=) to form a pair of antigen binding regions. These linear antibodies can be bispecific or monospecific, as described, for example, in Zapata et al., 1995, Protein Eng. 8(10): 1057-1062.

A humanized antibody or humanized antibody fragment is a specific type of chimeric antibody that includes an immunoglobulin amino acid sequence variant or fragment thereof that is capable of binding to a predetermined antigen and that includes one or more FRs having substantially the amino acid sequence of a human immunoglobulin, and one or multiple CDRs having essentially the amino acid sequence of a non-human immunoglobulin. This non-human amino acid sequence, often referred to as an import sequence, is typically taken from the import domain of an antibody, particularly the variable domain. In general, a humanized antibody includes at least a CDR or HVL of a non-human antibody inserted between the FRs of a human heavy or light chain variable domain. The present invention describes specific humanized anti-CD40 antibodies that contain CDRs derived from the murine monoclonal antibodies shown in table. 3 and 4, inserted between the FRs of the heavy and light chain variable domains of the human germline sequence. It should be understood that some residues of the mouse FR may be important for the function of humanized antibodies, and therefore some residues of the human germline sequence heavy and light chain variable domains are modified to be the same as those in the corresponding mouse sequence.

In another aspect, a humanized anti-CD40 antibody comprises substantially all of at least one, and typically two, variable domains (such as those contained in, for example, Fab, Fab', F(ab') ₂ , Fabc and Fv fragments) in which all or substantially all of the CDRs correspond to non-human immunoglobulin CDRs, and in particular, herein all of the CDRs are murine sequences, as detailed in Table. 1-4 herein below, and all or substantially all of the FRs are those from the human immunoglobulin consensus sequence or germline sequence. In another aspect, the humanized anti-CD40 antibody also includes at least a portion of the Fc region of an immunoglobulin, typically human immunoglobulin. Typically, an antibody will contain both a light chain and at least a heavy chain variable domain. Optionally, the antibody may also include one or more of the CH1, hinge, CH2, CH3 and/or CH4 heavy chain regions.

The humanized anti-CD40 antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG. IgG ₂ , IgG ₃ , IgG ₄ , IgA1 and IgA ₂ . For example, the constant domain may be a complement fixation constant domain if it is desired that the humanized antibody exhibit cytotoxic activity, and the isotype is typically IgGi. If such cytotoxic activity is undesirable, then the constant domain may be of a different isotype, for example IgG ₂ . An alternative humanized anti-CD40 antibody may contain sequences from more than one class or isotype of immunoglobulins, and the selection of specific constant domains to optimize desired effector functions is within the skill of ordinary skill in the art. In certain embodiments, the present invention provides antibodies that are IgG1 antibodies and, more specifically, are IgG1 antibodies that have knockout effector functions.

The FR and CDR or HVL of a humanized anti-CD40 antibody do not need to exactly match the parent sequences. For example, one or more residues in an imported CDR or HVL or consensus sequence or germline FR sequence may be altered (eg, mutagenized) by substitution, insertion, or deletion such that the resulting amino acid residue is no longer identical to the original residue at the corresponding position in any parent sequence, but, nevertheless, the antibody retains the function of binding to CD40. Such a change will usually not be extensive and will be conservative. Typically, at least 75%, more commonly at least 90%, and most often greater than 95%, or greater than 98%, or greater than 99% of the residues of the humanized antibodies will correspond to residues of the parent consensus sequence or germline FR sequence and imported CDR sequences.

Immunoglobulin residues that influence the interface between variable regions

- 12 046388 heavy and light chains (VL-VH interface), are residues that affect the proximity or orientation of the two chains relative to each other. Some residues that may be involved in interchain interactions include _VL residues 34, 36, 38, 44, 46, 87, 89, 91, 96, and 98 and VH residues 35, 37, 39, 45, 47, 91, 93. 95, 100, and 103 (using the numbering system outlined in Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987)). US Pat. No. 6,407,213 also discusses that residues such as _VL residues 43 and 85 and VH residues 43 and 60 may also be involved in this interaction. Although these residues are specified for human IgG only, they are applicable across species. Important antibody residues that are expected to participate in interchain interactions are selected for replacement in the consensus sequence.

The terms consensus sequence and consensus antibody refer to an amino acid sequence that includes the most frequently occurring amino acid residue at each location in all immunoglobulins of any particular class, isotype, or subunit structure, such as the human immunoglobulin variable domain. The consensus sequence may be based on immunoglobulins of a specific species or many species. By consensus sequence, structure, or antibody is meant a human consensus sequence, as described in certain embodiments, and to mean an amino acid sequence that includes the most commonly occurring amino acid residues at each location in all human immunoglobulins of any particular class, isotype, or subunit structure. Thus, the consensus sequence contains an amino acid sequence having at each position an amino acid that is present in one or more known immunoglobulins, but which cannot exactly duplicate the entire amino acid sequence of any individual immunoglobulin. The variable region consensus sequence is not derived from any naturally occurring antibody or immunoglobulin. Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., and variations thereof. The FR heavy and light chain consensus sequences and variants thereof provide useful sequences for the production of humanized anti-CD40 antibodies. See, for example, US Patent Nos. 6,037,454 and 6,054,297.

Human germline sequences occur naturally in the human population. The combination of these germline genes creates antibody diversity. The germline antibody sequences for the antibody light chain are derived from the conserved human kappa or lambda germline v and j genes. Likewise, heavy chain sequences are derived from the germline v-, d- and j-genes (LeFranc, M.-R., and LeFranc, G., The Immunoglobulin Facts Book, Academic Press, 2001).

As used herein, the terms variant, anti-CD40 variant, humanized anti-CD40 variant, or humanized anti-CD40 antibody variant each refer to a humanized anti-CD40 antibody having at least a heavy chain variable murine CDR from any of the sequences of SEQ ID NO: 1- 4 or a mouse light chain CDR sequence derived from a mouse monoclonal antibody as shown in any of SEQ ID NO: 5 through SEQ ID NO: 8, and FR sequences derived from human consensus sequences. Variants include those that contain one or more amino acid substitutions in one or both light chain or heavy chain variable domains, provided that the amino acid change does not significantly impair binding of the antibody to CD40. Exemplary humanized antibodies produced according to the present invention include the antibodies designated Antibody A, Antibody B, and Antibody C, and various heavy and light chain sequences thereof are shown in SEQ ID NO: 26 through SEQ ID NO: 40.

An isolated antibody is an antibody that has been identified and separated and/or isolated from a component of its natural environment. Contaminants in the natural environment of an antibody are those materials that may interfere with the diagnostic or therapeutic use of the antibody, and may be enzymes, hormones, or other protein or non-protein solutes. In one aspect, the antibody will be purified to at least greater than 95% recovery of the antibody by weight.

An isolated antibody includes the antibody in situ in the recombinant cells in which it is produced, since at least one component of the antibody's natural environment will not be present. Typically, however, the isolated antibody is produced by at least one purification step in which the recombinant cellular material is removed.

The term antibody characteristics refers to factors that contribute to an antibody's recognition of an antigen or the effectiveness of an antibody in vivo. Changes in the amino acid sequence of an antibody can affect properties of the antibody, such as folding, and can affect physical factors such as the initial rate of binding of the antibody to the antigen ( _ka ), the dissociation constant of the antibody from the antigen ( _kd ), the affinity constant of the antibody for the antigen (K _d ), antibody conformation, protein stability, and antibody half-life.

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The term epitope-tagged as used herein refers to an anti-CD40 antibody fused to an epitope tag.

An epitope tag is a polypeptide having sufficient amino acids to enable the epitope to produce an antibody, but designed so that it does not interfere with the desired activity of the humanized anti-CD40 antibody. The epitope tag is usually sufficiently unique such that an antibody raised against the epitope tag does not substantially cross-react with other epitopes. Suitable tag polypeptides typically contain at least 6 amino acid residues and typically contain about 8-50 amino acid residues or about 9-30 residues. Examples of epitope tags and an antibody that binds to the epitope include the flu HA tag polypeptide and its antibody 12CA5 (Field et al., 1988, Mol. Cell. Biol. 8:2159-2165; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies to them (Evan et al., 1985, Mol. Cell. Biol. 5(12):3610-3616; and the herpes simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al ., 1990, Protein Engineering, 3(6):547-553).In some embodiments, the epitope tag is a salvage receptor binding epitope. As used herein, the term salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (such as IgGi, IgG ₂ , IgG3 or IgG ₄ ), which is responsible for increasing the half-life of the IgG molecule in blood serum in vivo.

In some embodiments, antibodies of the present invention may be conjugated to a cytotoxic agent. It is any substance that inhibits or prevents cell function and/or causes cell destruction. The term is intended to include radioactive isotopes (such as I ¹³¹ , I ¹²⁵ , Y ⁹⁰ and Re ¹⁸⁶ ), chemotherapeutic agents and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, as well as fragments thereof. Such cytotoxic agents can be coupled to the humanized antibodies of the present invention using standard procedures and used, for example, to treat a patient who is a candidate for antibody therapy.

A chemotherapy drug is a chemical compound used in the treatment of cancer. There are many examples of chemotherapeutic agents that can be conjugated to therapeutic antibodies in accordance with the present invention. Examples of such chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbochon, meturedopa and uredopa; ethylene imines and methyl melamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including a synthetic analogue of topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin, auristatins (including analogs of monomethyl auristatin E and monomethyl auristatin F); duocarmycin (including synthetic analogues KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomafazine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine; trophosfamide, uracil mustard; nitrosureas nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics (eg, calicheamicin, especially calicheamicin gammaP and calicheamicin phiI1, see, for example, Agnew, Chem. Intl. Ed. Engl., 33:183186; dinemycin, including dinemycin A; bisphosphonates, such as clodronate; esperamycin; as well as neocarzinostatin chromophore and related chromoproteins enediyne antibiotic chromophores), aclacinomysins, actinomycin, outramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-dia zo-5-oxo- Lnorleucine, doxorubicin (Adriamycin™) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin and deoxidoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, pep lomicin, potfiromycin, puromycin, kelamicin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; dromostanolone propionate, epithiostanol, mepithiostane, testolactone; a folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; democolcine; diaziquon; elfomitin; elliptinium acetate; epothilone; ethoglucide; gallium nitrate; hydroxyurea; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone, mitoxantrone; mopidamole; nitracrine; pentostatin; phenomet; pirarubicin; lozok

- 14 046388 santron; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sisofuran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurine A, roridin A and anhydrine); urethane; vindesine; dacarbazine; mannomustin; mitabronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine Navelbine™); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Also included in this definition are antihormonal agents that act by regulating or inhibiting the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifene, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™); aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole ( Femara™) and anastrozole (Arimidex™); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Any one or more of these agents can be conjugated to humanized antibodies in accordance with the present invention to obtain a useful therapeutic agent for the treatment of various disorders.

Antibodies can 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 is capable of being enzymatically activated or converted to a more active form. See, for example, Wilman, 1986, Prodrugs in Cancer Chemotherapy, In Biochemical Society Transactions, 14, p. 375-382, ^615th Meeting Belfast and Stella et al., 1985, Prodrugs: A Chemical Approach to Targeted Drug Delivery, In: Directed Drug Delivery, Borchardt et al., (ed.), p. 247-267, Humana Press. Suitable prodrugs 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, prodrugs containing an optionally substituted phenoxyacetamide, and prodrug properties containing optional substituted phenylacetamide, 5-fluorocytosine, and other prodrugs of 5-fluorouridine, which can be converted into a more active, non-cytotoxic drug. Examples of cytotoxic drugs that can be converted into prodrug form include, but are not limited to, those chemotherapeutic agents described above.

For diagnostic as well as therapeutic monitoring purposes, antibodies according to the invention can also be conjugated to a label, either a single label or a label and an additional second agent (prodrug, chemotherapeutic agent, etc.). A label, unlike other second agents, refers to an agent that is a detectable compound or composition, and it can be conjugated directly or indirectly to the humanized antibody of the present invention. The label may itself be detectable (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical change in the substrate compound or composition that is detectable. Labeled humanized anti-CD40 antibodies can be produced and used in a variety of applications, including in vitro and in vivo diagnostics.

Antibodies in accordance with the present invention can be formulated as part of a liposomal preparation in order to influence its delivery in vivo. A liposome is a small vesicle composed of various types of lipids, phospholipids and/or surfactants. Liposomes are useful for delivering to a mammal a compound or composition, such as a humanized anti-CD40 antibody described herein, optionally in combination with or in combination with one or more pharmaceutically active agents and/or labels. The components of a liposome are typically arranged in a bilayer formation, similar to the arrangement of lipids in biological membranes.

In accordance with the present invention, some aspects relate to isolated nucleic acids that encode one or more domains of the humanized antibodies proposed in the present invention. An isolated nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant

- 15 046388 the host nucleic acid molecule to which it is typically associated in a natural source of antibody nucleic acid. The isolated nucleic acid molecule is different from the nucleic acid molecule that exists in natural cells.

In various aspects of the present invention, one or more domains of the humanized antibodies will be expressed recombinantly. Such recombinant expression may use one or more control sequences, i.e. polynucleotide sequences necessary for the 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. These control sequences can be used to express and produce a humanized anti-CD40 antibody in prokaryotic and eukaryotic host cells.

A nucleic acid sequence is operably linked when it is in operative relationship with another nucleic acid sequence. For example, a nucleic acid presequence or secretory leader is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to the coding sequence if it is positioned to facilitate translation. In general, operably linked means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers are not necessarily contiguous. Linking can be accomplished by ligation into convenient restriction sites. If such regions do not exist, then synthetic oligonucleotide adapters or linkers can be used.

As used herein, the expressions cell, cell line, and cell culture are used interchangeably, and all such designations include their progeny. Thus, transformants and transformed cells include the primary cell in question and the cultures derived from it, without regard to the number of transfers.

The term mammal for medical purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo animals, animals used in sports, or pets such as dogs, horses, cats, cows, etc. P. Preferably the mammal is human.

A disorder as used herein means any condition that can be improved by treatment with a humanized anti-CD40 antibody described herein. It includes chronic and acute disorders or diseases, including those pathological conditions that provoke the diseases in question in a mammal. Non-limiting examples or disorders to be treated according to the present invention include cancer, hematological malignancies, benign and malignant tumors, leukemias and lymphoid malignancies, as well as inflammatory, angiogenic, autoimmune and immunological disorders.

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

As used herein, the term CD40-related disorder or CD40-related disease refers to a condition in which there is modification or elimination of cells expressing CD40. These include CD40-expressing cells that exhibit abnormal proliferation, or CD40-expressing cells that are associated with cancerous or malignant growth. More specific examples of cancers that exhibit abnormal expression of the CD40 antigen include B lymphoblastoid cells, Burkitt's lymphoma, multiple myeloma, T-cell lymphomas, Kaposi's sarcoma, osteosarcoma, epidermal and endothelial tumors, pancreatic, lung, breast, ovarian, colon, prostate, head and neck, skin (melanoma), bladder and kidneys. Such disorders include, but are not limited to, leukemias, lymphomas, including B-cell lymphoma and non-Hodgkin's lymphoma, multiple myeloma, Waldenström's macroglobulinemia; solid tumors including sarcomas such as osteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinoma including ovarian adenocarcinoma, Kaposi's sarcoma/Kaposi's tumor and squamous cell carcinoma.

CD40-related disorder also includes diseases and disorders of the immune system, such as autoimmune disorders and inflammatory disorders. Such conditions include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, multiple sclerosis, psoriasis, inflammatory bowel diseases (eg, ulcerative colitis and Crohn's disease), pneumonia, asthma, and idiopathic thrombocytopenic pur

- 16 046388 puru (ITP).

The phrase growth arrest or growth inhibitor as used herein refers to inhibiting the growth or proliferation of a cell, especially a neoplastic cell type expressing the CD40 antigen. Thus, growth inhibition, for example, significantly reduces the percentage of neoplastic cells in S phase.

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

The term intravenous bolus or intravenous bolus refers to the administration of a drug into a vein of an animal or person such that the body receives the drug in approximately 15 minutes or less, usually in 5 minutes or less.

The term subcutaneous administration refers to the administration of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, through a relatively slow, sustained delivery from a drug container. When the skin is pinched or pulled upward and away from the underlying tissue, a pocket can form.

The term subcutaneous infusion refers to the administration 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 container over a period of time including, but not limited to, 30 minutes or less. or 90 minutes or less. Optionally, the infusion may be accomplished 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, such as 30 minutes, 90 minutes, or a period of time covering the duration of the treatment regimen.

The term subcutaneous bolus refers to the administration of a drug under the skin of an animal or human patient, where the bolus delivery of the drug is less than about 15 minutes; in another aspect less than 5 minutes and in yet another aspect less than 60 seconds. In yet another aspect, administration occurs within a pocket between the skin and the underlying tissue, where the pocket can be created by pinching or pulling the skin upward and away from the underlying tissue.

The term therapeutically effective amount is used to refer to an amount of active agent that reduces or improves one or more symptoms of the disease being treated. Moreover, it is precisely this amount that has a favorable outcome for the patient, for example, it stops growth or causes cell destruction. In one aspect, the therapeutically effective amount has apoptotic activity or is capable of causing 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 the progression of a disease. Effectiveness can be measured in conventional ways depending on the condition being treated. For example, in neoplastic diseases or disorders characterized by CD40-expressing cells, efficacy may be measured by assessing the time to disease progression or determining the rate of response.

The terms treatment and therapy, etc., as used herein are intended to include therapeutic as well as prophylactic or suppressive measures for a disease or disorder resulting in any clinically desirable or beneficial effect, including, but not limited to, mitigation or attenuation one or more symptoms, regression, slowing or stopping the progression of a disease or disorder. Thus, for example, the term treatment includes administering an agent before or after the onset of a symptom of a disease or disorder, thereby preventing or removing one or more symptoms of the disease or disorder. In addition, the administration of an agent after the onset and after the development of clinical symptoms, when the administration affects the clinical parameters of the disease or disorder, such as the degree of tissue damage or the amount or extent of metastasis, whether the treatment results in improvement or not, the disease includes yourself treatment or therapy in the context of this description. Moreover, as long as the compositions of the invention, either alone or in combination with another therapeutic agent, alleviate or reduce at least one symptom of the disease being treated compared to that symptom in the absence of use of the humanized antibody composition against CD40 result should be considered as an effective treatment of the underlying disease, regardless of whether all symptoms of the disorder are relieved or not.

The term package insert is used to refer to instructions typically included in packages of therapeutic products for sale that contain information regarding the indications, use, administration, contraindications and/or warnings regarding the use of such therapeutic products.

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Antibodies.

Described and disclosed herein are humanized anti-CD40 antibodies, as well as compositions and articles of manufacture containing one or more humanized anti-CD40 antibodies in accordance with the present invention. Antibodies in accordance with the present invention are also disclosed in US Patent No. 8,591,900 and WO 2011/123489, the contents of each of which are incorporated herein by reference. Binding agents that include an antigen binding fragment of a humanized anti-CD40 antibody are also described. Humanized anti-CD40 antibodies and binding agents may arrest cell growth, cause the elimination of CD40-expressing cells, or otherwise induce or cause a cytotoxic or cytostatic effect on target cells. Humanized anti-CD40 antibodies and binding agents can be used in the treatment of a variety of diseases or disorders characterized by the proliferation of cells expressing the CD40 surface antigen. The humanized anti-CD40 antibody and CD40 binding agent include at least a portion that specifically recognizes a CD40 epitope (ie, an antigen-binding fragment).

In initial characterization, mouse antibodies were selected based on CD40 binding characteristics.

From these initial studies, murine antibodies were selected that had the following heavy chain variable regions shown in Table 1. 1, and the light chain variable regions shown in table. 2:

Table 1

Lead mouse anti-CD40 antibodies VH sequences.

EVQLQQSGAELVRPGASVKLSCTASGFNIKDYYVHWVKQRPEKGLEWIGR IDPEDGDSKYAPKFQGKATMTADTSSNTAYLHLSSLTSEDTAVYYCTTSY 2Н11 YVGTYGYWGQGTTLTVSS (SEQ ID NO:1)

EVQLQQSGAELVRPGASVKLSCTASGFNIKDYYIHWVKQRPEKGLEWIGR IDPEDGDTKYDPKFQGKATMTADTSSNTAYLHLSSLTSEDTAVYYCTTSY 10F2 YVGTYGYWGQGTTLTVSS(SEQ ID NO:2)

EVQLQQSGAELVRPGASVQLSCTASGFNIKDYYVHWVKQRPEKGLEWIGR IDPEDGDTKFAPKFQGKATMTADTSSNTVYLHLSSLTSEDTAVYYCTTSY

19B10 YVGTYGYWGQGTTLTVSS(SEQ ID NO:3)

EVQLVESGGGLVKPGGSRKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAY ISSGNRIIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTALYYCARQD

20E2 GYRYAMDYWGQGTSVTVSS(SEQ ID NO:4)

table 2

Lead mouse anti-CD40 antibodies V _K sequences

QIVLTQSPAIMSASPGEKVTITCSASSSVSYMLWFQQKPGTSPKLWIYST SNLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRTFYPYTFGGG 2H11 TKLEIK (SEQ ID NO:5)

QIVLTQSPTIMSASPGEKVIITCSATSSVSYILWFQQKPGTSPKLWIYST SNLASGVPARFSGSGSGASYSLTISRMEAEDAATYYCQQRTFYPYTFGGG 10F2 TKLEIK (SEQ ID NO:6)

QIVLTQSPAIMSASPGEKVTITCSASSSVSYMLWFQQKPGTSPKLWIYST SNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRTFYPYTFGGG

19B10 TKLEIK (SEQ ID NO:7)

DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWHQQKPGQPP KLLIYWTSTRESGVPDRFTGSGSGTDFTLTISNLQAEDLAVYYCQNDYTY 20E2 PLTFGAGTKLELK (SEQ ID NO:8)

Human framework sequences were selected for each of the mouse lead antibodies based on framework homology, CDR structure, conserved canonical residues, conserved interface packaging residues, and other parameters.

The murine heavy chain and light chain CDRs of the various selected murine antibodies are shown in Table 1. 3 and 4 respectively:

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Table 3

Heavy chain CDR sequences

Name designs 2N11 H-CDR1 GFNIKDYYVH H-CDR2 RIDPEDGDSKYAPKFQG H-CDR3 SYYVGTYGY SEQ ID NO:9 SEQ ID NO:12 SEQ ID NO:16 10F2 GFNIKDYY/H RIPPED GD TKYDPKFQ G SYYVGTYGY 19B10 SEQ ID NO:10 GFNIKDYYVH SEQ ID NO:13 RIDPEDGDTKFA PKFQ G SEQ ID NO:16 SYYVGTYGY 20E2 SEQ ID NO:9 GFTFSDYGMH SEQ ID NO:14 YISSGNRIIYYADTVKG SEQ ID NO:16 QDGYRYAMDY SEQ ID NO:11 SEQ ID NO:15 SEQ ID NO:17 In the above H-CDR1 used sequence with using a numbering system

by Chothia (Al-Lazikani et al., (1997), JMB, 273, 927-948). Kabat numbering for the sequences is indicated in bold italic and IMGT numbering is shown in the underlined text of the residues in the table above for CDR1 and CDR2. The H-CDR3 sequence for each of 2H11, 10F2 and 19B10 is TTSYYVGTYGY (SEQ ID NO: 77), and for 20E2 is ARQDGYRYAMDY (SEQ ID NO: 78).

Table 4 Light chain CDR sequences Name

designsE-CDRl L-CDR2 STSNLAS L-CDR3 QORTFYPYT 2N11 SASSSVSYML 10F2 SEQ ID NO:18 SATSSVSYIL SEQ ID NO:22 STSNLAS SEQ ID NO:24 QORTFYPYT 19B10 SEQ ID NO:19 SASSSVSYML SEQ ID NO:22 STSNLAS SEQ ID NO:24 QORTFYPYT 20E2 SEQ ID NO:20 SEQ ID NO:22 KSSQSLLNSGNQKNYL T WTSTRES SEQ ID NO:24 ONDYTYPLT SEQ ID NO:21 SEQ ID NO:23 SEQ ID NO:25

Again, the Hotia numbering system is used in the table. 4, where the Kabat numbering for the sequences is shown in bold italic text and the IMGT numbering is shown in underlined text.

Fabs that showed better or equal binding compared to the chimeric parent Fab were selected for conversion to IgG. Clones from the 20E2 series were converted into two different formats: a) IgG4DM (double mutant) has two mutations in the Fc/hinge region, Ser228Pro, which reduces hemimolecule formation and Leu235Glu, which further reduces FcyR binding; b) IgG ₁ -KO (effector function knockout) has two mutations in the Fc region, Leu234Ala and Leu235Ala, which reduce effector function such as FcyR and complement fixation. Both IgG formats are described in the literature. Example 1 describes the humanization of three candidates in more detail. The result of this humanization is humanized antibody sequences that have the heavy and light chain sequences shown below.________________

Identity Subsequence SEQ ID NO: Antibody A (light chain) DIVMTQSPDSLAVSLGERATMSCKSSQSLLNSGNQKNYLTW HQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTIS 26 SLQAEDVAVYYCQNDYTYPLTFGGGTKVEIKRTVAAP S VFI

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FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC Antibody A (heavy chain, IgGlKO) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ 27 MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS NE D R EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody A (heavy chain, IgGl) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ 28 MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS NE D R EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody A (heavy chain, IgG4DM) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 29 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDT LMIS RT P EVT CVVVDVS Q E D P EVQ FNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody A (heavy chain, IgGlKOb) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP thirty GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS HE D P EVK FNWYVD GVEVH

- 20 046388

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (light chain) DIVMTQSPDSLAVSLGEKVTINCKSSQSLLNSGNQKNYL 31 TWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Antibody B (heavy chain, IgGlKO) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 32 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS NE D R EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (heavy chain, IgGl) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 33 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS NE D R EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (heavy chain, IgG4 DM) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 34 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDT LMIS RT P EVT CVVVDVS Q E D P EVQ FNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody B (heavy chain, IgGlKOb) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 35 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS HE D P EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

- 21 046388

Antibody C (light chain) DIQMTQSPSSLSSASVGDRVTITCSASSSVSYMLWFQ 36 QKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody C (heavy chain, IgGlKO) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 37 GQGLEWMGRID PED GD SKYAPKFQGKATMTAD TS T S TVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSAS T KG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 38 GQGLEWMGRID PED GD SKYAPKFQGKATMTAD TS T S TVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSAS T KG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (heavy chain, IgG4 DM) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 39 GQGLEWMGRID PED GD SKYAPKFQGKATMTAD TS T S TVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSAS T KG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPK DT LMIS RT P EVT CVVVDVS Q E D P EVQ FNWYVD GVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody C (heavy chain, IgGlKOb) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 40 GQGLEWMGRID PED GD SKYAPKFQGKATMTAD TS T S TVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSAS T KG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, the antigen binding moiety may, for example, block proliferation or otherwise arrest the growth of a cell or cause it to starve, die, or otherwise be eliminated, for example, by binding to the CD40 surface antigen. For example, in T- and B-cell malignancies, antitumor effects (eg, growth arrest with or without cell removal or apoptosis) often occur when malignant cells are exposed to stimuli that lead to activation of normal lymphocytes. This activation-induced growth arrest has been observed by signaling through antigen receptors or costimulatory receptors (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). Stimulation of CD40 through specific binding by either an antibody or a soluble ligand inhibits the growth of B-cell lymphoma (see, eg, Funakoshi et al., 1994, Blood, 83:27872794). Agents which thus inhibit the growth of malignant cells and which are directed against the CD40 surface antigen are examples of suitable agents.

CD40-specific agents include an antigen-binding fragment of a humanized anti-CD40 antibody that binds to CD40 (eg, human CD40 or a variant thereof). CD40-specific agents and antibodies may optionally be conjugated or fused to a cytotoxic or chemotherapeutic agent. In aspects where the humanized antibody binds to the CD40 surface antigen and causes depletion of cell types expressing CD40, the binding is typically characterized by homing to the cell of the CD40 surface antigen in vivo. Suitable binding agents bind CD40 antigen with sufficient affinity and/or avidity such that the specific agent is useful as a therapeutic agent by specifically targeting a cell expressing the antigen.

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

- 22 046388 or at least 80%, or at least 90%, or at least 95%.

In some embodiments, the humanized anti-CD40 antibodies, including antigen binding fragments thereof, such as heavy and light chain variable domains, comprise an amino acid sequence of residues derived from the Antibody A CDR (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) as described herein above, and amino acid residues derived from human immunoglobulin framework regions. Humanized anti-CD40 antibodies do not necessarily include specific amino acid substitutions in germline consensus or framework regions.

Specific substitution of amino acid residues at these framework positions can improve various aspects of antibody performance, including binding affinity and/or stability, compared with that demonstrated in humanized antibodies formed by direct replacement of CDRs or HVLs into human germline framework regions, as shown in examples below.

In some embodiments, the present invention discloses other monoclonal antibodies having heavy chain (VH) sequences as set forth in SEQ ID NO: 1SEQ ID NO: 4 and light chain (V _L ) sequences as set forth in SEQ ID NO: 5 SEQ ID NO: : 8 (see tables 1 and 2 above). The CDR sequence of these murine antibodies is shown in Table. 3 and 4, placement of such CDRs in the FRs of the human heavy and light chain consensus variable domains will yield useful humanized antibodies in accordance with the present invention.

In some specific embodiments, the humanized anti-CD40 antibodies described herein contain at least a heavy or light chain variable domain containing the CDR or HVL of the murine monoclonal antibodies, as shown in Table 1. 1-4 above and FR of the human germline heavy and light chain variable domains. In exemplary embodiments, the humanized antibodies generated in accordance with the invention are Antibody A, Antibody B, and Antibody C, and the various heavy and light chain sequences thereof are shown in SEQ ID NO: 26 through SEQ ID NO: 40.

In certain embodiments, antibodies are provided that have a heavy chain sequence of any of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30 in combination with a light chain sequence of SEQ ID NO: 26. Alternative antibodies include antibodies that have the heavy chain sequence of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35 in combination with the light chain sequence of SEQ ID NO: 31. In still further embodiments, humanized antibodies that have the heavy chain sequence of SEQ ID NO: 37, SEQ ID NO: 38; SEQ ID NO: 39 or SEQ ID NO: 40 in combination with the light chain sequence of SEQ ID NO: 36.

The CDRs of these sequences are shown in Table. 3 and 4. In certain embodiments, it is contemplated that chimeric antibodies with switched CDR regions (i.e., for example, switching one or two CDRs of Antibody A with a similar CDR of Antibody C) between these exemplary immunoglobulins may produce useful antibodies.

In some embodiments, the humanized anti-CD40 antibody is an antibody fragment. The various antibody fragments have been discussed generally above and there are methods that have been developed to obtain antibody fragments. Fragments can be obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods, 24:107-117; and Brennan et al., 1985, Science, 229:81). Alternatively, fragments can be produced directly in recombinant host cells. For example, Fab'-SH fragments can be directly isolated from E. coli and chemically coupled to form F(ab') ₂ fragments (see, for example, Carter et al., 1992, Bio/Technology, 10:163-167) . Using another approach, F(ab') ₂ fragments can be isolated directly from recombinant host cell cultures. Other methods for producing antibody fragments will be apparent to one skilled in the art.

Some embodiments include an F(ab') ₂ fragment of a humanized anti-CD40 antibody comprising a heavy chain sequence of any of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30 in combination with a light chain sequence chains SEQ ID NO: 26. Alternative antibodies include antibodies that have the heavy chain sequence SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35 in combination with the light chain sequence SEQ ID NO: 31. In still further embodiments, humanized antibodies are provided that have the heavy chain sequence of SEQ ID NO: 37, SEQ ID NO: 38; SEQ ID NO: 39 or SEQ ID NO: 40 in combination with the light chain sequence of SEQ ID NO: 36. Such embodiments may include an intact antibody containing such F(ab') ₂ .

In some embodiments, the antibody or antibody fragment includes a constant volume

- 23 046388 part that mediates the effector function. The constant region may mediate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC) responses against target cells expressing CD40. The effector domain(s) may be, for example, the Fc region of the Ig molecule. Typically, the CD40 binding agent attracts and/or activates cytotoxic leukocytes (eg, natural killer (NK) cells, phagocytotic cells (eg, macrophages), and/or serum complement components).

The antibody effector domain can be derived from any suitable vertebrate species and isotype. Isotypes from different animal species differ in their ability to mediate effector functions. For example, the ability of human immunoglobulin to mediate CDC and ADCC/ADCP is usually in the following order:

IgM~IgGi~IgG ₃ >IgG2>IgG..| and IgG ₁ »IgG ₃ >IgG2/IgM/IgG. ₁ respectively.

Mouse immunoglobulins mediate CDC and ADCC/ADCP, usually in the following order: mouse IgM»IgG ₃ >>IgG _2b >IgG _2a >>IgG ₁ and IgG _2b >IgG _2a >IgG ₁ >>IgG ₃ , respectively.

In another example, mouse IgG _2a mediates ADCC, while mouse IgG _2a and IgM mediate CDC.

Antibody modifications.

Humanized anti-CD40 antibodies and agents may include modifications of a humanized anti-CD40 antibody or an antigen-binding fragment thereof. For example, it may be desirable to modify an antibody with respect to effector function to increase the effectiveness of the antibody in treating cancer. One such modification is the introduction of cysteine residue(s) into the Fc region, which allows the formation of interchain disulfide bonds in this region. The homodimeric antibody thus produced may have improved internalization capacity and/or increased complement-mediated cell death and/or antibody-dependent cellular cytotoxicity (ADCC). See, for example, Caron et al., 1992, J. Exp. Med. 176:1191-1195; and Shopes, 1992, J. Immunol. 148:2918–2922. Homodimeric antibodies having enhanced antitumor activity can also be produced using heterobifunctional cross-linkers as described in Wolff et al., 1993, Cancer Research, 53:2560-2565. Alternatively, the antibody can be engineered to contain dual Fc regions, enhancing complement lysis and ADCC potential of the antibody. See Stevenson et al., 1989, Anti-Cancer Drug Design, 3:219230.

Antibodies with improved ability to support ADCC have been generated by modifying the glycosylation pattern of their Fc region. This is possible because antibody glycosylation at the asparagine residue, N297, in the C _H2 domain is involved in the interaction between IgG and Fcy receptors, which is a prerequisite for ADCC. Host cell lines have been engineered to express antibodies with altered glycosylation, such as increased N-acetylglucosamine bisection or decreased fucose content. Decreasing the amount of fucose provides a greater enhancement of ADCC activity than increasing the presence of bisected N-acetylglucosamine. Moreover, the enhancement of ADCC by low-fucose antibodies is independent of the FcYRIIIa V/F polymorphism.

Altering the amino acid sequence of the Fc region of antibodies is an alternative to glycosylation engineering to enhance ADCC. The human IgG ₁ binding site for Fcy receptors was determined by extensive mutational analysis. This has led to the development of humanized IgG ₁ antibodies with Fc mutations that increase binding affinity to FcYRIIIa and enhance ADCC in vitro. In addition, Fc variants have been produced with a variety of different binding properties, such as improved binding to specific FcyR receptors with unchanged or reduced binding to other FcyR receptors.

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

Chemotherapeutic agents suitable for the preparation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can 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, A-chain modeccin chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restricttocin, fenomycin, enomycin, tricothecenes and so on. Various radionuclides are available to produce radioactively conjugated humanized anti-CD40 antibodies. Examples include ²¹² Bi, ¹³¹ 1, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Conjugates of a humanized anti-CD40 antibody and a cytotoxic or chemotherapeutic agent

- 24 046388 These agents can be prepared by known methods using various bifunctional protein binding agents such as H-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine) , diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the immunotoxin ricin can be prepared as described in Vitetta et al., 1987, Science, 238:1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for radionucleotide-antibody conjugation. Conjugates can also be formed with a cleavable linker.

In another embodiment, the antibody may be conjugated to a receptor (such as streptavidin) for use in tumor pre-targeting. In this procedure, an antibody-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the bloodstream using a scavenger and then administration of a ligand that selectively binds the receptor (eg, avidin), the ligand being conjugated to a cytotoxic agent (eg, radionuclide).

The humanized anti-CD40 antibodies described herein can also be formulated as immunoliposomes. Antibody-containing liposomes are prepared by methods known in the art, such as those described in Epstein et al., 1985, Proc. Nail. Acad. Sci. USA, 82:3688; Hwang et al., 1980, Proc. Natl. Acad. Sci. USA, 77:4030; and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes having an extended circulation time are disclosed, for example, in US Pat. No. 5,013,556.

Particularly useful liposomes can be prepared by reverse phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol and a derivative and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of specific pore sizes to obtain liposomes of the desired diameter. Fab' fragments of an antibody disclosed herein can be conjugated to liposomes as described in Martin et al., 1982, J. Biol. Chem. 257:286-288 via disulfide exchange reaction. Optionally, the liposomes contain a chemotherapeutic agent (such as doxorubicin). See, for example, Gabizon et al., 1989, J. National Cancer Inst. 81(19): 1484.

The antibodies described and disclosed herein can also be used in ADEPT (antibody directed enzyme prodrug therapy) procedures by conjugating the antibody to a prodrug activating enzyme that converts the prodrug (eg, a peptidyl chemotherapy agent) into an active anticancer drug. See, for example, WO 81/01145, WO 88/07378 and US Patent No. 4,975,278. The enzyme component of an immunoconjugate suitable for ADEPT is an enzyme capable of acting on a prodrug in such a way as to convert it to its more active cytotoxic form. Specific enzymes that can be used in ADEPT include, but are not limited to, alkaline phosphatase, capable of converting phosphate-containing prodrugs into free drugs; arylsulfatase, capable of converting sulfate-containing prodrugs into free drugs; a cytosine deaminase capable of converting the non-toxic 5-fluorocytosine anticancer drug, 5-fluorouracil; proteases such as serratia pretease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L) to convert peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases for the conversion of prodrugs containing D-amino acid substituents; carbohydrate-degrading enzymes such as β-galactosidase and neuraminidase to convert glycosylated prodrugs into free drugs; β-lactamase for converting drugs derivatized by β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, to convert drugs derivatized at their amine nitrogen atoms with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity (abzymes) can be used to convert prodrugs into free active drugs (see, for example, Massey, 1987, Nature, 328:457-458). Antibody-abzyme conjugates can be prepared by known methods to deliver the abzyme to a population of tumor cells, for example, by covalently linking the enzyme to the humanized anti-CD40 antibody/heterobifunctional cross-linking reagents described above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody described herein linked to at least a functionally active portion of the enzyme as described above can be constructed using recombinant DNA techniques (see, e.g., Neuberger et al. , 1984, Nature, 312:604-608).

In some embodiments, it may be desirable to use a humanized anti-CD40 antibody fragment rather than the intact antibody, for example, to increase tumor penetration. It may be advisable to modify the antibody fragment to increase its half-life in serum. This can be achieved, for example, by including a rescue receptor binding epitope in the antibody fragment. In one method, the corresponding region of the antibody fragment can be changed (eg, mutated) or the epitope can be included in a peptide tag, which is then fused to the antibody fragment at either end or in the middle, for example, through DNA or peptide synthesis. See, for example, WO 96/32478.

Other embodiments also include covalent modifications of the humanized anti-CD40 antibody. Covalent modifications include modification of cysteinyl residues, histidyl residues, lysinyl and amino-terminal residues, arginyl residues, tyrosyl residues, pendant carboxyl groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues, or seryl or threonyl residues. Another type of covalent modification involves chemical or enzymatic coupling of glycosides to the antibody. Such modifications can be made by chemical synthesis or enzymatic or chemical degradation of the antibody, if applicable. Other types of covalent modifications of an antibody can be introduced into the molecule by reacting the target amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or amino- or carboxy-terminal residues.

Removal of any carbohydrate moieties present in antibodies can be accomplished chemically or enzymatically. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved using a variety of endo- and exoglycosidases, as described in Thotakura et al., 1987, Meth. Enzymol. 138:350.

Another type of useful covalent modification involves linking the antibody to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in a manner set forth in one or more of U.S. Pat. 37.

Humanization and amino acid sequence variants.

Variants of the amino acid sequence of the anti-CD40 antibody can be obtained by making appropriate nucleotide changes in the DNA of the anti-CD40 antibody or by peptide synthesis. Such variants include, for example, deletions from and/or insertions into, and/or substitutions of residues in the amino acid sequences of the anti-CD40 antibodies of the examples provided herein. Any combination of deletions, insertions and substitutions is made to produce the final construct, provided that the final construct has the desired characteristics. Amino acid changes may also alter post-translational processes of the humanized or variant CD40 antibody, such as changing the number or position of glycosylation sites.

A useful method for identifying specific residues or regions of an anti-CD40 antibody that are preferred sites for mutagenesis is called alanine scan mutagenesis, as described by Cunningham and Wells (Science, 244:1081-1085 (1989)). Here, a residue or group of target residues (eg, charged residues such as arg, asp, his, lys and glu) are identified and replaced with a neutral or negatively charged amino acid (usually alanine) to influence the interaction of the amino acids with the CD40 antigen. Those amino acid positions that exhibit functional sensitivity to substitution are then refined by introducing additional or different variants at or in place of the substitution sites. Thus, although the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the effectiveness of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed anti-CD40 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 100 residues or more, as well as intra-sequence insertions of one or more amino acid residues. Examples of end inserts include an anti-CD40 antibody fused to an epitope tag. Other insertion variants of the anti-CD40 antibody molecule include fusion to the N- or C-terminus of the anti-CD40 antibody with an enzyme or polypeptide that increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. In these embodiments, at least one amino acid residue in the anti-CD40 antibody molecule is deleted and another residue is inserted in its place. Sites of greatest interest for replacement mutagenesis include hypervariable regions, but FR changes are also considered. Conservative substitutions are shown in table. 5 under the heading Preferred Substitutions. If such substitutions result in a change in biological activity, then more significant changes may be made, called Example Substitutions, or, as further described below with reference to amino acid classes, producing 26 046388

you've been screened. Table 5 Original Approximate Preferred remainder replacements replacements Ala (A) val; leu; ile val Arg(R) lys; gin; asn lys Asn(N) gin; his; asp, lys; arg gin Asp (D) glu; asn glu Cys(C) ser; ala ser Gin (Q) asn; glu asn Glu(E) asp; gin asp Gly (G) ala ala His(H) arg; asn; gin; lys; arg He(I) leu; val; met; ala; phe; norleucine leu Leu (L) ile; norleucine; val; met; ala; phe ile Lys (K) arg; gin; asn arg Met(M) leu; phe; ile leu Phe(F) tyr; leu; val; ile; ala; tyr Pro (P) ala ala Ser(S) thr thr Thr(T) ser ser Trp(W) tyr; phe tyr Tyr (Y) phe;trp; thr; ser phe Val(V) leu; ile; met; phe ala; norleucine; leu

In protein chemistry, it is generally accepted that the biological properties of an antibody can be achieved by selecting substitutions that vary significantly in their effect on maintaining (a) the structure of the polypeptide backbone at the site of the substitution, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (in) the main part of the side chain. Naturally occurring residues are divided into groups based on general side chain properties:

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

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

(3) acidic: asp, glu;

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

(5) residues affecting chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail replacing a member of one of these classes with another class.

Any cysteine residue not involved in maintaining the proper conformation of the humanized or variant anti-CD40 antibody may also be replaced, typically with serine, to improve the oxidative stability of the molecule, prevent aberrant cross-linking, or provide established points of conjugation with a cytotoxic or cytostatic compound. Conversely, cysteine bond(s) may be added to the antibody to increase its stability (especially when the antibody is an antibody fragment, such as an Fv fragment).

The type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Typically, the resulting variants selected for further development will have improved biological properties compared to the parent antibody from which they are derived. A convenient way to generate such substitution variants is affinity maturation using phage display. Briefly, several hypervariable region sites (eg, 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus produced are displayed monovalently from the filamentous phage particles as a fusion with the M13 gene III product packaged in each particle. The variants presented on the phage are then screened for their biological activity (eg, binding affinity). To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or in addition, it may be useful to analyze

- 27 046388 to determine the crystal structure of the antigen-antibody complex to determine the points of contact between the antibody and human CD40. Such contact residues and adjacent residues are candidates for replacement according to the methods developed in the present invention. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more appropriate assays can be selected for further development.

Another type of amino acid variant of an antibody changes the original glycosylation pattern of the antibody. By change is meant the deletion of one or more carbohydrate moieties found in the antibody and/or the addition of one or more glycosylation sites that are 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 usually N-linked or O-linked. N-linking refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagineX-threonine, where X is any amino acid other than proline, are recognition sequences for enzymatically attaching a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the addition 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 can also be used. Thus, to glycosylate a given protein, such as an antibody, the amino acid sequence of the protein is designed to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The change can also be made by adding or replacing one or more serine or threonine residues in the sequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules that encode variants of the amino acid sequence of the anti-CD40 antibody are prepared by various methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or production by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier derived variant or non-variant version of the antibody against CD40.

Polynucleotides, vectors, host cells and recombinant methods.

Other embodiments include isolated polynucleotides that contain a sequence encoding a humanized anti-CD40 antibody, vectors and host cells containing the polynucleotides, and recombinant methods for producing the humanized antibody. The isolated polynucleotides may encode any desired form of anti-CD40 antibody, including, for example, full-length monoclonal antibodies, Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments .

Some embodiments include isolated polynucleotides comprising sequences that encode an antibody or antibody fragment having a heavy chain variable region amino acid sequence of any of SEQ ID NO: 1-4, 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 or SEQ ID NO: 40. Some embodiments include isolated polynucleotides comprising sequences that encode 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(s) encodes an antibody or antibody fragment having a heavy chain variable domain and a light chain variable region comprising the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 26, respectively; SEQ ID NO: 28 and SEQ ID NO: 26 respectively; SEQ ID NO: 29 and SEQ ID NO: 26 respectively;

SEQ ID NO: 30 and SEQ ID NO: 26 respectively; SEQ ID NO: 32 and SEQ ID NO: 31 respectively;

SEQ ID NO: 33 and SEQ ID NO: 31, respectively; SEQ ID NO: 34 and SEQ ID NO: 31 respectively;

SEQ ID NO: 35 and SEQ ID NO: 31, respectively; SEQ ID NO: 37 and SEQ ID NO: 36, respectively;

SEQ ID NO: 38 and SEQ ID NO: 36 respectively; SEQ ID NO: 39 and SEQ ID NO: 36, respectively;

SEQ ID NO: 40 and SEQ ID NO: 36 respectively.

The polynucleotide(s) that contain a sequence encoding a humanized anti-CD40 antibody, or a fragment or chain thereof, may be fused to one or more regulatory or control sequences as known in the art, and may be contained in suitable expression vectors or cells. the owner, as is known from the prior art. Each of the polynucleotide molecules encoding a heavy or light chain variable domain can be independently fused to a polynucleotide sequence encoding a constant domain, such as constant

- 28 046388 human domain, which allows the production of intact antibodies. Alternatively, the polynucleotides or portions thereof may be fused together to provide a template for the production of a single chain antibody.

For recombinant production, the polynucleotide encoding the antibody is inserted into a replicable vector for cloning (DNA amplification) or expression. A variety of suitable vectors for expression of the recombinant antibody are available. Vector components typically 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 element, a promoter, and a transcription termination sequence.

Humanized anti-CD40 antibodies can also be prepared 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 selected heterologous signal sequence is typically recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize or process the signal sequence of a humanized anti-CD40 antibody, the signal sequence can be replaced with a prokaryotic signal sequence. The signal sequence may be, for example, alkaline phosphatase, penicillinase, lipoprotein, thermostable enterotoxin II leaders, and the like. For yeast secretion, the native signal sequence can be replaced, for example, by a leader sequence derived from yeast invertase alpha factor (including Saccharomyces and Kluyveromyces afactor leaders), acid phosphatase, C. albicans glucoamylase, or the signal described in WO 90/13646. In mammalian cells, mammalian signal sequences can be used, as well as viral secretory leaders, such as the herpes simplex gD signal. The DNA for such a precursor region is ligated in frame to DNA encoding a humanized anti-CD40 antibody.

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

Expression and cloning vectors may contain a gene encoding a selectable marker to facilitate identification of expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, or alternatively are auxotrophic complement deficiencies, or in other alternatives supply certain nutrients that are not present in complex environments , for example the gene encoding D-alanine racemase for Bacilli.

In one example of a selection scheme, a drug is used to stop the growth of a host cell. Those cells that are successfully transformed by a heterologous gene produce a protein that confers drug resistance and thus survive the selection regime. Examples of such dominant selection include the drugs neomycin, mycophenolic acid and hygromycin. Common selectable markers for mammalian cells are those that identify cells capable of receiving a nucleic acid encoding a humanized anti-CD40 antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and -II (such as primate metallothionein genes), adenosine deaminases, ornithine decarboxylase, etc. Cells transformed with the DHFR selection gene are first identified by culturing all transformants in culture medium containing methotrexate methotrexate (Mtx), a competitive antagonist of DHFR. When using wild-type DHFR, a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (eg, DG44).

Alternatively, host cells (especially wild-type hosts that contain endogenous DHFR) transformed or cotransformed with DNA sequences encoding an anti-CD40 antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'phosphotransferase (APN) may be selected by growing cells in a medium containing a selection agent for a selection marker, such as an aminoglycoside antibiotic such as kanamycin, neomycin or G418. See, for example, US Patent No. 4,965,199.

If recombinant production is carried out in a yeast cell as a host cell, then the TRP1 gene, present in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature, 282:39), can be

- 29 046388 use as a selection marker. The TRP1 gene provides a selection marker for a mutant yeast strain lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics, 85:12). The presence of trp1 damage in the host yeast cell genome then provides an effective environment for detecting growth transformation in the absence of tryptophan. Similarly, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626 are complemented with known plasmids carrying the LEU2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces yeast. Alternatively, an expression system for large-scale production of recombinant calf chymosin has been described for K. lactis (Van den Berg, 1990, Bio/Technology, 8:135). Stable multicopy expression vectors for the secretion of mature recombinant human serum albumin by commercial Kluyveromyces strains have also been described (Fleer et al., 1991, Bio/Technology, 9:968-975).

Expression and cloning vectors typically contain a promoter that is recognized by the host and is operably linked to a nucleic acid molecule encoding an anti-CD40 antibody or polypeptide chain thereof. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgamo (S.D.) sequence operably linked to the DNA encoding the humanized anti-CD40 antibody.

Many eukaryotic promoter sequences are known. Almost all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream of the site where transcription begins. Another sequence found 70-80 bases upstream of the transcription start of many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the sequence AATAAA, which can be a signal for adding a poly-A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include promoters for 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.

Inducible promoters have the additional advantage that transcription is regulated by growth conditions. These include the yeast promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, derivative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Suitable vectors and promoters for use in yeast expression are further described in EP 73657. Yeast enhancers are also advantageously used with yeast promoters.

Transcription of humanized anti-CD40 antibody from vectors in mammalian host cells is controlled, for example, by promoters derived from the genomes of viruses such as polyoma virus, fowl pox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40), from heterologous mammalian promoters, such as the actin promoter or immunoglobulin promoter, or from heat shock promoters, provided that such promoters are compatible with host cell systems.

The SV40 early and late promoters are conveniently obtained as an SV40 restriction fragment that also contains the SV40 origin of replication. The human cytomegalovirus immediate early promoter is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is disclosed in US Pat. No. 4,419,446. A modification of this system is described in US Patent No. 4,601,978. See also Reyes et al., 1982, Nature, 297:598-601, revealing the expression of human p-interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

Another useful element that can be used in a recombinant expression vector is an enhancer sequence, which is used to enhance transcription of DNA encoding a humanized anti-CD40 antibody by higher eukaryotes. Currently, many enhancer sequences of mammalian genes are known (eg, globin, elastase, albumin, α-fetoprotein, and insulin). However, a eukaryotic cell virus enhancer is usually used. Examples include the SV40 enhancer on the late side of the origin of replication (100-270 bp

- 30 046388 bases), cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the origin of replication, and adenovirus enhancers. See also Yaniv, 1982, Nature, 297:17-18 for a description of enhancing elements for activation of eukaryotic promoters. The enhancer can be spliced into the vector at a position 5' or 3' relative to the sequence encoding the humanized anti-CD40 antibody, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms)? may also contain sequences necessary to terminate transcription and stabilize mRNA. Such sequences are usually available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-CD40 antibody. One useful component of transcription termination is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vector disclosed therein. In some embodiments, humanized anti-CD40 antibodies can be expressed using the CHEF system. (See, for example, US Pat. No. 5,888,809; the disclosure of which is incorporated herein by reference.)

Suitable host cells for cloning or expression of DNA in vectors in this case are prokaryotic, yeast or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example Enterobacteriaceae, such as Escherichia, for example E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (for example, B. licheniformis 41 P disclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa and Streptomyces. One of the preferred hosts for cloning E. coli is E. coli 294 (ATCC 31.446), although other strains such as E. coli B, E. coli X1776 (ATCC 31.537) and E. coli W3110 (ATCC 27.325) are suitable. These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for cloning or expression of humanized vectors encoding anti-CD40 antibody. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used lower eukaryotic microorganism host. However, a number of other genera, species and strains are commonly available and useful in this case, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, for example, K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906 ), K. thermotolerans and K. marxianus; yarrowia (EP 402226); Pichia pastors (EP 183070); Candida; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for expression of the glycosylated humanized anti-CD40 antibody are from multicellular organisms. Examples of invertebrate cells include plant and insect cells, including, for example, numerous strains and variants of baculoviruses and corresponding insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori (silkworm). A variety of viral strains for transfection are publicly available, such as Autographa californica NPV variant L-1 and Bombyx mori NPV strain Bm-5, and such viruses can be used in particular for transfecting Spodoptera frugiperda cells.

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

In another aspect, expression of a humanized anti-CD40 antibody is carried out in vertebrate cells. Propagating vertebrate cells in culture (tissue culture) has become a routine procedure and methods are widely available. Examples of suitable mammalian host cell lines are the monkey kidney line CV1 transformed with SV40 (COS-7, ATCC CRL 1651), the human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture (Graham et al., 1977, J. Gen Virol. 36:59), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA, 77 :4216; e.g. DG44), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1, ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), Buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL 51), TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68), MRC 5 cells, FS4 cells and human hepatoma lines (Hep G2).

Host cells are transformed with the expression or cloning vectors described above to

- 31 046388 production of humanized anti-CD40 antibodies and cultured in conventional culture media modified as required for induction of promoters, selection of transformants or amplification of genes encoding the desired sequences.

The host cells used to produce the humanized anti-CD40 antibody described herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma-Aldrich Co., St. Louis, Mo.), minimum essential medium (MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich Co.) is suitable for culturing host cells. In addition, any of the media described in one or more of the literature Ham et al., 1979, Meth. Enz. 58:44, Barnes et al., 1980, Anal. Biochem. 102:255, US Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, WO 90/103430 and WO 87/00195, can be used as a culture medium for host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace minerals (defined as inorganic compounds typically present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Other additives may also be included in suitable concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to one of ordinary skill in the art.

Using recombinant methods, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the cells may be disrupted to release the protein as a first step. Debris particles, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology, 10:163-167 describes a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 min. Remaining cells can be removed by centrifugation. When the antibody is secreted into the medium, the supernatants of such expression systems are typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of incidental contaminants. Various methods can be used to isolate an antibody from a host cell.

The antibody composition obtained from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being a typical purification method. 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 that are based on human gamma-1, gamma-2 or gamma-4 heavy chains (see, for example, Lindmark et al., 1983, J. Immunol. Meth. 62:1-13) . Protein G is recommended for all mouse isotypes and for human gamma-3 (see, eg, Guss et al., 1986, EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl)benzene provide higher flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, N.J.) can be used for purification. Other protein purification methods such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography, SEPHAROSE™ heparin chromatography, anionic or cation exchange resin chromatography (ACHR), chromatofocusing, SDS- PAGE and ammonium sulfate precipitation are also available depending on the antibody to be isolated.

After any preliminary purification step(s), the mixture containing the antibody of interest and impurities can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, typically performed at low salt concentrations (e.g. from about 0-0.25 M salt).

Also included are nucleic acids that hybridize under low, medium, and high stringency conditions, as defined herein, to all or part (e.g., a portion encoding a variable region) of a nucleotide sequence represented by isolated polynucleotide sequence(s) that encode an antibody or an antibody fragment having a heavy chain variable domain and a light chain variable region containing the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 26, respectively; SEQ ID NO: 28 and SEQ ID NO: 26 respectively; SEQ ID NO: 29 and SEQ ID NO: 26 respectively; SEQ ID NO: 30i

SEQ ID NO: 26 respectively; SEQ ID NO: 32 and SEQ ID NO: 31 respectively; SEQ ID NO: 33i

SEQ ID NO: 31 respectively; SEQ ID NO: 34 and SEQ ID NO: 31 respectively; SEQ ID NO: 35i

SEQ ID NO: 31 respectively; SEQ ID NO: 37 and SEQ ID NO: 36, respectively; SEQ ID NO: 38i

SEQ ID NO: 36 respectively; SEQ ID NO: 39 and SEQ ID NO: 36, respectively; SEQ ID NO: 40i

SEQ ID NO: 36 respectively.

The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (eg, 20, 25, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, such as at least 90%, at least 95%, or at least 98% identical to the sequence of a portion or all of the nucleic acid encoding an anti-CD40 polypeptide (e.g., variable heavy chain or light chain region) or its complement. Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer, such as a PCR primer, or a diagnostic probe.

Some embodiments include isolated polynucleotides, including sequences that encode an antibody or antibody fragment having a heavy chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95%, 98% or at least 99% identical to the amino acid sequence of any of SEQ ID NO: 1-4, 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, or SEQ ID NO: 40. Some embodiments include dedicated polynucleotides, including sequences that encode an antibody or antibody fragment having a light chain variable domain amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or is at least 99% identical to the amino acid sequence of any of SEQ ID NO: 5-8, SEQ ID NO: 26, SEQ ID NO: 31 or SEQ ID NO: 36.

In one aspect, the isolated polynucleotide sequence(s) encodes an antibody or antibody fragment having a heavy chain variable domain and a light chain variable region, each comprising an amino acid sequence that is 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 comprising the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 26, respectively ; SEQ ID NO: 28 and SEQ ID NO: 26 respectively;

SEQ ID NO: 29 and SEQ ID NO: 26 respectively; SEQ ID NO: 30 and SEQ ID NO: 26 respectively;

SEQ ID NO: 32 and SEQ ID NO: 31 respectively; SEQ ID NO: 33 and SEQ ID NO: 31, respectively;

SEQ ID NO: 34 and SEQ ID NO: 31 respectively; SEQ ID NO: 35 and SEQ ID NO: 31, respectively;

SEQ ID NO: 37 and SEQ ID NO: 36, respectively; SEQ ID NO: 38 and SEQ ID NO: 36 respectively;

SEQ ID NO: 39 and SEQ ID NO: 36, respectively SEQ ID NO: 40 and SEQ ID NO: 36, respectively.

In another aspect, the invention provides a polynucleotide in the embodiment described immediately above, wherein the heavy chain variable domain and the light chain variable region of the encoded antibody or antibody fragment comprise an amino acid sequence that is at least 95%, at least 98%, or is 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 comprising the amino acid sequences, in one embodiment, 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, respectively; 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, respectively; 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, respectively; 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.

As used herein, the terms identical or percent identity in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a certain percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum compliance. To determine percent identity, sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into the first amino acid or nucleic acid sequence for optimal

- 33 046388 alignment with a second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, % identity = # number of identical positions/total number of # positions (eg, overlapping positions) x 100). In some embodiments, the two sequences being compared have the same length after spaces are introduced into the sequences, as appropriate (eg, excluding additional sequence that extends beyond the sequences being compared). For example, when comparing variable region sequences, leader and/or constant domain sequences are not considered. For sequence comparisons between two sequences, the corresponding CDR refers to the CDR at the same location in both sequences (eg, CDR-H1 of each sequence).

Determining the percent identity or percent similarity between two sequences can be done using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm used to compare two sequences is that of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA, 87:2264-2268, modified from Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA, 90:5873-5877. This algorithm is included in the NBLAST and XBLAST programs according to Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. A BLAST protein search can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the corresponding protein. To obtain gap alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389–3402. Alternatively, PSI-Blast can be used to perform iterative searches that detect distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST, and PSI-BLAST programs, you can use the default settings of the corresponding programs (for example, XBLAST and NBLAST). Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is the Myers and Miller algorithm, CABIOS (1989). Such an algorithm is included in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional sequence analysis algorithms are known in the art and include ADVANCE and ADAM, as described in Torellis and Robotti , 1994, Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, then similar regions in the two sequences being compared are detected by looking at pairs of aligned residues; if ktup=1 then sibling amino acids are tested, ktup can be set to 2 or 1 for protein sequences or 1 to 6 for DNA sequences. The default if ktup is not specified is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignment can be performed using the CLUSTAL W algorithm as described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

Non-therapeutic use.

The antibodies described herein can be used as affinity purification agents. In this process, antibodies are 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 then washed with a suitable solvent that removes substantially all material in the sample except the CD40 protein, which is bound to the immobilized antibody. Finally, the carrier is washed with another suitable solvent, which releases the CD40 protein from the antibody.

Humanized anti-CD40 antibodies are also useful in diagnostic assays to detect and/or quantify CD40 protein, for example, to determine CD40 expression in specific cells, tissues, or serum.

In some embodiments, for example for diagnostic purposes, it will be useful to label the antibody with a detectable moiety. Numerous detectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels, and the like. The label can be indirectly conjugated to the antibody using various known techniques. For example, an antibody can be conjugated to biotin, and any of the three broad categories of labels mentioned above can be conjugated to avidin, or vice versa. Biotin from

- 34 046388 binds selectively to avidin, and thus the label can be conjugated to the antibody indirectly. Alternatively, to achieve indirect conjugation of a label to an antibody, the antibody can be conjugated to a small hapten (such as digoxin) and one of the various types of labels mentioned above is conjugated to an anti-hapten antibody (for example, an anti-digoxin antibody). In this way, indirect conjugation of the label to the antibody can be achieved.

Examples of radioisotope labels include ³⁵ S, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I. An antibody can be labeled with a radioisotope using methods described, for example, in Current Protocols in Immunology, vols. 1 and 2, 1991, Coligen et al., Wiley-Interscience, New York, NY, Pubs. Radioactivity can be measured, for example, using scintillation counting.

Examples of fluorescent labels include those derived from rare earth chelates (europium chelates), or available fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and Texas red. Fluorescent tags can be conjugated to the antibody by known methods, such as those disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.

There are various well-characterized enzyme-substrate tags known in the art (see, for example, US Pat. No. 4,275,149 for a review). The enzyme typically catalyzes a chemical change in a chromogenic substrate, which can be measured using a variety of methods. For example, the change may be a change in the color of the substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Methods for quantifying fluorescence changes are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and can then emit light, which can be measured, for example, using a chemiluminometer, or transfer energy to a fluorescent acceptor.

Examples of enzymes include lucifers, such as Lyuciferase Firefly and Bacterial Lucifers (US Patent No. 4,737,456), Luciferin, 2.3-dihydroftalazienia, malath dehydrogenase, ureaaz, peroxidase, such as the peroxidase of the crap (HRPO), alkaline phosphatezidase, β-g. Lactosidase, glucoamilasis , lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocidal oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase and the like. Methods for conjugating enzymes with antibodies are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academic press, N.Y., 73:147-166.

Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as the substrate, where the hydrogen peroxidase oxidizes a dye precursor such as orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB) ; alkaline phosphatase (AP) with p-nitrophenyl phosphate as the chromogenic substrate and e-D-galactosidase (β-D-Gal) with a chromogenic substrate such as p-nitrophenyl-β-E-galactosidase, or the fluorogenic substrate 4-methylumbelliferyl-β-O- galactosidase.

Many other enzyme-substrate combinations are available to those skilled in the art. For an overview of these, see US Patent Nos. 4,275,149 and 4,318,980.

In another embodiment, the humanized anti-CD40 antibody is used unlabeled and detected by a labeled antibody that binds the humanized anti-CD40 antibody.

The antibodies described herein can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, p. 147-158 (CRC Press, Inc. 1987).

Diagnostic kits.

The humanized anti-CD40 antibody can be used in a diagnostic kit, i.e. in a packaged combination of reagents in predetermined quantities with instructions for performing a diagnostic test. If the antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme, such as a substrate precursor that provides a detectable chromophore or fluorophore. In addition, other additives may be included, such as stabilizers, buffers (eg, blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents can be varied widely to provide reagent concentrations in solution that significantly optimize the sensitivity of the assay. Reagents may be provided in the form of dry powders, usually lyophilized, including excipients which, when dissolved, will yield a reagent solution of the appropriate concentration.

Therapeutic use.

In another embodiment, a humanized anti-CD40 antibody described herein is useful in the treatment of various disorders associated with CD40 expression as described herein.

- 35 046388

The humanized anti-CD40 antibody or agent is administered by any suitable route, including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal and, if desired for local immunosuppressive treatment, intralesional administration (including perfusion or other contact of the graft with the antibody prior to transplantation). The humanized anti-CD40 antibody or agent can be administered, for example, as an infusion or bolus. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the humanized anti-CD40 antibody is suitably administered by pulse infusion, especially with decreasing doses of the antibody. In one aspect, the dose is administered by injection, most preferably intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

For the prevention or treatment of a disease, the appropriate dose of antibody will depend on a variety of factors, such as the type of disease being treated as defined above, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history, and response to antibody, and at the discretion of the attending physician. The antibody is usually administered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, approximately 1 mcg/kg to 20 mg/kg (eg, 0.1-15 mg/kg) of antibody is a potential initial dose to administer to a patient, regardless of whether, for example, it is administered in one or several separate administrations, or by continuous infusion. A typical daily dose may range from about 1 mcg/kg to 100 mg/kg or more, depending on the factors mentioned above. With 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 applicable. The progress of this therapy is easy to monitor using conventional methods and tests. An exemplary dosage regimen is described in WO 94/04188.

The term suppression is used here in the same context as amelioration and alleviation to mean a reduction in one or more characteristics of a disease.

The antibody composition will be formulated, dosed and administered in accordance with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to the practitioner. A therapeutically effective amount of antibody administered will be determined by such considerations and represents the minimum amount necessary to prevent, alleviate, or treat a disorder associated with CD40 expression.

The antibody may optionally, but optionally, be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of humanized anti-CD40 antibody present in the formulation, the type of disease or treatment, and other factors discussed above. They are generally used in the same dosages and via the same routes of administration as suggested above or from about 1 to 99% of the dosages hitherto used.

CD40-related disorders.

Anti-CD40 antibodies or agents are useful for the treatment or prevention of CD40-expressing cancer or an immunological disorder characterized by CD40 expression, for example, by inappropriate activation of immune cells (eg, lymphocytes or dendritic cells). Such CD40 expression may be due, for example, to increased levels of cell surface CD40 protein and/or altered antigenicity of the expressed CD40. Treatment or prevention of an immunological disorder in accordance with the methods described herein is achieved by administering to a subject in need of such treatment or prevention an effective amount of an anti-CD40 antibody or agent, whereby the antibody (i) binds to activated immune cells that express CD40 and that associated with a painful condition; and (ii) have a cytotoxic, cytostatic or immunosuppressive effect on activated immune cells.

Immunological diseases that are characterized by inappropriate activation of immune cells and that can be treated or prevented by the methods described herein can be classified, for example, by the type(s) of hypersensitivity reaction(s) that underlie the disease. These reactions are generally classified into four types: anaphylactic reactions, cytotoxic (cytolytic) reactions, immune complex reactions, or cellular-mediated immunity (CMI) reactions (also called delayed-type hypersensitivity (DTH) reactions). (See, for example, Fundamental Immunology (William E. Paul ed., Raven Press, N.Y., 3rd ed. 1993).)

Specific examples of such immunological diseases include the following: rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, autoimmune demyelinating diseases (eg, multiple sclerosis, allergic encephalomyelitis), endocrine ophthalmopathy,

- 36 046388 uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disease, inflammatory bowel disease (eg Crohn's disease or ulcerative colitis), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis , Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, multiple endocrine insufficiency, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, malignant anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Rey phenomenon but, violation esophageal motility, sclerodactyly and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitoperistitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas disease, sarcoidosis, rheumatic fever, asthma , spontaneous abortion, antiphospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, poultry farmers' pulmonary allergy, toxic epidermal necrolysis, Alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sumpter syndrome, eczema, lymphomatoid granulomatosis, Behçet's disease, Kaplan syndrome, Kawasaki disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis , endophthalmitis, persistent elevated erythema, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuchs cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, graft rejection, cardiomyopathy , Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenström's macroglobulemia, Evan's syndrome, acute respiratory distress syndrome, pneumonia, osteoporosis, delayed hypersensitivity and autoimmune gonadal failure.

Accordingly, the methods described herein include the treatment of disorders associated with B lymphocytes (for example, systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis and type I diabetes), Th1 lymphocytes (for example, rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft-versus-host disease) or Th2 lymphocytes (eg, atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn syndrome, systemic sclerosis or chronic graft-versus-host disease). Typically, dendritic cell disorders involve TM lymphocyte or TH2 lymphocyte disorders.

Rheumatoid arthritis (RA) is one of the most common inflammatory autoimmune diseases, affecting approximately 1% of the population. Although effective treatments are available (eg, MTX and anti-TNF agents), there is a large unmet medical need, especially for those patients who do not respond well to anti-TNF therapy (about 30% of patients). In addition, up to 50% of patients discontinue treatment with TNF antagonists within 5 years, mainly due to side effects, but also because an increasing number of patients lose therapeutic benefit. Thus, it is important to develop effective treatments to combat inflammation and joint destruction in RA, but not rely solely on direct TNF inhibition. A very attractive approach is to target costimulatory cellular pathways. One of the key receptor ligand pairs in costimulation is CD40/CD40L. This system allows interaction between immune cells and between immune and non-immune cells, all of which are important in the pathogenesis of RA. Blockade of CD40 with an antagonist antibody according to the present invention may have one or more of the following effects in RA:

1) inhibit B-cell differentiation and antibody isotype switching;

2) inhibit the production of cytokines and chemokines and the upregulation of adhesion molecules in T cells and macrophages;

3) inhibit the activation of dendritic cells and

4) inhibit the production of proinflammatory cytokines, chemokines, matrix metalloproteinases, prostaglandins and downregulate adhesion molecules in non-immune cells (eg, epithelial, endothelial and mesenchymal cells).

Methods for achieving one or more of the foregoing effects are expressly provided herein. In addition to RA, compositions in accordance with the present invention will be particularly useful in methods of treating systemic lupus erythematosus, lupus nephritis, disseminated

- 37 046388 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 that occurs in approximately 1% of adults. The disease continues to cause significant morbidity and premature mortality (mortality is mainly associated with a high risk of developing cardiovascular disease). It is now established that joint damage occurs very early in the disease, with up to 30% of patients showing radiographic evidence of bone erosions at diagnosis, increasing to 60% after 1 year. Current guidelines recommend starting therapy with traditional disease-modifying antirheumatic drugs (DMARDs) within 3 months of definitive diagnosis. DMARDs can reduce or prevent joint damage and preserve joint function. Currently, rheumatologists select methotrexate (MTX) as the initial DMARD therapy for most patients.

The TNF antagonists etanercept (Enbrel®), infliximab (Remicade®), adalimumab (Humira®), the CTLA4 antagonist abatacept (Orencia®), the anti-IL-6 receptor mAb tocilizumab, and the anti-CD20 mAb rituximab (Rituxan®) are effective in the treatment of RA. Current guidelines generally recommend the use of biologic DMARDs to treat active RA after an inadequate response to traditional DMARDs.

Recent studies in patients with early aggressive RA without prior treatment with methotrexate showed that the combination of methotrexate and a TNF antagonist was superior to either when used as monotherapy. The most striking result was the significant radiological benefit of the combination therapy. Therefore, a combination of MTX and TNF inhibitors should be used in patients at greatest risk for aggressive disease and aggressive phenotype (eg, high activity score, functional impairment, rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) seropositivity, elevated CRP, X-ray erosions). However, the inventors expect that TNF antagonists will rarely be used as first-line therapy in clinical practice. A survey of American rheumatologists conducted in April 2005 found that the factors most influencing the decision to use a TNF antagonist were: failure of MTX or multiple DMARDs, physician global assessment, functional impairment, and radiographic worsening or erosion. Currently, approximately 20% of patients with RA in the United States are treated with TNF inhibitors.

A significant percentage of patients with RA are not adequately managed by available treatments, including biologic therapies, either due to drug intolerance and toxicity or lack of response. Up to 50% of patients discontinue treatment with TNF antagonists within 5 years, mainly due to side effects, but also because an increasing number of patients fail to respond.

In some embodiments, the immunological disorder is a T cell-mediated immunological disorder, such as a T cell disease, in which activated T cells associated with the disorder express CD40. Anti-CD40 antibodies or agents can be administered to deplete such CD40-expressing activated T cells. In a particular embodiment, administration of anti-CD40 antibodies or agents can deplete CD40-expressing activated T cells while resting T cells are not substantially depleted by the anti-CD40 antibody or agent. In this context, substantially non-exhausted means that less than about 60%, less than about 70%, or less than about 80% of resting T cells are not exhausted.

Antibodies against CD40 and the agents described herein are also useful for treating or preventing cancers that express CD40. Treatment or prevention of cancer expressing CD40, in accordance with the methods described herein, is achieved by administering to a subject in need of such treatment or prevention an effective amount of an anti-CD40 antibody or agent, whereby the antibody or agent (i) binds to cancer cells , expressing CD40; and (ii) has a cytotoxic or cytostatic effect to deplete or inhibit the proliferation of cancer cells expressing CD40.

CD40-expressing cancers that can be treated or prevented by the methods described herein include, for example, leukemia, such as acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (eg, myeloblastic, promyelocytic, myelomonocytic, monocytic or erythroleukemia), chronic leukemia , chronic myelocytic (granulocytic) leukemia or chronic lymphocytic leukemia; polycythemia vera; lymphoma (eg, Hodgkin's disease or non-Hodgkin's disease); multiple myeloma, Waldenström's macroglobulinemia; heavy chain disease; solid tumors such as sarcomas and carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma

- 38 046388 coma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangiondotheliosarcoma, synoviom, mesothelioma, Yinga tumor, leiomiosarcoma, rabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, cancer of the mall ovarian cancer, prostate cancer, plane cell cancer , basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cancer cervical, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma , meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma or esophageal carcinoma).

Pharmaceutical compositions and their administration.

A composition containing a CD40 binding agent (eg, an anti-CD40 antibody) may be administered to a subject at risk of developing an immunological disorder or cancer expressing CD40. The invention further provides the use of a CD40 binding agent (eg, an anti-CD40 antibody) in the manufacture of a medicament for the prevention or treatment of CD40-expressing cancer or an immunological disorder. As used herein, the term subject means any mammalian patient to which a CD40 binding agent can be administered, including, for example, humans and non-human mammals such as primates, rodents and dogs. Particularly preferred subjects for treatment using the methods described herein are humans. Antibodies or agents can be administered alone or in combination with other compositions for the prevention or treatment of an immunological disorder or cancer expressing CD40.

Preferred antibodies for use in such pharmaceutical compositions are those that contain a humanized antibody or antibody fragment having the heavy chain variable region amino acid sequence of any of SEQ ID NO: 1-4, 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 or SEQ ID NO: 40.

Some embodiments include isolated polynucleotides containing sequences that encode 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 an antibody fragment having a heavy chain variable domain and a light chain variable region containing the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 26, respectively; SEQ ID NO: 28 and SEQ ID NO: 26 respectively; SEQ ID NO: 29 and SEQ ID NO: 26 respectively;

SEQ ID NO: 30 and SEQ ID NO: 26 respectively; SEQ ID NO: 32 and SEQ ID NO: 31 respectively;

SEQ ID NO: 33 and SEQ ID NO: 31, respectively; SEQ ID NO: 34 and SEQ ID NO: 31 respectively;

SEQ ID NO: 35 and SEQ ID NO: 31, respectively; SEQ ID NO: 37 and SEQ ID NO: 36, respectively;

SEQ ID NO: 38 and SEQ ID NO: 36 respectively; SEQ ID NO: 39 and SEQ ID NO: 36, respectively;

SEQ ID NO: 40 and SEQ ID NO: 36 respectively.

The present invention contemplates isolated polynucleotides that encode any of the heavy chain sequences SEQ ID NO: 1-4, 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 provide isolated nucleic acids that encode a light chain sequence of any of SEQ ID NO: 5 - 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 some embodiments where treatment of RA is contemplated, compositions in accordance with the invention can be used in methods of reducing signs and symptoms, inducing a major clinical response, and reducing the progression of structural damage in patients with moderate to severe RA who do not respond adequately to MTX alone. A current example of such a therapy is: Enbrel/Humira (data with Humira and Enbrel were obtained in two different patient populations). The compositions of the present invention can be used instead of Enbrel/Humira therapy or in combination with Enbrel/Humira therapy for subjects who do not respond to treatment with MTX alone. Preferably, in such variants

- 39 046388 In each embodiment, the compositions in accordance with the invention will have higher efficacy than Enbrel + MTX in patients who have had an inadequate response to methotrexate, as determined, for example, by ACR20 at 6 months >85% for the compound plus MTX (GS: Enbrel + MTX 71% vs placebo + MTX 27%, Humira + MTX at 12 months 59% vs placebo + MTX 24%)*. Additional criteria for superior efficacy of the compositions of the invention may include: inhibition of progression of structural damage over a period of one year similar to Enbrel (at 52 weeks, mean modified Sharp score of humira+MTX 0.1 versus placebo+MTX 2.7 )*. In yet other embodiments, the compositions produce a good clinical response superior to Enbrel in patients who had an inadequate response to methotrexate as measured by ACR70 (20% for Humira + MTX, 4% for placebo + MTX)*.

In other embodiments, the compositions of the invention are indicated for reducing signs and symptoms by inducing good clinical response and reducing the progression of structural damage in patients with moderately to severely active RA who have had an inadequate response to anti-TNF agents. The current gold standard is biological therapy with non-TNF agents. Preferably, for such subjects, the compositions of the invention are noninferior to non-TNF biologic agents (eg, Orencia, Rituxan), when compared chronologically in patients who have had an inadequate response to an anti-TNF agent: ACR20 at 6 months >50% for compound plus DMARD (GS: Orencia + DMARD 50% vs. placebo + DMARD 20%). In yet other embodiments, the compositions of the invention inhibit the progression of structural damage over a period of one year, as assessed by conventional X-ray assessment methods for joint erosion and joint space narrowing, similar to Rituxan (at 52 weeks, the mean modified scale score Sharp Rituxan + MTX 1.0 versus placebo + MTX 2.31).

Various delivery systems are known that can be used to administer the CD40 binding agent. Routes of administration include, but are not limited to, intravenous, subcutaneous, intranasal, epidural and oral routes. The CD40 binding agent may be administered, for example, by infusion, bolus, or injection, and may be administered along with other biologically active agents, such as chemotherapeutic agents. Administration may be systemic or local. In preferred embodiments, administration is by subcutaneous injection. Formulations for such injections can be prepared, for example, in pre-filled syringes that can be administered once every two weeks.

The safety characteristics of antibodies in accordance with the invention will be determined and preferably include one or more characteristics, such as the absence of clinically significant adverse interactions with other drugs commonly used to treat rheumatoid arthritis (eg, DMARDs, steroids, NSAIDs); no higher incidence of drug discontinuation due to safety or tolerability issues compared with Enbrel; the incidence of serious infections is no higher than that of anti-TNF agents or other commonly used biologic agents; the frequency and/or severity of injection site or infusion reactions similar to Enbrel; absence or minimal development of drug resistance (less than 5%) with repeated courses of therapy; absence or minimal amount of neutralizing antibodies; no evidence of increased platelet aggregation/activation that could lead to thromboembolic events in vivo or platelet/endothelial dysfunction that could lead to bleeding.

In certain embodiments, the CD40 binding agent composition is administered by injection via a catheter, via a suppository, or via an implant, where the implant is a porous, non-porous or gel-like material, including a membrane such as a sialastic membrane or fiber. Typically, when administering the composition, materials are used that are not absorbed by the anti-CD40 antibody or agent.

In other embodiments, the anti-CD40 antibody or agent is delivered by a controlled release system. In one embodiment, a pump may be used (see, for example, Langer, 1990, Science, 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al. 1989 N. Engl. J. Med. 321:574). In another embodiment, polymeric materials may be used (see, for example, Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance ( Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science, 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Other controlled release systems are discussed, such as Langer's, see above.

The CD40 binding agent (eg, an anti-CD40 antibody) can be administered in the form of pharmaceutical compositions containing a therapeutically effective amount of the binding agent and

- 40 046388 one or more pharmaceutically compatible ingredients.

In typical embodiments, the pharmaceutical composition is formulated according to conventional procedures into a pharmaceutical composition adapted for intravenous or subcutaneous administration to humans. Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical preparation may also include a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the injection site. Typically, the ingredients are supplied either individually or mixed together in a unit dosage form, such as a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active ingredient. If the pharmaceutical drug is administered by infusion, it can be dispensed using an infusion bottle containing sterile water or pharmaceutical grade saline. If the pharmaceutical drug is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

Additionally, the pharmaceutical composition may be presented as a pharmaceutical kit comprising (a) a container containing a CD40 binding agent (eg, an anti-CD40 antibody) in lyophilized form; and (b) a second container containing a pharmaceutically acceptable diluent (eg, sterile water) for injection. A pharmaceutically acceptable diluent can be used to reconstitute or dilute the lyophilized anti-CD40 antibody or agent. Such container(s) may optionally be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting the agency's approval of the manufacture, use, or sale for administration to humans.

The amount of CD40 binding agent (eg, anti-CD40 antibody) that is effective in treating or preventing an immunological disorder or cancer expressing CD40 can be determined by standard clinical methods. In addition, in vitro assays may not necessarily be used to help determine optimal dosage ranges. The exact dosage used in the formulation will also depend on the route of administration and the stage of the immunological disorder or CD40-expressing cancer and should be determined in accordance with the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

For example, the toxicity and therapeutic efficacy of an anti-CD40 antibody or agent can be determined in cell cultures or experimental animals using standard pharmaceutical procedures for determining _ED50 (the dose therapeutically effective in 50% of the population). Preferred is a CD40 binding agent (eg, an anti-CD40 antibody) that exhibits a large therapeutic index. When a CD40 binding agent exhibits toxic side effects, a delivery system that targets the CD40 binding agent to an affected tissue site can be used to minimize potential damage to non-CD40 expressing cells and thereby reduce side effects.

Data obtained from cell culture assays and animal studies can be used to develop dose ranges for use in humans. The dosage of the CD40 binding agent is typically within a circulating concentration range that includes the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any CD40 binding agent used in the method, a therapeutically effective dose can be initially estimated from cell culture assays. The dose can be formulated in animal models to achieve a range of circulating plasma concentrations that includes the IC ₅₀ (ie, the concentration of test compound at which half-maximal symptom suppression is achieved) as determined in cell culture. Such information can be used to more accurately determine applicable human doses. Plasma levels can be measured, for example, by high performance liquid chromatography, ELISA, etc.

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

Exemplary doses include, but are not limited to, 1 ng/kg to 100 mg/kg. In some embodiments, the dose is about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, approximately 7 mg/kg, approximately 8 mg/kg, approximately 9 mg/kg, approximately 10 mg/kg, approximately 11 mg/kg, approximately 12 mg/kg, approximately 13 mg/kg, approximately 14 mg/kg, approximately 15 mg/kg or approximately 16 mg/kg. The dose can be administered

- 41 046388 e.g. daily, once a week (weekly), twice a week, three times a week, four times a week, five times a week, six times a week, weekly or monthly, every two months or every three month.

In certain embodiments, the dosage 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, approximately 6 mg/kg/week, approximately 7 mg/kg/week, approximately 8 mg/kg/week, approximately 9 mg/kg/week, approximately 10 mg/kg/week, approximately 11 mg/week 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 dosage ranges from about 1 to about 15 mg/kg/week.

In some embodiments, pharmaceutical compositions containing a CD40 binding agent may further comprise a therapeutic agent, either conjugated or unconjugated to the binding agent. An anti-CD40 antibody or CD40 binding agent may be administered in combination with one or more therapeutic agents for the treatment or prevention of immunological disorders or CD40-expressing malignancies. For example, the combination therapy may include a cytostatic, cytotoxic, or immunosuppressive agent. Combination therapy may also include, for example, administration of an agent targeting a receptor or receptor complex other than CD40 on the surface of activated lymphocytes, dendritic cells, or cancer cells expressing CD40. An example of such an agent includes a second, non-CD40 antibody that binds to a molecule on the surface of an activated lymphocyte, dendritic cell, or cancer cell expressing CD40. Another example involves a ligand targeting such a receptor or receptor complex. Typically, such an antibody or ligand binds to a cell surface receptor on activated lymphocytes, dendritic cells or cancer cells expressing CD40 and enhances the cytotoxic or cytostatic effect of the anti-CD40 antibody by delivering a cytostatic or cytotoxic signal to the activated lymphocytes, dendritic cells or cancer cells expressing CD40 .

Such administration of combination therapy may have an additive or synergistic effect on disease parameters (eg, symptom severity, number of symptoms, or relapse rate).

With respect to combinatorial therapeutic regimens, in a particular embodiment, the anti-CD40 antibody or CD40 binding agent is administered simultaneously with the therapeutic agent. In other specific embodiments, the therapeutic agent is administered before or after administration of the anti-CD40 antibody or CD40 binding agent for at least 1 hour and up to several months, such as for at least 1, 5, 12 hours, one day, a week , month or three months, before or after administration of an anti-CD40 antibody or CD40 binding agent.

Useful classes of cytotoxic or immunosuppressive agents include, for example, antitubulin agents, auristatins (e.g., MMAE or MMAF), DNA minor groove binders, inhibitors of DNA replication, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum) ), bis-(platinum) and trinuclear complexes of platinum and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapeutic sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, transforming compounds, purine antimetabolites, puromycins, sensitizers radiation, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc.

Selected cytotoxic or immunosuppressive agents include, for example, androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide , cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, estrogen, 5-fluorodeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU) , mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin, tenoposide, 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 (for example, auristatin E, AFP, MMAF, MMAE, AEB or AEVB), DNA minor groove binders (for example, enediins and lexitropsins), duocarmycins, taxanes (for example, paclitaxel and docetaxel), Barvinka, SS-1065, SN-38, topotsan, morpholinocorubicin, rhizoxin, cyanomorpholin-codesorubicin, echinomicin, commercial, non-nitropsin, epotylon A and B, estramoustine, popinzinoids, aminzinoids, discodermolate, discodermoteine Robin or Mitoxantron.

In some embodiments, the cytotoxic agent is a conventional chemotherapeutic agent, such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. In addition, potent drugs such as

- 42 046388 CC-1065 analogs, calicheamicin, maytansine, dolastatin 10 analogs, rhizoxin and palytoxin, may be associated with anti-CD40 antibodies or agents thereof.

In certain embodiments, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Typically, the auristatin E derivative is, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can react with para-acetylbenzoic acid or benzoylvaleric acid to form AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF and MMAE. The synthesis and structure of auristatin E and its derivatives are described, for example, in US Patent Application Publications No. 2004-0157782 A1 and 2005-0238649; International Patent Application No. PCT/US03/24209, International Patent Application No. PCT/US02/13435 and US Patent No. 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 descriptions of which are incorporated herein by reference.

In certain embodiments, the cytotoxic agent is a DNA minor groove binder (see, for example, US Pat. No. 6,130,237.) For example, in some embodiments, the minor groove binder is a CBI compound. In other embodiments, the minor groove binding agent is enediyne (eg, calicheamicin).

Examples of antitubulin agents include, but are not limited to, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs (eg, epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, discodermolide and eleutherobin.

In some embodiments, the cytotoxic agent is a maytansinoid, another group of antitubulin agents. For example, in certain embodiments, the maytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131).

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

In some embodiments, the cytotoxic or immunosuppressive agent is an antimetabolite. The antimetabolite may be, for example, a purine antagonist (eg, azothioprine or mycophenolate mofetil), a dihydrofolate reductase inhibitor (eg, methotrexate), acyclovir, ganciclovir, zidovudine, vidarabine, ribavarine, azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscamet or trifluridine .

In other embodiments, the cytotoxic or immunosuppressive agent is tacrolimus, cyclosporine, or rapamycin. In additional embodiments, the cytotoxic agent is aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene, bexarotene, calusterone, capecitabine, celecoxib, cladribine, darbepoetin alfa, denileukin diftitox, dexrazoxane, dromostanolone propionate , epirubicin, epoetin alfa, estramustine, exemestane, filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin, goserelin, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a, irinotecan, letrozole, leucovorin, levamisole, meclorethamine or nitrogen mustard , megestrol, mesna , methotrexate, methoxalen, mitomycin C, mitotane, nandrolone phenpropionate, oprelvekin, oxaliplatin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pentostatin, pipobromane, plicamycin, sodium porfimer, procarbazine, quinacrine, rasburicase, Revlimid, sargramostim, strep tozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine and zoledronate.

In additional embodiments, the drug is a humanized anti-HER2 monoclonal antibody; RITUXAN (rituximab; Genentech, Inc., South San Francisco, CA); chimeric monoclonal antibody against CD20); OVAREX (AltaRex Corporation, MA); PANOREX (Glaxo Wellcome, NC; mouse IgG _2a antibody); cetuximab erbitux (Imclone Systems Inc., NY; anti-EGFR IgG chimeric antibody); Vitaxin (Medlmmune, Inc., MD); Campath I/H (leukositis, MA; humanized IgG ₁ antibody); Smart MI95 (Protein Design Labs, Inc., CA; humanized anti-CD33 IgG antibody); LymphoCide (Immunomedics, Inc., NJ; humanized anti-CD22 IgG antibody); Smart ID10 (Protein Design Labs, Inc., CA; humanized anti-HLA-DR antibody); Oncolim (Techniclone, Inc., CA; radiolabeled mouse anti-HLA-Dr10 antibody); Allomune (BioTransplant, CA; humanized anti-CD2 mAb); Avastin (Genentech, Inc., CA; humanized anti-VEGF antibody); Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA; anti-CD22 antibody) and CEAcide (Immunomedics, NJ; humanized anti-CEA antibody).

Other useful antibodies include, but are not limited to, antibodies against the following an

- 43 046388 tigens: CA125, CA15-3, CA19-9, L6, Lewis Y., Lewis X., alpha-fetoprotein, CA 242, placental alkaline phosphatase, prostate-specific antigen, prostatic acid phosphatase, epidermal growth factor, MAGE-1 , MAGE-2, MAGE-3, MAGE-4, anti-transferrin receptor, p97, MUC1-KLH, CEA, gp100, MARTI, prostate specific antigen, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, mucin, P21, MPG and Neu oncogene product.

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

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

Suitable lipoxygenase inhibitors include redox inhibitors (e.g. catecholbutane derivatives, nordihydroguaiaretic acid (NDGA), masoprocol, phenidone, ianopalen, indazolinones, naphasatrom, benzofuranol, alkylhydroxylamine) and non-redox inhibitors (e.g. hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and their derivatives, methoxytetrahydropyran, boswellic acids and acetylated derivatives of boswellic acids and quinolinemethoxyphenylacetic acids substituted with cycloalkyl radicals and precursors of redox inhibitors).

Other useful lipoxygenase inhibitors include antioxidants (e.g., phenols, propyl gallate, flavonoids and/or naturally occurring substrates containing flavonoids, hydroxylated flavone derivatives, flavonol, dihydroquercetin, luteolin, galangin, orobol, chalcone derivatives, 4,2',4'- trihydroxychalcone, orthoaminophenols, N-hydroxyureas, benzofuranols, ebselen and compounds that increase the activity of reducing selenoenzymes), iron chelating agents (for example, hydroxamic acids and their derivatives, N-hydroxyureas, 2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C, catechol, naphthol, sulfasalazine, zileuton, 5-hydroxyanthranilic acid and 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containing compounds (for example, ketoconazole and itraconazole), phenothiazines and benzopyran derivatives.

Still other suitable lipoxygenase inhibitors include eicosanoid inhibitors (eg, octadecatetraenoic, eicosatetraenoic, docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and their esters, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol, 15-monohydroxyeicosatetraenoic, 15-monohydroxy eicosatriene and 15- monohydroxyeicosapentaenoic acids and leukotrienes B5, C5 and D5), calcium flux interfering compounds, phenothiazines, diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid, caffeic acid, 5,8,11,14-eicosatetraenoic acid (ETYA), hydroxyphenylretinamide, lonapalene , esculin, diethylcarbamazine, phenanthroline, baicalein, proxicromil, thioethers, diallyl sulfide and di-(1-propenyl) sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, Bayer Bau-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark

ETH-615, Lilly LY-293111, Ono ONO-4057, Terumo TMK-688, Boehringer

Ingleheim BI-RM-270, Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono

ONO LB457, Pfizer 105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP

66153, SmithKline Beecham SB-201146, SmithKline Beecham SB-201993,

SmithKline Beecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American

Home Products WAY 121006, Bayer Bay-o-8276, Warner-Lambert CI-987, WarnerLambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283, MacroNex

MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, Ono ONO-LB-448,

Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893, Rhone-Poulenc Rorer

RP 66364, Rhone-Poulenc Rorer RP 69698, Shionoogi S-2474, Searle SC-41930,

Searle SC-50505, Searle SC-51146, Searle SC-52798, SmithKline Beecham SK and F104493, Leo Denmark SR-2566, Tanabe T-757 and Teijin TEI-1338.

Industrial products

In another aspect, a manufactured article is provided containing materials useful for treating the disorders described above. A manufactured product includes a container and a label. Suitable containers are, for example, bottles, vials, syringes and test tubes. Containers can be made from various materials such as glass or plastic.

- 44 046388

The container contains a composition that is effective for treating the condition and may have a sterile access opening. For example, the container may be an intravenous solution bag or a vial having a stopper pierced by a hypodermic needle. The active agent in the composition is a humanized anti-CD40 antibody. The label on or associated with the container indicates that the composition is used to treat the selected condition. The article may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may additionally include other materials required from a commercial and user point of view, including other buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

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

Examples

Example 1: Production of Humanized Anti-CD40 Antibody.

Mouse antibodies 20E2 and 2H11 are presented in table. 1 and 2 herein above. Humanization of clones 20E2 and 2H11 has been completed. A library was created in which the human and mouse residues were rearranged so that at any given position there could be a human or mouse residue. Such a library was created for those amino acids that differed between the human germline and the mouse antibody. Only clones that retain the function of the parental mouse antibody were selected.

Thus, Antibody A, Antibody B, and Antibody C were humanized antibodies derived from mouse antibody 20E2 (Antibody A and Antibody B) or 2H11 (Antibody C) cloned into human IgG ₁ -KO (KO)/kappa backbone . IgG ₁ -KO has two mutations in the Fc region, Leu234Ala and Lue235Ala, to reduce FcyR and complement binding.

As a result of this humanization, various humanized heavy and light chain variable sequences were obtained, as follows:

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

DIVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSG

SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIК

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

EVQLVKSGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTI

SRDNAKNSLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS

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

DIVMTQSPDSLAVSLGERATMSCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSG

SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIК

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

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTI

SRDNAKNSLYLQMNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSS

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

DIVMTQSPDSLAVSLGEKVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSG

SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGAGTKVEIK

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

EVQLVESGGGLVKPGGSRRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTI

SRDNAKNSLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS.

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

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSG

SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIК

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

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTI

SRDNAKNSLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS

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

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSG

SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGAGTKVEIК

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

EVQLVESGGGLVKPGGSRRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTI

SRDNAKNSLYLQMNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSS

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

DIVMTQSPDSLAVSLGEKVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSG

SGSGTDFTLTISSLQAEDLAVYYCQNDYTYPLTFGAGTKVEIK.

- 45 046388

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

DIVMTQSPDSLAVSLGEKVTINCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRESGVPDRFSG

SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

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

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYADTVKGRFTI

SRDNAKNSLYLQMNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSS

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

QIQMTQSPSSSLSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLWIYSTSNLASGVPARFSGSGSGTDF

TLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

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

DIQMTQSPSSLSSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLLIYSTSNLASGVPARFSGSGSGTDF

TLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

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

DIQMTQSPSSLSSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDF

TLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

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

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDSKYAPKFQGKATM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS.

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

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWMGRIDPEDGDSKYAPKFQGKATM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

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

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDSKYAPKFQGKVTM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS.

SEQ ID NO: 60 (Heavy Chain Variable Region Established Sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWIGRIDPEDGDSKYAPKFQGKATM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

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

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQAPGQGLEWMGRIDPEDGDSKYAPKFQGKATM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

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

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDTKFAPKFQGKATM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

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

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDTKFAPKFQGKVTM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

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

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWIGRIDPEDGDTKFAPKFQGKATM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

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

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWMGRIDPEDGDTKFAPKFQGKATM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

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

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQAPGQGLEWMGRIDPEDGDTKFAPKFQGKATM

TADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

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

EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYDPKFQGRVTM

TADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

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

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYDPKFQGRVTM

TADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

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

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYDPKFQGKVTM

TADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

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

- 46 046388

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQAPGKGLEWMGRIDPEDGDTKYDPKFQGKATM

TADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

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

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYDPKFQGKATM

TADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

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

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQAPGKGLEWIGRIDPEDGDTKYDPKFQGKATM

TADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

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

EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYIHWVQQAPGKGLEWMGRIDPEDGDTKYDPKFQGRVTM

TADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO: 74 (light chain variable region sequence) 1 from 10F2Hum antibody:

DIQMTQSPSSLSSASVGDRVTTITCSATSSVSYILWFQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDF

TLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO: 75 (light chain variable region sequence) 2 from antibody 10F2Hum:

DIQMTQSPSSLSSASVGDRVTTITCSATSSVSYILWFQQKPGKAPKLLIYSTSNLASGVPARFSGSGSGTDF

TLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIК

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

QIQMTQSPSSLSSASVGDRVTTITCSATSSVSYILWFQQKPGKAPKLWIYSTSNLASGVPARFSGSGSGTDF

TLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIК

Exemplary humanized antibodies in accordance with the present invention are antibodies that have the heavy and light chain sequences shown in the following table. Sequences in bold and underlined in the following table are variable domains, while normal, ununderlined sequences are constant domains:

Identity Subsequence SEQ ID NO: Antibody A (light chain) DIVMTQSPDSLAVSLGERATMSCKSSQSLLNSGNQKNYLTW HQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTIS 26 SLQAEDVAVYYCQNDYTYPLTFGGGTKVEIKRTVAAP S VFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC Antibody A (heavy chain, IgGlKO) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ 27 MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTP EVT СVVVDVS NE D РEVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNK ALPAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody A (heavy chain, IgGl) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ 28 MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS NE D R EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody A (heavy chain, IgG4DM) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 29 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD

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HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDT LMIS RT Р EVT CVVVDVS Q E D P EVQ FNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody A (heavy chain, IgGlKOb) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP thirty GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVT CVVVDVS HE D P EVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (light chain) DIVMTQSPDSLAVSLGEKVTINCKSSQSLLNSGNQKNYL 31 TWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Antibody B (heavy chain, IgGlKO) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 32 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS HE D P EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (heavy chain, IgGl) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 33 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS HE D P EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (heavy chain, IgG4 DM) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 34 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDT LMIS RT P EVT CVVVDVS Q E D P EVQ FNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT

- 48 046388

VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody B (heavy chain, IgGlKOb) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP 35 GKGLEWVAYIS S GNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTP EVT CVVVDVS NE D R EVK FNWYVD GVEVH NAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (light chain) DIQMTQSPSSLSSASVGDRVTITCSASSSVSYMLWFQ 36 QKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody C (heavy chain, IgGlKO) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 37 GQGLEWMGRID PED GD SKYAPKFQGKATMTAD T S TS TVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 38 GQGLEWMGRID PED GD SKYAPKFQGKATMTAD T S T S TVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (heavy chain, IgG4 DM) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 39 GQGLEWMGRID PED GD SKYAPKFQGKATMTAD T S T S TVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPK DT LMIS RT P EVT CVVVDVS Q E D P EVQ FNWYVD GVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody C (heavy chain, QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 40 GQGLEWMGRID PED GD SKYAPKFQGKATMTAD T S T S TVYME IgGlKOb) LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The variable regions have been subcloned into one or two different suitable IgG expression vectors:

A) human IgG1-KO (knockout)/kappa format with double mutation Leu234Ala, Leu235Ala in the Fc region to reduce effector function such as FcyR and complement binding;

B) human IgG ₄ -DM (double mutant)/kappa format with a Ser228Pro mutation in the hinge region to reduce the occurrence of IgG ₄ hemimolecules and a Leu235Glu mutation to further reduce FcyR binding.

Two candidates Antibody A and Antibody B were purified and evaluated according to the following criteria:

CCF (clouding) appearance;

filtering properties of CCF;

rProteinA output;

turbidity during elution and neutralization;

soluble aggregates (SEC);

clean/stain sample (SDS);

- 49 046388 charge diagram (IEF).

Example 2: In vitro data.

Antibody A, Antibody B, and Antibody C were characterized along with antibodies 4D11 (Kirin/Astellas) and PG-102 (PanGenetics), which were derived from published sequences. Data for Antibody A, Antibody B, Antibody C, and 4D11 are shown below. PG-102 exhibited agonistic activity and only incomplete inhibition of B cell proliferation (not shown). In table 6 summarizes the obtained data. A more detailed description of the data is presented in table. 6.

Table 6

Summary of in vitro data for Antibodies A, Antibodies B and Antibodies C and Kirin 4D11 anti-CD40 antibodies

Parameter/Analysis Antibody A Antibody B Antibody C 4D11 Kd +/- pers. serum <100 pM <100 pM <100 pM <100 pM Cell binding (EC50/nM ± SD 1.2 (±0.28) 1.5 (±0.68) 1.7 (±0.28) 0.9 (±0.3) B cell proliferation: antagonism (IC50/HM+SD) 0.3 (±0.13) 0.2 (±0.10) 0.1 (±0.004) 0.03 (±0.02) B cell proliferation:agonism (SI*) (IC50/HM+SD) No agonism (SI<2) No agonism (SI<2) No agonism (SI <2) No agonism (SI <2) Dendritic cells/IL12/23p40 Antagonism (1C50/nM ± SD) < 1 nM < 1 nM < 1 nM < 1 nM Dendritic cells /IL12/23p40 Agonism No agonism No agonism No agonism No agonism Cross reactivity types: Hu/Cyno binding (EC50 ratios**) 3 2 1 Not tested

*SI: stimulation index;

** ratio >1 indicates increased binding to Cyno compared to Human.

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

Specific binding of humanized antibodies to cellular CD40 was analyzed by flow cytometry using human HEK293 cells transfected with CD40. Concentration-dependent binding of Antibody A, Antibody B, and Antibody C was observed. The antibodies showed a similar binding profile. The _EC50 values of the antibodies of the present invention and the Kirin 4D11 antibodies are in the same range of ~1 nM, which is most likely at the limit of assay sensitivity due to the high levels of CD40 in the transfected cells. Specific binding of humanized antibodies to cellular CD40 on human Ramos cells also demonstrated concentration-dependent binding. The antibodies showed slightly different binding profiles and EC ₅₀ values between 0.21-1.22 nM. Binding was not detected on CD40-negative cells, such as untransfected HEK293 cells or the HSB-2 T cell line, confirming selective binding to CD40 (data not shown).

The binding affinities of Antibody A, Antibody B and Antibody C to human CD40-Fc protein were measured using ForteBio Octet and dissociation constants (K _D ) <100 pM were detected. Due to the bivalency of antibodies and CD40-Fc, avidity effects prevent accurate determination of Kds below 100 pM. Additionally, binding to CD40-Fc was assayed 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 the humanized antibodies was tested in a B cell proliferation assay in which human B cells derived from peripheral blood were stimulated with recombinant CD40L in the presence of IL-2 and IL-4. Antibody A, Antibody B, and Antibody C showed potent inhibition of B cell proliferation. Comparison with the inhibition curves and IC ₅₀ values of the BI antibody and the Kirin 4D11 antibody indicates that the 4D11 antibody has higher potency when tested on multiple donors. When tested for agonistic activity in the absence of CD40L, antibodies such as Antibody B, Antibody A and Antibody C did not induce any B cell proliferation above background levels at concentrations up to 10 μg/ml (67 pM), similar to antibody 4D11.

Competitive antibody 4D11 was found to be slightly more potent, with an average IC ₅₀ of ~0.02 nM and no agonistic effects. Data for three antibodies against BI and 4D11 are summarized in Table. 6 above. Another competitive antibody, PG-102 (derived from clone 5D12), also tested in this assay, showed significant agonist effects stimulating B cell proliferation in the absence of CD40L. Therefore, the lack of agonist activity in the lead candidates of the invention clearly distinguishes them from PG-102.

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In a second assay, the antibodies were assessed for inhibition of CD86 upregulation in human B cells. In this case, the assay can be performed with human whole blood or with purified B cells, as in the presence of exogenous CD40L. Consistent with the B cell proliferation data, Antibody B, Antibody A, and Antibody C tested in human whole blood showed potent inhibition of CD40-mediated CD86 upregulation as measured by flow cytometry. Antibody C showed similar potency to 4D11 in this assay, while the potency of Antibody B and Antibody A was slightly weaker. Comparison of Antibody B and 4D11 on purified B cells or in whole blood shows that the effectiveness of Antibody B (IC ₅₀ and IC ₉₀ values) is relatively unchanged for purified B cells compared to B cells in the presence of other cells bearing CD40 or serum, while 4D11 undergoes a dramatic change in activity in whole blood conditions.

Similar data were obtained when Antibody B, Antibody A, and Antibody C were assessed for inhibition of CD86 upregulation on cynomolgus B cells using whole blood samples. Antibody B, Antibody A, and Antibody C tested in cynomolgus monkey whole blood showed potent inhibition of CD40-mediated CD86 upregulation as measured by flow cytometry. Thus, all of these antibodies exhibit functional cross-reactivity to cynomolgus CD40 with similar potency to human CD40.

The activity of Antibody B IgG ₁ KOb and Antibody B IgG ₁ WT was assessed for the ability to mediate antibody-dependent cellular cytotoxicity. In this assay, RAMOS cells were incubated with human PBMCs at a 50:1 ratio of effector cells to target cells. Antibody B IgG1KOb and Antibody B IgG1WT were titrated from 20 μg/ml and the degree of cell death was monitored by LDH release. Data shown are from one representative experiment. Data indicate that Antibody A IgG1Wt 20E2-12-RIgG1WT is an effective mediator of ADCC and that Antibody B IgG1KOb containing mutations that eliminate effector function does not have ADCC activity.

Example 3: Pharmacokinetic/pharmacodynamic studies.

A. Single intravenous administration of antibody A and antibody B at a dose of 1 or 10 mg/kg in cynomolgus monkeys.

Antibody A and Antibody B were administered at a dose of 1 and 10 mg/kg intravenously to male cynomolgus monkeys (K=3)/dose. Blood samples were collected from 0 to 504 hours (3 weeks), serum was collected and samples were stored at -20°C until analysis. Samples were analyzed by sandwich ELISA as described above. Serum concentration profiles of both antibodies in monkeys after intravenous doses and pharmacokinetic parameters are summarized in Table 1. 7 (Antibody A) and 8 (Antibody B) shown below. Both antibodies demonstrated dose-dependent pharmacokinetics, suggesting that at the low dose, clearance is predominantly due to target-mediated disposition, whereas at the higher dose, the antibody is cleared primarily through catabolism. Similar dose-dependent pharmacokinetic profiles were observed for other MAbs targeting membrane-associated targets (eg, CD19, CD20, EGFR, CD146, and HER2). Clearance for antibody A was 0.8 and 0.1 ml/h/kg for doses of 1 and 10 mg/kg, respectively. Clearance for antibody B was 0.7 and 0.1 ml/h/kg for doses of 1 and 10 mg/kg, respectively. Likewise, the half-lives of Antibody A were 1 and 13 days for the 1 and 10 mg/kg doses, respectively, and the half-lives of Antibody B were 2 and 13 days for the same respective doses. Although Antibody B had a slightly longer half-life at a lower dose compared to the same dose for Antibody A, this difference would not be expected to result in longer effects when administered chronically. The AUC for both compounds was disproportionate, and the volume of distribution (Vss) for both compounds approximated plasma volume (~40 mL/kg), demonstrating the limited tissue distribution typically observed for large, polar protein therapeutics. Overall, there were no noticeable differences in pharmacokinetic parameters between the two antibodies.

Table 7

Pharmacokinetic parameters of antibody A in male cynomolgus monkeys (N 3) dose after a single intravenous dose of 1 and 10 mg/kg

Dose (mg/kg) CLp (ml/h/kg) Vss (ml/kg) AUC (µM.h) T1/2 (days) MRT (days) 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

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Table 8

Pharmacokinetic parameters of antibody B in male cynomolgus monkeys (N 3) dose after a single intravenous dose of 1 and 10 mg/kg

Dose (mg/kg) CLp (ml/h/kg) Vss (ml/kg) AUC (µM.h) T1/2 (days) MRT (days) 1 0.7 ± 0.16 40 ± 2 10.1 ± 2.7 1.5± 0.2 2.6 ± 0.8 10 0.09 ± 0.01 41 ± 6 744 ± 55 13.3 ± 3.0 19.3 ± 4.2

B. Ex vivo pharmacodynamic study.

As part of the PK study described above, we analyzed the pharmacodynamic effects of anti-CD40 antibodies. To do this, whole blood samples were incubated with recombinant CD40L overnight and the increase in CD86 expression on B cells was determined by flow cytometry. Samples were analyzed on day 0 (pre-treatment), days 2, 7 and 14 post-dose. Although the increase in CD86 expression is relatively small (~5-20%), a dose-dependent effect was observed. In the group of animals treated with 10 mg/kg Antibody A and Antibody B, CD86 induction was completely inhibited after 2, 7 and 14 days, consistent with long-term exposure to this dose. Animals dosed at 1 mg/kg showed complete inhibition on day 2, partial inhibition on day 7, and no inhibition on day 14. Loss of pharmacodynamic effect over time correlates with faster clearance of the antibody in the low dose group.

Example 4: Toxicology Studies: CD40 on Platelets.

CD40 is constitutively expressed on human platelets (Henn, et al., 2001) and (Inwald, et al., 2003), whereas CD40L is rapidly and transiently expressed on the cell surface of activated platelets (Henn, et al., 2001). Although anti-CD40 antibodies without FcyR binding would not be expected to have an effect on platelets, it is important to directly demonstrate that this is the case. Flow cytometry studies were performed to demonstrate binding of anti-CD40 lead candidates to human and cynomolgus platelets.

It was previously demonstrated by flow cytometry that G28.5 and the anti-CD40 mAb 89 bind to resting human platelets (Henn, et al., 2001). This was confirmed using the FITC-labeled antibody G28.5. 5-fold serial dilutions of G28.5 were prepared and incubated ranging from 0.5 μg/ml to 0.32 ng/ml in 100 μl of platelets obtained from humans (2 donors) or cynomolgus monkeys (3 donors) for 30 min at room temperature. In addition, APC-labeled anti-CD45 mAbs were used to identify platelets associated with other CD40+ cell types in order to exclude these cells from the analysis. After antibody staining, platelets were washed and fixed with Optilyse C and flow cytometry was performed. Mean fluorescence intensity (MFI) was determined as a measure of antibody binding to CD45 ⁻ platelets.

Commercially available 5c3 and selected mouse anti-CD40 mAbs according to the invention were labeled with FITC. Binding to Ramos cells was confirmed. The number of FITC molecules per antibody molecule varied from 2 to 4 FITC per antibody molecule. Five-fold serial dilutions of commercial and candidate anti-CD40 mAbs ranging from 0.5 μg/ml to 0.32 ng/ml were prepared and incubated with human (3 donors) and cynomolgus (2 donors) platelets for 30 min at room temperature.

A representative graph showing the binding of a murine anti-CD40 mAb candidate to human platelets was previously shown in US Pat. No. 8,591,900. Four candidate monoclonal antibodies exhibited specific binding to human platelets compared to a FITC-labeled isotype control antibody. 10F2, 2H11, 19B10 and 20E2 showed comparable binding to platelets. A similar trend was observed for cynomolgus monkey platelets (data not shown).

In addition to these studies, directly labeled B and 4D11 antibodies were compared for their ability to bind platelets and B cells in human and cynomolgus monkey whole blood samples. 4D11 exhibited similar binding (as demonstrated by EC ₅₀ ) to both B cells and platelets in human and cynomolgus monkey blood samples. Antibody B showed a similar pattern, but with much weaker binding capacity.

Example 5: In vivo pharmacological studies in the NSG mouse model.

The effectiveness of the humanized antibody, Antibody A, was assessed in an antibody production model in which human PBMC were administered to immunodeficient NSG mice to generate graft-versus-host disease. Significant production of human IgM (hIgM) and IgG (hIgG) can be detected 2 weeks after engraftment. Antibody A treatment at doses of 5 and 1 mg/kg significantly inhibited the hIgG and hIgM responses at 2 and 3 weeks postengraftment. A comparator antibody (4D11) was evaluated at a single dose of 5 mg/kg and also demonstrated an abrogation response. In the second study, all antibodies - Antibody A, Antibody B and Antibody C - were tested at a single dose

- 52 046388 mg/kg and showed complete inhibition of the IgM and IgG response at 2 weeks.

Example 6. Biomarker analysis.

Receptor upregulation: CD40L-induced receptor upregulation can be measured using flow cytometry. Human whole blood can be stimulated with an optimized concentration of soluble CD40L, and the full percentage of CD20 + receptor + cells can be measured using flow cytometry. The change in percentage of CD86 expression on CD20-positive cells was measured in parallel with a PK study in cynomolgus monkeys assessing antibodies A and B. Data show inhibition of CD86 upregulation at time points corresponding to antibody exposure.

Targeted proteomics: Increased protein secretion upon CD40 stimulation in whole blood can be used as potential biomarkers. Optimized soluble CD40L concentration and stimulation timing were established using the Luminex multiplex bead platform detecting MDC/CCL22 and several other secreted proteins. Clinical samples will be evaluated from human whole blood across the full dose range of anti-CD40 mAbs.

Receptor occupancy: CD40 receptor occupancy can be determined in an in vitro or ex vivo assay based on flow cytometry analysis of human whole blood B cells. The current antibody candidates of the present invention and the non-competitive anti-CD40 antibody 5C3 will be used for quantitative receptor occupancy analysis.

Example 7 Antitumor activity of humanized anti-CD40 antibody.

In some cases, it may be appropriate to determine the antitumor properties of antibodies in accordance with the present invention. Such determination can be made by analyzing the antitumor activity of a humanized anti-CD40 antibody in a xenograft model of SCID mouse lymphoma. In such a SCID model, cancer cells can be introduced to represent a tumor, for example, 5 x ^{10 6} million tumor cells can be injected subcutaneously into SCID mice (10/group) 13 days before drug treatment. Mouse anti-CD40 antibodies in accordance with the present invention or for comparison (eg, control or other humanized antibody) are administered intraperitoneally 3 times a week (4 mg/kg/dose) with 8 or 5 doses administered. The development and growth of tumors in mice is monitored, and tumor volume can be measured weekly during a selected study period, such as a 14-day study period. Advantageously, the results will show a 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater increase in tumor growth in control mice compared to mice treated with antibodies in accordance with the present invention. Preferably, during the treatment period, tumor growth in mice treated with antibodies in accordance with the invention will be negligible. Such data may confirm that the tested humanized antibody effectively suppresses tumor growth in this xenograft mouse model of B lymphoma.

Example 8 Prolonged survival due to humanized anti-CD40 antibody.

The effectiveness of a humanized anti-CD40 antibody on the survival of tumor-bearing mice such as those described above can be assessed in a xenograft model of SCID mouse lymphoma. SCID mice (10/group) were inoculated intravenously with 1x 10 ⁶ million tumor cells three days before antibody treatment. Mice are then treated with murine or humanized anti-CD40 antibodies of the present invention or control Ig, administered intraperitoneally twice weekly (4 mg/kg/dose) for a total of five doses. The mouse cells can then be monitored daily for mortality to determine the level of effectiveness of the antibodies in prolonging the survival of the subject having cancer.

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

Example 9 Safety, pharmacokinetics and pharmacodynamics of multiple escalating doses of the antibody of the present invention, an anti-CD40 antagonist antibody, in healthy subjects: Possible new treatment for autoimmune diseases.

The purpose of this randomized, placebo-controlled, double-blind study was to determine the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of 4 weeks of repeated once-weekly subcutaneous administration of 80, 120, 180, or 240 mg of the antibody of the present invention. in healthy subjects.

Methods.

Study design.

This phase 1 study was approved by the participating site's Independent Ethics Committee and the New Zealand health authorities, and all participants provided informed consent. The study was sponsored by Boehringer Ingelheim and conducted jointly

- 53 046388 venous research center in Auckland, New Zealand, by Auckland Clinical Studies Ltd.

The study was a randomized, placebo-controlled, double-blind, dose-limited group study.

Multiple increasing subcutaneous doses of 80-240 mg of the antibody of the present invention were tested in healthy subjects once a week over a 4-week treatment period.

Doses were selected based on safety, PK, and PD data from a single dose escalation study. In this study, the maximum intravenous dose tested (120 mg) was well tolerated and produced 7 times the maximum observed concentration ( _Cmax ) and 3 times the area under the concentration-time curve (AUC) than the 120 mg subcutaneous dose tested. Based on these PK data, it was calculated that exposure from a 120 mg IV dose in a single ascending dose study would cover the exposure expected from a 240 mg SC dose.

Eligible subjects were randomized to receive the antibody of the present invention or placebo in a 4:1 ratio using the Interactive Response Technology tool in four sequential subcutaneous dose groups (80, 120, 180 and 240 mg), with dose groups separated by at least for 7 days. Each dose group consisted of 10 subjects (8 active, 2 placebo) (Fig. 1). The decision to increase to the next dose level was made by an independent data monitoring committee based on an assessment of safety, tolerability, PK and PD data.

All subjects received the last dose of treatment on day 22. Subjects in the 80, 120, and 180 mg subcutaneous dose groups were followed for 42 days after their last dose, for a total study duration of 64 days. Subjects in the 240 mg subcutaneous dose group were followed for 56 days for a total study duration of 78 days.

Subjects, investigators, and sponsoring personnel remained blinded to the study treatment. Initial database closure was performed after patients in the 80–180 mg subcutaneous dose groups completed the study, and all data were released after completion of the 240 mg subcutaneous dose group.

Blood samples (2.7 ml) for PK analysis were taken from a vein of the forearm using an indwelling catheter into tubes with an anticoagulant - tripotassium salt of ethylenediaminetetraacetic acid.

Samples were collected pre- and post-dose at 1, 8 and 12 hours, and on days 1, 2, 3 (morning and evening, 12 hours apart), 4 (morning and evening, 12 hours apart), 5, 6 , 7 (before the second dose), 14 (before the third dose), 21 (before the fourth dose and 1 and 12 hours after the fourth dose), 22, 23, 24, 25, 26, 27, 28, 29, 31, 34 , 38, 42, 49, 56, 63 (80-180 mg dose group only) and 77 (240 mg dose group only) after the first dose.

Blood samples for antibody assessment (ADA) versus antibodies of the present invention were collected predose and after the first dose on day 21 (prior to the fourth dose), 38, 63 (80-180 mg dose groups only ) and 77 (240 mg group only) after the first dose.

Within 30 min of sample collection, blood samples were immediately placed on ice after collection and centrifuged at 4°C for 10 min. Plasma was transferred into two polypropylene sample vials (0.5 ml each) and stored at -20°C before being sent to the analytical laboratory.

Blood samples (4.9 ml) for PD analysis were collected from a forearm vein using an indwelling catheter into heparin anticoagulant tubes, predose and on days 3, 7 (before the second dose), 21 (before the fourth dose), 24. 28, 38, 63 (80-180 mg dose group only) and 77 (240 mg dose group only) after the first dose and were immediately taken to the laboratory for analysis. Validation of CD40 RO and CD54 upregulation assays has shown that whole blood can be left at room temperature for up to 24 h and 6 h, respectively, before analysis.

Study participants.

The study included eligible subjects aged 18 to 60 years with a body mass index of 18.5 to 29.9 kg/m ² . Female participants were required to be postmenopausal, have undergone surgical sterilization, abstain from sexual intercourse, have a vasectomized sexual partner, or have been using accepted contraceptive methods for <30 days before study drug administration and within 30 days after completion of the study, and test results for pregnancy were negative during the study.

Subjects were excluded if they had any evidence of clinically significant abnormalities identified by physical examination or laboratory testing; concomitant disease; any gastrointestinal, hepatic, renal, respiratory, cardiovascular, metabolic, immunological or hormonal disorders; disease of the central nervous system; orthostatic hypotension, fainting or temporary loss of consciousness; or allergies or hypersensitivity reactions to drugs. Subjects were also excluded if they had taken any other drugs with a long half-life ( ^t1 / ₂ ; >24 hours) within 30 days or less than 10 half-lives before randomization; took medications that could affect the study results within 10 days before the first day of study; received any study drug during

- 54,046,388 days prior to the first dosing day of the study; were blood donors within 30 days prior to the first study dose day; there was evidence of drug abuse or excessive use of alcohol or cigarettes; test positive for human immunodeficiency virus, hepatitis B, hepatitis C, tuberculosis, or chronic or acute infection; intention was to initiate a new exercise regimen within 1 week prior to the first study dosing day. Women who were breastfeeding or who planned to become pregnant within 30 days of completing the study were also excluded.

Analytical methods.

Plasma concentrations of the antibody of the present invention were analyzed using a validated sandwich enzyme-linked immunosorbent assay (ELISA) with a lower limit of quantitation of 30 ng/ml. 96-well microtiter plates were first coated with an antibody comparable to the antibody of the present invention, blocked and washed. The plates were then incubated with test samples, calibrators, or quality control samples and washed again. Binding of an antibody of the present invention was detected using a biotinylated antibody against an antibody of the present invention, then streptavidin conjugated to horseradish peroxidase, and finally to the peroxidase substrate tetramethylbenzidine. The plates were read colorimetrically and the data were analyzed using a 5-parameter logistic fit. The quantitative range was 30-800 ng/ml. Adequate accuracy and precision were assessed during routine analysis with quality control samples at three concentrations—low (50 or 100 ng/mL), medium (126 or 200 ng/mL), and high (500 or 590 ng/mL). The reproducibility of the ELISA was verified by re-analysis of the samples, in which 93% of the samples met the acceptance criteria (deviation <30% from the mean).

Antibodies in accordance with the present invention were analyzed in plasma samples using a validated bridging electrochemiluminescence method. All sample data submitted met the assay-specific acceptance criteria. Validation of the ADA assay showed that 250 ng/ml positive control ADA antibody could be detected in the presence of plasma concentrations of 50 μg/ml antibody in accordance with the present invention. In addition, the subject's true positive response was characterized using additional titer quantification assays. Titers were determined by analyzing serial 2-fold dilutions of the sample. The reported titers were the highest fold dilution that produced a mean electrochemiluminescence value that was greater than or equal to the plate cutoff point.

Both determinations of antibody concentrations in accordance with the present invention and ADA assessments were performed at Covance Laboratories, Inc. (Chantilly, Virginia, USA).

To measure CD40 RO, whole blood samples were incubated with excess fluorescein isothiocyanate (FITC)-labeled antibody of the present invention and anti-CD19 allophycocyanin (APC; for B cell gating) for 20 min at room temperature in the dark. Fluorescence-activated cell sorting (FACS) lysis solution was added and the tubes were incubated for 15 min at room temperature in the dark, followed by centrifugation (1300 rpm) at 4°C for 6 min and removal of the supernatant. CellFix was added and the tubes were shaken and stored at 4°C in the dark until FACS analysis, which was performed within 24 hours of CellFix addition. All samples were kept on ice during measurements.

To measure inhibition of CD54 upregulation, whole blood was incubated with either interleukin-4 (IL-4) alone - unstimulated FACS tubes or MegaCD40L + IL-4 stimulated FACS tubes. The tubes were shaken and incubated in the dark at 37°C in a humidified incubator for 23-26 hours. Ahtu-CD19-APC and anti-CD54-phycoerythrin (PE) were added to each tube and the tubes were shaken and incubated for 20 minutes in the dark at room temperature. FACS lysis solution was added and the tubes were incubated for 15 min at room temperature in the dark, followed by centrifugation (1300 rpm) at 4°C for 6 min and removal of the supernatant. CellFix was added and the tubes were vortexed and stored at 4°C in the dark until FACS analysis was performed within 2 h of CellFix addition. All samples were kept on ice during measurements.

With both CD40 RO and CD54, up-regulation assays were quasi-quantitative. Results of the analysis were based on percent change (ie, the on-treatment samples were related to the pre-dose sample).

To examine the possibility of thromboembolic events, the following assessments were performed: international normalized prothrombin time ratio (PT-INR), activated partial thromboplastin time (aPTT), antithrombin III, fibrinogen, S and C proteins, platelet count, bleeding time (measured by the Duke method) and D-dimers.

Pharmacokinetic assessment.

Plasma concentration-time data for the antibody of the present invention

- 55 046388 was analyzed by a non-compartmental approach using WinNonlin™ (version 5.02, Gary, NC, USA). Parameters determined included: _Cmax , time to reach _Cmax ( _tmax ), terminal elimination constant ^z) and final ^t1 / ₂ using the standard WinNonlin™ procedure. The area under the concentration-time curve during the uniform dosing interval τ (AUC _0-T ) after the last (fourth) dose was calculated using the WinNonlin™ algorithm with linear increase and decrease. Accumulation coefficients (RA, C _max based on C _max ; RA, AUC based on AUC _0-T ) were calculated as the ratio of the value after the fourth dose to the value after the first dose.

Pharmacodynamic assessment.

Pharmacodynamic evaluation included assessment of CD40 RO using the antibody of the present invention and inhibition of B cell activation as measured by megaCD40L-induced CD54 up-regulation in whole blood using the above-mentioned validated FACS assays. The relationship between antibody dose of the present invention and CD40 RO inhibition and CD54 upregulation has previously been examined using standard sigmoidal E _max models and described in Albach et al. Eur. J. Clin. Pharmacol. 2018; 74(2):161-169.

Safety and tolerability.

The safety and overall tolerability of the antibody of the present invention were assessed by monitoring treatment-emergent adverse events (AEs), physical examinations, vital signs (blood pressure and pulse), 12-lead electrocardiogram (ECG), and clinical laboratory tests (blood count, biochemical analysis and urine analysis).

Statistical analysis.

No formal sample size determination was made; 8 subjects in each dose group were considered sufficient for pharmacokinetic and safety analyses. Study results were analyzed using descriptive safety, PK and PD statistics. The safety population included all subjects who received study drug (antibody of the present invention or placebo). The PK and PD populations included all subjects who received study drug and provided evaluable data for the PK and PD analyses, respectively. Dose proportionality of AUC _0-T and _Cmax after the fourth dose was assessed using a power-law model. The 95% confidence interval (CI) for the slope was calculated. Ideal dose proportionality was defined by a slope parameter (β) of 1. Descriptive analyzes including graphical presentation of concentration data were performed to assess whether steady state had been achieved.

Results.

Subjects.

A total of 40 healthy subjects were randomized and treated in the study. Subjects received repeated subcutaneous treatment once a week with placebo (n=8), 80 mg antibody of the present invention (n=8), 120 mg antibody of the present invention (n=8), 180 mg antibody of the present invention with the present invention (n=8) or 240 mg of an antibody in accordance with the present invention (n=8) over a 4-week period. All 40 subjects completed the planned follow-up period, and there were no premature terminations. The majority of subjects were male (83%) and white (73%), with a mean (standard deviation [SD]) age of 30 (10.8) years and a mean (SD) body mass index of 25 (3.1) kg/m ² . There were no significant demographic differences between treatment groups.

Pharmacokinetics.

The geometric mean (gMean) of selected PK parameters for the antibody of the present invention after administration of the first subcutaneous dose (day 1) and the last subcutaneous dose (fourth) is presented in table. 9. After the first dose, the median _tmax increased with each weekly dose, but _tmax did not show any clear dose relationship after the fourth dose ( _tmax , ₄ ). The maximum plasma concentration and AUC normalized for the administered dose (Cmax, _norm , 4 and AUC _T , _norm , ₄ , respectively) were lower for the 80 mg dose group of the antibody of the present invention and similar for the three higher dose groups , suggesting a more than proportional increase in exposure from 80 to 120 mg, but nearly dose-proportional kinetics for doses >120 mg. _Cmax or AUC (RA, _Cmax , ₄ and RA, _AUC , ₄ , respectively) was determined to evaluate the accumulation of the antibody in accordance with the present invention after four repeated doses. After four once-weekly subcutaneous doses of 80 mg, RA, _Cmax , ₄ and RA, _AUC , ₄ values were 8.3- and 11.6-fold higher, respectively, than after a single dose, indicating antibody accumulation consistent with the present invention. gMean accumulation was lower for the three higher doses (range: 3.7-4 for _RA , _Cmax , ₄ and 4.9-5.8 for RA, _AUC , ₄ ). The final t _1/2 of the antibody in accordance with the present invention ranges from 156 to 199 hours (6-8 days). Visual inspection of trough concentrations showed that steady state was not achieved for any of the doses: trough plasma concentrations for all groups continued to increase with each subsequent dose (Fig. 2).

- 56 046388

Table 9

Selected PK parameters of the antibody of the present invention after the first dose (day 1) and the last dose (after 4 subcutaneous administrations once a week)

BI 655064

After the first dose 80 mg (n = 8) 120 mg (n = 8) 180 mg (n = 8) 240 mg (n = 8) Stakh, MKG/ML 1.6(492) 7.7 (29.5) 9.9 (67.8) 18.0(46.3) ds.pogt, mcg/ml/mg 0.02 (492) 0.06 (29.5) 0.05 (67.8) 0.08 (46.3) tmax, H 66(12-168) 78 (48-108) 108 (72-144) 156(108-168) ACCo-tL μg-h/ml 154 (683) 850 (34.4) 919(78.9) 1630 (42.6) AUCo-T.norm, MCG-H/ML/MG After the fourth dose ³ 1.9 (683) 7.1 (34.4) 5.1 (78.9) 6.8 (42.6) Stakh, 4, MKG/ML 13.1 (59.1) 28.7 (35.6) 39.8 (37.4) 68.4(21.9) Sshah.pogt, 4, mcg/ml/mg 0.16(59.1) 0.24 (35.6) 0.22 (37.4) 0.29(21.9) tmax, 4, H 96.0 (96-144) 84.1 (12-144) 96.0 (72-192) 108 (96-504) AUC ₀ . _T , 4? µg-h/ml 1790 (56.4) 4140 (35.4) 5470 (37.4) 9460 (22.4) AUCo-T.norm, 4, 22.3 (56.4) 34.5 (35.4) 30.4 (37.4) 39.4 (22.4) mcg-h/ml/mg tl/2, 4, Ch 186 (39.4) 156(28.3) 171 (39.6) 199 (28.4) R _A ,Cmax, 4 8.3 (226) 3.7(31.9) 4.0 (46.9) 3.8 (23.4) Ra,AUC, 4~ 11.6 (289) 4.9 (27.7) 6.0 (45.3) 5.8 (25.4)

AUC, area under the curve;

_Cmax , maximum observed concentration;

PK, pharmacokinetic;

RA, accumulation ratio;

t _1/2 , half-life;

tmax, time to reach Cmax.

Data are presented as geometric mean (geometric coefficient of variation, %), except for tmax data, which are presented as median (range).

^and PK parameters analyzed after the fourth dose of the antibody of the present invention are indicated by a superscript 4 (eg, tmax, 4 ⁾ ;

^b AUC ₀ - _T is synonymous with AUC ₀ . _{168 h} ;

^c RA,A _UC is equal to the AUC _0-T after the fourth dose divided by the AUC _0-T after the first dose.

Dose proportionality analysis over the 80-240 mg subcutaneous dose range showed that the slope for C _max and AUC _0-T was significantly different from unity, indicating that the effect of the antibody of the present invention was not dose proportional (C _max : power law slope model β=2.1 [95% CI 1.2-2.9] after first dose; slope β=1.4 [95% CI 1.1-1.8] after last dose; n=32; AUC _0-T : slope β=1.4 [95% CI 1.1 -1.8] after the last dose; n=32). However, for higher doses (120-240 mg), a trend towards dose proportionality was observed.

Pharmacodynamics.

Administration of an antibody according to the present invention resulted in a dose-dependent CD40 RO and inhibition of CD54 upregulation (FIG. 3).

Following a single subcutaneous dose of the antibody of the present invention, the arithmetic mean CD40 RO had already reached near maximum values for each dose level at the first post-dose measurement (72 hours) (FIG. 3A). At this time point, the 80 mg dose resulted in approximately 89% CD40 RO. For the 120-240 mg dose groups, CD40 RO stabilized at 94-95%; because this was the detection limit of the assay, it was not possible to determine whether higher occupancy levels were achievable. These CD40 RO levels were maintained until the end of the study.

- 57 046388

After the last (fourth) weekly subcutaneous administration of the antibody of the present invention, the CD40 RO was >90% at all time points measured until day 39 (17 days after the last administration) for all doses (80-240 mg). For the 180 mg dose group, 79% (geometric coefficient of variation [gCV] 23%), CD40 RO was still detectable at day 64, and for the 240 mg dose group, 68% (gCV 29%) CD40 RO was determined at day 78, indicating long-term persistent binding to the receptor, although variability was greater at these later time points. No noteworthy CD40 RO was observed in the placebo group.

Inhibition of CD54 upregulation after administration of a single dose of an antibody according to the present invention followed a pattern similar to that observed for CD40 RO, with the 80 mg dose providing 87% inhibition in the first post-dose measurement (72 hours), and >90% inhibition. observed for higher doses at this time point (all dose groups; Fig. 3B). For the 80 mg dose group, inhibition increased further to 95% on day 7 post-dose, whereas for all other doses, 95% inhibition was observed after 72 hours; in the placebo group, inhibition of CD54 upregulation varied between -20 and 30%. After the last (fourth) weekly subcutaneous administration of the antibody of the present invention, inhibition of CD54 upregulation was >90% for all doses up to day 39 (17 days after the last administration). The inhibitory effect was still detectable for the 180 mg dose group (89% at day 64) and for the 240 mg dose group (51% at day 78).

Safety.

The overall incidence and intensity of CR were similar in the antibody treatment groups of the present invention (all doses of antibodies of the present invention [78%] and placebo group [88%]). No serious adverse reactions, severe adverse reactions, or adverse reactions leading to discontinuation or death were reported. Although the number of subjects was small, no relationship was found between the dose of the antibody of the present invention, treatment-related ADRs, or the frequency and intensity of ADRs. Infections were reported in 8 subjects (25%) receiving the antibody of the present invention and in 5 subjects (63%) receiving placebo. There were no thromboembolic events. The most common treatment-related adverse event was headache in 4 subjects (13%) receiving the antibody of the present invention and 2 subjects (25%) receiving placebo. All ADRs were mild or moderate in severity, and all of them were eliminated.

For individual subjects in the 80 and 120 mg treatment groups, significant increases in creatine kinase (CK) were observed both at baseline and after treatment with either an antibody of the present invention or placebo (range 1.1-88 times the upper limit of normal [ULN ]). Overall, this increase in CK was determined to be primarily associated with intense exercise. Introducing more stringent exercise restrictions to the higher dose groups (180–240 mg) resulted in decreased CK levels (maximum 3 times ULN).

Of all subjects receiving the antibody of the present invention, mild and transient leukopenia and neutropenia were observed in 12 (37.5%) and 14 (43.8%) subjects, respectively. Only 1 subject receiving placebo had mild and transient neutropenia. Values below the lower limit of normal (LLN) were observed in 4 of 12 subjects (33.3%) with mild transient leukopenia and in 5 of 14 subjects (35.7%) with mild transient neutropenia before treatment. In all subjects, white blood cell (WBC) and absolute neutrophil counts returned to normal values (WBC normal range: 4-11 x ¹⁰ /L and neutrophil absolute normal range: 1.9-7.5 x 10 /L) or reached pre-treatment levels by the end of the study , except for 1 subject with a WBC count of 3.77 x ^{10 9} /L at the end of the study and another subject with a low WBC count and an absolute neutrophil count of 3.58 x ^{10 9} /L and 1.69 x 10 /L, respectively, at the end of the study visit. Moreover, there was no increase in the number of subjects with leukopenia or neutropenia when increasing the dose of the antibody antibody in accordance with the present invention.

There were no clinically significant changes in bleeding time, platelet count, or coagulation parameters including D-dimers, antithrombin III, fibrinogen, and proteins S and C.

There were no clinically significant findings or treatment differences between groups regarding vital signs, ECGs, or physical examinations. Evaluation of local tolerability showed that all doses of the antibody in accordance with the present invention were well tolerated, and there were no differences compared with the placebo group.

Pre-existing ADA reactions were observed in 4 subjects (10%), 3 of whom subsequently received antibodies in accordance with the present invention, and 1 of whom received placebo. The ADA titer was increased in only 1 of these subjects receiving a 240 mg dose of the antibody of the present invention (treatment-boosted ADA [pre-existing ADA that was increased to a higher level after biological administration]).

Seroconversion was observed in 16 subjects (50%) after treatment with an antibody in accordance with the present invention; onset was mainly in the post-study samples (15 subjects

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[47%]). At that time, antibody levels of the present invention were already very low (gMean plasma concentrations of antibody of the present invention ranged from 0.182-10.5 μg/ml for the 80-240 mg dose groups).

Treatment-induced ADAs (ADAs produced de novo following biological administration) or treatment-enhanced responses to ADAs were observed in 5 subjects (62.5%) in the 80 mg dose group and 6 subjects (75%) in the 120 mg dose group. Overall median titers in the 80 and 120 mg dose groups were 20 and 8, respectively. Treatment-induced or treatment-enhanced responses to ADA were observed in fewer subjects in the higher dose groups; 2 (25%) and 4 (50%) subjects in the 180 and 240 mg dose groups, respectively, with overall mean titers of 4 and 8, respectively. The maximum titer in an individual subject was 640, which was observed in the 120 mg dose group.

Discussion.

The objectives of this study were to examine the effects of 4 weeks of escalating subcutaneous doses of the antibody of the present invention (80-240 mg per week) in healthy subjects. Evaluation of PK parameters showed nearly proportional kinetics for subcutaneous doses of 120-240 mg of the antibody of the present invention, but over-proportional kinetics for subcutaneous doses of 80-120 mg due to target-mediated drug clearance as observed in a previous study after single intravenous doses of the antibody in accordance with the present invention. The effect is enhanced by the wide distribution of CD40 receptors (especially platelets with their short t1 _/2 ), as previously reported in other studies with antagonistic CD40 antibodies. An almost proportional dose-content relationship was observed in the range of subcutaneous doses of 120-240 mg of the antibody in accordance with the present invention for C _max (after the first and last dose) and for AUC _0-T (after the last dose), with a slope of β = 1.2 for both parameters, possibly indicating that CD40 RO approaches saturation at these doses. The plasma levels achieved in this study after the first 80 and 120 mg subcutaneous doses of the antibody of the present invention were similar to those observed in a previous single dose escalation study in healthy volunteers, where the mean gMean AUC values were 120 and 888 μg/h /ml were obtained after a single subcutaneous dose of 80 and 120 mg of the antibody in accordance with the present invention, respectively. The accumulation of antibodies in accordance with the present invention was observed at all dose levels after multiple administrations compared to the administration of a single dose (first dose). However, less accumulation was observed for doses of 120-240 mg, with relatively constant gMean RA, _Cmax values of 3.7 to 4 and gMean RA, _AUC values of 4.9 to 6. Steady state was not achieved over the 4-week period. period for any of the administered doses. Simulations have shown that it may take up to 12 weeks to reach steady state at a dosage of 120 mg of the antibody of the present invention once a week (unpublished in preparation). The prediction of achieving steady state within about 12 weeks was confirmed by treating patients with rheumatoid arthritis with 120 mg of the antibody of the present invention subcutaneously once a week for 12 weeks, demonstrating that steady state PK is achieved within about 10-12 weeks. Thus, this provides a rationale for using a loading dose to reach steady state more quickly in future clinical studies. For the AUC and C _max content parameters, interindividual variability was higher for the 80 mg dose (gCVs: 56.4-59.1%), moderate for the 120 and 180 mg doses (gCVs: 35.4-37.4%) and lower for dose 240 mg (gCVs: 21.9-22.4%). The antibody of the present invention was slowly absorbed from the subcutaneous injection site, with the mean _tmax increasing with dose after the first dose; after the fourth dose there was no relationship between doses and _tmax , ₄ . After administration of multiple doses of antibodies in accordance with the present invention, the estimated final t _1/2 was in the range of 6 to 8 days with no apparent difference between doses.

Evaluation of CD40 RO and inhibition of CD54 upregulation showed that single subcutaneous doses of 120 to 240 mg of the antibody of the present invention resulted in >90% CD40 RO and >90% inhibition of CD54 upregulation 72 hours post-dose. After the last (fourth) dose of the antibody of the present invention, >90% CD40 RO and inhibition of CD54 upregulation was maintained for at least 408 hours (17 days) after dosing in the 80-240 mg subcutaneous dose range. These results suggest the possibility of continuous complete inhibition of CD40 agonistic ligation when administered subcutaneously with the antibody of the present invention. Modeling has shown that a dosage of the antibody of the present invention of 120 mg subcutaneously once a week for 3 weeks followed by once every other week will result in a continuous >90% CD40 RO. In patients with inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, or lupus nephritis, the CD40 receptor is expected to be highly expressed and activated on a variety of immune cells and resident cells (eg, mesangial cells in lupus nephritis); thus, higher doses of the antibody of the present invention may be required to completely block the CD40 receptor in patients with autoimmune diseases

- 59 046388 than those that result in 90% receptor occupancy by B cells in healthy subjects. Clinical studies of the antibody of the present invention in these patients will need to evaluate whether longer dosing intervals can achieve clinical efficacy or whether weekly dosing is necessary.

Increasingly multiple subcutaneous doses of the antibody of the present invention have been found to be safe and have been shown to be generally well tolerated in healthy subjects. All adverse events were mild or moderate in severity and there were no adverse events reported that would have resulted in discontinuation of study participation. Serum CK values exceeding the upper limit of normal were reported in some subjects at baseline and after treatment in both the antibody group of the present invention and the placebo groups, but these were associated with excessive exercise; similar to what was previously reported in the literature. Introducing stricter exercise restrictions to the 180 and 240 mg dose groups resulted in decreased CK concentrations. A few subjects experienced mild and transient leukopenia and neutropenia following treatment with an antibody of the present invention. However, values below the LLN were already observed before treatment in 33.3% of leukopenic subjects and, correspondingly, in 35.7% of neutropenic subjects. Transient neutropenia is very common in healthy people and in some cases is associated with concomitant viral infections. Neutropenia has previously been reported in subjects participating in high-intensity sports, and the majority of subjects included in this study performed intense exercise, as supported by the observed significant increase in serum CK values. In addition, it has recently been shown that exercise-induced muscle damage causes a rapid local inflammatory response and that local accumulation of leukocytes is associated with muscle weakness. The elevated CK levels observed in this study indicate that these subjects had some muscle damage; therefore, redistribution of leukocytes from the circulation toward muscle may have contributed to the observed transient leukopenia and neutropenia. In general, there is no clear relationship between the observed neutropenia and treatment with the antibody in accordance with the present invention. However, changes in leukocytes and neutrophils will be carefully monitored in subsequent clinical studies with the antibody of the present invention.

No clinically significant vital signs, ECG evaluations, or physical examination findings were reported. Consistent with observations following administration of single intravenous and subcutaneous doses of the antibody of the present invention, there were no reports of thromboembolic events with repeated administration, and there were no clinically significant changes in platelet or coagulation parameters. Previous platelet aggregometry and human platelet binding studies have shown that blocking CD40 has no apparent effect on platelet function (unpublished data); In toxicological studies, it was demonstrated that the antibody of the present invention bound to platelets in cynomolgus monkeys did not affect platelet count or function. Overall, these results indicate that when bound to platelets, the antibody of the present invention does not appear to alter platelet activation, aggregation or function. These data, as well as data from other anti-CD40 or anti-CD40L antibodies lacking the functional Fc region, 17, 19, 20 support the interpretation that the risk of thromboembolic events, as observed with earlier anti-CD40L antibodies, 16 can be avoided by eliminating Fc function of antibodies.

Treatment-induced or treatment-enhanced ADA responses in 50% of subjects receiving an antibody of the present invention did not cause any clinical symptoms (no association with CR or content change) or resulted in observable changes in PK after multiple doses. White blood cell and neutrophil counts did not change and were within normal limits or reached pre-treatment levels except in two subjects (these two subjects were negative for ADA). There were more cases of ADA in the 80 and 120 mg dose groups than in the higher dose groups (180 and 240 mg). Plasma concentrations of the antibody of the present invention were close to the lower limit of assay quantitation at the time of onset of response to ADA (end of study visit); the antibody of the present invention was largely eliminated, and circulating levels of the antibody of the present invention were below the drug tolerance of the ADA assay. In addition, the small number of subjects in this study did not allow definitive assessment of the dose of the antibody of the present invention or the manifestation or titer of ADA. Based on the mechanism of the antibody of the present invention inhibiting CD40 receptors and thereby blocking antibody production, the occurrence of ADA after administration of the antibody of the present invention and isotope switching of the antibody is not expected. At time point 1512 hours (63 days) (80-180 mg dose group) or 1848 hours (77 days) (240 mg dose group), CD40 RO had already decreased below 90%.

Moreover, it is expected that the levels of antibodies in accordance with the present invention will be even lower in germinal centers; Thus, antibody concentrations in accordance with this ISO

- 60 046388 shaving may have been too low to block ADA formation. This hypothesis is also supported by preclinical evaluations of antibodies in accordance with the present invention in cynomolgus monkeys, where all doses showed >90% CD40 RO for peripheral B cells, but the lowest group (1 mg/kg) did not show full pharmacological effect in the germinal centers and generated by the ADA (21 and unpublished data).

Conclusion.

Following escalating multiple weekly subcutaneous doses of an antibody of the present invention over a 4-week period in healthy subjects, PK increased disproportionately due to target-mediated clearance for doses between 80 and 120 mg, but was nearly proportional for doses >120 mg. The dose-dependent accumulation of the antibody of the present invention supports the use of a loading dose to more quickly achieve steady state in future clinical studies. The antibody of the present invention demonstrated a high potential for blocking the CD40-CD40L pathway, with consistent inhibition of CD40L-induced CD54 up-regulation. Thus, further studies are needed to evaluate whether a longer dosing interval may be clinically effective in patients suffering from an autoimmune disease such as rheumatoid arthritis, systemic lupus erythematosus, or lupus nephritis. Increasing multiple subcutaneous doses of the antibody of the present invention in the range of 80-240 mg were generally well tolerated, and no corresponding signs of acute immune reaction were observed.

The application of the solutions disclosed herein should not be limited in scope to the specific embodiments described herein. Indeed, various modifications will be within the capabilities of one skilled in the art in light of the decisions contained herein and the accompanying examples. Such modifications are intended to fall within the scope of the appended claims.

Claims (1)

A method of treating autoimmune diseases in a subject, comprising administering a therapeutically effective amount of an anti-CD40 antibody comprising SEQ ID NO: 35 heavy chain and SEQ ID NO: 31 light chain, in a loading dose that is greater than 120 mg.