EA042934B1 - AGONISTIC ANTIBODIES THAT BIND TO HUMAN CD40 AND OPTIONS FOR THEIR APPLICATION - Google Patents

Description

The present application claims priority under U.S. Provisional Application No. 62/324,170, filed April 18, 2016. The contents of the above application are incorporated herein by reference.

State of the art

Interactions between T cells and antigen presenting cells involve many accessory molecules that facilitate the development of an immune response. One such molecule is CD40, a member of the tumor necrosis factor receptor (TNF-R) superfamily, which binds to CD40L (Ranheim EA, et al., Blood. 1995 June 15; 85(12):3556-65). CD40 is a 43-48 kDa transmembrane glycoprotein consisting of 277 amino acid residues (Braesch-Andersen et al., 1989). CD40 is expressed by antigen presenting cells (APCs) and binding of its natural ligand (CD40L) on T cells activates APCs, including dendritic cells and B cells (Khalil and Vonderhide (2007) Update Cancer Ther, 2(2): 61-65) thereby enhancing immune responses. CD40 is also expressed on many tumor cells, and in this case, its ligation causes a direct cytotoxic effect, for example, the involvement of CD40 on tumor cells leads to apoptosis in vitro and disruption of tumor growth in vivo (Tai et al. (2004) Cancer Res. 64(8):2846-52).

Monoclonal antibodies against CD40 provide a number of potential therapeutic targets, including the treatment of various types of cancer. For example, anti-CD40 agonist antibodies have been shown to replace T-cell assistance provided by CD4 lymphocytes in mouse models of T-cell mediated immunity, and in tumor-bearing hosts, CD40 agonists have been shown to trigger effective immune responses against tumor-associated antigens (Bennett et al. ( 1998) Nature, 393(6684):478-80). In addition, anti-CD40 antibodies are very promising for use in vaccines (Fransen et al. (2014) Vaccine 32:1654-1660). However, there are potential adverse effects associated with agents that strongly modulate the immune system (Sandin et al. (2014) Cancer Immunol Res, 2:80-90). Accordingly, there is a need for a better understanding of the specific properties and mechanisms that make anti-CD40 antibodies therapeutically effective, as well as for improved therapeutic anti-CD40 antibodies that can be used to treat and/or prevent diseases.

Brief description of the invention

The present invention relates to isolated anti-CD40 antibodies having specific functional properties that may be associated with useful and desirable therapeutic effects. In particular, anti-CD40 agonist monoclonal antibodies (MATs) have been developed and characterized that are capable of increasing the immune response to an antigen (eg, an antigen expressed on a cell). As used herein, the term antibody refers to full-length antibodies and their antigen-binding portions.

In one embodiment of the present invention, anti-CD40 antibodies enhance immune responses against the antigen, for example, by enhancing T-cell mediated immune responses, B-cell activation, and/or cytokine production. The antibodies may be administered alone or as part of various combination therapies (eg, in combination with vaccine therapy and/or chemotherapy).

According to another embodiment of the present invention, anti-CD40 antibodies are capable of increasing the immune response to an antigen without inducing antibody-dependent cellular cytotoxicity (ADCC) of CD40-expressing cells and/or complement-dependent cellular cytotoxicity (CDC) of CD40-expressing cells.

According to another embodiment of the present invention, the antibodies contain a constant region that does not have effector functions. In one embodiment of the present invention, the constant region is an IgG2 isotype (eg, human IgG2).

According to another embodiment of the present invention, anti-CD40 antibodies exhibit one or more of the following properties:

(a) are unable to block CD40L binding to human CD40, regardless of Fc receptor binding;

(b) capable of blocking CD40L binding to human CD40, independent of Fc receptor binding;

(c) activate human CD40 expressed on an antigen presenting cell (APC) independent of Fc receptor binding;

(d) induce apoptosis of the tumor cell;

(e) have activity aimed at stimulating T cells;

(f) capable of enhancing B cell activation; and/or (g) capable of acting synergistically with CD40L.

Preferably, the antibodies act independently of interaction with the Fc receptor. Preferably the antibodies are of the IgG2 isotype.

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According to one implementation variant of the present invention, agonist antibodies are able to increase the immune response, regardless of binding to the Fc receptor. For example, antibodies can exhibit strong agonistic features without cross-linking to an Fc receptor such as FcyR. Such agonistic features include, for example, an increase in T cell activity and/or an increase in B cell activation, as measured, for example, based on an increase in the expression of cell surface markers selected from the group consisting of HLA-DR V450, CD54FE, CD86- APC, CD83 BV510, CD19 V500, CD54-PE, HLA-DR V450, CD23 PerCP-Cy5.5, CD69-APC, CD86-APC, CD38 and CD71-PE.

According to another embodiment of the present invention, the antibodies block the binding of CD40 to CD40L (CD154) on CD40-expressing cells. In specific embodiments, the antibodies inhibit the binding of soluble CD40L to CD40-expressing cells by at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 96, 97, 98, 99 or 100%. In a specific embodiment of the present invention, an anti-CD40 antibody inhibits CD40L binding by at least about 70% as measured by, for example, fluorescence activated cell sorting (FACS), biolayer interferometry (BLI), or the Biacore system. In another embodiment, the anti-CD40 antibody inhibits CD40L binding by at least about 80% as measured by, for example, FACS, BLI, or the Biacore system.

According to another embodiment of the present invention, the antibodies induce cell apoptosis as measured, for example, based on an increase in CD95 expression. Antibodies can also be designed to include an Fc region that is specific for a particular Fc receptor (e.g., FcyRI (CD64), FcyRIIA (CD32), FcyRIIB1 (CD32), FcyRIIB2 (CD32), FcyRIIIA (CD16a), FcyRIIIB (CD16b ), FceRI, FcεRII (CD23), FcaRI (CD89), Fcα/μR and FcRn).

According to another embodiment of the present invention, the antibodies are capable of binding to human CD40 with an equilibrium dissociation constant Kd of 10 ^-10 M or less, preferably 10 ^-10 M or less, and/or cross-reacting with cynomolgus CD40.

Specific anti-CD40 antibodies of the present invention include the 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, 3B6-NS antibodies and related embodiments described below.

In one embodiment of the present invention, the antibodies comprise a heavy chain variable region hypervariable region (CDR) 3 sequence selected from the group consisting of SEQ ID NOs: 9, 10, 23, 24, 37, 38, 51, 52, 65, 66, 65, 66, 79, 80, 93, 94, 107, 108, including conservative modifications of these sequences (eg, conservative amino acid substitutions). The antibodies may further comprise a light chain variable region CDR3 sequence selected from the group consisting of SEQ ID NOs: 15, 16, 29, 30, 43, 44, 57, 58, 71, 72, 85, 86, 99, 100, 113 , 114, including conservative modifications of these sequences. According to another embodiment of the present invention, the heavy chain CDR2 and/or CDR1 sequences are selected from the sequences shown in SEQ ID NOs: 7, 8, 21, 22, 35, 36, 49, 50, 63, 64, 77, 78, 91, 92, 105, 106 and SEQ ID NOs: 5, 6, 19, 20, 33, 34, 47, 48, 61, 62, 61, 62, 75, 76, 89, 90, 103, 104, respectively, including conservative modifications of these sequences. According to another embodiment of the present invention, the light chain CDR2 and/or CDR1 sequences are selected from the sequences shown in SEQ ID NOs: 13, 14, 27, 28, 41, 42, 55, 56, 69, 70, 84, 85, 97, 98, 111, 112 and SEQ ID NOs: 11, 12, 25, 26, 40, 41, 53, 54, 67, 68, 81, 82, 95, 96, 109, 110, respectively, including conservative modifications of these sequences.

According to another embodiment of the present invention, the antibodies comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 17, 31, 45, 59, 73, 87, 101, including conservative modifications of these sequences. The antibodies may further comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 18, 32, 46, 60, 74, 88, 102, including conservative modifications of these sequences.

According to another embodiment of the present invention, the antibodies comprise heavy and/or light chain variable regions, respectively, containing the following amino acid sequences (including conservative sequence modifications):

(a) SEQ ID NO: 3 and/or 4;

(b) SEQ ID NO: 17 and/or 18;

(c) SEQ ID NO: 31 and/or 32;

(d) SEQ ID NO: 45 and/or 46;

(e) SEQ ID NO: 59 and/or 60;

(f) SEQ ID NO: 73 and/or 74;

(g) SEQ ID NO: 87 and/or 88; or (h) SEQ ID NO: 101 and/or 102.

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The present invention also relates to antibodies that contain heavy and light chain variable regions whose sequences are at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or more identical to any of the above sequences. Ranges that are intermediate to those listed above, such as heavy and light chain variable regions that are at least 80-85%, 85-90%, 90-95%, or 95-100% identical in sequence to any of the above sequences are also included in the scope of the present invention.

In another embodiment, the antibodies bind to human CD40 and comprise CDR sequences from the heavy and light chain variable regions, respectively, comprising the amino acid sequences set forth in (a) SEQ ID NOs: 3 and 4;

(b) SEQ ID NOs: 17 and 18;

(c) SEQ ID NOs: 31 and 32;

(d) SEQ ID NOs: 45 and 46;

(e) SEQ ID NOs: 59 and 60; or (f) SEQ ID NOs: 73 and 74;

(g) SEQ ID NOs: 87 and 88; or (h) SEQ ID NOs: 101 and 102 (in each case including one conservative sequence modification, two conservative sequence modifications, or up to three, up to four, or up to five conservative sequence modifications in one or more CDRs).

According to another embodiment of the present invention, the antibodies bind to human CD40 and contain:

(a) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 5, 7, 9, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 11, 13, 15, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 19, 21, 23, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 25, 27, 29, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 33, 35, 37, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 39, 41, 43, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 47, 49, 51, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 53, 55, 57, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 61, 63, 65, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 67, 69, 71, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 75, 77, 79, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 81, 83, 85, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 89, 91, 93, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 95, 97, 99, respectively; or (h) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 103, 105, 107, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 109, 111, 113, respectively (in each case, optionally including one conservative sequence modification, two conservative sequence modifications, or up to three, up to four, or up to five conservative sequence modifications in one or more of said CDRs).

According to another embodiment of the present invention, the antibodies bind to human CD40 and contain:

(a) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 6, 8, 10, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 12, 14, 16, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 20, 22, 24, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 26, 28, 30, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 34, 36, 38, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 40, 42, 44, respectively;

- 3 042934 (d) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 48, 50, 52, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 54,

56, 58, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 62, 64, 66, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 68, 70, 72, respectively; or (f) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 76, 78, 80, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 82, 84, 86, respectively ;

(g) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 90, 92, 94, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 96, 98, 100, respectively; or (h) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 104, 106, 108, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 110, 112, 114, respectively (in each case, optionally including one conservative sequence modification, two conservative sequence modifications, or up to three, up to four, or up to five conservative sequence modifications in one or more of said CDRs).

According to another aspect, the present invention relates to antibodies that compete for CD40 binding with the specific antibodies described above. In one embodiment of the present invention, an antibody competes for binding to CD40 with an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences shown in SEQ ID NOs: 3 and 4, SEQ ID NOs: 17 and 18, SEQ ID NOs : 31 and 32, SEQ ID NOs: 45 and 46, SEQ ID NOs: 59 and 60, SEQ ID NOs: 73 and 74, SEQ ID NOs: 87 and 88, SEQ ID NOs: 101 and 102, respectively.

According to another aspect, the present invention relates to antibodies that bind to the same epitope as an epitope on CD40 recognized by the specific antibodies described above. In one embodiment, the antibody binds to an epitope on CD40 recognized by an antibody comprising heavy and/or light chain variable regions comprising the amino acid sequences shown in SEQ ID NOs: 3 and 4, SEQ ID NOs: 17 and 18, SEQ ID NOs: 31 and 32, SEQ ID NOs: 45 and 46, SEQ ID NOs: 59 and 60, SEQ ID NOs: 73 and 74, SEQ ID NOs: 87 and 88, SEQ ID NOs: 101 and 102, respectively. In some embodiments, the antibody binds to the same epitope as the 3C3 or 3G5 antibody.

According to another aspect, the present invention relates to antibodies that bind to one or more residues within the region containing amino acid residues 1-5 and 33-36 of the extracellular domain (ECD) of human CD40 (SEQ ID NO: 133). In some embodiments, the antibodies also bind to one or more amino acids selected from the group consisting of amino acids 25, 26, 28, and 30 of human CD40 EVA (SEQ ID NO: 133). In some embodiments, the antibodies bind to one or more amino acids selected from the group consisting of amino acids 5, 33, 34, and 36 of human CD40 EVA (SEQ ID NO: 133). In some embodiments, the antibodies bind to amino acids 5, 33, and 36 of human CD40 EVA (SEQ ID NO: 133). In some embodiments, the antibodies bind to amino acids 5, 33, 34, and 36 of human CD40 EVA (SEQ ID NO: 133).

According to any of the above aspects, the present invention relates to antibodies, in which the replacement of alanine with threonine at position 5 of human CD40 EVA (SEQ ID NO: 133) reduces antibody binding by at least 30% compared to binding to human CD40 EVA (SEQ ID NO : 133). In some embodiments of the present invention, the replacement of alanine with threonine at position 5 of human CD40 VVD reduces antibody binding by at least 50% compared to human CD40 VVV binding (SEQ ID NO: 133). In some embodiments of the present invention, substitution of alanine for threonine at position 5 of human CD40 VVD reduces antibody binding by at least 80% compared to human CD40 VVV binding (SEQ ID NO: 133).

According to any of the above aspects, the present invention relates to antibodies that exhibit a synergistic effect with CD40L, which may be endogenous CD40L. In some embodiments of the present invention, the synergistic effect is an increased induction of CD95 expression upon incubation with Ramos cells.

In some embodiments of the present invention, the synergistic effect is an increase in B cell proliferation upon incubation with human B cells. According to some embodiments of the present invention, the synergistic effect is an increased induction of IL-12p40 expression upon incubation with dendritic cells. According to some variants of implementation of the present invention, the synergistic effect is measured based on the expression of CD95.

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According to another aspect, the present invention relates to antibodies that bind to one or more residues within a region containing amino acid residues 13-15 and 33-36 of the EVA.

Human CD40 (SEQ ID NO: 133). In some embodiments, the antibodies bind to one or more amino acids selected from the group consisting of amino acids 33, 34, and 36 of human CD40 EVA (SEQ ID NO: 133).

The antibodies of the present invention may be full-length, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD and IgE antibodies or variants thereof. In another embodiment, the antibodies may be fragments such as Fab, F(ab')2, Fv, single chain Fv, isolated hypervariable region (CDR), or a combination of two or more isolated CDRs. Antibodies may be of any known type or species of antibody, including, but not limited to, fully human, humanized, and chimeric antibodies. Preferably the antibodies are IgG2 antibodies. It should be understood that some modifications may be made to the IgG2 sequence, such as deletion of the N-terminal lysine and/or various other mutations known in the art. Therefore, an IgG2 antibody includes, for example, antibodies containing constant domains, the sequences of which are at least 90%, preferably at least 95%, preferably at least 97% and preferably at least 99% identical to the native sequence of IgG2 person.

The present invention also relates to molecular conjugates containing an antibody of the present invention coupled to an antigen (including fragments, epitopes, and antigenic determinants), such as a tumor antigen, self-antigen, or pathogen component. For example, an antigen may include a tumor antigen such as ehCG, gp100 or Pmel17, CEA, gp100, TRP-2, NY-BR-1, NY-CO-58, MN (gp250), idiotype, tyrosinase, telomerase, SSX2, MUC -1, MAGE-A3 and melanoma associated high molecular weight antigen (HMW-MAA) MART1, melan-A, NY-ESO-1, MAGE-1, MAGE-3, WT1, Her2, mesothelin or melanoma associated high molecular weight antigen (HMW -MAA).

According to another embodiment of the present invention, the molecular complex further comprises a therapeutic agent, such as a cytotoxic agent, an immunosuppressive agent, or a chemotherapeutic agent.

In another aspect, the present invention relates to bispecific molecules comprising antibodies of the present invention coupled to a second functional moiety having a different binding specificity. For example, in one embodiment of the present invention, the second molecule can bind to a T cell receptor (eg, CD3, CD40, or CTLA-4), an NK cell receptor (eg, CD56), a B cell receptor (eg, CD20), or another tumor necrosis factor receptor (eg CD95).

The present invention also relates to compositions containing the antibodies, molecular conjugates or bispecific molecules described herein, formulated with a pharmaceutically acceptable carrier. The compositions may further comprise an adjuvant, an immunostimulatory agent (e.g., CD40 ligand, FLT 3 ligand, cytokines, colony stimulating factors, an anti-CTLA-4 antibody (including, but not limited to, ipilimumab), an anti-PD1 antibody (including, but not limited to, MPDL3280A or durvalumab), anti-41BB antibody, anti-OX-40 antibody, LPS (endotoxin), ssRNA, dsRNA, Bacillus Calmette-Guerin (BCG), levamisole hydrochloride, intravenous immune globulins, and a Toll-like receptor (TLR) agonist (eg, a TLR3 agonist such as a poly-IC, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist)), an immunosuppressive agent, another antibody or antigen, or a STING agonist.

Tumor antigens that may be included in the molecular conjugates or compositions of the present invention (e.g., in a vaccine used in combination with an anti-CD40 antibody of the present invention) include any antigen or antigenic determinant that is present (or associated with it) on the tumor cell but not on normal normal cells, or an antigen or antigenic determinant that is present on or associated with tumor cells in greater numbers than on normal (non-tumor) cells, or an antigen or antigenic determinant that is present on tumor cells in a form other than on normal (non-tumor) cells. Such antigens include tumor-specific antigens, including tumor-specific membrane antigens, tumor-associated antigens, including tumor-associated membrane antigens, embryonic antigens on tumors, growth factor receptors, growth factor ligands, and any other type of antigen that is associated with cancer. The tumor antigen can be, for example, an epithelial cancer antigen (e.g., breast cancer, gastrointestinal cancer, lung cancer), prostate specific antigen (PSA) or prostate specific membrane antigen (PSMA), bladder cancer antigen, lung cancer antigen (eg, small cell lung cancer), rectal cancer antigen, ovarian cancer antigen, brain cancer antigen, stomach cancer antigen, kidney carcinoma antigen, pancreatic cancer antigen, liver cancer antigen, esophageal cancer antigen, head and neck cancer antigen or colorectal cancer antigen. For example, an antigen may include a tumor antigen such

- 5 042934 as ehCG, gp100 or Pmel17, CEA, gp100, TRP-2, NY-BR-1, NY-CO-58, MN (gp250), idiotype, tyrosinase, telomerase, SSX2, MUC-1, MAGE-A3 and melanoma-associated high molecular weight antigen (HMW-MAA) MART1, melan-A, EGFRvDI, NY-ESO-1, MAGE-1, MAGE-3, WT1, Her2, or mesothelin. Other antigens useful in the present invention (e.g., in a vaccine used in combination with an anti-CD40 antibody of the present invention) include those of pathogens that cause infectious diseases such as viruses, bacteria, parasites, and fungi, examples of which are disclosed herein.

The present invention also relates to nucleic acid molecules encoding all or parts of the heavy and/or light chain variable regions of the antibodies of the present invention, as well as expression vectors containing such nucleic acids, and host cells containing such expression vectors. According to one embodiment of the present invention, the nucleic acid sequences are selected from the group consisting of SEQ ID NOs: 87-112, respectively, or nucleic acid sequences that are, for example, at least 85%, 90%, or 95% identical to said nucleic acid sequences.

The present invention also relates to methods for enhancing an immune response (e.g., a T cell mediated immune response and/or an NK cell mediated response and/or a B cell mediated immune response) against an antigen in a subject using the agonist antibodies described herein. . In one embodiment of the present invention, antibodies bind to human CD40 (which is expressed on a variety of immune cell types), thereby triggering the proliferation and activation of antigen presenting cells (APCs) and activating B cells as well as effector and memory T cells, resulting in to enhance immune responses, for example, against tumor cells. Accordingly, according to one embodiment of the present invention, the methods comprise administering an antibody (e.g., a full-length antibody or an antigen-binding portion thereof), a composition, or a bispecific molecule of the present invention in an amount effective to induce or enhance an immune response against the antigen. According to another embodiment of the present invention, the methods further comprise administering the antigen, for example, simultaneously, separately, or after the antibody, composition, or bispecific molecule.

The present invention also relates to methods for inhibiting the proliferation of CD40 expressing cells (eg, in the treatment of cancer). For example, the agonist antibodies of the present invention have been shown to increase the expression of cell surface molecules that attract immune effector cells, leading to cell death, such as apoptosis. Therefore, according to another embodiment of the present invention, the method comprises administering or bringing into contact with cells an antibody (e.g., a full length antibody or an antigen-binding portion thereof), a composition or a bispecific molecule of the present invention in an amount effective to inhibit the proliferation of CD40-expressing cells.

The present invention also provides methods for targeting an antigen to a cell, e.g., a cell capable of presenting the antigen (e.g., peripheral blood mononuclear cells (PBMCs), monocytes (such as THP-1), B-lymphoblastic cells (such as C1R.A2, 1518 B-LCL) and DCs of monocytic origin) in a subject by introducing a molecule that binds to a receptor on the cell (eg, previously described anti-CD40 antibodies) coupled to an antigen.

The methods described herein can be used in the treatment of various diseases, in particular, various types of cancer (for example, cancer selected from the group including leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, such as myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphoblastic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma, marginal zone B-cell lymphoma, Osler's disease, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma , Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilm's tumor, cervical cancer, 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, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, cancer

- 6 042934 bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer and neck, stomach cancer, intraepithelial neoplasm, kidney cancer, larynx cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancer (eg, lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary tract cancer). Specific cancers include CD40-expressing tumors selected from the group consisting of chronic lymphoblastic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma, and B-cell marginal cell lymphoma.

According to another embodiment of the present invention, the methods can be used to treat or prevent a bacterial, fungal, viral, or parasitic infection.

CD40-expressing cells include any and all CD40-expressing cells, including, but not limited to, antigen presenting cells (APCs), including dendritic cells (DCs), B cells, macrophages, and monocytes. CD40 is also expressed on other cell types such as epithelial cells, endothelial cells and platelets. CD40 expression has been demonstrated in a variety of tumor cells, including B-cell lymphoma and kidney cancer cells. According to a specific embodiment of the present invention, CD40-expressing cells include cell lines such as Jurkat cells, Raji cells, Ramos cells, and Daudi cells. According to another embodiment of the present invention, the CD40-expressing cells are tumor cells or cancer cells. According to another embodiment of the present invention, CD40-expressing cells include B cells, NK cells, T cells found to be infiltrating tumor or cancer cells, also referred to as tumor infiltrating lymphocytes.

In another embodiment, the present invention relates to the use of an antibody, composition, or bispecific molecule described herein in the manufacture of a medicament for inducing or enhancing an immune response against an antigen (eg, tumor antigen) in a subject. According to other embodiments, the present invention relates to the use of an antibody or composition described herein in the manufacture of a medicament for (1) increasing the immune response to an antigen, (2) inhibiting the proliferation of CD40-expressing cells, and/or (3) targeting the antigen on the APK.

The present invention also provides methods for detecting the presence or absence of CD40 in a biological sample by (1) contacting the biological sample with an antibody described herein (wherein the antibody is labeled with a detectable substance) and (2) detecting an antibody bound to CD40.

The present invention also relates to kits containing compositions (eg, antibodies and/or bispecific molecules) according to the present invention, and optionally instructions for use. The kit may further contain at least one additional reagent, such as a cytokine or complement, or one or more additional antibodies according to the present invention.

Other features and advantages of the present invention will be apparent from the following detailed description and claims.

Brief description of the drawings

In FIG. Figure 1 shows the equilibrium dissociation constant (KD), kinetic association rate constant (kon), and dissociation rate constant (koff) for 3C3, 3G5, 1B4, 3B6, and 6H6 antibodies determined by biolayer interferometry (BLI) using the Octet™ QK ^e instrument. (Pall ForteBio, Menlo Park, CA, USA) according to manufacturer's recommendations.

In FIG. 2 is a graph showing the results of a concentration dependence study of the binding of anti-human CD40 antibodies (including 3C3, 3G5, 1B4, 3B6 and 6H6) to microtiter plates coated with purified recombinant human CD40 as assessed by absorbance (OD ₄₅₀ ) by the solid phase method. enzyme immunoassay (ELISA).

In FIG. Figure 3 is a graph showing the concentration dependence of the binding of anti-human CD40 antibodies (3C3, 3G5, 1B4, 3B6 and 6H6) to purified human PBMC (left) and cynomolgus monkey PBMC (right), presented as mean fluorescence intensity (MFI) values. ) measured by flow cytometry.

In FIG. 4A and 4B are graphs showing the effect of anti-human CD40 antibodies on soluble CD40 ligand (sCD40L) binding to CD40 protein, as measured by ELISA.

In FIG. 5 shows the results of a study of the binding of antibodies to human CD40 (3C3, 3G5, 1B4, 3B6 and 6H6) to CD40 on Raji cells expressing human CD40 on their surface, measured by flow cytometry.

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In FIG. 6 shows the results of a binding study of antibodies to human CD40 (3C3, 3G5,

1B4, 3B6 and 6H6) with CD40 on Ramos cells expressing human CD40 on their surface as measured by flow cytometry.

In FIG. 7A and 7B are graphs showing the results of the induction study.

CD95 on Ramos cells under the action of antibodies to human CD40.

In FIG. 8A and 8B are graphs showing the results of a study of the activation of dendritic cells (DC) by antibodies to human CD40 (3C3 and 3G5) based on changes in the expression level of the following markers: CD54, HLA-DR, CD86, CD83 and the percentage of CD83+ cells, as indicated on the charts.

In FIG. 9A and 9B are graphs showing the results of a study of IL-12p40 induction by anti-human CD40 antibodies (3C3 and 3G5).

In FIG. 10A and 10B are graphs showing the results of a study of B cell activation by anti-human CD40 antibodies (3C3 and 3G5) based on changes in the expression level of the following markers: CD54, HLA-DR, CD23, percentage of CD23+ cells, CD69, CD86, CD38 and CD71 as indicated on the graphs.

In FIG. 11A and 11B are graphs showing the results of a study of NFkB activation by anti-human CD40 antibodies using a CD40-expressing cell line carrying a luciferase reporter.

In FIG. 12 is a graph showing the results of a tumor growth and survival study in a mouse tumor model of SCID (Raji cell line) after treatment with anti-human CD40 antibody clones, 3C3 and 3G5, by intraperitoneal injection at 0.3 mg/dose.

In FIG. 13 is a graph showing the results of a tumor growth and survival study in a SCID mouse tumor model (Ramos cell line) after treatment with anti-human CD40 antibody clones, 3C3 and 3G5, at 0.3 mg/dose intraperitoneally.

In FIG. 14A and 14B are graphs showing T cell proliferation assay results for labeled PBMCs incubated with anti-CD40 antibodies as indicated or isotype control antibody (IgG2).

In FIG. 15 is a graph showing the results of a CD40 binding study, independent of Fc receptor interaction, using anti-CD40, 3C3, and 3G5 antibodies.

In FIG. 16 is a graph showing the results of an NFkB activation study using anti-CD40, 3C3, and 3G5 antibodies.

In FIG. 17 is a graph showing the results of a CD95 induction study on Ramos cells using anti-CD40, 3C3, and 3G5 antibodies.

In FIG. 18 is a schematic representation of an exemplary APC-targeted vaccine construct containing an anti-CD40 antibody/antigen hybrid.

In FIG. 19 is a graph showing the results of a synergistic study of the anti-CD40 antibody, 3C3, in combination with soluble CD40L on CD95 expression in Ramos cells.

In FIG. 20 is a cDNA diagram of soluble CD40 encoding a full-length extracellular domain (ECD) spanning amino acid residues 1-173 with an N-terminal kappa light chain and a Flag C-terminal tag.

In FIG. 21 shows the amino acid sequence alignment of human CD40 ECV (SEQ ID NO: 133) with the monkey CD40 ECV (SEQ ID NO: 139) amino acid sequence (upper panel) and with the mouse CD40 ECV amino acid sequence (lower panel). The resulting fragments are indicated (nucleotides 1-94 in SEQ ID NO: 133, SEQ ID NOS: 140-147).

In FIG. 22 are graphs showing the results of an assay of binding of an anti-CD40 antibody, 3C3, to human CD40 EVA fragment A (amino acid residues 1-5, upper panel) or fragment D (amino acid residues 33-36, lower panel) containing various point mutations. or their combinations.

In FIG. 23A-23C are graphs showing aspartate aminotransferase (ACT; 23A), alanine aminotransferase (ALT; 23B), and creatine kinase (23C) levels measured in monkeys before and after anti-CD40, 3C3, or 3G5 antibody treatment at the indicated time points. .

In FIG. 24 is a graph showing the results of a study of the levels of IL-12 (pg/ml), measured in the blood of monkeys treated with antibodies to CD40, 3C3 or 3G5, at the indicated time points.

In FIG. 25A-25C are graphs showing the results of leukocyte (25A), neutrophil (25B), and lymphocyte (25C) counts in monkeys before and after treatment with anti-CD40, 3C3, or 3G5 antibodies at the indicated time points.

In FIG. 26 is a graph showing the results of assessing the percent change from baseline in B cell counts in monkeys treated with anti-CD40, 3C3, or 3G5 antibodies over time (days).

- 8 042934

In FIG. 27 are graphs showing the results of the assessment of HLA-DR expression on B cells, relative to baseline, after administration of 2 mg (left) or 0.2 mg (right) of anti-CD40 antibody 3C3 (square), 3G5 (diamonds), or saline solution (circles).

In FIG. 28 is a graph showing the results of a B cell proliferation assay when cells were cultured in the presence of an anti-CD40, 3C3 mAb.

In FIG. 29 and 30 are graphs showing the results of a synergistic study of the combination of mAbs against CD40, 3C3, and CD40L in B cells.

In FIG. 31 is a table showing cytokine responses in whole blood when incubated with an anti-CD40, 3C3 mAb.

Detailed description of the invention

The present invention provides anti-CD40 antibodies that exhibit specific functional properties correlated with significant therapeutic benefits associated with activation of immune function (e.g., T-cell mediated immune response as in various vaccine therapies, NK cell activation in various cancer therapies). ), inhibition of cell proliferation (eg, in cancer therapy), and/or enhanced APC antigen processing and presentation (eg, in vaccine therapy). Such functional features include, for example, an increased immune response to an antigen independent of Fc receptor binding and/or without induction of antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cellular cytotoxicity (CCC). Additional functional features include, for example, (1) inhibition (eg, complete or partial blocking) of CD40L (CD154) binding to CD40-expressing cells by at least 50%, at least 60%, or at least 70%; (2) blocking CD40L binding to human CD40 independent of Fc receptor binding; (3) induction of cell apoptosis (eg, as measured by an increase in CD95 expression); (4) increased activity aimed at stimulating T cells (eg, as measured by increased expression of IL-12p40); and/or (5) an increase in B cell activation (e.g., as measured by an increase in the expression of at least one cell surface marker selected from the group consisting of HLA-DR V450, CD54-FE, CD86-APC, and CD83 BV510, CD19 V500, CD54-PE, HLA-DR V450, CD23 PerCP-Cy5.5, CD69-APC, SP86-APC, CD38 PerCP-Cy5.5 and CD71PE.

For a better understanding of the present invention, some terms are first defined. Additional definitions are set forth in the text of the detailed description.

The term CD40 (also called CD40 molecule, Bp50, CDW40, TNFRSF5, p50, CD40 B cell surface antigen, B cell-associated molecule, CD40 antigen, TNF 5 receptor superfamily member, type II CD40 isoform, CD40L receptor, B-lymphocyte activation molecule , related to the nerve growth factor receptor or a member of the tumor necrosis factor receptor superfamily 5) refers to a receptor that is a member of the TNF receptor superfamily that binds to the CD40L ligand (also referred to as CD154). CD40 mediates a wide range of immune and inflammatory responses, including T cell-dependent immunoglobulin class switching and memory B cell production. The term CD40 includes any CD40 variants or isoforms that are naturally expressed by cells (eg, human CD40 deposited with GenBank® Accession No. P25942). Accordingly, the antibodies of the present invention can cross-react with CD40 from non-human species. In another embodiment, the antibodies may be specific for human CD40 and may not be cross-reactive with other species. CD40 or any of its variants and isoforms can be isolated from cells or tissues that naturally express them, or recombinantly produced using methods well known in the art and/or described herein. Preferably, the antibodies target human CD40 (4CD40), which has a naturally occurring glycosylation pattern.

The amino acid sequence of human CD40 deposited with GenBank® (Accession No. P25942) is shown below (SEQ ID NO: 1).

MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPI IFGILFAILL VLVFIKKVAK I<PTNI<APHPI< QEPQEINFPD DLPGSNTAAP

VQETLHGCQP VTQEDGKESR ISVQERQ

The term CD40L (also called CD40 ligand, CD407L or CD154) refers to a ligand for CD40 (see, for example, Schonbeck and Libby (2001) Cell Mol Life Sci, 58(1):4-43). CD40L is expressed primarily on activated T cells and is a member of the TNF superfamily of molecules. It binds to CD40 on antigen presenting cells (APCs) resulting in multiple effects depending on the type of target cells (Parham, Peter (2004). The Immune System

- 9 042934 ( ^2nd ed.). Garland Science. pp. 169-173).

The amino acid sequence of human CD40L deposited with GenBank® (Accession No. NP_000065) is shown below (SEQ ID NO: 2):

MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL DKIEDERNLH EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ FEGFVKDIML NKEETKKENS FEMQKGDQNP QIAAHVISEA SSKTTTSVLQW AEKGYYTMSN NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN VTDPSQVSHG

TGFTSFGLLK

As used herein, the term antibody includes whole antibodies and any antigen-binding fragment (ie, antigen-binding portion) or single chain thereof. An antibody refers, according to one preferred embodiment, to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V _H ) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V _L ) and a light chain constant region. The light chain constant region consists of a single CL domain. The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) that alternate with more conserved regions called framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs arranged in the amino to carboxy direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

As used herein, the term antigen-binding portion of an antibody (or simply antibody portion) refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (eg, human CD40). Such fragments are, for example, from about 8 to about 1500 amino acids in length, suitably from about 8 to about 745 amino acids in length, suitably from about 8 to about 300 amino acids in length, for example, from about 8 to about 200 amino acids in length, or from about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments included in the term antigen-binding portion of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, V _H , CL and CH1 domains; (ii) an F(ab') ₂ fragment, a divalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of V _H and CH1 domains; (iv) an Fv fragment consisting of the V _L and V _H domains of one antibody arm; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V _H domain; and (vi) isolated hypervariable region (CDR); or (vii) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. In addition, although the two domains of the Fv fragment, Vl and Vh, are encoded by separate genes, they can be connected using recombinant methods using a synthetic linker that allows them to form a single protein chain in which the Vl and Vh regions pair. to form monovalent molecules (known as single chain Fv (scFv), see e.g. Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 :5879-5883). Such single chain antibodies are also meant to be included in the term antigen-binding portion of an antibody. Such antibody fragments are prepared using conventional techniques known to those skilled in the art and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

A bispecific or bifunctional antibody is an artificial hybrid antibody containing two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be made in a variety of ways, including hybridoma fusion or Fab' fragment joining. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

As used herein, the term monoclonal antibody refers to an antibody that exhibits a single kind of binding specificity and affinity for a particular epitope. Accordingly, the term human monoclonal antibody refers to an antibody that exhibits a single kind of binding specificity and that contains variable and optional constant regions derived from human germline immunoglobulin sequences.

According to one embodiment of the present invention, human monoclonal antibodies are generated by a hybridoma that includes a B cell derived from a transgenic non-human animal, e.g., a transgenic mouse having a genome containing a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

As used herein, the term human recombinant antibody includes all human antibodies that are produced, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for human immunoglobulin genes , or from a hybridoma derived from such an animal, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies obtained, expressed, created or isolated by any other means, which involves the splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies contain variable and constant regions that use specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region contains an antigen-binding domain that is encoded by various genes that rearrange to form an antibody specific for a foreign antigen. In addition to the rearrangement, the variable region can be further modified with several single amino acid changes (called a somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region will change with the subsequent antigen response (i.e., an isotype switch will occur). Therefore, rearranged and somatically mutated nucleic acid molecule sequences that encode immunoglobulin light chain and heavy chain polypeptides in response to an antigen may not be identical to the sequences of the original nucleic acid molecules, but, alternatively, will be substantially identical or similar to them (i.e. have at least 80% identity).

The term human antibody includes antibodies containing variable and constant regions (if present) from human germline immunoglobulin sequences. Human antibodies of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or in vivo somatic mutation) (see Lonberg, N et al (1994) Nature 368(6474):856-859); Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546). However, the term human antibody does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human backbone sequences (ie, humanized antibodies).

As used herein, the term heterologous antibody is defined in relation to a transgenic non-human organism that produces such an antibody. The term refers to an antibody containing an amino acid sequence or a nucleic acid coding sequence corresponding to that found in a non-human transgenic animal, and generally refers to an antibody from a non-human transgenic animal species.

As used herein, the term isolated antibody is intended to mean an antibody that is substantially free of other antibodies having different types of antigenic specificity (e.g., an isolated antibody that specifically binds to human CD40 is essentially free of antibodies that specifically bind to antigens). other than human CD40). However, an isolated antibody that specifically binds to an epitope may be cross-reactive with other CD40 proteins from different species. However, the antibody preferably always binds to human CD40. In addition, the isolated antibody is typically substantially free of other cellular materials and/or chemicals. In one embodiment of the present invention, a combination of isolated antibodies having different CD40 specificities is combined in a well established composition.

The term epitope or antigenic determinant refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from adjacent amino acids and from non-adjacent amino acids brought together when the protein is folded into a tertiary structure. Epitopes formed from contiguous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed when a protein is folded into a tertiary structure are usually lost when treated with denaturing solvents. An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, quantitative immunoblotting and immunoprecipitation studies in which overlapping or contiguous peptides from CD40 are tested to determine their ability to react with this anti-CD40 antibody. Methods for determining the spatial conformation of epitopes include techniques known in the art and described herein, such as x-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed (1996)).

Accordingly, antibodies that bind to the same epitope, or an epitope on CD40 that contains all or part of an epitope recognized by the specific antibodies described herein (e.g., the same or overlapping region, or a region that is within or covers the region of an epitope) are also provided according to the present invention. Antibodies that bind to the same epitope or an epitope that contains all or part of an epitope recognized by a particular antibody can be identified using standard techniques. Such techniques include, for example, epitope mapping methods, such as X-ray examination of crystals of antigen:antibody complexes, which provide atomic resolution of the epitope. Other methods allow control of antibody binding to fragments of an antigen or mutated variants of an antigen, with loss of binding due to modification of an amino acid residue in the antigen sequence often taken as an indication of an epitope component. In addition, computational combinatorial methods can also be used to map epitopes. These methods rely on the ability of the antibody of interest to provide affinity isolation of specific short peptides from combinatorial phage display peptide libraries. The peptides are then considered as a means to establish an epitope corresponding to the antibody used to screen the peptide library. Computational algorithms have also been developed for epitope mapping and have been shown to map conformational discontinuous epitopes.

The present invention also relates to antibodies that compete for binding to human CD40 with the antibodies described herein. Antibodies that compete for binding can be identified using standard techniques. Suitable techniques include, for example, immunoassays that show the ability of one antibody to block the binding of another antibody to a target antigen, i.e. quantitative study of competitive binding. Competitive binding is determined in a quantitative assay in which the immunoglobulin being tested inhibits the specific binding of a reference antibody to a common antigen such as CD40. Numerous types of quantitative competitive binding assays are known, for example: direct or indirect radioimmunoassay (RIA), direct or indirect enzyme-linked immunosorbent assay (EIA), quantitative competition binding assay in sandwich format (see Stahli et al., Methods in Enzymology 9:242 (1983)); biotin-avidin direct solid phase EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); direct solid phase label assay, direct solid phase label assay in sandwich format (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); direct solid phase RIA using the I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); biotin-avidin direct solid phase EIA (Cheung et al., Virology 176:546 (1990)); and direct RIA using a label (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such assay involves the use of a purified antigen bound to a solid surface, or cells bearing either of an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of the immunoglobulin being tested. Typically, the immunoglobulin tested is present in excess. Typically, if the competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more.

As used herein, the term specific binding, selective binding, selective binding, and specific binding refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody will bind to an equilibrium dissociation constant (Ko) of less than about 10 ^-7 M, e.g., less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M, or even lower, as determined by biolayer interferometry (BLI) using the Octet™ QK ^e instrument or Surface Plasmon Resonance (SPR) technology on the BIACORE 2000 instrument using recombinant human CD40 as analyte and antibody as ligand, and binds to a predetermined antigen with an affinity that is at least twice as high than its binding affinity for a non-specific antigen (eg, BSA, casein) other than a predetermined antigen or a closely related antigen. Present

- 12 042934 document, the terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably with the term "antibody that specifically binds to an antigen".

Also included within the scope of the present invention are antibodies that bind to human CD40 and are capable of enhancing the immune response, independent of binding to the Fc receptor. For example, such antibodies exhibit strong agonistic features without crosslinking to an Fc receptor such as FcyR. Such agonistic features include, for example, an increase in T cell activity and/or an increase in B cell activation as measured, for example, based on an increase in the expression of cell surface markers.

As used herein, the term KD is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. Typically, human antibodies of the present invention bind to CD40 with an equilibrium dissociation constant (KD) of about 10 ^-8 M or less, such as less than 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, or 10 ^-12 M or even lower than determined by biolayer interferometry (BLI) using the Octet™ QK ^e instrument or by surface plasmon resonance (SPR) on the BIACORE 2000 instrument using recombinant human CD40 as analyte and antibody as ligand.

As used herein, the term kd is intended to refer to the rate constant of the reverse reaction for the dissociation of an antibody from an antibody/antigen complex.

As used herein, the term ka is intended to refer to the forward reaction rate constant for association of an antibody with an antigen.

As used herein, the term EC ₅₀ refers to the concentration of an antibody, or antigen-binding portion thereof, that induces a response, both in vitro and in vivo, that is 50% of the maximum response, i. the magnitude of the response is in the middle of the curve with endpoints corresponding to the maximum response and the response at the initial conditions.

As used herein, the term isotype refers to the class of antibodies (eg, IgM or IgG1) that is encoded by heavy chain constant region genes. According to one embodiment of the present invention, the human monoclonal antibody of the present invention is of the IgG1 isotype. According to another embodiment of the present invention, the human monoclonal antibody of the present invention is of the IgG2 isotype.

The term binds to immobilized CD40 refers to the ability of a human antibody of the present invention to bind to CD40, eg expressed on a cell surface or attached to a solid support.

As used herein, the term cross-react refers to the ability of an antibody of the present invention to bind to CD40 from different species. For example, an antibody of the present invention that binds to human CD40 can also bind to CD40 from another species. As used herein, cross-reactivity is measured by detecting specific reactivity for a purified antigen in quantitative binding assays (eg, SPR, ELISA) or binding or other functional interaction with cells expressing CD40. Methods for determining cross-reactivity include the standard quantitative binding assays described herein, such as Biolayer Interferometry (BLI) using the Octet™ QK ^e instrument or Surface Plasmon Resonance (SPR) using the Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometry techniques.

As used herein, the term isotype switch refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

As used herein, the term non-switched isotype refers to an isotype class of a heavy chain that is produced in the absence of isotype switching; the CH gene encoding an unswitched isotype is usually the first CH gene located immediately after the functionally rearranged VDJ gene. Isotype switching is classified as classical or non-classical isotype switching. Classical isotype switching occurs through recombination events that involve at least one region of the switch sequence in the transgene. Non-classical isotype switching can occur, for example, by homologous recombination between human σμ and human Σμ (σ-linked deletion). Alternative non-classical switching mechanisms such as intertransgenic and/or interchromosomal recombination, among others, can also occur and lead to isotype switching.

As used herein, the term switch sequence refers to those DNA sequences that are responsible for recombination during the switch step. The switch-donor sequence, typically the μ-switch site, will be located 5'-end (ie, forward) relative to the construct site to be removed during recombination in the switch step. The switch acceptor site will lie between the construct site to be removed and the replacement constant region (eg, y, s, etc.). Because it doesn't exist

- 13 042934 a specific site at which recombination always occurs, the final gene sequence generally cannot be predicted based on the construct.

As used herein, the term glycosylation profile is defined as the profile of carbohydrate blocks that are covalently attached to a protein, more specifically an immunoglobulin protein. The glycosylation profile of a heterologous antibody can be characterized as substantially similar to the glycosylation profiles that occur naturally on antibodies produced by a non-human transgenic animal species, when one skilled in the art determines that the glycosylation profile of a heterologous antibody is more similar to the indicated glycosylation profile in the non-human species of the transgenic animal than in the species from which the CH genes of the transgene were derived.

In this document, the term natural, when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in the body (including viruses), that can be isolated from a source in nature, and that has not been intentionally modified by humans in the laboratory, is naturally occurring.

As used herein, the term rearranged refers to a configuration of an immunoglobulin heavy chain or light chain locus where the V segment is located immediately adjacent to the DJ or J segment in a conformation encoding a substantially full length V _H or VL domain, respectively. The rearranged immunoglobulin gene locus can be identified by comparison with germline DNA; the rearranged locus will contain at least one recombined heptamer/nonamer homologous element.

As used herein, the term non-rearranged or germline configuration with respect to a V segment refers to a configuration in which the V segment is not recombined to be directly adjacent to the D or J segment.

As used herein, the term nucleic acid molecule is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single or double stranded, but is preferably double stranded DNA.

As used herein, the term isolated nucleic acid molecule, as used in relation to nucleic acids encoding antibodies or antibody portions (e.g., V _H , VL, CDR3) that bind to CD40, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or part of an antibody, do not contain other nucleotide sequences encoding antibodies or parts of antibodies that bind antigens other than CD40, while these other sequences can flank the nucleic acid in human genomic DNA.

Also included within the scope of the present invention are conservative sequence modifications of the sequences shown in SEQ ID NOs: 3-132, ie. modifications of the nucleotide and amino acid sequences that do not eliminate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as additions and deletions of nucleotides and amino acids. For example, modifications can be introduced into the sequences shown in SEQ ID NO: 3-148 using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), with acidic side chains (e.g., aspartic acid, glutamic acid), with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), with non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine). Therefore, a predicted non-essential amino acid residue in an anti-human CD40 antibody is preferably replaced by another amino acid residue from the same side chain family. Methods for detecting conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12 (10):879-884 (1999), and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

Conservative substitutions can be made, for example, in accordance with the table below. For example, amino acids in the same block in the second column, and preferably in the same row in the third column, can substitute for each other._____________________________

Aliphatic non-polar gap

- 14 042934

ILV Polar uncharged CSTM NQ Polar charged DE KR aromatic HFWY

Alternatively, according to another embodiment of the present invention, mutations can be introduced randomly throughout or in part of the coding sequence of an anti-CD40 antibody, for example, by saturation mutagenesis, and the resulting modified anti-CD40 antibodies can be screened for binding activity.

In the case of nucleic acids, the term significant homology indicates that two nucleic acids, or specially selected sequences thereof, when optimally aligned and compared, are identical, with corresponding nucleotide insertions or deletions, for at least about 80% of the nucleotides, usually in at least about 90 to 95% nucleotides, and more preferably at least about 98 to 99.5% nucleotides. In another embodiment, significant homology exists if the segments will hybridize under selective hybridization conditions to a complementary strand partner.

The percent identity between two sequences depends on the number of identical positions present in the sequences (i.e. % homology = number of identical positions / total number of positions * 100), taking into account the number of gaps and the length of each gap that must be entered to optimally align the two sequences . Comparing sequences and determining percent identity between two sequences can be performed using the mathematical algorithm described in the non-limiting examples below.

Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com) using the NWSgapdna.CMP matrix, gap penalty 40, 50, 60, 70 or 80 and a gap extension penalty of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), which was built into the ALIGN program (version 2.0) using the PAM120 residue substitution weight table, a gap extension penalty of 12, and a gap insertion penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman algorithm. and Wunsch (J. Mol. Biol. (48):444-453 (1970)), which was built into the GAP program in the GCG software package (available at http://www.gcg.com) using the matrix Blossum 62 or PAM250 matrix, and a gap penalty of 16, 14, 12, 10, 8, 6, or 4 and a gap gap penalty of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention can then be used as a query sequence to search public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST (version 2.0) programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. A BLAST protein search can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default settings for the respective programs (eg, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. The nucleic acid is isolated or converted to substantially pure when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, using standard techniques, including alkali/SDS treatment, CsCl gradient separation, column chromatography, electrophoresis in agarose gel and other methods well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, although often presented as a native sequence (with the exception of modified restriction sites, etc.), from cDNA, genomic DNA, or mixtures thereof, can be mutated according to the standard

- 15 042934 using standard techniques to provide gene sequences. In the case of coding sequences, such mutations can affect the amino acid sequence, if desired. In particular, included within the scope of the present invention are DNA sequences substantially homologous to or derived from native V-, D-, J-, constant sequences, switches, and other such sequences described herein (where occurring indicates that the sequence identical to another sequence or obtained by modifying it).

A nucleic acid is operably linked if it is operably linked to another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. In relation to transcription control sequences, operably linked means that the linked DNA sequences are contiguous and, if two protein-coding regions are to be joined, contiguous and in the same reading frame. In the case of switch sequences, functional linkage indicates that the sequences are capable of recombining during the switch step.

As used herein, the term vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a plasmid, which refers to a double-stranded DNA circular loop into which additional DNA segments can be ligated. Another type of vector is the viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell and thereby replicate along with the host genome. Moreover, some vectors are capable of directing the expression of the genes to which they are operably linked. In this document, such vectors are referred to as recombinant expression vectors (or simply expression vectors). Typically, expression vectors suitable for use in recombinant DNA methods are often in the form of plasmids. In the present description, the plasmid and vector can be used interchangeably, since the plasmid is the most commonly used form of the vector. However, other forms of expression vectors, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), that perform equivalent functions, are intended to be included within the scope of the present invention.

As used herein, the term recombinant host cell (or simply host cell) is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to a particular cell of a subject, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to both mutations and environmental influences, such progeny may not actually be identical to the parent cell, however, they are still included within the scope of the term host cell as used herein.

As used herein, the term antigen refers to any natural or synthetic immunogenic substance such as a protein, peptide, or hapten. Suitable antigens for use in the present invention (e.g., in a vaccine in combination with an anti-CD40 antibody of the present invention) include, for example, infectious disease antigens and tumor antigens against which protective or therapeutic immune responses are desired, e.g. cell or pathogenic organism, or antigens of infectious diseases. For example, suitable antigens include tumor-associated antigens for preventing or treating cancer. Examples of tumor-associated antigens include, but are not limited to, sequences containing all or portions of the ehCG, gp100 or Pmel17, HER2/neu, WT1, mesothelin, CEA, gp100, MARTI, TRP-2, melan-A, NYESO antigens. -1, NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3, tyrosinase, telomerase, SSX2 and MUC-1, as well as tumor-derived antigens from germ cells. Tumor-associated antigens also include blood group antigens, eg Le ^a , Le ^b , LeX, LeY, H-2, B-1, B-2 antigens. In another embodiment, more than one antigen may be included in the antigen-antibody constructs of the present invention. For example, the MAGE antigen can be combined with other antigens such as melanin A, tyrosinase, and gp100, along with adjuvants such as GM-CSF or IL-12, and linked to an anti-APK antibody.

Other suitable antigens include viral antigens for the prevention or treatment of viral diseases. Examples of viral antigens include, but are not limited to, HIV-1 gag, HIV-1 env, HIV-1 nef, HBV (surface or core antigens), HPV, FAS, HSV-1, HSV-2, p17, ORF2, and ORF3. Examples of bacterial antigens include, but are not limited to, Toxoplasma gondii or Treponema pallidum. Antibody-bacterial antigen conjugates according to

- 16 042934 The present invention can be used in the treatment or prevention of various bacterial diseases such as anthrax, botulism, tetanus, chlamydia, cholera, diphtheria, Lyme disease, syphilis and tuberculosis. Other suitable antigens from infectious pathogens such as viruses, bacteria, parasites and fungi are described below.

The sequences of the above antigens are well known in the art. For example, an exemplary MAGE-3 cDNA sequence is provided in US Pat. No. 6,235,525 (Ludwig Cancer Research Institute); examples of the nucleic acid sequence and protein sequence of NY-ESO-1 are presented in US patent No. 5804381 and 6069233 (Ludwig Cancer Research Institute); examples of the nucleic acid sequence and the melan-A protein sequence are given in US Pat. Nos. 5,620,886 and 5,854,203 (Ludwig Cancer Research Institute); examples of the nucleic acid sequence and protein sequence of NY-BR-1 are given in US Pat. ; an exemplary amino acid sequence of the HER-2/neu protein is available from GenBank® under accession number AAA58637; and the nucleotide sequence (mRNA) for human carcinoembryonic antigen-like protein 1 (CEA-1) is available from GenBank® under accession number NM_020219.

The HPV antigen that can be used in the compositions and methods of the present invention may include, for example, HPV-16 antigen, HPV-18 antigen, HPV-31 antigen, HPV-33 antigen and/or HPV-35 antigen; HPV-16 antigen and/or HPV-18 antigen is also acceptable. The HPV-16 genome is described in Virology, 145:181-185 (1985), and the DNA sequences encoding HPV-18 are described in US Pat. No. 5,840,306, the disclosure of which is incorporated herein by reference in its entirety. HPV-16 antigens (eg, seroreactive regions of HPV-16 E1 and/or E2 proteins) are described in US Pat. No. 5840306, the disclosure of which is incorporated herein by reference. Similarly, the complete HBV genome is available from GenBank® under accession number NC_003977, the disclosure of which is incorporated herein. The HCV genome is described in European Patent Application No. 318216, the disclosure of which is incorporated herein. PCT/US90/01348, incorporated herein by reference, discloses information on the sequence of HCV genome clones, amino acid sequences of HCV viral proteins, and methods for preparing and using such compositions for HCV vaccines containing HCV proteins and peptides derived therefrom.

Antigenic peptides of proteins (ie, those containing T-cell epitopes) can be detected by various methods well known in the art. For example, T-cell epitopes can be predicted by protein sequence analysis using predictive algorithms (BIMAS & SYFPEITHI) available on the Internet to generate potential MHC class I and II binding peptides that match an internal database of 10,000 well-characterized MHC- binding peptides previously identified by CTL. Peptides with high scores can be categorized and selected as interesting based on high affinity for a given MHC molecule.

Another way to detect antigenic peptides containing T-cell epitopes is to divide the protein into non-overlapping peptides of the desired length or overlapping peptides of the desired length, which can be obtained by recombinant, synthetic methods or, in certain limited situations, by chemical cleavage of the protein, and testing immunogenic properties, such as the ability to induce T cell responses (ie, proliferation or secretion of lymphokines).

In order to determine precise T-cell epitopes of a protein, for example by precise mapping techniques, a peptide having T-cell stimulation activity and therefore containing at least one T-cell epitope as determined by T-cell biology techniques, can be modified by addition or deletion of amino acid residues at both the amino-terminus and the carboxy-terminus of the peptide, and tested to determine the change in T-cell reactivity towards the modified peptide. If two or more peptides that have a region of overlap in the native protein sequence are found to have activity to stimulate human T cells as determined by T cell biology techniques, then additional peptides can be prepared containing such complete peptides or part of them, and these additional peptides can be tested using a similar procedure. Following this methodology, peptides are selected and produced recombinantly or synthetically. Peptides are selected based on various factors, including the intensity of the T cell response to the peptide (eg, stimulation index). The physical and chemical properties of such selected peptides (eg, solubility, stability) can then be examined to determine the suitability of these peptides for use in therapeutic compositions or the need for modification of the peptides.

The term antigen-presenting cell or APC refers to a cell that presents on its surface a foreign antigen that forms a complex with MHC. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (such as THP-1), B-lymphoblastic cells (such as C1R.A2, 1518 B- LCL) and dendritic cells of monocytic origin (DC). Some APCs internalize antigens by phagocytosis or receptor-mediated endocytosis. Examples of APC receptors include, but are not limited to, C-type lectins such as the human dendritic or epithelial cell receptor 205 (DEC-205) and the human macrophage mannose receptor.

The term antigen presentation refers to the process by which APCs take up antigens and make them recognized by T cells, eg as a component of an MHC-I and/or MHC-II conjugate.

MHC molecules include two types of molecules, MHC class I and MHC class II. Class I MHC molecules present antigen to specific CD8+ T cells, and class II MHC molecules present antigen to specific CD4+ T cells. Antigens delivered to the APC exogenously are primarily processed for association with MHC class II. In contrast, antigens delivered endogenously to the APC are primarily processed for association with MHC class I.

As used herein, the term immunostimulatory agent includes, but is not limited to, compounds capable of stimulating APC such as DCs and macrophages. For example, suitable immunostimulatory agents for use in the present invention are capable of stimulating APC such that APC maturation is accelerated, APC proliferation is increased, and/or recruitment or release of co-stimulatory molecules (e.g., CD80, CD86, ICAM-1, MHC molecules, and CCR7) is enhanced, and pro-inflammatory cytokines (eg, IL-1b, IL-6, IL-12, IL-15 and IFNγ). Suitable immunostimulatory agents are also capable of increasing T cell proliferation. Such immunostimulatory agents include, but are not limited to, CD27 ligand; FLT 3 ligand; cytokines such as IFN-α, IFN-β, IFN-γ and IL-2; colony stimulating factors such as G-CSF (granulocyte colony stimulating factor) and GM-CSF (granulocyte macrophage colony stimulating factor); an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-41BB antibody, or an anti-OX-40 antibody; LPS (endotoxin); ssRNA; dsRNA; bacillus Calmette-Guerin (BCG); levamisole hydrochloride; and intravenous immune globulins. In one embodiment of the present invention, the immunostimulatory agent may be a Toll-like receptor (TLR) agonist. For example, the immunostimulatory agent may be a TLR3 agonist, such as a double-stranded polynucleotide inosine:cytosine (poly-1:C, for example, available as Ampligen™ from Hemispherx Bipharma, PA, USA, or poly-IC:LC, available from Oncovir) or poly-A:U; a TLR4 agonist such as monophosphoryl lipid A (MPL) or RC-529 (eg available from GSK, UK); a TLR5 agonist such as flagellin; a TLR7 or TLR8 agonist such as an imidazoquinoline, a TLR7 or TLR8 agonist such as imiquimod (eg Aldara™) or resiquimod and related imidazoquinoline agents (eg available from 3M Corporation); or a TLR 9 agonist such as a deoxynucleotide with unmethylated CpG motifs (so-called CpGs, eg available from Coley Pharmaceutical). A preferred immunostimulatory agent is a TLR3 agonist, preferably poly-1:C. Such immunostimulatory agents may be administered simultaneously, separately, or sequentially with the antibodies and constructs of the present invention, and may also be physically coupled to the antibodies and constructs.

As used herein, the term connected refers to the association of two or more molecules. The bond may be covalent or non-covalent. The relationship may also be genetic (ie, recombinantly hybridized). Such linkages can be achieved using a wide variety of techniques conventional in the art, such as chemical conjugation and recombinant protein production.

As used herein, the term cross-antigen presentation refers to the presentation of exogenous protein antigens to T cells by MHC class I and class II molecules on APCs.

As used herein, the term T cell mediated response refers to any T cell mediated response, including effector T cells (eg CD8 cells) and helper T cells (eg CD4 cells). T cell mediated responses include, for example, cytotoxicity and T cell proliferation.

As used herein, the term cytotoxic T lymphocyte (CTL) refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8 ⁺ T cells.

As used herein, the terms inhibit or block (eg, in relation to inhibition/blocking of CD40L binding to CD40 on cells) are used interchangeably and include both partial and complete inhibition/blocking. Inhibition/blocking of CD40L preferably reduces or alters the normal level or type of activity that occurs when CD40L binds without inhibition or blocking. It is also intended that inhibition and blocking include any measurable reduction in the binding affinity of CD40L when contacted with an anti-CD40 antibody, compared to CD40L not contacted with an anti-CD40 antibody, by

- 18 042934 example, inhibition of CD40L binding by at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96 , 97, 98, 99 or 10o%. In a specific embodiment of the present invention, an anti-CD40 antibody inhibits CD40L binding by at least about 70% as measured by, for example, BLI or SPR (Biacore). In another embodiment, the anti-CD40 antibody inhibits CD40L binding by at least about 80%.

As used herein, the term inhibits proliferation (e.g., in relation to cells) is intended to include any measurable reduction in cell proliferation, e.g., inhibition of cell proliferation by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 , 99 or 100%.

The terms immune response induction, immune response enhancement, and immune response enhancement are used interchangeably and refer to the stimulation of an immune response (ie, both passive and adaptive) to a particular antigen.

The terms induce and increase, as used in relation to the induction of CDC or ADCC, refer to the stimulation of specific mechanisms of direct cell killing. For example, in one embodiment of the present invention, the antibody at 10 μg/mL induces at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% lysis of CD40-expressing cells by CSC. According to a preferred embodiment of the present invention, the antibody at a concentration of 10 μg/ml induces at least about 40% lysis of CD40-expressing cells by CSC. In another embodiment, the antibody at 10 μg/mL induces at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% lysis of CD40-expressing cells through ADCC (i.e. specific lysis). In one embodiment of the present invention, the antibody at a concentration of 10 μg/ml induces at least about 40% lysis of CD40-expressing cells by ADCC.

As used herein, the terms treat, treating, and treatment refer to the therapeutic or prophylactic measures described herein. Methods of treatment employ administering to a subject in need of such treatment a human antibody of the present invention, e.g., a subject in need of an enhanced immune response against a particular antigen, or a subject who may eventually develop such a disorder, to prevent, cure, delay , reduce the severity or improve one or more symptoms of a disorder or relapsing disorder, or in order to prolong the subject's survival beyond that expected in the absence of such treatment.

The term effective dose or effective dosage is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term therapeutically effective dose is defined as an amount sufficient to cure or at least partially relieve a disease and its complications in a patient already suffering from the disease. Amounts effective for such use will depend on the severity of the disorder being treated and the general condition of the patient's own immune system.

As used herein, the term synergistic means that the administration of two drugs has a greater effect when used in combination than would be expected from the summation of the individual effects of the two components, e.g. more than two times, more than three times, more than five times or more than ten times what would be expected from the summation of the individual effects of the two components. For example, drug interactions can be studied using the commercial Calcusyn software package, which is based on the median effect model described by Chou and Talalay (Chou, T.C. & Talalay, P. (1984) Adv. Enzyme Regul. 22, 27-55. Quantatative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors). A combined index (C.I.) of 1 indicated an additive drug interaction, while a C.I. greater than 1 indicated an antagonistic interaction, and a score below 1 indicated a synergistic interaction. Definitions of K.I. are as follows: 1.45-1.2 is moderately antagonistic, 1.2-1.1 is slightly antagonistic, 1.1-0.9 is additive, 0.9-0.85 is slightly synergistic, 0.85 -0.7 is moderately synergistic and 0.7-0.3 is synergistic.

The term patient includes human and other mammalian subjects who are receiving either prophylactic or therapeutic treatment.

As used herein, the term subject includes any human or non-human animal. For example, the methods and compositions of the present invention may be used to treat a subject with an immune disorder. The term non-human includes all vertebrates, for example, mammals and non-mammals such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

Various aspects of the present invention are described in more detail in the subsections below.

I. Obtaining antibodies to CD40.

- 19 042934

Anti-CD40 antibodies of the present invention can be generated using a number of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures can be used, in general, other methods for producing monoclonal antibodies can be used, for example, viral or oncogenic transformation of B-lymphocytes, phage display techniques using human antibody gene libraries.

In a specific (exemplary) embodiment of the present invention, a mouse (e.g., H2L2 Harbor® transgenic mice) or other suitable host animal is immunized with a suitable antigen to induce the production of lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, the lymphocytes may be immunized in vitro. The lymphocytes can then be fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). The culture medium in which the hybridoma cells propagate is examined to determine the production of monoclonal antibodies directed against the antigen. Once hybridoma cells have been identified that produce antibodies with the desired specificity, affinity, and/or activity, clones can be subcloned using the serial dilution method and propagated using standard methods (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)). Culture media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be propagated in vivo as ascitic tumors in an animal. Monoclonal antibodies secreted by subclones can be separated from the culture medium, ascitic fluid, or serum using conventional immunoglobulin purification procedures such as, for example, purification on protein A-sepharose, hydroxyapatite carrier chromatography, gel electrophoresis, dialysis, or affinity chromatography. .

According to another embodiment of the present invention, antibodies directed against CD40 are generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. In one embodiment, the present invention uses transgenic mice, herein referred to as HuMAb mice, that contain human immunoglobulin gene miniloci that encode non-rearranged immunoglobulin heavy chain (μ and γ) and κ light chain sequences, together with targeted mutations that inactivate endogenous μ and k chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856-859). Accordingly, mice exhibit reduced expression of mouse IgM or κ and, in response to immunization, introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), see above, reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101, Lonberg, N. and Huszar, D. (1995) Intern Rev. Immunol Vol 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci 764:536-546). The production of HuMAb mice is detailed in section II below and in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg et al. (1994) Nature 368(6474):856859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Taylor, L. et al. (1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93; Harding, F. and Lonberg, N. (1995) Ann. NY. Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851. See also U.S. Patent Nos. 5,545,806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299; and 5770429; all issued by Lonberg and Kay, and GenPharm International; US Pat. No. 5,545,807 to Surani et al.; international publication WO 98/24884 published June 11, 1998; WO 94/25585 published November 10, 1994; WO 93/1227 published June 24, 1993; WO 92/22645 published December 23, 1992; WO 92/03918 published March 19, 1992.

According to another embodiment of the present invention, antibodies that bind to human CD40 can be isolated from antibody phage libraries generated using the techniques described, for example, in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624628 (1991), Marks et al., J. Mol. Biol, 222:581-597 (1991) and Hoet et al (2005) Nature Biotechnology 23, 344348; US Pat. No. 5,223,409; 5403484; and 5571698 issued by Ladner et al.; US Pat. Nos. 5,427,908 and 5,580,717 issued to Dower et al.; US Pat. Nos. 5,969,108 and 6,172,197 issued to McCafferty et al; and US Pat. Nos. 5,885,793; 6521404; 6544731; 6555313; 6582915 and 6593081 issued by Griffiths et al. In addition, the chain shuffling method (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and recombination in vivo in as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)).

In a specific embodiment of the present invention, an antibody that binds to human CD40 is generated using the phage display technique described by Hoet et al., supra. This technique involves generating a human Fab library having a unique combination of immunoglobulin sequences isolated from human donors and having synthetic diversity in the heavy chain CDRs. The library is then screened for Fabs that bind to human CD40.

A preferred animal system for generating hybridomas that produce antibodies of the present invention is the mouse system. The production of hybridomas in mice is well known in the art, including immunization protocols and techniques for isolating and fusion of immunized splenocytes.

Preparation of transfectomas producing anti-CD40 monoclonal antibodies.

The antibodies of the present invention can also be generated in a host cell transfectome using, for example, a combination of recombinant DNA techniques and gene transfection techniques well known in the art (Morrison, S. (1985) Science 229:1202).

For example, in one embodiment of the present invention, the gene(s) of interest, e.g., human antibody genes, can be ligated into an expression vector, such as a eukaryotic expression plasmid, such as the GS gene-based expression system disclosed in WO 87/ 04462, WO 89/01036 and EP 338841, or other expression systems well known in the art. The purified plasmid with cloned antibody genes can be introduced into eukaryotic host cells such as CHO cells or NSO cells, or alternatively other eukaryotic cells such as plant-derived, fungal or yeast cells. The method used to introduce such genes may be a method described in the art, such as electroporation, lipofectin, lipofectamine, or others. Once such antibody genes are introduced into host cells, cells expressing the antibody can be identified and selected. These cells are transfectomes, which can then be amplified to assess the level of expression, and their number can be increased to produce antibodies. Recombinant antibodies can be isolated and purified from the supernatants of said cultures and/or cells.

In another embodiment, such cloned antibody genes may be expressed in other expression systems such as E. coli or in complete organisms, or may be expressed synthetically.

Use of partial antibody sequences for the expression of intact antibodies.

Antibodies interact with target antigens primarily through amino acid residues that are located in the six hypervariable regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDR are more diverse within individual antibodies than those outside the CDR.

Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific natural antibodies by constructing expression vectors that contain CDR sequences from a specific natural antibody grafted onto framework sequences from another antibody with differing properties (see, e.g., Riechmann, L. et al., 1998, Nature 332:323327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C. et al., 1989, Proc. Natl. Acad. See U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that contain germline antibody gene sequences. Such germline sequences will differ from mature antibody gene sequences because they will not contain the fully assembled variable genes that are generated by V(D)J fusion during B cell maturation. Germline gene sequences will also differ from high affinity secondary repertoire antibody sequences at the individual level and evenly across the variable region. For example, somatic mutations are relatively rare in the amino-terminal portion of the framework. For example, somatic mutations are relatively rare in the amino-terminal portion of framework region 1 and in the carboxy-terminal portion of framework region 4. In addition, many somatic mutations do not significantly affect the binding properties of an antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties that are similar to those of the original antibody (see PCT/US99/05535, filed March 12, 1999). For this purpose, a partial sequence of heavy and light chains spanning the CDRs is usually sufficient. The partial sequence is used to determine which variable and concatenated germline gene segments have contributed to the recombined antibody variable genes. The germline sequence is then used to fill in the missing portions of the variable regions. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add

- 21 042934 missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, a complete variable region can be synthesized as a set of short, overlapping oligonucleotides and combined by PCR amplification to create a fully synthetic variable region clone. This process has certain advantages, such as elimination or inclusion of specific restriction sites, or optimization of specific codons.

The nucleotide sequences of the heavy and light chain transcripts from the hybridoma are used to design an overlapping set of synthetic oligonucleotides to generate synthetic V sequences with identical amino acid coding abilities as the natural sequences. Synthetic heavy chain and kappa light chain sequences can differ from natural sequences in three ways: strands of repetitive nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are built according to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266:19867-19870); and HindIII sites are designed in the 5' direction relative to translation initiation sites.

In the case of both heavy and light chain variable regions, the optimized sequences of the coding and corresponding non-coding chains are split into 30-50 nucleotides approximately in the middle of the respective non-coding oligonucleotide. Therefore, for each strand, the oligonucleotides can be assembled into overlapping double strand sets that span segments of 150-400 nucleotides. The pools are then used as templates to obtain PCR amplification products containing 150-400 nucleotides. Typically, a single set of variable region oligonucleotides will be split into two pools, which are amplified separately to create two overlapping PCR products. These overlapping products are then combined by PCR amplification to create a complete variable region. It may also be desirable to include an overlapping heavy or light chain constant region fragment (including the BbsI site of the kappa light chain or the AgeI site in the case of the heavy gamma chain) in PCR amplification to produce fragments that can be easily cloned into expression vector constructs.

The reconstituted heavy and light chain variable regions are then combined with cloned promoter, leader sequence, translation initiation site, leader sequence, constant region, 3' untranslated region, polyadenylation sequence and transcription termination signal sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, sequentially transfected, or individually transfected into host cells which are then fused to form a host cell expressing both chains.

Plasmids for use in constructing expression vectors were designed such that PCR-amplified kappa chain V-heavy chain and kappa-chain V-light cDNA sequences could be used to regenerate complete heavy and light chains. These plasmids can be used to fully express human IgG1K or IgG4K antibodies. The fully human and chimeric antibodies of the present invention also include IgG2, IgG3, IgE, IgA, IgM and IgD antibodies. Similar plasmids can be constructed to express other heavy chain isotypes or to express antibodies containing lambda light chains.

Accordingly, in another aspect of the present invention, the structural features of the anti-CD40 antibodies of the present invention are used to generate structurally related anti-CD40 antibodies that retain at least one functional property of the antibodies of the present invention, such as, for example:

(a) inducing or enhancing an immune response to an antigen, independent of Fc receptor binding;

(b) inducing or enhancing an immune response to an antigen without inducing antibody-dependent cellular cytotoxicity (ADCC) of CD40-expressing cells;

(c) inducing or enhancing an immune response to an antigen without inducing complement-dependent cellular cytotoxicity (CDC) of CD40-expressing cells; and/or (d) the ability to act synergistically with CD40L; and/or additional features may include, for example:

(d) no inhibition or blocking of CD40L binding;

(e) inhibition or blocking of CD40L binding;

(f) inhibiting or blocking the binding of CD40L to human CD40, independent of binding to the Fc receptor;

(g) induction or enhancement of cellular apoptosis of the tumor cell;

(h) inducing or enhancing the activity of a cell aimed at stimulating T cells (eg, as measured by increased expression of IL-12p40); and/or (i) induction or enhancement of B cell activation (e.g., as measured by an increase in

- 22 042934 expression of at least one cell surface marker selected from the group consisting of HLA-DR V450, CD54-FE, CD86-APC and CD83 BV510, CD19 V500, CD54-FE, HLA-DR V450, CD23

PerCP-Cy5.5, CD69-APC, CD86-APC, CD38 PerCP-Cy5.5 and CD71-PE.

In one embodiment of the present invention, one or more CDRs of antibodies of the present invention can be recombinantly combined with known framework regions and CDRs to generate additional recombinantly modified anti-CD40 antibodies of the present invention. Framework regions of the variable regions of the heavy and light chains can be obtained from the sequences of the same or different antibodies. The antibody sequences may be natural antibody sequences or may be consensus sequences of multiple antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) and Carter et al., WO 92/22653.

Accordingly, according to another embodiment, the present invention provides a method for producing an anti-CD40 antibody comprising: producing an antibody comprising (1) heavy chain framework regions and a heavy chain CDR, wherein at least one of the heavy chain CDRs comprises an amino acid sequence selected from amino acid sequences CDRs shown in SEQ ID NOs: 5, 6, 7, 8, 9, 10, 19, 20, 21, 22, 23, 24, 33, 34, 35, 36, 37, 38, 47, 48, 49, 51, 52, 61, 62, 63, 64, 65, 66, 75, 76, 77, 78, 79, 80, 89, 90, 91, 92, 93, 94, 103, 104, 105, 106, 107, 108; and (2) light chain framework regions and light chain CDRs, wherein at least one of the light chain CDRs comprises an amino acid sequence selected from the amino acid sequences of the CDRs shown in SEQ ID NOs: 11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 29, 30, 39, 40, 41, 42, 43, 44, 53, 54, 55, 56, 57, 58, 67, 68, 69, 70, 71, 72, 81, 82, 83, 84, 85, 86, 95, 96, 97, 98, 99, 100, 109, 110, 111, 112, 113, 114; while said antibody retains its ability to bind to CD40. The ability of an antibody to bind to CD40 can be determined using standard quantitative binding assays such as those indicated in the examples (eg, ELISA or FLISA).

It is well known in the art that the CDR3 domains of the heavy and light chains of an antibody play a particularly important role in the binding specificity/affinity of an antibody for an antigen (see Hall et al., J. Imunol., 149:1605-1612 (1992); Polymenis et al., J. Immunol, 152:5318-5329 (1994), Jahn et al., Immunobiol, 193:400-419 (1995), Klimka et al., Brit. J. Cancer, 83:252-260 (2000); Beiboer et al., J. Mol. Biol, 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA, 95:8910-8915 (1998); Barbas et al. al., J. Am. Chem. Soc, 116:2161-2162 (1994); Ditzel et al, J. Immunol, 157:739-749 (1996)). Accordingly, the recombinant antibodies of the present invention prepared as described above preferably contain CDR3 from the heavy and/or light chains of the 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK and 3B6NS antibodies. The antibodies may also contain CDR2 from antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK and 3B6NS. The antibodies may also contain CDR1 from antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK and 3B6NS. The antibodies may further comprise any combination of CDRs.

Accordingly, in another embodiment, the present invention also provides anti-CD40 antibodies comprising: (1) heavy chain framework regions, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region, wherein said heavy chain CDR3 region is selected from CDR3 antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, and (2) light chain framework regions, light chain CDR1 region, light chain CDR2 region, and light chain CDR3 region, said light chain CDR3 region being selected from the CDR3 of antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK or 3B6NS, wherein said antibody binds to CD40. The antibody may further comprise a heavy chain CDR2 and/or a light chain CDR2 of antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK or 3B6-NS. The antibody may further comprise a heavy chain CDR1 and/or a light chain CDR1 of antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK or 3B6-NS.

Obtaining antibodies having modified sequences.

According to another embodiment of the present invention, the sequences of the variable regions, or portions thereof, of the anti-CD40 antibodies of the present invention are modified to generate structurally related anti-CD40 antibodies that retain the ability to bind (i.e., with the same epitope as the unmodified antibody) and , therefore, are functionally equivalent. Methods for identifying residues that can be altered without eliminating antigen binding are well known in the art (see e.g. Marks et al. (Biotechnology (1992) 10(7):779-83 light chain then heavy chain variable regions with fixed CDR3 sequence changes), Jespers et al (1994) Biotechnology 12(9):899-903 (selection of human antibodies directed to a single antigen epitope from the phage display repertoire), Sharon et al. al (1986) PNAS USA 83(8):2628-31 (Site-directed mutagenesis of an invariant amino acid residue in the junction of antibody V-D segments) Casson et al (1995) J Immunol 155(12):5647-54 (evolution of loss and change in specificity as a result of random mutagenesis of the variable region of the heavy chain of the antibody).

Accordingly, according to one aspect of the present invention, the CDR1, 2 and/or 3 regions of the modified antibodies described above may contain the exact amino acid sequence(s), such as the antibody sequences 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5- NK

- 23 042934 or 3B6-NS disclosed in this document. However, according to other aspects of the present invention, the antibodies contain derivatives of the exact sequences of the CDRs of antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK or 3B6-NS, but still retain the ability to bind to CD40. Such sequence modifications may include one or more amino acid additions, deletions, or substitutions, such as the conservative sequence modifications described above. Sequence modifications may also be based on the consensus sequences described above for specific CDR1, CDR2 and CDR3 sequences of antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK or 3B6-NS.

Accordingly, according to another embodiment of the present invention, the modified antibody may consist of one or more CDRs that are, for example, 90%, 95%, 98%, or 99.5% identical to one or more of the CDRs from antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK or 3B6-NS. Ranges that are intermediate to the above values, such as CDRs that are 90-95%, 95-98%, or 98-100% identical to one or more of the above sequences, are also intended to be included within the scope of the present invention.

According to another embodiment of the present invention, one or more CDR residues can be altered to modify binding to achieve a more favorable forward binding rate, a more favorable reverse binding reaction rate, or both, such that an ideal binding constant is achieved. Using this strategy, an antibody having an ultra-high binding affinity, for example, 10 ¹⁰ M ^-1 or more, can be obtained. Affinity maturation techniques well known in the art and described herein can be used to modify the CDR region(s) followed by screening the resulting binding molecules for the desired binding change. Accordingly, as the CDR site(s) is altered, changes in binding affinity as well as immunogenicity can be monitored and assessed so as to produce an antibody optimized for better combined binding and low immunogenicity.

In addition to or in lieu of modifications to the CDRs, modifications may be made within one or more of the framework regions, FR1, FR2, FR3 and FR4, heavy and/or light chain variable regions of the antibody, as long as such modifications do not eliminate the binding affinity antibodies. For example, one or more non-germline amino acid residues in the framework regions of the heavy and/or light chain variable regions of an antibody of the present invention are replaced by a germline amino acid residue, i. the corresponding amino acid residue in the human germline sequence for the heavy or light chain variable region with which the antibody has significant sequence identity. For example, an antibody chain can be aligned with a germline antibody chain with which it has significant sequence identity, and amino acid residues that do not match between the antibody backbone sequence and the germline chain backbone can be replaced with the corresponding residues from the germline sequence. If an amino acid differs between the framework region of the antibody variable region and the equivalent framework region of the human germline sequence variable region, then the amino acid of the antibody framework region should generally be replaced with an equivalent amino acid from the human germline sequence if there is reason to believe that the amino acid falls into one of the following categories :

(1) an amino acid residue that binds directly non-covalently to the antigen;

(2) an amino acid residue that is adjacent to the CDR site;

(3) an amino acid residue that otherwise interacts with the CDR region (eg, within about 3-6 A of the CDR region, as determined by computer simulation); or (4) an amino acid residue that is involved in creating the VL-VH interface.

Residues that bind directly non-covalently to the antigen include amino acids in framework regions at positions that have a high likelihood of interacting directly with amino acids on the antigen according to established chemical forces, e.g., hydrogen bonding, van der Waals forces, hydrophobic interactions and so on. Accordingly, in one embodiment of the present invention, an amino acid residue in the framework region of an antibody of the present invention is replaced with a corresponding germline amino acid residue that binds directly non-covalently to the antigen.

Residues that are adjacent to a CDR region include amino acid residues at positions immediately adjacent to one or more CDRs in the primary sequence of an antibody, e.g., at positions immediately adjacent to a Kabat-defined CDR or a Chothia-defined CDR (See, for example, Chothia and Lesk J. Mol. Biol. 196:901 (1987)). Accordingly, according to one embodiment of the present invention, an amino acid residue in the framework region of an antibody of the present invention is replaced by a corresponding germline amino acid residue that is adjacent to the CDR region.

Residues that otherwise interact with the CDR include those residues that

- 24 042934 which, as determined by secondary structure examination, are in a spatial orientation sufficient to affect the CDR region. Such amino acids typically have a side chain atom within about 3 angstroms (A) of some atom in the CDR and must contain an atom that can interact with CDR atoms according to established chemical forces such as those listed above. Accordingly, according to one embodiment of the present invention, an amino acid residue in the framework region of an antibody of the present invention is replaced by a corresponding germline amino acid residue that otherwise interacts with the CDR region.

Amino acids at several positions in the framework are known to be important in determining CDR conformation (eg, being able to interact with CDRs) in many antibodies (Chothia and Lesk, supra, Chothia et al., supra, and Tramontano et al., J. Mol. Biol 215:175 (1990), all of which are incorporated herein by reference). The authors of these literature sources have identified conserved framework residues important for CDR conformation by examining the structures of several known antibodies. The antibodies studied were included in a limited number of structural or canonical classes based on the CDR conformation. Conservative frame residues in sequences of members of a canonical class are called canonical residues. Canonical residues include residues 2, 25, 29, 30, 33, 48, 64, 71, 90, 94 and 95 of the light chain and residues 24, 26, 29, 34, 54, 55, 71 and 94 of the heavy chain. Additional residues (eg, residues defining the CDR structure) can be identified according to the methodology of Martin and Thorton (1996) J. Mol. Biol. 263:800. Notably, amino acids at positions 2, 48, 64, and 71 of the light chain and 26-30, 71, and 94 of the heavy chain (Kabat numbering) are known to be able to interact with CDRs in many antibodies. The amino acids at positions 35 on the light chain and 93 and 103 on the heavy chain are also likely to interact with CDRs. Additional residues that may influence CDR conformation can be identified according to the methodology of Foote and Winter (1992) J. Mol. Biol. 224:487. Such residues are referred to as vernier residues and are residues in the framework region located immediately below (ie forming a platform underneath) the CDR.

Residues that participate in the formation of the VL-VH interface or packing residues include residues at the VL and VH interface as defined by, for example, Novotny and Haber, Proc. Natl. Acad. sci. USA, 82:4592-66 (1985) or Chothia et al., supra.

There is sometimes some controversy as to whether a particular amino acid falls into one or more of the above categories. In such cases, alternative antibody variants are obtained, one of which contains this particular substitution and the other does not. Alternative antibodies produced in this way can be tested in any of the quantitative studies described herein to determine the desired activity, with the selection of the preferred antibody.

Additional candidates for substitution in the framework region are amino acids that are unusual or rare for the antibody at that position. Such amino acids may be replaced by amino acids from an equivalent position in the human germline sequence or from equivalent positions in more typical antibodies. For example, a substitution may be desirable if the amino acid in the antibody framework is rare at that position and the corresponding amino acid in the germline sequence is common at that position in immunoglobulin sequences; or if the amino acid in the antibody is rare for that position and the corresponding amino acid in the germline sequence is also rare compared to other sequences. It is contemplated that an antibody can be made less immunogenic by replacing an unusual amino acid with an amino acid from a germline sequence that is typical of antibodies. A substitution may also be desirable, for example in the case of mismatched cysteine residues or putative N-glycosylation sites.

As used herein, the term rare means an amino acid present at a given position in less than about 20%, preferably less than about 10%, more preferably less than about 5%, even more preferably less than about 3%, even more preferably less than than about 2%, and even more preferably, less than about 1% of sequences in a representative set of sequences, and as used herein, the term ordinary means an amino acid present in more than about 25%, but typically more than about 50%, of sequences in a representative set of sequences. . For example, all light and heavy chain variable region sequences are appropriately grouped into subsets of sequences that are particularly homologous to each other and contain the same amino acids at certain critical positions (Kabat et al., supra). When determining whether an amino acid in an antibody sequence is rare or common among sequences, it will generally be preferable to consider only those sequences that are in the same subgroup as the antibody sequence.

In general, the framework regions of antibodies are usually substantially identical, and more usually identical.

- 25 042934 framework regions of human germline sequences from which they were derived. Indeed, many amino acids in the framework region have little effect on the specificity or affinity of the antibody. Therefore, many individual conservative framework substitutions can be tolerated without appreciable change in the specificity or affinity of the resulting immunoglobulin. Accordingly, according to one embodiment of the present invention, the sequence of the framework region of the variable region of the antibody is at least 85% identical to the sequence of the framework region of the human germline variable region or the consensus region of such sequences. According to another embodiment of the present invention, the antibody variable region framework sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the human germline variable region framework sequence or a consensus region of such sequences.

In addition to simply binding to CD40, an antibody can be selected based on its ability to retain other functional properties of the antibodies of the present invention, such as, for example:

(a) inducing or enhancing an immune response to an antigen, independent of Fc receptor binding;

(b) inducing or enhancing an immune response to an antigen without inducing antibody-dependent cellular cytotoxicity (ADCC) of CD40-expressing cells;

(c) inducing or enhancing an immune response to an antigen without inducing complement-dependent cellular cytotoxicity (CDC) of CD40-expressing cells; and/or (d) the ability to act synergistically with CD40L.

Additional features may include, for example:

(e) no blocking of CD40L binding to human CD40, independent of Fc receptor binding;

(e) blocking CD40L binding to human CD40, independent of Fc receptor binding;

(g) activation of human CD40 expressed on APC, independent of Fc receptor binding;

(h) induction of apoptosis of the tumor cell;

(i) activity aimed at stimulating T cells; and/or (j) enhanced B cell activation.

Characterization of monoclonal antibodies to CD40.

Monoclonal antibodies according to the present invention can be characterized in relation to binding to CD40 using a variety of known techniques. Typically, antibodies are first characterized by ELISA. In general terms, microtiter plates can be coated with purified CD40 in phosphate buffered saline (PBS) and then blocked using off-target proteins such as bovine serum albumin (BSA) diluted in PBS. Plasma dilutions from mice immunized with CD40 are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS/tween-20 and then incubated with alkaline phosphatase conjugated goat anti-human IgG Fc polyclonal reagent for 1 hour at 37°C. After washing, the staining in the plates is developed using the ABTS substrate and the optical density is evaluated at a wavelength of 405 nm (OD ₄₀₅ ). Preferably, cells from mice that have developed the highest titers will be used for fusions.

The quantitative ELISA assay described above can be used to screen for antibodies, and therefore hybridomas, that produce antibodies that show positive reactivity with the CD40 immunogen. Hybridomas that bind, preferably with high affinity, to CD40 can then be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the original cells (as measured by ELISA) can then be selected for cell bank preparation and antibody purification.

To purify anti-CD40 antibodies, selected hybridomas can be expanded in roller bottles, 2 liter shake flasks, or other culture systems. Supernatants can be filtered and concentrated prior to affinity chromatography on a Protein Asepharose support (Pharmacia, Piscataway, NJ, USA) to purify the protein. After exchanging buffer for PBS, the concentration can be determined from OD ₂₈₀ values using an extinction coefficient of 1.43, or preferably by nephelometric analysis. IgG can be tested using gel electrophoresis and antigen-specific method.

To determine if selected anti-CD40 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL, USA). Binding of biotinylated MAT can be detected using a streptavidin-labeled probe. To determine the isotype of purified antibodies, an isotype ELISA can be performed using techniques generally recognized in the art. For example, the wells of microtiter plates can be coated with 10 μg/ml anti-Ig overnight at 4°C. After blocking with 5% BSA, the plates are reacted with 10 μg/ml monoclonal antibody or purified isotype control antibody at ambient temperature for two hours. The wells can then be reacted with either IgG1 or other isotype-specific conjugated probes. Plate stains are developed and examined as described above.

Binding of monoclonal antibodies to live cells expressing CD40 can be tested using flow cytometry. In general, cell lines and/or human PBMC expressing membrane-bound CD40 (grown under standard culture conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with a fluorescein-labeled anti-IgG antibody under conditions similar to those for staining with the primary antibody. Samples can be examined with the FACScan instrument using the light and side scatter properties to pass individual cells and determine the binding of labeled antibodies. An alternative quantification method using fluorescence microscopy can be used in addition to or instead of flow cytometry. Cells can be stained using a method identical to that described above and examined using fluorescence microscopy. This method allows visualization of individual cells, but may have reduced sensitivity depending on the density of the antigen.

The ability of anti-CD40 IgGs to react with the CD40 antigen can also be tested by Western blotting. In general terms, cell extracts from cells expressing CD40 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 20% mouse serum and probed using the monoclonal antibodies to be tested. IgG binding can be detected using an alkaline phosphatase-conjugated anti-IgG antibody and staining can be developed using BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO, USA).

Methods for studying the binding affinity, cross-reactivity, and binding kinetics of various anti-CD40 antibodies include standard quantitative assays known in the art, such as Biacore Surface Plasmon Resonance (SPR) using the Biacore 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or interferometry biolayers (BLI) using the Octet™ QKe instrument as described in the examples.

The present invention also provides anti-CD40 agonist antibodies that bind to the same epitope as anti-CD40, 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK and 3B6-NS antibodies (as determined by specific epitope mapping technique). For example, as described in Example 17, antibodies of the present invention (e.g., antibody 3C3) bind to one or more residues within the region containing amino acid residues 1-5 and 33-36 of the extracellular domain (ECD) of human CD40 (SEQ ID NO: 133), for example, amino acids 5, 33, 34 and/or 36 of human CD40 EVA (SEQ ID NO: 133). The 3C3 antibody has been shown to additionally bind to one or more amino acids 26, 28 and/or 30 of the human CD40 EVA (SEQ ID NO: 133), e.g., amino acids 5, 33, 34 and 36 of the human CD40 EVA (SEQ ID NO: 133) or amino acids 5, 33 and 36 of human CD40 EVA (SEQ ID NO: 133).

Other antibodies of the present invention (e.g. antibody 3G5) bind to one or more residues within the region containing residues amino acids 13-15 and 33-36 of human CD40 EVA (SEQ ID NO: 133), e.g. amino acids 33, 34 and 36 ECV human CD40 (SEQ ID NO: 133).

Antibodies that bind to the epitopes on human CD40 described herein (eg, the same epitopes as exemplary antibodies) exhibit therapeutically useful properties. For example, as shown in Example 16 and 20, 3C3 antibodies exhibit synergistic agnostic activity with soluble CD40 ligand (sCD40L), as measured, for example, by increased induction of CD95 expression when incubated with Ramos cells, increased B cell proliferation when incubated with B human β-cells and/or increased induction of IL-12p40 expression upon incubation with dendritic cells.

Accordingly, antibodies that bind to the same epitope as 3C3 are capable of synergistic action with other therapeutic agents, including those that bind to the human CD40 ligand binding site. Typical synergistic effects include, for example, enhancement of immune function (eg, T-cell mediated immune responses as in various vaccine therapies, NK cell activation in various cancer therapies), inhibition of cell proliferation (eg, in cancer therapy), and/or increased processing and presentation of the APC antigen (for example, during vaccine therapy).

As described herein, techniques for detecting antibodies that bind to the same epitope on CD40 as the antibodies described herein include, for example, epitope mapping methods such as X-ray examination of crystals of antigen:antibody complexes, which provide atomic resolution of the epitope. Other methods allow you to control the binding of antibodies to fragments of the antigen or mutated variants of the antigen,

- 27 042934 while the loss of binding due to modification of the amino acid residue in the antigenic sequence is often considered as an indication of an epitope component. In addition, computational combinatorial methods can also be used to map epitopes. Methods can also be based on the ability of the antibody of interest to provide affinity isolation of specific short peptides (either in native three-dimensional form or in denatured form) from combinatorial peptide libraries for phage display. The peptides are then considered as a tool to determine the epitope corresponding to the antibody used to screen the peptide library. Computational algorithms have also been developed for epitope mapping and have been shown to provide mapping of conformational discontinuous epitopes.

II. Molecular conjugates/immunotoxins.

The present invention relates to various therapeutic molecular conjugates (eg, conjugate vaccines) that contain an antigen, such as a tumor or viral antigen, coupled to an antibody that binds to a receptor on APC, eg, an antibody that binds to CD40. This allows antigen to be targeted to APCs such as CD40 expressing cells (eg dendritic cells, B cells and macrophages) to enhance processing, presentation and ultimately immune response against the antigen(s). A schematic representation of such a conjugate is shown in Fig. 18, in which, for example, the antigen is genetically hybridized to the CH3 domain of each of the heavy chains of a substantially complete anti-CD40 antibody. However, it should be understood that the antigen may alternatively be coupled to other parts of such an antibody or fragment thereof, and that other forms of conjugation may also be used, such as chemical conjugation, as discussed below.

Suitable antigens for use in molecular conjugates include, for example, infectious disease antigens and tumor antigens against which protective or therapeutic immune responses are desired, such as antigens expressed by a tumor cell or pathogenic organism, or infectious disease antigens. For example, suitable antigens include tumor-associated antigens for preventing or treating cancer. Examples of tumor-associated antigens include, but are not limited to, sequences containing all or part of the ehCG, gp100 or Pmel17, HER2/neu, WT1, mesothelin, CEA, gp100, MARTI, TRP-2, melan-A, NY antigens -ESO-1, NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MaGe-3, MAGE-A3, tyrosinase, telomerase, SSX2 and MUC-1, and tumor antigens derived from germ cells. Tumor-associated antigens also include blood group antigens, eg Le ^a , Le ^b , LeX, LeY, H-2, B-1, B-2 antigens. Alternatively, more than one antigen may be included in an antigen-antibody construct of the present invention. For example, the MAGE antigen can be combined with other antigens such as melanin A, tyrosinase, and gp100, along with adjuvants such as GM-CSF or IL-12, and linked to an APC-targeted antibody.

Other suitable antigens include viral antigens for the prevention or treatment of viral diseases. Examples of viral antigens include, but are not limited to, HIV-1 gag, HIV-1 env, HIV-1 nef, HBV (surface or core antigens), HPV, FAS, HSV-1, HSV-2, p17, ORF2, and ORF3 . Examples of bacterial antigens include, but are not limited to, Toxoplasma gondii or Treponema pallidum. The antibody-bacterial antigen conjugates of the present invention can be used in the treatment or prevention of various bacterial diseases such as anthrax, botulism, tetanus, chlamydia, cholera, diphtheria, Lyme disease, syphilis and tuberculosis.

The sequences of the antigens described above are well known in the art. For example, an example of a MAGE-3 cDNA sequence is given in US Pat. No. 6,235,525 (Ludwig Cancer Research Institute); examples of the nucleic acid sequence and protein sequence of NY-ESO-1 are given in US patent No. 5804381 and 6069233 (Ludwig Cancer Research Institute); examples of a nucleic acid sequence and a melan-A protein sequence are given in US Pat. Nos. 5,620,886 and 5,854,203 (Ludwig Cancer Research Institute); examples of the nucleic acid sequence and protein sequence of NY-BR-1 are given in US Pat. ; an exemplary amino acid sequence of the HER-2/neu protein is available from GenBank® under accession number AAA58637; and the nucleotide sequence (mRNA) for human carcinoembryonic antigen-like protein 1 (CEA-1) is available from GenBank® under accession number NM_020219.

In one embodiment of the present invention, the antigen is an HPV antigen, such as HPV-16 antigen, HPV-18 antigen, HPV-31 antigen, HPV-33 antigen and/or HPV-35 antigen. The HPV-16 genome is described in Virology, 145:181-185 (1985), and the DNA sequences encoding HPV-18 are described in US Pat. No. 5,840,306, the disclosure of which is incorporated herein in its entirety.

- 28 042934 document by reference. HPV-16 antigens (eg, seroreactive regions of HPV-16 E1 and/or E2 proteins) are described in US Pat. 5840306, the disclosure of which is incorporated herein by reference. Similarly, the complete HBV genome is available from GenBank® under accession number NC_003977, the disclosure of which is incorporated herein. The HCV genome is described in European Patent Application No. 318216, the disclosure of which is incorporated herein. PCT/US90/01348, incorporated herein by reference, discloses the sequence of HCV genome clones, amino acid sequences of HCV viral proteins, and methods for making and using such compositions for HCV vaccines containing HCV proteins and peptides derived therefrom.

Antigenic peptides of proteins (ie, those containing T-cell epitopes) can be detected by various methods well known in the art. For example, T-cell epitopes can be predicted by probing the protein sequence using predictive algorithms available online (BIMAS & SYFPEITHI) to generate potential MHC class I and II binding peptides that match an internal database of 10,000 well-characterized MHC binding peptides previously identified by CTL. Peptides with high scores can be divided into classes and selected as interesting based on high affinity for a given MHC molecule.

Another way to detect antigenic peptides containing T-cell epitopes involves separating the protein into non-overlapping peptides of the desired length or overlapping peptides of the desired length, which can be obtained by recombinant, synthetic methods or, in certain limited situations, by chemical cleavage of the protein, and tested for detection immunogenic properties, for example, the ability to induce a T cell response (ie proliferation or secretion of lymphokines).

In order to determine the exact T-cell epitopes of a protein, for example, using precise epitope mapping techniques, a peptide having activity to stimulate T cells, and therefore containing at least one T-cell epitope, determined using T -cells can be modified by adding or deleting amino acid residues at the amino or carboxy terminus of the peptide and tested to determine the change in T cell reactivity towards the modified peptide. If two or more peptides that have an area of overlap in the native protein sequence are found to have human T cell stimulation activity as determined by T cell biology techniques, additional peptides can be made containing such complete peptides or a portion thereof. , and such additional peptides can be tested using a similar procedure. Following this methodology, peptides are selected and produced by recombinant or synthetic methods. Peptides are selected based on various factors, including the intensity of the T cell response to the peptide (eg, stimulation index). The physical and chemical properties of such selected peptides (eg, solubility, stability) can then be examined to determine the suitability of the peptides for use in therapeutic compositions or the need for modification of the peptides.

In addition, the conjugate vaccine may contain one or more immunostimulatory agents which also enhance the immune response against the antigen. The antibody-antigen conjugate vaccines of the present invention may be prepared by genetic or chemical means. In any case, the antibody portion of the conjugate may consist of the entire antibody or an antibody portion such as a Fab fragment or a single chain Fv. In addition, more than one antigen and/or immunostimulatory agent may be included in the conjugate.

Chemically engineered antibody-antigen conjugates can be prepared using a number of well known and readily available crosslinking reagents. Suitable crosslinking agents may be homofunctional or heterofunctional compounds such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-3-acetylthioacetate (SATA), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (sulfo-SMCC), 5,5'-dithiobis2-nitrobenzoic acid (DTNB), which form covalent bonds with various reactive amino acid or carbohydrate side chains on the DC-targeted antibody and the selected antigen. Other binding and cross-linking agents can also be used to form covalent bonds, such as protein A, carbodiimide, and o-phenylenedimaleimide (oPDM); (See, for example, Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, M.A. et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie et al. (J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Illinois, USA). Immunostimulatory agents may also be chemically coupled to the molecular conjugates of the present invention using the same coupling methods as described above.

According to another embodiment of the present invention, the antibodies of the present invention

- 29 042934 of the invention are connected to a therapeutic moiety such as a cytotoxin, a drug or a radioisotope. When antibodies are conjugated to a cytotoxin, such antibody conjugates are called immunotoxins. A cytotoxin or cytotoxic agent includes any agent that is detrimental (eg, causes death) to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, or docaine, propranolol and puromycin, as well as their analogues or homologues. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5fluorouracil decarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU) , cyclophosphamide, busulfan, dibromomannite, streptozotocin, mitomycin C, and platinum(II) dichlorodiamine (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and antimitotic agents (eg vincristine and vinblastine). An antibody of the present invention can be conjugated to a radioisotope, such as radioactive iodine, to produce cytotoxic radiopharmaceuticals for the treatment of a dendritic cell-associated disorder, such as an autoimmune or inflammatory disease, or graft-versus-host disease.

The antibody conjugates of the present invention can be used to modify a particular biological response and the drug moiety should not be construed as being limited to classic chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Suitable proteins may include, for example, an enzymatically active toxin or active fragment thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth factors.

Techniques for conjugating such a therapeutic moiety to antibodies are well known, see, for example, Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., Antibodies For Drug Delivery, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol. Rev. 62:119-58 (1982).

III. Compositions.

According to another implementation variant of the present invention, a composition is provided, for example, a composition containing one or more monoclonal antibodies according to the present invention, prepared together with a carrier (eg, a pharmaceutically acceptable carrier). The present invention also relates to compositions containing bispecific molecules that contain an antibody according to the present invention. According to one implementation variant of the present invention, the compositions contain a combination of several (for example, two or more) selected antibodies according to the present invention. Preferably, each of the antibodies of the composition binds to a separate, pre-selected CD40 epitope.

Pharmaceutical compositions according to the present invention can also be administered as a combination therapy, i. in combination with other agents. For example, combination therapy may include a composition of the present invention with at least one or more additional therapeutic agents such as anti-inflammatory agents, DMARDs (disease modifying antirheumatic drugs), immunosuppressive agents, and chemotherapeutic agents. The pharmaceutical compositions of the present invention may also be administered in combination with radiation therapy. Co-administration with other antibodies is also included within the scope of the present invention.

As used herein, the terms carrier and pharmaceutically acceptable carrier include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, ie. antibody, bispecific and polyspecific molecule, can be coated with a material to protect the compound from the action of acids and other

- 30 042934 environmental conditions that can inactivate the connection.

Examples of adjuvants that can be used with antibodies and constructs according to the present invention include: incomplete Freund's adjuvant and complete Freund's adjuvant (Difco Laboratories, Detroit, Michigan, USA); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ, USA); AS-2 (SmithKline Beecham, Philadelphia, PA, USA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; cytokines such as GM-CSF, interleukin-2, -7, -12 and the like; 3D-MFL; CpG oligonucleotide; and monophosphoryl lipid A, e.g. 3-de-O-acylated monophosphoryl lipid A.

MFL adjuvants are available from Corixa Corporation (Seattle, Washington, USA, see, for example, US patents No. 4436727, 4877611, 4866034 and 4912094). CpG-containing oligonucleotides (in which the CpG dinucleotide is not methylated) are well known and are described, for example, in WO 96/02555, WO 99/33488 and US Pat. Nos. 6,008,200 and 5,856,462. Science 273:352, 1996.

Other alternative adjuvants include, for example, saponins such as Quil A or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA, USA); eskin; digitonin; or Gypsophila or Chenopodium quinoa saponins; Montanide ISA 720 (Seppic, France); SAF (Chiron, California, USA); ISCOMS (CSL), MF-59 (Chiron); the SBAS adjuvant series (eg SBAS-2 or SBAS-4 available from SmithKline Beecham, Riksansart, Belgium); detox (enhansin™) (Corixa, Hamilton, Montana, USA); RC-529 (Corixa, Hamilton, Montana, USA) and other aminoalkyl glucosaminide-4-phosphates (AGP); polyoxyethylene ether adjuvants such as those described in WO 99/52549A1; synthetic imidazoquinolines such as imiquimod [S26308, R-837] (Harrison, et al., Vaccine 19: 1820-1826, 2001) and resiquimod [S-28463, R-848] (Vasilakos, et al., Cellular immunology 204 : 64-74, 2000); Schiff bases of carbonyls and amines that are constitutively expressed on antigen presenting cells and T cell surfaces, such as tucaresol (Rhodes, J. et al., Nature 377: 71-75, 1995), cytokines, chemokines, and costimulatory molecules such as protein or a peptide including, for example, pro-inflammatory cytokines such as interferon, GMCSF, IL-1 alpha, IL-1 beta, TGF-alpha and TGF-beta, Th1 inducers such as interferon-gamma, IL2, IL-12, IL -15, IL-18 and IL-21, Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13 and other chemokines, and costimulatory genes such as MCP-1, MIP -1 alpha, MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD70; immunostimulatory agents targeting ligands such as CTLA-4 and Lselectin, apoptosis stimulating proteins and peptides such as Fas; synthetic lipid-based adjuvants such as vaxfectin (Reyes et al., Vaccine 19: 3778-3786, 2001), squalene, alpha-tocopherol, polysorbate 80, DOPC, and cholesterol; endotoxin, [LPS], (Beutler, B., Current Opinion in Microbiology 3:2330, 2000); ligands that trigger Toll receptors to produce Th1-inducing cytokines, such as synthetic mycobacterial lipoproteins, mycobacterial p19 protein, peptidoglycan, teichoic acid, and lipid A; and XT (cholera toxin, subunits A and B) and LT (thermolabile enterotoxin from E. coli, subunits A and B), the heat shock protein (HSP) family, and LLO (listeriolysin O, WO 01/72329). These and various other Toll-like receptor (TLR) agonists are described, for example, in Kanzler et al., Nature Medicine, May 2007, Vol 13, No 5. A preferred immunostimulatory agent for use in combination with an anti-CD40 antibody of the present invention is an agonist TLR3 such as poly-IC.

A pharmaceutically acceptable salt refers to a salt that retains the desired biological activity of the parent compound and does not have an undesirable toxic effect (see, for example, Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphoric and the like, as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chlorprocaine, choline. , diethanolamine, ethylenediamine, procaine, etc.

The composition according to the present invention can be entered in various ways known in the art. One of skill in the art will appreciate that the route and/or mode of administration will vary depending on the desired results. Active compounds can be formulated with carriers that will protect the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyortho esters, and polylactic acid can be used. Many methods for preparing such formulations are patented or known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery

Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In order to administer a compound of the present invention via certain routes of administration, it may be necessary to coat the compound or co-administer the compound with a material to prevent its inactivation. For example, the compound may be administered to the subject in a suitable vehicle, such as liposomes, or a diluent. Acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Carriers include sterile aqueous solutions or dispersions and sterile powders for immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except where any conventional media or agent is not compatible with the active compound, the scope of the present invention includes their use in the pharmaceutical compositions of the present invention. Additional active compounds may also be included in the compositions.

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases it is preferred to include isotonic agents, eg sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Sustained absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as needed, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and other necessary ingredients selected from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, vacuum drying and freeze-drying (lyophilization) are the preferred methods of preparation, which make it possible to obtain a powder of the active ingredient plus any additional desired ingredient from a solution previously sterilized by filtration.

Dosing regimens are adjusted to provide the optimum desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over a period of time, or the dose may be proportionally reduced or increased as required by the therapeutic situation. For example, the antibodies of the present invention may be administered once or twice a week by subcutaneous or intramuscular injection, or once or twice a month by subcutaneous or intramuscular injection.

It is particularly useful to formulate compositions for parenteral administration in unit dosage form for ease of administration and dosage uniformity. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The description of the unit dosage form according to the present invention is due to and depends directly on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the original limitations of the technology of mixing such an active compound for the treatment of sensitivity in individuals.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In the case of therapeutic compositions, the compositions of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. For convenience, the compositions can be presented

- 32 042934 in the form of a standard dosage form and can be obtained by any methods known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to form a unit dosage form will vary depending on the subject being treated and the particular route of administration. The amount of active ingredient that can be combined with a carrier material to form a unit dosage form will generally be that amount of the composition that provides a therapeutic effect. Typically, out of one hundred percent, such an amount will range from about 0.001% to about ninety percent of the active ingredient, preferably from about 0.005% to about 70%, most preferably from about 0.01% to about 30%.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or aerosol formulations containing carriers known in the art. Dosage forms for topical or transdermal administration of the compositions of the present invention include powders, aerosols, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers or propellants that may be required.

As used herein, the terms parenteral administration and parenterally administered means methods of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

Suitable compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured either by the sterilization procedures described above or by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the pharmaceutical injectable form can be provided by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

When the compounds of the present invention are administered as pharmaceutical preparations to humans and animals, they may be administered singly or as a pharmaceutical composition containing, for example, from 0.001 to 90% (more preferably from 0.005 to 70%, for example, from 0.01 to 30%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

Regardless of the route of administration chosen, the compounds of the present invention which may be administered in a suitable hydrated form and/or pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may vary so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration without toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors including the potency of the particular compositions of the present invention or their ester, salt or amide, route of administration, time of administration, rate of release of the particular compound employed, duration of treatment, other drugs, compounds and/or materials. used in combination with the specific compositions used, age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in medicine. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian may begin administering dosages of the compounds of the present invention used in a pharmaceutical composition at lower levels than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Usually a suitable daily dose of the composition according to the present invention

- 33 042934 niyu will be the amount of the compound, which is the lowest dose effective to obtain a therapeutic effect. Such an effective dose will usually depend on the factors described above. The preferred route of administration includes intravenous, intramuscular, intraperitoneal or subcutaneous administration, preferably proximal to the target site. If necessary, the effective daily dose of the therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally as unit dosage forms. Although the compound of the present invention may be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

Therapeutic compositions may be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition of the present invention may be administered using a needleless hypodermic injection device, such as those disclosed in US Pat. Known implants and modules that can be used in the present invention include: US Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dosing a drug at a controlled rate; US patent No. 4486194, which discloses a therapeutic device for the introduction of drugs through the skin; US Pat. No. 4,447,233, which discloses an infusion pump for administering a drug at a precise infusion rate; US patent No. 4447224, which discloses an implantable infusion device with adjustable flow rate for continuous drug delivery; US Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other suitable implants, delivery systems and modules are known to those skilled in the art.

According to some variants of implementation of the present invention, the antibodies according to the present invention can be prepared to ensure proper distribution under in vivo conditions. For example, the blood-brain barrier (BBB) does not allow many highly hydrophilic compounds to pass through. To ensure that the therapeutic compounds of the present invention will cross the BBB (if necessary), they can be formulated, for example, in liposomes. Methods for obtaining liposomes are described, for example, in US patent No. 4522811; 5374548; and 5,399,331. Liposomes may contain one or more moieties that are selectively transferred to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, for example, US Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); a surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), various types of which may contain formulations of the inventions as well as components of the invention molecules; p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; J.J. killion; I.J. Fidler (1994) Immunomethods 4:273. According to one embodiment of the present invention, the therapeutic compounds of the present invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In the most preferred embodiment, the therapeutic compounds in liposomes are delivered by bolus injection to a site proximate to the tumor or site of infection. The composition must be fluid to the extent that it is permeable through the needle. It must be stable under the conditions of manufacture and storage and must be resistant to the contaminating action of microorganisms such as bacteria and fungi.

The ability of a compound to inhibit cancer can be assessed in an animal model system that predicts efficacy in human tumors. In another embodiment, this property of the composition can be assessed by examining the inhibitory ability of the compound, such inhibition can be assessed in vitro using quantitative testing methods known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can reduce the size of a tumor or otherwise improve symptoms in a subject. One skilled in the art will be able to determine such amounts based on such factors as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The composition must be sterile and fluid to the extent that the composition can be delivered via syringe. In addition to water, the carrier may be isotonic buffered saline, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases it is preferred to include isotonic agents, for example sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the compositions. Long-term absorption of injectable compositions can be achieved by

- 34 042934 inclusion in the composition of an agent that slows down the absorption, for example, aluminum monostearate or gelatin.

When the active compound is suitably protected as described above, the compound may be administered orally, for example with an inert diluent or an assimilable edible carrier.

IV Applications and methods according to the present invention.

The antibodies, molecular conjugates, bispecific molecules, and compositions of the present invention can be used to treat and/or prevent (eg, immunize against) a number of diseases and conditions.

One of the main indications for treatment is cancer. Cancer types include, but are not limited to, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia such as myeloid, promyelocytic, myelomonocytic, monocytic and erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphoblastic leukemia, mantle cell lymphoma , primary central nervous system lymphoma, Burkitt's lymphoma and marginal B-cell lymphoma, Osler's disease, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma , chrondrosarcoma, osteosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, cancer prostate , squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, penialoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer , cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasm, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancer (eg, lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer and urinary system cancer. Specific cancers include CD40-expressing tumors selected from the group consisting of chronic lymphoblastic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma, and B-cell marginal cell lymphoma.

The antibodies and conjugates of the present invention can also be used to treat bacterial, fungal, viral and parasitic infections.

When used in therapy, the antibodies of the present invention may be administered to a subject directly (ie, in vivo), either as monotherapy or with other therapies such as an immunostimulatory agent, vaccine, chemotherapy, or radiation therapy. In all cases, antibodies, conjugates, bispecific molecules, compositions, and immunostimulatory agents and other therapies are administered in an effective amount to effect the desired therapeutic effect. The term effective amount refers to that amount which is necessary or sufficient to effect the desired biological effect. For example, an effective amount may be the amount needed to eliminate a tumor, cancer, or bacterial, viral, or fungal infection. The effective amount for any particular use may vary depending on such factors as the disease or condition being treated, the particular antibody administered, the size of the subject, or the severity of the disease or condition. One skilled in the art can empirically determine the effective amount of a particular molecule without undue experimentation.

Preferred routes of administration include, for example, injection (eg, subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal). The injection can be given as a bolus or continuous infusion. Other routes of administration include oral administration.

According to another embodiment of the present invention, an antibody is administered in combination with a vaccine antigen to enhance the immune response against the vaccine antigen, such as a tumor antigen (thereby enhancing the immune response against the tumor) or an antigen from an infectious disease agent (thereby enhancing the immune response against the infectious agent). - 35 042934 Levia). A vaccine antigen can be any antigen or antigenic composition capable of eliciting an immune response against a tumor or against an infectious disease agent such as a virus, bacterium, parasite or fungus. It may also be, for example, a neoantigen, such as those obtained by sequencing patient tumors. The antigen or antigens may be, for example, peptides/proteins, polysaccharides and/or lipids, or may be administered as nucleic acids (such as DNA) encoding peptide or protein antigens that can be expressed under in vivo conditions. An antigen or antigens derived from tumors, such as various tumor antigens, are described above herein. In another embodiment, the antigen or antigens may be derived from pathogens, such as viruses, bacteria, parasites, and/or fungi, such as the various pathogenic antigens described herein above. Additional examples of suitable pathogenic antigens include, but are not limited to, the following:

Viral antigens or antigenic determinants can be obtained, for example, from: cytomegalovirus (especially human cytomegalovirus, eg gB or derivatives thereof); Epstein-Barr virus (eg gp350); flaviviruses (eg, yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus); hepatitis virus such as hepatitis B virus (e.g. hepatitis B surface antigen such as the PreS1, PreS2 and S antigens described in EP-A-414 374; EP-A-0304 578 and EP-A-198474), hepatitis virus A, hepatitis C virus and hepatitis E virus; HIV-1 (such as tat, nef, gp120 or gp160); human herpes viruses such as gD or derivatives thereof or immediate early protein such as ICP27 from HHV 1 or HHV 2; human papillomaviruses (for example, HPV 6, 11, 16, 18); influenza virus (whole live or inactivated virus, split influenza virus grown in eggs or MDCK cells, or Vero cells, or whole virus virosomes (as described by Gluck, Vaccine, 1992, 10, 915-920) or their purified or recombinant proteins , such as NP, NA, HA, or M proteins); measles virus; mumps virus; parainfluenza virus; rabies virus; respiratory syncytial virus (eg, F and G proteins); rotavirus (including live viruses with reduced virulence); smallpox virus; varicella zoster virus (eg gp1, II and IE63); and HPVs responsible for cervical cancer (e.g., early E6 or E7 proteins hybridized with D-carrier protein to form D-E6 or E7 protein fusions from HPV 16, or combinations thereof; or combinations of E6 or E7 with L2 (see e.g. WO 96/26277).

Bacterial antigens or antigenic determinants can be obtained, for example, from Bacillus spp., including B. anthracis (eg, botulinum toxin); Bordetella spp., including B. pertussis (eg pertactin, pertussis toxin, filamentous hemagglutinin, adenylate cyclase, fimbriae); Borrelia spp., including B. burgdorferi (e.g. OspA, OspC, DbpA, DbpB), B. garinii (e.g. OspA, OspC, DbpA, DbpB), B. afzelii (e.g. OspA, OspC, DbpA, DbpB), B. andersonii (eg OspA, OspC, DbpA, DbpB), B. hermsii; Campylobacter spp., including C. jejuni (eg toxins, adhesins and invasins) and C. coli; Chlamydia spp., including C. trachomatis (eg, MOMPs, heparin-binding proteins), C. pneumonie (eg, MOMPs, heparin-binding proteins), C. psittaci; Clostridium spp., including C. tetani (such as tetanus toxin), C. botulinum (such as botulinum toxin), C. difficile (such as clostridial toxins A or B); Corynebacterium spp., including C. diphtheriae (eg, diphtheria toxin); Ehrlichia spp., including E. equi and human granulocytic ehrlichiosis agent; Rickettsia spp., including R. rickettsii; Enterococcus spp., including E. faecalis, E. faecium; Escherichia spp., including enterotoxic E. coli (eg, colonization factors, heat-labile toxin or derivatives thereof, or heat-resistant toxin), enterohemorrhagic E. coli, enteropathogenic E. coli (eg, toxin like whitefish toxin); Haemophilus spp., including H. influenzae type B (e.g., PRP), non-typable H. influenzae, e.g., OMP26, high molecular weight adhesins, P5, P6, protein D, and lipoprotein D, and fimbrin and fimbrin-derived peptides (see example US 5843464); Helicobacter spp., including H. pylori (eg urease, catalase, vacuolating toxin); Pseudomonas spp., including P. aeruginosa; Legionella spp., including L. pneumophila; Leptospira spp., including L. interrogans; Listeria spp., including L. monocytogenes; Moraxella spp., including M. catarrhalis, also known as Branhamella catarrhalis (eg high and low molecular weight adhesins and invasins); Morexella catarrhalis (including their outer membrane vesicles and OMP106 (see eg WO97/41731)); Mycobacterium spp., including M. tuberculosis (eg, ESAT6, antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Neisseria spp., including N. gonorrhea and N. meningitidis (eg, capsular polysaccharides and their conjugates, transferrin-binding proteins, lactoferrin-binding proteins, PilC, adhesins); Neisseria meningitidis B (including their outer membrane vesicles and NspA (see e.g. WO 96/29412), Salmonella spp. including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis, Shigella spp., including S sonnei, S. dysenteriae, S. flexnerii, Staphylococcus spp., including S. aureus, S. epidermidis, Streptococcus spp., including S. pneumonie (eg, capsular polysaccharides and their conjugates, PsaA, PspA, streptolysin, choline-binding proteins) and pneumolysin protein antigen (Biochem Biophys Acta, 1989,67,1007; Rubins et al., Microbial Pathogenesis, 25,337-342) and their mutated detoxified derivatives (see e.g. WO 90/06951, WO 99/03884), Treponema spp., including T. pallidum (eg, outer membrane proteins), T. denticola, T. hyodysenteriae, Vibrio spp., including V. cholera (eg, cholera toxin), and Yersinia spp., including Y. enterocolitica (e.g. Yop protein), Y. pestis, Y. pseudotuberculosis.

Parasitic/fungal antigens or antigenic determinants can be obtained, for example, from: Babesia spp., including B. microti; Candida spp., including C. albicans., Cryptococcus spp., including C. neoformans; Entamoeba spp., including E. histolytica; Giardia spp., including G. lamblia; Leshmania spp., including L. major; Plasmodium falciparum (MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and their analogues in Plasmodium spp.); Pneumocystis spp., including P. carinii; Schisostoma spp., including S. mansoni; Trichomonas spp., including T. vaginalis; Toxoplasma spp., including T. gondii (eg SAG2, SAG3, Tg34); Trypanosoma spp., including T. cruzi.

It should be understood that in accordance with this aspect of the present invention, antigens and antigenic determinants can be used in many different forms. For example, antigens or antigenic determinants may be present as isolated proteins or peptides (for example, in so-called subunit vaccines) or, for example, as cell-associated or virus-associated antigens or antigenic determinants (for example, both in live pathogenic strains and and in dead pathogenic strains). Live pathogens will preferably be attenuated in a known manner. In another embodiment, antigens or antigenic determinants can be obtained from a subject in situ using a polynucleotide encoding an antigen or antigenic determinant (as in so-called DNA vaccination), although it should be understood that polynucleotides that can be used with this approach are not are limited to DNA and may also include RNA and modified polynucleotides as discussed above.

When used in therapy, the molecular conjugates (ie, conjugate vaccines) of the present invention may be administered to a subject directly (ie, in vivo), either alone or with an immunostimulatory agent. In one aspect, the immunostimulatory agent is coupled to a conjugate. Alternatively, the conjugates may be administered to the subject indirectly by bringing the first conjugate (eg, by culture or incubation) into contact with APCs, such as dendritic cells, and then introducing the cells to the subject (ie, ex vivo). Contacting and delivering the conjugates to the APC so that they are processed and presented to the APC prior to administration is also referred to as antigen or cell loading. Techniques for loading antigens into APCs are well known in the art and include, for example, those described in Gunzer and Grabbe, Crit Rev Immunol 21(1-3):133-45 (2001) and Steinman, Exp Hematol 24(8) :859-62 (1996).

In all cases, conjugate vaccines and immunostimulatory agents are administered in an amount effective to achieve the desired therapeutic effect.

Antibodies, molecular conjugates, bispecific molecules and compositions of the present invention may also be co-administered with adjuvants and other therapeutic agents. As used herein, the term co-administered is to be understood to include any or all of the simultaneous, separate, or sequential route of administration of antibodies and conjugates of the present invention with adjuvants and other agents, including administration as part of a dosing regimen. Antibodies are usually prepared alone in a carrier or in combination with such agents. Examples of suitable carriers include solutions, solvents, dispersion media, retarding agents, emulsions, and the like. The use of such media for pharmaceutically active substances is well known in the art. Any other standard carrier suitable for use with these molecules is included within the scope of the present invention.

Agents suitable for co-administration with antibodies, conjugates, bispecific molecules and compositions include other antibodies, cytotoxins and/or drugs, as well as adjuvants, immunostimulatory agents and/or immunosuppressive agents. In one embodiment of the present invention, the agent is a chemotherapeutic agent. Antibodies, bispecific molecules and compositions can be administered in combination with radiation.

Chemotherapeutic agents suitable for co-administration with the antibodies and conjugates of the present invention in the treatment of tumors include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin , dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, as well as their analogues or homologues. Additional agents include, for example, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannite, streptozotocin, mitomycin C, and platinum(II) cis-dichlorodiamine (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agents (eg vincristine and vinblastine) and temozolomide.

Agents that abolish or inhibit immunosuppressive activities, such as immune cells (eg, regulatory T cells, NKT cells, macrophages, myeloid-derived suppressor cells, immature or suppressor dendritic cells) or suppressor factors produced by tumor cells or host cells in the local tumor microenvironment (eg, TGF-β, indolamine-2,3-dioxygenase - IDO), can also be administered with antibodies and conjugates according to the present invention. Such agents include antibodies and small molecule drugs such as IDO inhibitors such as 1-methyltryptophan or derivatives thereof.

Agents suitable for co-administration with antibodies, conjugates and bispecific molecules according to the present invention to induce or enhance an immune response include, for example, adjuvants and/or immunostimulatory agents, non-limiting examples of which have been disclosed above. A preferred immunostimulatory agent is a TLR3 agonist such as poly-IC.

V. Types of combination therapy.

The anti-CD40 antibodies described herein can also be used in combination therapy, for example for the treatment of cancer. Accordingly, the present invention relates to combination therapy methods in which an anti-CD40 antibody is administered together with one or more additional agents, for example, small molecule drugs, antibodies or antigen-binding portions thereof and/or protein ligands, which effectively stimulate immune responses, thereby further enhance, stimulate or activate immune responses in the subject. Moreover, as shown in the examples herein, administration of an anti-CD40 agonist antibody and a soluble CD40 ligand has a synergistic effect in inducing T cell receptor-mediated signals, for example, as evidenced by an increase in CD95 expression in tumor cells.

For example, an anti-CD40 antibody, such as described herein, can be combined with (i) an agonist of a stimulatory (e.g., costimulatory) molecule (e.g., a receptor or ligand) and/or (ii) an antagonist of an inhibitory signal or molecule (e.g., a receptor or ligand) on immune cells, such as T cells, both of which result in enhanced immune responses, such as those of antigen-specific T cells. In some aspects, the immuno-oncological agent is (i) an agonist of a stimulatory (including co-stimulatory) molecule (e.g., a receptor or ligand) or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., a receptor or ligand) on cells involved in innate immunity, e.g. , NK cells, and at the same time, the immunooncological agent enhances innate immunity. Such immuno-oncological agents are often referred to as immune checkpoint regulators, such as an immune checkpoint inhibitor or immune checkpoint stimulant.

In one embodiment of the present invention, an anti-CD40 antibody is administered with an agent that targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin superfamily (IgSF). For example, anti-CD40 antibodies, such as those described herein, can be administered to a subject with an agent that targets a member of the IgSF family to increase the immune response. For example, an anti-CD40 antibody can be administered with an agent that targets (or specifically binds to) a member of the B7 family of membrane-bound ligands, which includes B7-1, B7-2, B7-H1 (PD-L1), B7- DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6, or a co-stimulatory or co-inhibitory receptor that specifically binds to a member of the B7 family.

An anti-CD40 antibody can also be administered with an agent that targets a member of the TNF and TNFR family of molecules (ligands or receptors), such as CD40 and CD40L (e.g., human CD40 and human CD40L), OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTeR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDA1, EDA2, TNFR1, lymphotoxin α/TNFe, TNFR2, TNFa, LTeR, lymphotoxin α 1β2, FAS, FASL, RELT, DR6, TROY and NGFR (see, for example, Tansey (2009) Drug Discovery Today 00:1).

T cell responses can be stimulated with a combination of the anti-CD40 antibodies described herein, e.g., 3C3 and 3G5, and one or more antagonists (an inhibitor or blocking agent) of a protein that inhibits T cell activation (e.g., immune checkpoint inhibitors). ), such as CTLA-4, PD-1, PD-L1, PD-L2 and LAG-3 as described above, and any of the following proteins: TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4, and/or one or more agonists of a protein that stimulates activation T cells such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and can be combined with CD40 agonist antibodies, such as those described herein, for the treatment of cancer include: Yervoy™ (ipilimumab) or Tremelimumab (anti-CTLA4), galiximab (against B7.1), BMS-936558/nivolumab (against PD-1), MK

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3475/pembrolizumab (against PD-1), AMP224 (against B7DC), BMS-936559 (against B7H1), MPDL3280A/atezolizumab (against B7-H1), MEDI-570 (against ICOS), AMG557 ( against B7H2), MGA271 (against B7H3), IMP321 (against LAG-3), BMS-663513 (against CD137), PF-05082566 (against CD137), CDX-1127 (against CD27), anti-OX40 (Providence Health Services) , huMAbOX40L (against OX40L), Atacicept (against TACI), CP-870893 (against CD40), Lucatumumab (against CD40), Dacetuzumab (against CD40), Muromonab (against CD40), Muromonab (against CD3), ipilimumob (Ipilumumab) (against CTLA-4).

Other molecules that can be combined with anti-CD40 agonist antibodies for cancer treatment include inhibitory receptor antagonists on NK cells or activating receptor agonists on NK cells. For example, anti-CD40 agonist antibodies can be combined with KIR antagonists (eg, lirilumab).

T cell activation can also be regulated by soluble cytokines, and anti-CD40 antibodies can be administered to a subject, such as a cancer patient, with cytokine antagonists that inhibit T cell activation or cytokine agonists that stimulate T cell activation.

According to another embodiment of the present invention, anti-CD40 antibodies can be used in combination with (i) antagonists (or inhibitors or blocking agents) of the IgSF family or B7 family or TNF family proteins that inhibit T cell activation, or cytokine antagonists that inhibit activation T cells (eg, IL-6, IL-10, TGF-β, VEGF, immunosuppressive cytokines), and/or (ii) agonists of the IgSF family, B7 family, or TNF family excitatory receptors, or cytokines that stimulate T- cells to stimulate an immune response, for example in the treatment of proliferative diseases such as cancer.

Other agents for various combination therapies include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF1R antagonists such as CSF-IR antagonists, including RG7155 (WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716, WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

Anti-CD40 antibodies can also be administered with agents that inhibit TGF-β signaling.

Additional agents that can be combined with an anti-CD40 antibody include agents that enhance the presentation of tumor antigens, such as dendritic cell-based vaccines, GM-CSF-secreting cell vaccines, CpG oligonucleotides, and imiquimod, or therapies that enhance the immunogenicity of tumor cells. (eg anthracyclines).

Other therapies that can be combined with an anti-CD40 antibody include therapies that deplete or block regulatory T cells, such as an agent that specifically binds to CD25.

Another therapy that can be combined with an anti-CD40 antibody is a therapy that inhibits a metabolic enzyme such as indolamindioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase.

Another class of agents that can be used with an anti-CD40 antibody includes agents that inhibit the formation of adenosine or inhibit the adenosine A2A receptor.

Other therapies that can be combined with an anti-CD40 antibody for the treatment of cancer include therapies that reverse/prevent T cell anergy or depletion and therapies that trigger innate immune activation and/or inflammation at the tumor site.

An anti-CD40 antibody can be combined with more than one immuno-oncological agent and can be used, for example, in combination with a combinatorial approach that targets multiple elements of the immune pathway, such as one or more of the following: a therapy that enhances tumor antigen presentation (e.g., a vaccine based on dendritic cells, cellular vaccines secreting GM-CSF, CpG oligonucleotides, imiquimod); therapy that inhibits immune downregulation, for example, by inhibiting CTLA-4 and/or the PD1/PDL1/PD-L2 pathway and/or depleting or blocking regulatory T cells or other immunosuppressive cells; therapy that stimulates positive immune regulation, for example, using agonists that stimulate the CD-137, OX-40 and/or GITR pathway and/or stimulate T cell effector function; therapy that systemically increases the frequency of antitumor T cells; therapy that depletes or inhibits regulatory T cells, such as regulatory T cells in tumors, eg using a CD25 antagonist (eg, daclizumab) or by depleting CD25 using CD25-specific beads ex vivo; therapy that affects the function of suppressor myeloid cells in the tumor; therapy that enhances the immunogenicity of tumor cells (eg, anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells, such as cells modified with chimeric antigen receptors (CAR-T therapy); therapy that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase; therapy that reverses/prevents anergy or T-cell depletion; therapy that triggers activation of the innate immune response and/or inflammation in the tumor site; the introduction of immunostimulatory cytokines; or blocking immunosuppressive cytokines.

The anti-CD40 agonist antibodies described herein can be used together with one or more agonist agents that ligate positive co-stimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of antitumor T cells, agents that traverse various immune suppressive pathways in the tumor microenvironment (eg, block inhibitory receptor involvement (eg, PD-L1/PD-1 interactions), deplete or inhibit regulatory T cells (eg, using a monoclonal antibody to CD25 (eg, daclizumab) or by depleting CD25 using CD25-specific granules), inhibit metabolic enzymes such as IDO or reverse/prevent anergy or T cell depletion), and agents that trigger activation of the innate immune response and/ or inflammation in tumor foci.

The present invention provides methods for stimulating an immune response in a subject, comprising administering to the subject an anti-CD40 agonist molecule, e.g., an antibody, and one or more additional immunostimulatory antibodies, such as a PD-1 antagonist, e.g. an antagonist antibody, a CTLA-4 antagonist, eg, an antagonist antibody, and/or a LAG3 antagonist, eg, an antagonist antibody, such that an immune response is stimulated in the subject, eg, to inhibit tumor growth or stimulate an antiviral response. In one embodiment of the present invention, the additional immunostimulatory antibody (eg, anti-PD-1 antagonist antibody, anti-PD-L1 antagonist antibody, anti-CTLA-4 antagonist antibody, and/or anti-LAG3 antagonist antibody) is a human antibody.

The present invention also provides methods for treating a hyperproliferative disease (eg, cancer) comprising administering an anti-CD40 agonist antibody and an anti-PD-1 antagonist antibody to a subject. According to one embodiment of the present invention, the subject is a human. According to another embodiment of the present invention, an anti-PD-1 antibody is a monoclonal antibody comprising a human antibody sequence, and an anti-CD40 antibody is a monoclonal antibody comprising a human antibody sequence, such as an antibody containing the CDRs or variable regions from 3C3 and 3G5 described herein, or another CD40 agonist antibody described herein.

Suitable PD-1 antagonists for use in the methods described herein include, but are not limited to, ligands, antibodies (eg, monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment of the present invention, the PD-1 antagonist is a fusion protein, for example an Fc fusion protein such as AMP-244. In one embodiment of the present invention, the PD-1 antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibody that contains the CDRs or variable regions of one of the antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In some embodiments, the anti-PD1 antibody is MK-3475 (lambrolizumab) as described in WO 02012/145493; and AMP-514 described in WO 2012/145493. Other known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO 2011/161699, WO 2012/145493, patents US No. 7635757 and 8217149 and US Patent Publication No. 2009/0317368. You can also use any of the antibodies to PD-1 described in WO 2013/173223. An anti-PD-1 antibody that competes for binding and/or binds to the same epitope on PD-1 as one of these antibodies can also be used in combination treatments. Another approach to target the PD-1 receptor is a recombinant protein consisting of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224.

The present invention provides methods for treating a hyperproliferative disease (eg, cancer) comprising administering an anti-CD40 agonist antibody and a PD-L1 antagonist antibody to a subject. According to one embodiment of the present invention, the subject is a human. According to another embodiment of the present invention, an anti-PD-L1 antibody is a monoclonal antibody comprising a human antibody sequence, and an anti-CD40 antibody is a monoclonal antibody comprising a human antibody sequence, such as an antibody containing the CDRs or variable regions from 3C3 and 3G5 described herein, or another CD40 agonist antibody described herein.

In one embodiment of the present invention, the anti-PD-L1 antibody is BMS-936559 (referred to as 12A4 in WO 2007/005874 and US Pat. No. 7,943,743), or an antibody that contains CDRs or variable regions from 3G10, 12A4, 10A5, 5F8, 10H10 , 1B12, 7H1, 11E6, 12B7, and 13G4, which are described in PCT publication WO 07/005874 and US Pat. No. 7,943,743.

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MPDL3280A (also known as RG7446), MSB0010718C (WO2013/79174) or rHigM12B7. You can also use any of the antibodies to PD-L1 described in WO2013/173223, WO2011/066389,

WO2012/145493, US Pat. Nos. 7,635,757 and 8,217,149 and US Publications 2009/145493. Anti-PD-L1 antibodies that compete and/or bind to the same epitope as any of these antibodies can also be used in combination treatments.

The present invention provides methods for treating a hyperproliferative disease (eg, cancer) comprising administering an anti-CD40 antibody described herein and an anti-CTLA-4 antagonist antibody to a subject. According to one embodiment of the present invention, the subject is a human. In another embodiment, the anti-CTLA-4 antibody is an antibody selected from the group consisting of: ervoy™ (ipilimumab or 10D1 antibody described in PCT publication WO 01/14424), tremelimumab (formerly ticilimumab, CP675206), a monoclonal antibody, or an anti-CTLA-4 antibody as described in any of the following publications: WO 98/42752; WO00/37504; US patent No. 6207156; Hurwitz et al. (1998) Proc. Natl. Acad. sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): abstract 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodies described in WO2013/173223 can also be used.

The present invention also provides methods for treating a hyperproliferative disease (eg, cancer) comprising administering an anti-CD40 antibody and an anti-LAG-3 antibody to a subject. According to one embodiment of the present invention, the subject is a human. According to another embodiment of the present invention, an anti-PD-L1 antibody is a monoclonal antibody comprising a human antibody sequence, and an anti-CD40 antibody is a monoclonal antibody comprising a human antibody sequence, such as an antibody containing CDRs or variable regions from 3C3 or 3G5 as described herein, or another CD40 agonist antibody described herein. Examples of anti-LAG3 antibodies include antibodies containing the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5, as described in US Patent Publication US2011/0150892, WO10/19570 and WO2014/008218. In one embodiment of the present invention, the anti-LAG-3 antibody is BMS986016. Other anti-LAG-3 antibodies well known in the art that can be used include IMP731 and IMP-321 described in US 2011/007023, WO08/132601 and WO09/44273. Anti-LAG-3 antibodies that compete with and/or bind to the same epitope as any of these antibodies can also be used in combination treatments.

Administration of anti-CD40 antibodies described herein and antagonists, e.g. antagonist antibodies, to one or more target antigens such as LAG-3 and/or CTLA-4 and/or PD-1 and/or PD-L1 , may enhance the patient's immune response to cancer cells. Cancers whose growth can be inhibited using the antibodies of the present invention include cancers that typically respond to immunotherapy and those that typically do not respond to immunotherapy. Typical examples of cancers for treatment using combination therapy according to the present invention include those cancers listed herein.

According to some embodiments of the present invention, the combination of therapeutic antibodies discussed herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions in which each antibody is in a pharmaceutically acceptable carrier. According to another embodiment of the present invention, the combination of therapeutic antibodies may be administered sequentially. For example, anti-CTLA-4 antibody and anti-CD40 antibody can be administered sequentially, for example, anti-CTLA-4 antibody is administered first and then anti-CD40 antibody is administered, or anti-CD40 antibody is administered first and then anti-CTLA-4 antibody is administered. Additionally or alternatively, the anti-PD-1 antibody and the anti-CD40 antibody can be administered sequentially, for example, the anti-PD-1 antibody is administered first and then the anti-CD40 antibody is administered, or the anti-CD40 antibody is administered first and then the anti-PD-1 antibody is administered. Additionally or alternatively, the anti-PD-L1 antibody and the anti-CD40 antibody can be administered sequentially, for example, the anti-PD-L1 antibody is administered first and then the anti-CD40 antibody is administered, or the anti-CD40 antibody is administered first and then the anti-PD-L1 antibody is administered. Additionally or alternatively, the anti-LAG-3 antibody and the anti-CD40 antibody can be administered sequentially, for example, the anti-LAG-3 antibody is administered first and then the anti-CD40 antibody is administered, or the anti-CD40 antibody is administered first and then the anti-LAG-3 antibody is administered.

In addition, if more than one dose of combination therapy is administered sequentially, then the order of sequential administration may be reversed or kept unchanged at each administration time point, sequential administrations may be combined with simultaneous administrations, or any combination thereof. For example, the first administration of the combination of anti-CTLA-4 antibody and anti-CD40 antibody may be simultaneous, the second administration may be sequential, with the anti-CTLA-4 antibody first and then the anti-CD40 antibody, and the third administration may be sequential, with first an anti-CD40 antibody is administered and then an anti-CTLA-4 antibody is administered, and so on. Additionally or alternatively, the first administration of the combination of anti-PD-1 antibody and anti-CD40 antibody may be simultaneous, the second administration may be sequential, with the anti-PD-1 antibody first and then the antibody

- 41 042934 to CD40, and the third administration may be sequential, with the anti-CD40 antibody being administered first and then the anti-PD-1 antibody being administered, and so on. Additionally or alternatively, the first administration of the combination of anti-PD-L1 antibody and anti-CD40 antibody may be simultaneous, the second administration may be sequential, with the anti-PD-L1 antibody first and then the anti-CD40 antibody, and the third administration may be sequential, in this case, the anti-CD40 antibody is administered first, then the anti-PD-L1 antibody is administered, and so on. Additionally or alternatively, the first administration of the combination of the anti-LAG-3 antibody and the anti-CD40 antibody may be simultaneous, the second administration may be sequential, wherein the anti-LAG-3 antibody is administered first and then the anti-CD40 antibody is administered, and the third administration may be sequential, wherein the anti-CD40 antibody is administered first, and then the anti-LAG-3 antibody is administered, and so on. Another exemplary dosing regimen may include a first administration that is sequential, with anti-CD40 antibody administered first and then anti-CTLA-4 antibody (and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-PD-L1 antibody to LAG-3), and subsequent administrations may be simultaneous.

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulating the immune system, such as cancer or an infectious disease, is treated by administering to said subject an anti-CD40 antibody and an immuno-oncological agent, wherein the immuno-oncological agent is a CD137 agonist ( 4-1BB), such as the CD137 agonist antibody. Suitable anti-CD137 antibodies include, for example, urelumab or PF-05082566 (WO12/32433).

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulating the immune system, such as cancer or an infectious disease, is treated by administering to said subject an anti-CD40 antibody and an immuno-oncological agent, wherein the immuno-oncological agent is an OX40 agonist, such as an anti-OX40 agonist antibody. Suitable antibodies to OX40 include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; WO06/029879).

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulating the immune system, such as cancer or an infectious disease, is treated by administering to said subject an anti-CD40 antibody and an immuno-oncological agent, wherein the immuno-oncological agent is a second CD40 agonist. , such as another anti-CD40 agonist antibody.

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulating the immune system, such as cancer or an infectious disease, is treated by administering to said subject an anti-CD40 antibody and an immuno-oncological agent, wherein the immuno-oncological agent is a CD27 agonist, such as an anti-CD27 agonist antibody. Suitable anti-CD27 antibodies include, for example, varlilumab (CDX-1127).

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulating the immune system, such as cancer or an infectious disease, is treated by administering to said subject an anti-CD40 antibody and an immuno-oncological agent, wherein the immuno-oncological agent is MGA271 (against B7H3) (WO11/109400).

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulating the immune system, such as cancer or an infectious disease, is treated by administering to said subject an anti-CD40 antibody and an immuno-oncological agent, wherein the immuno-oncological agent is a KIR antagonist, such as lirilumab.

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to said subject an anti-CD40 antibody and an immuno-oncological agent, wherein the immuno-oncological agent is an IDO antagonist. Suitable IDO antagonists include e.g. 2237) or F001287.

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to said subject an anti-CD40 antibody and an immuno-oncological agent, wherein the immuno-oncological agent is a Toll- a similar receptor, eg a TLR2/4 agonist (eg, Bacillus Calmette-Guérin); a TLR7 agonist (eg hiltonol or imiquimod); a TLR7/8 agonist (eg, resiquimod); or a TLR9 agonist (eg CpG7909).

According to one embodiment of the present invention, a subject having a disease that can be beneficially affected by stimulating the immune system, such as cancer or an infectious disease, is treated by administering an anti-CD40 antibody to said subject.

- 42 042934 and an immuno-oncological agent, wherein the immuno-oncological agent is an inhibitor

TGF-β e.g. GC1008, LY2157299, TEW7197 or IMC-TR1.

In one aspect, the anti-CD40 antibody is administered sequentially prior to the administration of the second agent, eg, an immuno-oncological agent. In one aspect, the anti-CD40 antibody is administered concurrently with a second agent, such as an immuno-oncological agent. In another aspect, the anti-CD40 antibody is administered sequentially after administration of the second agent. The administration of the two agents can be started at time points that are separated by a time interval of, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks, or administration of the second agent can be started after, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours , 48 hours, 3 days, 5 days, 7 days, or one or more weeks after administration of the first agent.

In some aspects, the anti-CD40 antibody and the second agent, eg, an immuno-oncological agent, are administered simultaneously, eg, administered to the patient simultaneously over a period of, eg, 30 or 60 minutes. In another embodiment, the anti-CD40 antibody may be co-produced with a second agent, such as an immuno-oncological agent.

Optionally, an anti-CD40 antibody is administered as the sole immunotherapeutic agent, or in combination with an anti-CD40 antibody, and one or more additional immunotherapeutic antibodies (e.g., an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD- L1 and/or anti-LAG-3 antibody) can be further combined with an immunogenic agent such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells and cells transfected with genes encoding immunostimulatory cytokines (He et al (2004) J Immunol 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides from melanoma antigens such as gp100 peptides, MAGE, Trp-2, MART1 and/or tyrosinase antigens, or tumor cells transfected to express the cytokine GMCSF (discussed in more detail below). An anti-CD40 antibody and one or more additional antibodies (e.g. anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3) can also be further combined. with standard cancer treatments. For example, an anti-CD40 antibody and one or more additional antibodies (e.g., anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies and/or anti-LAG-3 antibodies) can be effectively combined with chemotherapy regimens. In these cases, the dose of the other chemotherapeutic agent administered with the combination of the present invention can be reduced (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is the combination of an anti-CD40 agonist antibody (with or without an additional antibody such as anti-CTLA-4 and/or anti-PD-1 and/or anti-PDL1 and/or anti-LAG-3) in combination with decarbazine for the treatment of melanoma. Another example is the combination of an anti-CD40 antibody (with or without anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies) in combination with interleukin-2. (IL-2) for the treatment of melanoma. The scientific rationale for the combined use of an anti-CD40 antibody and an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody and/or an anti-LAG-3 antibody with chemotherapy is that the cell death that is a consequence of the cytotoxic effect of most chemotherapeutic compounds, should lead to an increase in the levels of tumor antigen in the antigen presentation pathway. Other combination therapies that may provide synergy with anti-CD40 antibody (with or without anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3) ), include radiation therapy, surgery, or hormonal depletion. Each of these protocols creates a source of tumor antigen in the host organism. Angiogenesis inhibitors can also be used in combination with a combination of an anti-CD40 antibody and an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PDL1 antibody and/or an anti-LAG-3 antibody. Inhibition of angiogenesis results in tumor cell death, which may be the source of the tumor antigen introduced in the antigen presentation pathway in the host.

An anti-CD40 agonist antibody as the sole immunotherapeutic agent or a combination of CD40 agonists and blocking antibodies against CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 can also be used in combination with bispecific antibodies that target effector cells expressing the Fca or Fcy receptor to tumor cells (see, for example, US Pat. Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. The T-cell mediated steps of these reactions will be enhanced by the combination of an anti-CD40 antibody and an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody and/or an anti-LAG-3 antibody. .

In another example, an anti-CD40 agonist antibody as the sole immunotherapeutic agent, or a combination of an anti-CD40 antibody and an additional immunostimulatory agent, e.g., an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody, and/or an anti-LAG-3 agent, such as an antibody, can be used in conjunction with an anti-neoplastic antibody such as

- 43 042934 rituxan (Rituxan®) (rituximab), herceptin (Herceptin®) (trastuzumab), bexar (Bexxar®) (tositumomab), zevalin (Zevalin®) (ibritumomab), campath (Campath®) (alemtuzumab), lymphocide ( Lymphocide®) (eg, eprtuzumab), Avastin® (bevacizumab) and Tarceva® (erlotinib), etc. By way of example and without wishing to be bound by theory, the present inventors believe that treatment with an anti-cancer antibody or a toxin-conjugated anti-cancer antibody can result in the death of cancer cells (e.g., tumor cells), which can enhance the immune response mediated by an immunostimulatory agent, eg an anti-CD40, CTLA-4, PD-1, PD-L1 or LAG-3 agent, eg an antibody. In an exemplary embodiment of the present invention, treatment of a hyperproliferative disease (e.g., cancer) may include an anti-cancer agent, e.g., an antibody, in combination with an anti-CD40 antibody and optionally an additional immunostimulatory agent, e.g., an anti-CTLA-4 agent and/or an anti-PD1 agent, and/or an anti-PD-L1 agent and/or an anti-LAG-3 agent, such as an antibody, which can be administered simultaneously or sequentially, or by any combination of these types of administration, which can enhance anti-tumor immune responses in the host.

Tumors evade the host's immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins that are expressed by tumors and that are immunosuppressive. Such proteins include, in particular, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200 ), and Fas ligand (Hahne et al. (1996) Science 274:1363-1365). Antibodies to each of these molecules can be further combined with an anti-CD40 antibody with or without an additional immunostimulatory agent, e.g. an anti-CTLA-4 agent and/or an anti-PD-1 agent and/or an anti-PD-L1 agent and/or against LAG-3, such as an antibody, to counteract the effects of immunosuppressive agents and promote the development of an antitumor immune response in the host.

Other agents, such as antibodies, which can be used to activate the responsiveness of the host's immune system, can also be used in combination with an anti-CD40 antibody with or without an additional immunostimulatory agent, such as an anti-CTLA-4 antibody and/or an anti-PD-1 antibody, and/or an anti-PD-L1 antibody and/or an anti-LAG-3 antibody. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. An anti-CD40 antibody (Ridge et al., supra) can be used in combination with an anti-CD40 antibody and optionally an additional immunostimulatory agent, e.g. an anti-CTLA-4 agent and/or an anti-PD-1 agent and/or an anti-PD-L1 agent, and/or an anti-LAG-3 agent, eg an antibody. Other activating antibodies to costimulatory T cell molecules (Weinberg et al., supra, Melero et al., supra, Hutloff et al., supra) can also provide increased levels of T cell activation.

As discussed above, bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. Immunotherapy with anti-CD40 antibodies alone or in combination with anti-CTLA-4 and/or anti-PD-1 and/or anti-PDL1 and/or anti-LAG-3 can be used to increase the efficacy of transplanted donor tumor-specific T cells. .

Several experimental treatment protocols include ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to recipients in order to provide antigen-specific T cells directed against the tumor (Greenberg & Riddell, supra). These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation is expected in the presence of an anti-CD40 antibody with or without additional immunostimulatory therapy, such as anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti- LAG-3 will increase the frequency and activity of adoptively transferred T cells.

The present invention relates to methods for modifying an adverse event associated with the treatment of a hyperproliferative disease (eg, cancer) using an immunostimulatory agent, comprising administering an anti-CD40 antibody to a subject in combination with an anti-CTLA-4 agent and/or an anti-PD-1 agent, and/ or an anti-PD-L1 agent and/or an anti-LAG-3 agent, eg an antibody, or without these agents. For example, the methods described herein provide a method for reducing the incidence of colitis or diarrhea induced by an immunostimulatory therapeutic antibody by administering a nonabsorbable steroid to a patient. As used herein, the term non-absorbable steroid is a glucocorticoid that is characterized by extensive first pass metabolism such that after metabolic conversion in the liver, the bioavailability of the steroid is low, i.e. less than about 20%. According to one embodiment of the present invention described herein, the non-absorbable steroid is budesonide. Budesonide is a topical glucocorticosteroid that is extensively metabolized, primarily by the liver, following oral administration. Entocort EC (ENTOCORT EC®) (Astra-Zeneca) is an oral formulation with a release that controls

- 44 042934 is a pH and time dependent medium designed to optimize drug delivery to the ileum and throughout the colon. Entocort EC® is approved in the US for the treatment of mild to moderate Crohn's disease involving the ileum and/or ascending colon. The usual oral dose of Entocort EC® for the treatment of Crohn's disease is 6 to 9 mg/day. Entocort EC® is released in the intestine until absorbed and retained in the intestinal mucosa. After passing through the intestinal mucosa, which is the target tissue, Entocort EC® is extensively metabolized in the liver by the cytochrome P450 system, while its metabolites have negligible glucocorticoid activity. Therefore, its bioavailability is low (about 10%). The low bioavailability of budesonide results in an improved therapeutic ratio compared to other glucocorticoids with less first pass metabolism. Budesonide produces fewer adverse events, including less suppression of the hypothalamic-pituitary axis, than systemic corticosteroids. However, chronic administration of Entocort EC® may result in systemic glucocorticoid effects such as hypercorticism and adrenal suppression. See PDR ^58th ed. 2004; 608-610.

In more detailed embodiments of the present invention, an anti-CD40 antibody with or without immunostimulatory therapeutic anti-CD40 antibodies and optionally anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti- LAG-3, in combination with a non-absorbable steroid, can be further combined with salicylate. Salicylates include 5-ASA agents such as, for example, sulfasalazine (azulfidine (AZULFIDINE®), Pharmacia &UpJohn); olsalazine (DIPENTUM®, Pharmacia &UpJohn); balsalazid (colazal (COLAZAL®), Salix Pharmaceuticals, Inc.); and mesalamine (Azacol (ASACOL®), Procter & Gamble Pharmaceuticals, Pentasa (PENTASA®), Shire US, Canasa (CANASA®), Axcan Scandipharm, Inc, Rovasa (ROWASA®), Solvay).

According to the methods described herein, salicylate is administered in combination with an anti-CD40 antibody, together with an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody and/or an anti-LAG- 3 or not, and a non-absorbable steroid to reduce the incidence of immunostimulatory antibody-induced colitis. Accordingly, for example, methods for reducing the incidence of colitis induced by immunostimulatory antibodies described herein include administering salicylate and a non-absorbable steroid simultaneously or sequentially (e.g., salicylate is administered 6 hours after administration of a non-absorbable steroid) or any combination thereof. In addition, the salicylate and the nonabsorbable steroid may be administered via the same route of administration (for example, both are administered orally) or via different routes (for example, the salicylate is administered orally and the nonabsorbable steroid is administered rectally), which may differ from the route(s) used to administer an anti-CD40 antibody and an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody and/or an anti-LAG-3 antibody.

The anti-CD40 antibodies and antibody-based combination therapies described herein can also be used in combination with other well-known therapies that are selected based on their particular utility for the indication being treated (eg, cancer). The anti-CD40 antibody combinations described herein can be used sequentially with known pharmaceutically acceptable agent(s).

For example, the anti-CD40 antibodies and antibody-based combination therapy methods described herein can be used in combination (e.g., simultaneously or separately) with an additional treatment such as radiation, chemotherapy (e.g., using camptothecin (CPT-11 ), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (taxol), doxorubicin, 5fluorouracil, or camptothecin+apo21/TRAIL (6X combo)), one or more proteasome inhibitors (eg, bortezomib or MG132), one or more Bcl-2 inhibitors (eg, BH3I-2 (bcl-xl inhibitor), indolamindioxygenase-1 inhibitor (eg, INCB24360, indoxymod, NLG-919, or F001287) AT-101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax) or MCL-1 antagonists (myeloid leukemia cell differentiation protein-1)), iAP antagonists (apoptosis protein inhibitor) (e.g. smac7, smac4, small molecule smac mimetics, synthetic smac peptides (see Fulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC inhibitors, anti-CD20 antibodies (eg, rituximab), angiogenesis inhibitors (eg, bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (eg, Avastin), synthetic triterpenoids (see Hyer et al., Cancer Research 2005; 65:4799-808), c-FLIP modulators (eg, natural and synthetic PPARy/ (peroxisome proliferator-activated receptor γ), 5809354 or 5569100), kinase inhibitors (eg, sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus, bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors , lenalildomide, GSK3P inhibitors, IAP inhibitors, and/or genotoxic drugs.

The anti-CD40 antibodies and antibody combination therapy methods described herein can also be used in combination with one or more antiproliferative cytotoxic agents. Classes of compounds that can be used as antiproliferative cytotoxic agents include, but are not limited to, the following:

Alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas and triazenes): uramustine, chlormethine, cyclophosphamide (Cytoxan™), phosphamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoamine, busulfan, carmustine, lomustine , streptozocin, dacarbazine and temozolomide.

Antimetabolites (including, but not limited to, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

Suitable antiproliferative agents for combination with anti-CD40 agonist antibodies include, but are not limited to, taxanes, paclitaxel (paclitaxel is commercially available as Taxol™), docetaxel, discodermolide (DDM), dictiostatin (DCT), peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, deoxyepothilone B1, [17]-dehydrodeoxyepothilone B, [18]-dehydrodeoxyepothilone B, C12,13 cyclopropylepothilone A, C6-C8 bridged epothilone A, trans-9, 10-dehydroepothilone D, cis-9,10dehydroepothilone D, 16-desmethylepothilone B, epothilone B10, discoderomolid, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (discodermolide), TZT-1027 (Soblidotin), ILX-651 (Tazidotin Hydrochloride), Halichondrin B, Eribulin Mesylate (E-7389), Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoid Immunoconjugates (DM-1) , MKC-1, ABT-751, T1-38067, T900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin, 17-beta-acetoxy-2ethoxy-6-oxo-B- homo-estra-1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7dehydroxy-14,16-didemethyl-(+)-discodermolides and cryptotilone 1, in addition to others microtubule stabilizing agents known in the art.

In cases where it is necessary to put aberrantly proliferative cells into a resting state in combination with treatment with anti-CD40 antibodies described herein, or before the start of such treatment, hormones and steroids (including synthetic analogues), such as 17a-ethinylestradiol, diethylstilbestrol, testosterone , prenison, fluoxometeron, proponant of dromostanolone, testolactone, megestrolacetate, methyl -prescript, methyltestosterone, prednisolone, triamcinolon, chlortrianizen, hydroxyprogesterone, aminoglutetimide, erastemustine, medoxyprogesteronacoronacreonacia, and lepril, flutamide, flutamide, flutamide, laplitamide, and leprosy Toremifen, Zodex ™, can also be introduced to the patient. When using the methods or compositions described herein, other agents used to modulate tumor growth or metastasis in the clinical setting, such as antimimetics, may also be administered as needed.

Those skilled in the art are aware of methods for safely and effectively administering chemotherapeutic agents. In addition, their administration is described in the standard literature. For example, the administration of many chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., 1996 ed. (Medical Economics Company, Montvale, N.J. 07645-1742, USA); the contents of which are incorporated herein by reference.

The chemotherapeutic agent(s) and/or radiation therapy may be administered according to therapeutic protocols well known in the art. Those skilled in the art will appreciate that the administration of the chemotherapeutic agent(s) and/or radiation therapy may be varied depending on the disease being treated and the known effects of the chemotherapeutic agent(s) and/or radiation therapy on that disease. In addition, according to the skill of the skilled physician, therapeutic protocols (eg, number of doses and time of administration) may vary depending on the observed effect of the administered therapeutic agents on the patient and taking into account the observed responses of the disease to the administered therapeutic agents.

VI. Results.

As shown in the examples herein, coadministration of an anti-CD40 antibody with one or more additional therapeutic agents (e.g., soluble CD40 ligand or other antibody such as anti-PD-1, anti-PD-L1, anti-CTLA-4 and/or an anti-LAG-3 antibody) provides improved efficacy over treatment with the antibody alone or with one or more additional therapeutic agents in the absence of antibody therapy. Preferably, the combination of the anti-CD40 antibody with one or more additional therapeutic agents exhibits a therapeutic synergistic effect.

Therapeutic synergy refers to the phenomenon whereby treatment of patients with a combination of therapeutic agents exhibits a therapeutically more favorable outcome than that achieved with each individual component of the combination at the optimum dose (T. H. Corbett et al., 1982 , Cancer Treatment Reports, 66, 1187). In this case, a therapeutically more favorable outcome is one in which patients either a) have a lower incidence of adverse events, while receiving a therapeutic benefit that is similar to or exceeds the benefit in patients to whom the individual components of the combination are administered as monotherapy at the same dose, in which they are part of the combination, or b) have no dose-limiting toxicity, obtaining a therapeutic benefit that is similar to or greater than that in patients who receive each individual component of the combination, and each individual component is administered at the same doses in which they are included in the composition of the combination (combinations), when they are introduced in the form of individual components. In xenograft models, a combination used at the maximum tolerated dose, in which each of the individual components will be present at a dose that usually does not exceed the individual maximum tolerated dose for that component, exhibits a therapeutic synergistic effect when, for example, a reduction in tumor growth is achieved when the combination is administered , which is greater than the reduction in tumor growth caused by the most effective component, when this component is administered as monotherapy.

Therefore, in combination, the components of such combinations have an additive or super-additive effect in suppressing tumor growth, compared to anti-CD40 antibody monotherapy or treatment with additional therapeutic agent(s) in the absence of antibody therapy. The term additive is understood to mean a result that exceeds in degree (for example, in the degree of reduction in the mitotic index of the tumor or tumor growth or degree of tumor shrinkage or the frequency and/or duration of periods without symptoms or with fewer symptoms) the best individual result achieved with each application. a single component as monotherapy, while the term superadditive is used to indicate an outcome that is greater than the sum of such individual outcomes. According to one implementation variant of the present invention, the additive effect is measured as slowing down or stopping the growth of the tumor. An additive effect can also be measured, for example, as a reduction in tumor size, a decrease in tumor mitotic index, a decrease in the number of metastatic lesions over time, an increase in overall response rate, or an increase in median survival or overall survival. According to another embodiment of the present invention, the additive effect is measured as an increase in the induction of CD95 expression when incubated with Ramos cells, an increase in the proliferation of B cells incubated with human B cells, and/or an increase in increased induction of IL-12p40 expression when incubated with dendritic cells.

One non-limiting example of a measure by which the effectiveness of a therapeutic treatment can be quantified is the calculation of log10 cell death, which is determined according to the following equation loglO cell death = TC (days)/3.32xTc1 where TC is the delay in cell proliferation, expressed as the average time in days for tumors in the treated group (T) and tumors in the control group (C) to reach a predetermined value (1 g, or 10 ml, for example), and Td is the time period in days required to double the tumor volume in control animals. Using this measure, a product is considered active if the log10 of cell death is greater than or equal to 0.7, and a product is considered very active if the log10 of cell death is greater than 2.8. In applying this measure, the combination used at its maximum tolerated dose, in which each of the components is present at a dose that is usually less than or equal to its maximum tolerated dose, exhibits a therapeutic synergistic effect if the log10 cell death value exceeds the log10 cell death value for the most effective a separate component when administered as monotherapy. In an exemplary case, the log10 cell death of the combination exceeds the log10 cell death of the most effective single component by at least 0.1 log cell death, at least 0.5 log cell death, or at least 1.0 log cell death.

The present invention is further illustrated by the following examples, which should not be construed as a further limitation. The contents of the Sequence Listing, Figures, and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

Examples.

Example 1

Obtaining CD40-specific human monoclonal antibodies.

Human CD40-specific monoclonal antibodies were obtained by immunizing H2L2 Harbor® transgenic mice with soluble human CD40 antigen. Harbor® transgenic mice had deletion of the heavy chain (HC) and kappa light chain (κ chain) DNA sequences and had human variable (V) and rat constant (C) region sequences stably inserted into their genome.

- 47 042934

Antigen and immunization: The antigen was a soluble CD40 extracellular domain fusion protein fused to the Fc domain of an antibody (R&D Systems) or a recombinant human-msG2a CD40 chimeric protein (in-house). The antigen was mixed with complete Freund's adjuvant (Sigma) for the first immunization. The antigen was then mixed with incomplete Freund's adjuvant (Sigma). Additional mice were immunized with soluble CD40 protein in MPL with a TDM adjuvant system (Sigma). 5-25 µg of soluble recombinant CD40 antigen in PBS or 5x10 ⁶ NSO cells transfected for surface expression of human CD40 in PBS were mixed with adjuvant in a ratio of 1:1. Mice were injected with 200 μl of prepared antigen by injection into the peritoneal cavity every 14 days. Animals that developed anti-CD40 antibodies received 5-10 micrograms of soluble recombinant CD40 antigen by intravenous injection three to four days prior to fusion. Mouse spleens were harvested and the isolated splenocytes were used to prepare a hybridoma.

Hybridoma Preparation: The murine myeloma cell line P3x63Ag8.653 (ATCC CRL 1580) was used for fusions. Myeloma cells were cultured using RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS). Additional media auxiliaries were added to hybridoma growth media which contained: up to 10% hybridoma growth promoter (Sigma), 10% PBS (Sigma), L-glutamine (Gibco) 0.1% gentamicin (Gibco), 2-mercaptoethanol (Gibco) with HAT (Sigma, 1.0x10 ⁴ M hypoxanthine, 4.0x10' ⁷ M aminopterin, 1.6x10'5 M thymidine media).

Spleen cells were mixed with P3x63Ag8.653 myeloma cells in a 6:1 ratio and pelleted by centrifugation. The polyethylene glycol was added dropwise and mixed gently to facilitate fusion. Hybridomas were grown for one to two weeks until visible colonies appeared. The supernatant was collected and used for initial screening for rat IgG by ELISA using soluble human CD40 fusion protein and specific rat Fc detection. The IgG positive supernatants were then quantified for CD40 specificity by flow cytometry. Hybridomas were also screened for cross-reactivity with cynomolgus monkey CD40, with all hybridomas binding positive.

Hybridoma cells were expanded and the cell mass was frozen for RNA isolation and sequencing. The V _H and VL coding regions of human monoclonal antibodies were identified using RNA from the respective hybridomas. The RNA was reverse transcribed to obtain cDNA, the V-coding regions were amplified by NRP, and the NRP product was sequenced, inserted into a vector expressing human IgG2, transiently expressed, and purified by protein A carrier column chromatography, which allowed the isolation of a number of antibodies representing of special interest, which were designated as 3C3, 3G5, 1B4, 3B6, 6H6, 6H6, 2E1.2, 1B5NK (in the latter case after modification of N75K to heavy chain FR3) and 3B6-NS (after N63S modification to light chain FR3 of antibody 3B6 to remove the N-glycosylation site).

In table. 1, 2 and 3 summarize the information on the germline and amino acid sequences of the human VH and V _L MAT regions (in the case of amino acid sequences, hypervariable regions (CDRs) have been highlighted). The corresponding nucleic acid sequences are shown in the sequence table entitled Summary of the Sequence Listing at the end of these examples.

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Table 1. Germline data

MAT VH/VL germ line V D J 3G5 H IGHV3-33*O1 F (VH3-33) IGHD3-10*01 F (D310) IGHJ4*02 F (JH4b) L IGKV3-15*O1 F (L2) IGKJ5*01 F (JK5) ZSZ H IGHV3-33*O1 F (VH333) IGHD3-10*02 F (D4b) IGHJ4*02 F (JH4b) L IGKV1-27*01 F (A20) IGKJ3*01 F (JK3) SW6 H IGHV3-23*01 F (VH323) IGHD2-15*01 F (D215) IGHJ6*02 F (JH6b) L IGKV2-28*01 F (A19) IGKJ1*O1F(JK1) 6H6 H IGHV3-33*01 F (VH333) IGHD3-10*01 F (D310) IGHJ4*02 F (JH4b) L IGKV3-15*01 F (L2) IGKJ4*01 F (JK4) 1B4 H IGHV3-23*01 F (VH3- 23) IGHDl-26*01 F (D215) IGHJ6*02 F (JH6b) L IGKV2-28*01 F (A19) IGKJ1*O1 F (JK1) 1B5-NK H IGHV3-33*03 F (VH3- 33) IGHD6-19*01 F (D215) IGHJ2*01 F (JH2) L IGKV1-27*01 F (A20) IGKJ2*01 F (JK2) 2E1.2 H IGHV3-33*01 F (VH333) IGHD3-10*01 F (D310) IGHJ4*02 F (JH4B) L2 IGKV3-15*01 F (L2) IGKJ4*01 F (JK4) 3B6-NS H IGHV3-23*01 F (VH3- 23) IGHD2-15*01 F (D215) IGHJ6*02 F (JH6b) L2 IGKV2-28*01 F (A19) IGKJ1*O1 F (JK1)

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Table 2. CDR sequences

MAT VH/VL CDR according to Kabat (Chothia) CDR1 SEQID NO CDR2 SEQID NO CDR3 SEQID NO 3G5 H SNGIH (GFTFSSN) 5, 6 VIWSDGSNKFYAD SVKG (WSDGSN) 7, 8 ASGSGSYYNFF DY (ASGSGSYYNFF DY) 9, 10 L RASQSVRSN LA (RASQSVRSN LA) H.12 GASTRAT (GASTRAT) 13, 14 QQHNKWIT (QQHNKWIT) 15, 16 ZSZ H RYGMY (GFIFSRY) 19, 20 VIWYDGSYKYYA DSVKG (WYDGSY) 21, 22 ESPWYYFDY (ESPWYYFDY) 23.24 L RASQGISNYL A (RASQGISNY LA) 25, 26 AASTLQS (AASTLQS) 27, 28 QKYKSAPFT (QKYKSAPFT) 29, 30 SW6 H SYAMS (GFTFSSY) 33, 34 GITGTGGSTYYAD SVKG (TGTGGS) 35, 36 RAGGSFYYYYG MDV (RAGGSFYYYY GMDV) 37.38 L RSSQSLLHST GYNYLD (RSSQSLLHS TGYNYLD) 39, 40 LGSNRAS (LGSNRAS) 41, 42 MQALQTPWT (MQALQTPWT) 43 ,44 6H6 H SYGMH (GFTLSSY) 47, 48 VIWDDDGSNKYYA DSVKG (WDDGSN) 49, 50 AGGSGRYYNYF DY (AGGSGRYYNY FDY) 51.52 L RASQSVRSN LA (RASQSVRSN LA) 53, 54 GASTRAT (GASTRAT) 55, 56 QQHNNWLT (QQHNNWLT) 57.58 1B4 H SYAMT (GFTFSSY) 61, 62 GITGSGANTFYTD SVKG (TGSGAN) 63, 64 RNGGSYYYYYG MDV (RNGGSYYYYY GMDV) 65, 66 L RSSQSLLHSS GYNYLD (RSSQSLLHS SGYNYLD) 67, 68 LGSNRAS (LGSNRAS) 69, 70 MQALQIPWT (MQALQIPWT) 71.72 1B5NK H SFGMH (GFTFSSF) 103, 104 LIWFDGSSKYYA DSVKG (WFDGSS) 105, GFAAVAGWYF 106 DF (GFAAVAGWY FDF) 107, 108 L RASQGVRK YLA (RASQGVR KYLA) 109, 110 AASTLQS (AASTLQS) 111 112 QKYFSAPYT (QKYFSAPYT) out, 114 2E1.2 H SYGMH (GFTFSSY) 89, 90 VIWDDDGSNKYY ADSVKG (WDDGSN) 91, AGSSGRYYNY 92 FDY (AGSSGRYYNY FDY) 93, 94 L RASQSVRS NLA (RASQSVRS NLA) 95, 96 GASTRAT (GASTRAT) 97, 98 QQYNKWLI (QQYNKWLI) 99, 100 ZV6- NS H SYAMS (GFTFSSY) 75, 76 GITGTGGSTYYAD SVKG (TGTGGS) 77, RAGGSFYYYYG 78 MDV (RAGGSFYYYY GMDV) 79, 80 L RSSQSLLHST GYNYLD (RSSQSLLHS TGYNYLD) 81, 82 LGSNRAS (LGSNRAS) 83, 84 MQALQTPWT (MQALQTPWT) 85, 86

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Table 3. Full-length variable region sequences

MAT VH/VL SEQ ID NO Subsequence 3G5 H 3 OVOLVESGGGVVOPGKSLRLSCAASGFTFSSNGIHWVRQAPGKGL EWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCARASGSGSYYNFFDYWGQGTLVTVSS L 4 EIVMTOSPATLSVSPGERATLSCRASOSVRSNLAWYOQKPGOAPR LLIYGASTRATGIPARFSGSGSGTEFTLTINSLOSEDFAVYYCOOH NKWITFGQGTRLEIK ZSZ H 17 OVOLVESGGGVVQPGRSLRLSCAGSGFIFSRYGMYWVROAPGKG LEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARESPWYYFDYWGQGTLVTVSS L 18 DIOMTOSPSSLSASVGDRVTITCRASOGISNYLAWYOQKPGKVPK LLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLOPEDVATYYCOKY KSAPTFGPGTKVDIK SW6 H 31 EVOLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVROAPGKGL EWVSGITGTGGSTYYADSVKGRFTISRDNSKNTLYVQMNSLRAE DTAVYYCAKRAGGSFYYYYGMDVWGQGTTVTVSS L 32 DIVMTOSPLSLPVTPGEPASISCRSSOSLLHSTGYNYLDWYLQKPG OSPOLLIYLGSNRASGVPDRFNGSGSGTDFTLKISRVEAEDFGVYY CMOALOTPWTFGHGTKVEIK 6H6 H 45 OVOLVESGGGVVOPGRSLRFSCAASGFTLSSYGMHWVRQAPGKG LEWVAVIWDDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARAGGSGRYYNYFDYWGQGTLVTVSS L 46 EIVMTOSPATLSVSPGERATLSCRASOSVRSNLAWYOQKPGOAPR LLIYGASTRATGIPARFSGSGSGTDFTLTISSLOSEDFAVYYCOOHN NWLTFGGGTKVEIK 1B4 H 59 EVOLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVROVPGKGL EWVSGITGSGANTFYTDSVKGRFTISRDNSNNSLYLQMNSLRAD DT AVYYC AKRNGGS YYYYYGMD VWGQGTT VT VS S L 60 DIVMTOSPLSLPVTPGEPASISCRSSOSLLHSSGYNYLDWYLQKPG OSPOLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMOALOIPWTFGQGTKVEIK 1B5- H 101 OVOLVESGGGVVOPGRSLRLSCAASGFTFSSFGMHWVRQAPGKG LEWVTLIWFDGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCVRGFAAVAGWYFDFWGRGTLVTVSS NK L 102 DIQMTOSPSSLASVGDRVTITCRASOGVRKYLAWYOQKPGKVP KLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLOPEDVATYYCQK YFSAPYTFGQGTKLEIK 2E1. H 87 OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG LEWVAVIWDDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARAGSSGRYYNYFDYWGQGTLVTVSS 2 L 88 EIVMTOSPATLSVSPGERATLSCRASOSVRSNLAWYOQKPGOAPR LLIYGASTRATGIPDRFSGSGSGTEFTLTISSLOSEDFAVYHCOOYN KWLIFGGGTKVEIK ZV6NS H 73 EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL EWVSGITGTGGSTYYADSVKGRFTISRDNSKNTLYVQMNSLRAE DTAVYYCAKRAGGSFYYYYGMDVWGQGTTVTVS S L 74 DIVMTOSPLSLPVTPGEPASISCRSSOSLLHSTGYNYLDWYLQKPG OSPOLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDFGVYY CMOALOTPWTFGHGTKVEIK

The full length amino acid sequences of the heavy and light chains of the 3C3 antibody were as follows.

Light Chain Sequence (Leader Deleted) (SEQ ID NO: 136)

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSR

FSGSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGTKVDIKRYNNNVSNVIVVPSVK^

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy Chain Sequence (Leader Deleted) (SEQ ID NO: 135)

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QVQLVESGGGWQPGRSLRLSCAGSGFIFSRYGMYWVRQAPGKGLEWVAVIWYDGSYKYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARESPWYYFDYWGQGTLVTVSSASYKGVS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSNFG TQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPMLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In each case, the variable sequence is in italics and the constant domain is in bold. The constant domain sequence is an IgG2 sequence from which the C-terminal lysines have been removed.

The identical constant domain sequence was used for other antibodies with their respective variable sequences as above.

Example 2

Determination of the affinity and kinetic constants of human MATs using biolayer interferometry (BLI).

The binding affinity and binding kinetics of various anti-human CD40 antibodies were examined by biolayer interferometry (BLI) using the Octet™ QKe instrument (Pall ForteBio, Menlo Park, CA, USA) according to the manufacturer's recommendations.

Purified antibodies from Example 1 were captured on Anti-Human Fc Capture (AHC) biosensors (Fortebio, catalog number 18-5060). Each antibody was prepared in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg/ml BSA + 0.5% Tween 20, pH 7.2) to a final concentration of 0.5 µg/ml and loaded onto newly hydrogenated AHC biosensors for 35-50 s at 25°C and shaking the plates at 1000 rpm to achieve the target response of 0.2 nm. Low levels of ligand were captured to limit any analyte mass transfer effects on kinetic parameters. To conduct one quantitative study, eight biosensors were loaded with the same antibody.

Binding was determined by exposing six of the antibody-loaded biosensors to the analyte: soluble human CD40-MsIgG2a (Celldex, 60 kDa according to SDS-PAGE gel electrophoresis). Affinity parameters were determined using 2-fold serial dilutions of the analyte ranging from 3.13 to 0.098 nM in dilution buffer at 25°C and a plate shaking speed of 1000 rpm. The association of the antibody-loaded biosensors with the analyte in the wells was carried out for 1200 seconds, then the biosensors were placed in the wells with dilution buffer for 2.5 h (9000 s) to evaluate the dissociation parameters.

Appropriate controls were run in each case, keeping the two remaining biosensors with captured antibody in wells with dilution buffer for the association and dissociation steps. The data for the control biosensors was used to subtract the non-specific signal, as well as to take into account the drift of the biosensor and the dissociation of antibodies from the biosensors.

Fortebio's data analysis software version 8.2.0.7 (Pall ForteBio, Menlo Park, CA, USA) was used to obtain the kinetic parameters for binding to the captured antibody from a series of analyte concentrations in dilution buffer in each case. Association and dissociation curves were fitted using a 1:1 binding model using data analysis software according to the manufacturer's recommendations.

The determined affinity and kinetic parameters (after non-specific signal subtraction) are shown in FIG. 1 where kon=association rate constant, kdis=dissociation rate constant, and K _D =equilibrium dissociation/binding constant given by kdis/kon.

Example 3

Quantitative studies to characterize the binding of human MATs to CD40.

Microtiter plates were coated with recombinant human CD40-Fc in PBS and then blocked with 5% bovine serum albumin in PBS. Human MAb after protein A purification from Example 1 and isotype control were added at various concentrations and incubated at 37°C. The plates were washed with PBS/tween and then incubated with horseradish peroxidase (HRP) conjugated goat polyclonal reagent specific for F(ab') ₂ human IgG at 37°C. After washing, staining in plates was developed using HRP substrate and analyzed at OD450-650 using a microtiter plate reader. Typical binding curves are shown in FIG. 2.

To establish whether cynomolgus monkeys are a suitable model for anti-CD40 MAT testing, purified macaque or human PBMCs were incubated with various concentrations of anti-human CD40 MATs for 20 minutes at room temperature on a plate shaker. The cells were then washed twice with PBS containing 0.1% BSA and 0.05% NaN ₃ (FSB-A). Next, goat anti-human Fc IgG conjugated with phycoerythrin (PE) was added and incubated for 20 minutes at room temperature on a plate shaker. B cells were detected by post-staining with an anti-CD20 antibody conjugated to allophycocyanin (APC). Cells were examined by flow cytometry and the binding curves are shown in FIG. 3 showing similar binding to CD40 from macaque and human.

Example 4

Study of sCD40L binding blocking by ELISA.

The effect of the human MAbs from Example 1 on the binding of soluble CD40 ligand (sCD40L) to the CD40 protein was measured by ELISA. The microtiter plate was coated with 2 μg/ml soluble recombinant human CD40 and Fc region chimeric protein available from R&D Systems, then blocked with 5% PBS-A. Anti-CD40 antibodies ([final concentration]=100 μg/ml) were added to the plate and then soluble recombinant human CD40L biotin available from Immunex ([final concentration]=0.5 μg/ml) was added. CD40-captured rCD40L was detected using streptavidin-HRP and Super Blue TMB substrate. The results are shown in FIG. 4A and B together with control sample data as indicated.

Example 5

Binding to CD40-expressing cells.

The ability of human anti-CD40 MAbs to bind to CD40 on cells expressing human CD40 on their surface was examined by flow cytometry as described below.

Investigated the binding of antibodies from example 1 with human cell lines expressing human CD40 on their surface. Human MAbs purified with protein A, 3C3, 3G5, 1B4, 3B6 and 6H6 were incubated with Raji and Ramos cell lines expressing human CD40 at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN ₃ (PBS-A) and bound antibodies were detected by incubating the cells with phycoerythrin-labeled goat Fc IgG specific probe antibody. Excess antibody probe was washed from the cells with PBS-A and cell-bound fluorescence intensity was determined using a FACSCanto II™ instrument (BD Biosciences, NJ, USA) according to the manufacturer's directions.

As shown in FIG. 5 (binding to Raji cell line) and FIG. 6 (binding to Ramos cells), human MAbs showed high levels of binding to cells expressing human CD40 that depended on antibody concentration.

Example 6

Induction of CD95 on Ramos cells.

Ramos cells were incubated overnight at 37° C., 6% CO ₂ with 2 μg/ml human anti-CD40 mAbs from Example 1. The following day, cells were washed once with PBS-A and stained with phycoerythrin-conjugated anti- CD95 (Becton Dickinson) for 20 minutes at room temperature with shaking. Excess labeled antibody was washed away and samples were read on a FACSCanto II™ instrument (BD Biosciences, New Jersey, USA). As shown in FIG. 7A and B ((in which hatched plots represent untreated/control cells and black lines represent cells treated with antibodies as indicated), 3C3 and 1B5-NK antibodies caused an increase in CD95 expression, and other antibodies 3G5, 1B4, 3B6, 6H6, 2E1.2 and 3B6-NS were able to induce a strong increase in CD95 surface expression.

Example 7

activation of dendritic cells.

Dendritic cells were obtained from human monocytes as follows.

PBMCs were added to T175 cm ² flasks and incubated for monocyte adhesion for ~2 hours at 37°C, 6% CO2. Cells were removed and monocytes were cultured for 7 days in RPMI containing 10% PBS, 10 ng/ml IL-4 (R&D Systems) and 100 ng/ml GM-CSF (R&D Systems). Cells were harvested and their identity to dendritic cells was confirmed based on CD11c expression (data not shown).

Cells were then incubated in the presence of 10 μg/ml anti-human CD40 antibodies 3C3 and 3G5 from Example 1 and appropriate controls at 37°C, 6% CO2. After 72 hours, the cells were harvested and the supernatant was collected and stored for cytokine testing. Cells were stained with the following labeled antibodies for 20 minutes at room temperature with shaking: HLA-DR V450, CD54-FE, CD86-APC and CD83 BV510 (all from BD). The cells were then washed twice and examined on the FACSCanto II™ instrument (BD Biosciences, New Jersey, USA). In FIG. 8A shows the level of expression for each of the listed markers when incubated with the indicated antibody or control.

IL-12p40 induction was assessed in supernatants from these 72 hour cultures by ELISA (R&D Systems). In FIG. 9 shows the increase in IL-12p40 production by anti-CD40 antibodies 3C3 and 3G5 relative to controls as indicated.

In another experiment, cells were incubated in the presence of 10, 1 and 0.1 μg/ml of anti-human CD40 antibodies 3C3 and 3G5 from Example 1 and respective controls at 37° C., 6% CO ₂ . After 48 hours the cells

- 53 042934 was collected and the supernatant was collected and stored for cytokine testing. Cells were stained with labeled anti-CD54 antibody (BD) for 20 minutes at room temperature with shaking. The cells were then washed twice and examined on the FACSCanto II™ instrument (BD Biosciences, New Jersey, USA). In FIG. 8B shows the expression level of CD54 when incubated with the indicated antibody or control.

IL-12p40 induction was assessed in supernatants from these 48 hour cultures by ELISA (R&D Systems). In FIG. 9B shows the increase in IL-12p40 production by anti-CD40 antibodies 3C3 and 3G5 relative to controls as indicated.

Example 8

B-cell activation.

Whole blood was incubated with 10 μg/ml of 3C3 and 3G5 anti-CD40 antibodies from Example 1 overnight at 37°C, 6% CO ₂ . The next day, the following labeled antibodies were used to stain B cells and activation markers: CD54-PE, HLA-DR V450, CD23 PerCP-Cy5.5, CD69-APC, CD86-APC, CD38 PerCP-Cy5.5, and CD71-PE . Cells were stained for 20 minutes at room temperature with shaking, then washed twice and parameters were read on a FACSCanto II™ instrument (BD Biosciences, New Jersey, USA). In FIG. 10A shows the change in expression level for each of these markers relative to controls, as indicated.

In another experiment, whole blood was incubated with 10, 1, and 0.1 μg/ml of anti-CD40, 3C3, and 3G5 antibodies from Example 1 overnight at 37°C, 6% CO2. The next day, the following labeled antibodies were used to stain B cells and activation markers: CD19 V500, HLA-DR V450, CD86APC (all from BD). Cells were stained for 20 minutes at room temperature with shaking, then washed twice and parameters were read on a FACSCanto II™ instrument (BD Biosciences, New Jersey, USA). In FIG. 10B shows the change in expression level for each of these markers relative to controls, as indicated.

Example 9

NFkB activation.

A cell line carrying a luciferase reporter expressing CD40 was incubated for 6 hours at 37° C., 6% CO2 with various concentrations of anti-human CD40 antibodies from Example 1. Luciferase expression was detected using the Promega Luciferase Assay System as recommended. manufacturer. In FIG. 11A and 11B show the high level of NFkB activation induced by antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK and 3B6NS as a function of antibody concentration.

Example 10

Tumor eradication in a xenograft tumor model in SCID mice harboring Raji cells.

CB.17 SCID mice (purchased from Taconic Biosciences, Inc.) were kept under sterile conditions in a mouse vivarium. Raji lymphoma cells (1x106) were injected subcutaneously into SCID mice, 5 mice per group. On days 1, 5, and 11, these mice were injected with a clone of human anti-CD40, 3C3, and 3G5 MAT by intraperitoneal injection at a dose of 0.3 mg. Tumor growth was measured with calipers 2 times a week. The results of the tumor growth evaluation and survival analysis are shown in FIG. 12, which shows that in mice infected with tumor cells, treatment with anti-CD40 antibodies inhibits tumor growth and significantly prolongs survival compared to saline-treated controls.

Example 11.

Tumor eradication in a xenograft tumor model in SCID mice harboring Ramos cells.

CB.17 SCID mice (purchased from Taconic Biosciences, Inc.) were kept under sterile conditions in a mouse vivarium. Ramos human lymphoma cells (1x106) were injected subcutaneously into SCID mice on day 0, 5 mice per group. On days 1, 5 and 11, these mice were injected with human anti-CD40, 3C3 or 3G5 mAbs by intraperitoneal injection at a dose of 0.3 mg. Tumor growth was measured with calipers 2 times a week.

The results shown in FIG. 13 indicate that anti-CD40 mAbs significantly inhibited tumor expansion compared to saline treated controls, resulting in 100% (3G5) or 80% (3C3) survival of mice infected with tumor cells.

Example 12.

Proliferation of T-cells.

Human peripheral blood mononuclear cells (PBMCs) isolated from buffy coat preparations were labeled using 0.5 μM carboxyfluorescein succinimidyl ester (CFSE) at room temperature with rotation for 5 minutes. CFSE-labeled PBMCs (1.5x106) were dispensed into wells dry-coated with anti-CD3 antibody (OKT3) at a concentration of 0.2 µg/ml.

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Anti-CD40 antibodies (3G5, 3C3, 1412) or isotype control antibody (IgG2) were dispensed into wells in soluble form at a final concentration of 10 μg/ml. The plates were incubated at 37°C (5% CO ₂ ). On day 6, cells were harvested and stained with APC-conjugated anti-CD3 antibody or isotype control and examined by flow cytometry. Typical plots are shown in Fig. 14A, from which it follows that antibodies significantly increased T cell proliferation, as evidenced by the reduced intensity of CFSE staining in the CD3+ gate. The results of the repeated experiment are shown in Fig. 14B, which show an increase in the number of dividing cells under the influence of antibodies to CD40, compared with the isotype control antibody.

Example 13

Binding to CD40 independent of Fc receptor interaction.

Microtiter plates were coated with recombinant human CD40-Fc in PBS and then blocked with 5% bovine serum albumin in PBS. Human MAbs purified on protein A (total IgG and F(ab') ₂ fragments as indicated) were added at various concentrations and incubated at 37°C. The plates were washed with PBS/tween and then incubated with horseradish peroxidase conjugated goat polyclonal reagent specific for F(ab') ₂ human IgG at 37°C. After washing, plate staining was developed with HRP substrate and examined at OD450-650 using a microtiter plate reader. The results are shown in FIG. 15. IgG2 variants and F(ab') ₂ variants of each antibody showed a similar concentration dependence of binding to CD40-Fc.

Example 14

CD40 activation independent of Fc receptor interaction.

The cell line carrying the CD40-expressing luciferase reporter from Example 9 was incubated for 6 hours at 37°C, 6% CO ₂ with various concentrations of anti-CD40 human antibodies (both total IgG and F(ab') fragments ₂ as indicated), Luciferase expression was detected using the Promega Luciferase Assay System according to the manufacturer's recommendations. The results are shown in FIG. 16. These results indicate that binding to the Fc receptor is not required for CD40-mediated activation of the reporter cell line by 3C3 and 3G5 antibodies, since both intact antibodies with Fc domains and their corresponding F(ab') ₂ variants that do not contain domains Fc were able to activate NFkB in the reporter cell line.

Example 15

CD95 induction independent of Fc receptor interaction.

Ramos cells were incubated overnight at 37° C., 6% CO ₂ with various concentrations of human anti-CD40 mAbs (both total IgG and F(ab')2 fragments as indicated). The next day, cells were washed once with PBS-A and stained with PE-conjugated anti-CD95 antibody (Becton Dickinson) for 20 minutes at room temperature with shaking. Excess labeled antibody was washed away and sample parameters were read on a FACSCanto II™ instrument (BD Biosciences, New Jersey, USA). The results are shown in FIG. 17. These data indicate that interactions with the Fc receptor are not required for the 3G5 antibody to induce CD95 expression on the Ramos CD40+ human lymphoblastic cell line.

Example 16

Synergistic action with sCD40L.

Ramos cells were incubated overnight with 3C3 antibody with or without 0.1 mg/ml of soluble CD40 ligand. The cells were then stained with PE-conjugated anti-CD95 antibody and examined by flow cytometry. The results are shown in FIG. 19 and indicate that the anti-CD40 3C3 antibody acts synergistically with sCD40L. Accordingly, the 3C3 antibody (and anti-CD40 antibodies that bind to the same epitope as 3C3) exhibits a synergistic agonistic effect with the soluble CD40 ligand (sCD40L) and therefore has the ability to interact synergistically with other therapeutic agents, including those that bind with the binding site of the human CD40 ligand. Typical synergistic effects include, for example, increased activity of the immune system (eg, T-cell mediated immune responses, as in vaccine therapy, activation of NK cells in various cancer therapies), inhibition of cell proliferation (eg, in cancer therapy), and/or enhanced processing and presentation of the APC antigen (for example, in vaccine therapy).

Example 17.

Epitope mapping of antibodies to human CD40, 3C3 and 3G5, and sCD40.

i) Obtaining truncated and mutated fragments of soluble CD40 (sCD40).

Soluble CD40 (sCD40) cDNA encoding the full-length extracellular domain (ECD) spanning amino acid residues 1-173 (SEQ ID NO: 133) and three smaller fragments encoding amino acids 1-94, 36-130 and 84-173 was synthesized from GenScript and inserted open reading frame into a mammalian expression vector with an N-terminal kappa light chain and a C-terminal Flag. The resulting kappa-sCD40-Flag fusion proteins were expressed using

- 55 042934 transient transfection into ExpiCHO-S (SAFC) cells. Since the anti-CD40 antibody, 3C3, recognizes human and monkey CD40, but not mouse CD40, a range of mutated sCD40aa 1-94 cDNA was constructed based on differences between human and mouse sequences, as shown in the sequence alignment schemes in FIG. 20 and 21. Mutated variants were synthesized and cloned by GenScript. All of these truncated or mutated fragments were cloned into the same vector and expressed in the same cell line as mentioned above.

ii) Determination of binding by ELISA.

3C3 binding to a series of sCD40 fragments was tested by ELISA. 1 µg/ml of purified kappa-sCD40-Flag fusion proteins or CHO cell supernatants containing sCD40 fusion proteins were captured on microtiter plates that were pre-coated with 5 µg/ml mouse anti-FLAG antibody (Sigma) in PBS and blocked with using 5% bovine serum albumin in PBS. After incubation with anti-CD40 antibody, microtiter plates were washed with PBS/tween and incubated with horseradish peroxidase conjugated polyclonal goat human Fc IgG specific reagent. After washing, plate staining was developed with HRP substrate and examined at OD450-650 using a microtiter plate reader. ELISA using HRP-conjugated goat anti-human Fab2 IgG to measure kappa chain binding was run in parallel to test for expression of the sCD40 fusion protein in samples from different transfections.

ELISA studies using ~1 μg/ml of full-length sCD40 and three truncated fragments determined that the N-terminal residues of sCD40 1-94 are significant and sufficient for binding to 3C3, since the fragment encoding amino acid residues 1-94 binds to 3C3, as well as the full-length VKD, but the fragments encoding amino acid residues 36-130 or 84-173 of this sequence did not bind to any extent (see Table 4).

Table 4

Average OD Fragment amino acid residues ZSZ aFab2HRP 1-173 1.264 1.264 1-94 1.803 1.720 36-130 0.024 1.695 84-173 0.024 1.669 Mutated fragment of amino acids 1-94 A(1-5) 0.189 1.718 At (13-15) 2.032 1,730 C (25, 26, 28, 30) 1.487 1.685 D(33-36) 0.092 1.631

Based on the results obtained, it was established that the critical recognition sites for 3C3 are in the range of amino acids 1-35.

In order to additionally identify regions and amino acid residues critical for the conformational organization of the 3C3 binding site, ∼2 µg/mL of 13 mutated sCD40 fragments (amino acid residues 1-94) (4 regional multiple mutations and 9 single mutations) were tested by ELISA (see Table 1). Tables 5 and 6, which present the results of individual experiments, and Fig. 22).

- 56 042934

Table 5

Average OD Fragment amino acid residues ZSZ aFab2HRP 1-94 2.157 1.473 Mutated fragment of amino acids 1-94 A(1-5) 0.167 1.489 D(33-36) 0.124 1.429 point mutation E1G 1.965 1.487 P2Q 2.077 1,490 P3S 2.011 1.489 T4V 2.152 1.519 A5T 1.126 1.517 EZZA 1.620 1.521 F34L 1.883 1,500 T35E 2.072 1.487 E36K 1.369 1.433 RVA 0.031 0.011

Table 6

Average OD Fragment amino acid residues ZSZ 3G5 aFab2HRP Full size fragment 1-173 2.364 2.214 1.525 1-94 2.151 2.170 1.755 36-130 0.029 0.048 1.716 84-173 0.024 0.038 1.599 Mutated fragment of amino acids 1-94 A(1-5) 0.250 2.139 1.699 At (13-15) 2.375 1.876 1.710 C (25, 26, 28, 30) 2.016 2.161 1.604 D(33-36) 0.233 0.042 1.548 point mutation E1G 2.011 2.083 1.720 P2Q 2.197 2.158 1.754 P3S 2.012 2.188 1.712 T4V 2.213 2.210 1.664 A5T 1.511 2.201 1.698 EZZA 1.695 0.074 1.709 F34L 1.845 1.192 1.686 T35E 2.102 2.128 1.682 E36K 1.689 1,930 1.674

<0.25

0.25<х<1.2

1.2<х<1.9

Multiple mutations of residues 1-5 almost completely prevented binding to 3C3. Point mutations of residues 1-4 did not decrease binding to 3C3. Point mutation of residue 5 significantly reduced binding, but not to the extent that was observed with multiple mutations of the protein.

Multiple mutations of residues 13-15 did not decrease binding to 3C3. Multiple mutations of residues 25, 26, 28 and 30 caused a slight decrease in binding to 3C3. Point mutations in these regions were not tested.

- 57 042934

Multiple mutations of residues 33-36 almost completely prevented binding to 3C3. Point mutation of residue 35 did not affect binding. Point mutations 33, 34, and 36 reduced binding to 3C3, but not to the extent seen with multiple protein mutations. The binding of an alternative anti-CD40 antibody, 3G5, was tested with all fragments and mutants and was shown to be different from 3C3 binding (Table 6). Multiple mutations of residues 1-5 did not reduce binding, while mutation of residues 33-36 abolished binding. In contrast to 3C3, point mutation of residue 33 completely abolished binding, and mutation of 34 significantly reduced binding.

Example 18.

Biological profile and toxicity profile.

An experimental non-NLP study was performed on naïve cynomolgus monkeys. This study was designed to provide preliminary data on the biological and toxicity profile of 3C3. An alternative anti-CD40 antibody (3G5) was also evaluated. The test products were administered by intravenous injection into the saphenous vein on day 1 (0.2 mg/kg or vehicle) and again on day 29 (2 mg/kg or vehicle). Animals also received a subcutaneous (1 mg) injection of keyhole limpet hemocyanin (KLH) on days 1 and 29. Estimates of potential effects associated with the test product were based on clinical signs, body temperature, laboratory diagnostic parameters (haematology, coagulogram, blood chemistry and urinalysis), presence of anti-drug antibodies, cytokine levels, T-cell dependent antibody response (TDAR), flow cytometry and toxicokinetic parameters. Body weight was recorded once before drug testing and weekly thereafter. This study was designed as a survival study without scheduled autopsy.

In this study, administration of 3C3 or 3G5 was well tolerated in cynomolgus monkeys, with none of the toxicity parameters significantly exceeding control levels. Of note are slight increases in aspartate aminotransferase (ACT), alanine aminotransferase (ALT), and creatinine kinase activity in 3C3-treated monkeys (FIGS. 23A-23C). Pharmacological reductions in IL-12 (Fig. 24), leukocytes (Fig. 25A), neutrophils (Fig. 25B) and lymphocytes (Fig. 25C) levels were observed in both anti-CD40 treated animals, with the most significant being the transient reduction in B -cells (FIGS. 26 and 27). In conclusion, under the conditions of this study, 3C3 and 3G5 showed minimal evidence of toxicity.

Example 19.

Proliferation of B-cells independent of interaction with the Fc receptor.

Human B cells were isolated from peripheral blood mononuclear cells by magnetic selection using beads specific for CD19. Cells were labeled with 0.5 μM carboxyfluorescein succinimidyl ether (CFSE) at room temperature and rotating for 5 minutes. Labeled cells were cultured in the presence of an anti-CD40 mAb, 3C3, or an isotype control antibody (both total IgG and F(ab') ₂ fragment) for 6 days. The cells were then harvested and examined by flow cytometry to assess proliferation. The results are shown in FIG. 28 and suggest that binding to the Fc receptor is not required for CD40-mediated proliferation by 3C3, since intact antibodies with Fc domains and their corresponding Fc domain-free F(ab') ₂ variants were able to induce B- cells.

Example 20.

Synergistic action with CD40L in human B cells.

Human B cells were isolated and labeled as described in Example 19. An anti-CD40, 3C3, or isotype control mAb at 0.1 μg/ml was incubated with cells for 6 days in the presence of 0.1 μg/ml soluble CD40L (Immunex ) or without it. In FIG. 29 shows that no significant proliferation was observed when using 3C3 alone or the isotype control antibody in combination with CD40L, however, the combination of CD40L and 3C3 induced proliferation in culture.

Dendritic cells were prepared and cultured with 0.5 μg/ml 3C3 as described in Example 7 with or without 0.1 μg/ml soluble CD40L. The production of IL-12p40 was measured by ELISA (R&D Systems). In FIG. 30 shows that, compared to the low production that was induced by 3C3 alone or the CD40L isotype control antibody, the combination of 3C3 and CD40L induced higher levels of IL-12p40.

Example 21.

Cytokine response in whole blood.

Whole blood was incubated overnight with 10 μg/ml isotype control antibody or 3C3 or LPS as a positive control. The next day, plasma was collected and cytokines were measured by ELISA (R&D Systems). The results are shown in FIG. 31 and indicate the absence of significant production of inflammatory cytokines.

Equivalents.

Those skilled in the art will be aware of, or be able to ascertain using routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be included within the scope of the following claims.

Sequence Listing Summary

SEQID NO: DESCRIPTION 1 Human CD40 (GenBank accession number: P25942) MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPI IFGILFAILL VLVFIKKVAK I<PTNI<APHPI< QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 2 Human CD40L (GenBank accession number: NP 000065) MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL DKIEDERNLH EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ FEGFVKDIML NKEETKKENS FEMQKGDQNP QIAAHVISEA SSKTTSVLQW AEKGYYTMSN NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN REASSQAPFIASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN VTDPSQVSHG TGFTSFGLLK 3 3G5-VH QVQLVESGGGVVQPGKSLRLSCAASGFTFSSNGIHWVRQAPGKGLEWVAVIWSDGS NKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSGSYYNFFDYW GQGTLVTVSS 4 3G5-VL EIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAPRLLIYGASTRATGI PARFSGSGSGTEFTLTINSLQSEDFAVYYCQQHNKWITFGQGTRLEIK 5 3G5 - VH CDR1 (KABAT) SNGIH 6 3G5 - VH CDR1 (CHOTIA) GFTFSSN 7 3G5 - VH CDR2 (KABAT) VIWSDGSNKFYADSVKG

- 59 042934

8 3G5 - VH CDR2 (CHOTIA) WSDGSN 9 3G5 - VH CDR3 (KABAT) ASGSGSYYNFFDY 10 3G5 - VH CDR3 (CHOTIA) ASGSGSYYNFFDY AND 3G5 - VL CDR1 (KABAT) RASQSVRSNLA 12 3G5 - VL CDR1 (CHOTIA) RASQSVRSNLA 13 3G5 - VL CDR2 (KABAT) GASTRAT 14 3G5 - VL CDR2 (CHOTIA) GASTRAT 15 3G5 - VL CDR3 (KABAT) QQHNKWIT 16 3G5 - VL CDR3 (CHOTIA) QQHNKWIT 17 3C3-VH QVQLVESGGGVVQPGRSLRLSCAGSGFIFSRYGMYWVRQAPGKGLEWVAVIWYDG SYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARESPWYYFDYWGQ GTLVTVSS 18 3C3-VL DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGV PSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGTKVDIK 19 3C3 - VH CDR1 (KABAT) RYGMY 20 3C3 - VH CDR1 (CHOTIA) GFIFSRY 21 3C3 - VH CDR2 (KABAT) VIWYDGSYKYYADSVKG

- 60 042934

22 ZSZ - VH CDR2 (CHOTIA) WYDGSY 23 ZSZ - VH CDR3 (KABAT) ESPWYYFDY 24 ZSZ - VH CDR3 (CHOTIA) ESPWYYFDY 25 ZSZ - VL CDR1 (KABAT) RASQGISNYLA 26 ZSZ - VL CDR1 (CHOTIA) RASQGISNYLA 27 ZSZ - VL CDR2 (KABAT) AASTLQS 28 ZSZ - VL CDR2 (CHOTIA) AASTLQS 29 ZSZ - VL CDR3 (KABAT) QKYKSAPFT thirty ZSZ - VL CDR3 (CHOTIA) QKYKSAPFT 31 3B6-VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGTGG STYYADSVKGRFTISRDNSKNTLYVQMNSLRAEDTAVYYCAKRAGGSFYYYYGMD VWGQGTTVTVSS 32 3B6-VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSTGYNYLDWYLQKPGQSPQLLIYLGSNR ASGVPDRFNGSGSGTDFTLKISRVEAEDFGVYYCMQALQTPWTFGHGTKVEIK 33 3B6 - VH CDR1 (KABAT) SYAMS 34 3B6 - VH CDR1 (CHOTIA) GFTFSSY 35 3B6 - VH CDR2 (KABAT) GITGTGGSTYYADSVKG

- 61 042934

36 3B6 - VH CDR2 (CHOTIA) TGTGGS 37 3B6-VH CDR3 (KABAT) RAGGSFYYYYGMDV 38 3B6 - VH CDR3 (CHOTIA) RAGGSFYYYYGMDV 39 3B6-VL CDR1 (KABAT) RSSQSLLHSTGYNYLD 40 3B6 - VL CDR1 (CHOTIA) RSSQSLLHSTGYNYLD 41 3B6-VL CDR2 (KABAT) LGSNRAS 42 3B6 - VL CDR2 (CHOTIA) LGSNRAS 43 3B6-VL CDR3 (KABAT) MQALQTPWT 44 3B6 - VL CDR3 (CHOTIA) MQALQTPWT 45 6H6-VH Q VQLVESGGGVVQPGRSLRF SC AASGFTLS S YGMHWVRQ APGKGLEWVAVIWDDG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGGSGRYYNYFD YWGQGTLVTVSS 46 6H6-VL EIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAPRLLIYGASTRATGI PARFSGSGSGTDFTLTISSLQSEDFAVYYCQQHNNWLTFGGGTKVEIK 47 6H6 - VH CDR1 (KABAT) SYGMH 48 6H6 - VH CDR1 (CHOTIA) GFTLSSY 49 6H6 - VH CDR2 (KABAT) VIWDDDGSNKKYYADSVKG

- 62 042934

50 6H6 - VH CDR2 (CHOTIA) WDDGSN 51 6H6 - VH CDR3 (KABAT) AGGSGRYYNYFDY 52 6H6 - VH CDR3 (CHOTIA) AGGSGRYYNYFDY 53 6H6 - VL CDR1 (KABAT) RASQSVRSNLA 54 6H6 - VL CDR1 (CHOTIA) RASQSVRSNLA 55 6H6 - VL CDR2 (KABAT) GASTRAT 56 6H6 - VL CDR2 (CHOTIA) GASTRAT 57 6H6 - VL CDR3 (KABAT) QQHNNWLT 58 6H6 - VL CDR3 (CHOTIA) QQHNNWLT 59 1B4-VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQVPGKGLEWVSGITGSGA NTFYTDSVKGRFTISRDNSNNSLYLQMNSLRADDTAVYYCAKRNGGSYYYYYGMD VWGQGTTVTVSS 60 1B4-VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSSGYNYLDWYLQKPGQSPQLLIYLGSNR ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQIPWTFGQGTKVEIK 61 1B4 - VH CDR1 (KABAT) SYAMT 62 1B4 - VH CDR1 (CHOTIA) GFTFSSY 63 1B4 - VH CDR2 (KABAT) GITGSGANTFYTDSVKG

- 63 042934

64 1B4 - VH CDR2 (CHOTIA) TGSGAN 65 1B4 - VH CDR3 (KABAT) RNGGSYYYYYGMDV 66 1B4 - VH CDR3 (CHOTIA) RNGGSYYYYYGMDV 67 1B4 - VL CDR1 (KABAT) RSSQSLLHSSGYNYLD 68 1B4 - VL CDR1 (CHOTIA) RS SQSLLHS SGYNYLD 69 1B4 - VL CDR2 (KABAT) LGSNRAS 70 1B4 - VL CDR2 (CHOTIA) LGSNRAS 71 1B4 - VL CDR3 (KABAT) MQALQIPWT 72 1B4 - VL CDR3 (CHOTIA) MQALQIPWT 73 3B6-NS-VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGTGG STYYADSVKGRFTISRDNSKNTLYVQMNSLRAEDTAVYYCAKRAGGSFYYYYGMD VWGQGTTVTVSS 74 3B6-NS-VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSTGYNYLDWYLQKPGQSPQLLIYLGSNR ASGVPDRFSGSGSGTDFTLKISRVEAEDFGVYYCMQALQTPWTFGHGTKVEIK 75 3B6-NS-VH CDR1 (KABAT) SYAMS 76 3B6-NS - VH CDR1 (CHOTIA) GFTFSSY 77 3B6-NS-VH CDR2 (KABAT) GITGTGGSTYYADSVKG

- 64 042934

78 3B6-NS - VH CDR2 (CHOTIA) TGTGGS 79 3B6-NS - VH CDR3 (KABAT) RAGGSFYYYYGMDV 80 3B6-NS - VH CDR3 (CHOTIA) RAGGSFYYYYGMDV 81 3B6-NS - VL CDR1 (KABAT) RSSQSLLHSTGYNYLD 82 3B6-NS - VL CDR1 (CHOTIA) RSSQSLLHSTGYNYLD 83 3B6-NS - VL CDR2 (KABAT) LGSNRAS 84 3B6-NS - VL CDR2 (CHOTIA) LGSNRAS 85 3B6-NS - VL CDR3 (KABAT) MQALQTPWT 86 3B6-NS - VL CDR3 (CHOTIA) MQALQTPWT 87 2E1.2-VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWDDG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGSSGRYYNYFDY WGQGTLVTVSS 88 2E1.2 - VL2 EIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAPRLLIYGASTRATGI PDRFSGSGSGTEFTLTISSLQSEDFAVYHCQQYNKWLIFGGGTKVEIK 89 2E1.2 - VH CDR1 (KABAT) SYGMH 90 2E1.2 - VH CDR1 (CHOTIA) GFTFSSY

- 65 042934

91 2E1.2 - VH CDR2 (KABAT) VIWDDDGSNKKYYADSVKG 92 2E1.2 - VH CDR2 (CHOTIA) WDDGSN 93 2E1.2 - VH CDR3 (KABAT) AGSSGRYYNYFDY 94 2E1.2 - VH CDR3 (CHOTIA) AGSSGRYYNYFDY 95 2E1.2 - VL2 CDR1 (KABAT) RASQSVRSNLA 96 2E1.2 - VL2 CDR1 (CHOTIA) RASQSVRSNLA 97 2E1.2 - VL2 CDR2 (KABAT) GASTRAT 98 2E1.2 - VL2 CDR2 (CHOTIA) GASTRAT 99 2E1.2-VL2 CDR3 (KABAT) QQYNKWLI 100 2E1.2 - VL2 CDR3 (CHOTIA) QQYNKWLI 101 1B5-NK-VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVTLIWFDGS SKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRGFAAVAGWYFDFW GRGTLVTVSS 102 1B5-NK-VL DIQMTQSPSSLSASVGDRVTITCRASQGVRKYLAWYQQKPGKVPKLLIYAASTLQSG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYFSAPYTFGQGTKLEIK 103 1B5-NK - VH CDR1 (KABAT) SFGMH 104 1B5-NK - VH CDR1 (CHOTIA)

- 66 042934

GFTFSSF 105 1B5-NK-VH CDR2 (KABAT) LIWFDGSSKYYADSVKG 106 1B5-NK - VH CDR2 (CHOTIA) WFDGSS 107 1B5-NK-VH CDR3 (KABAT) GFAAVAGWYFDF 108 1B5-NK - VH CDR3 (CHOTIA) GFAAVAGWYFDF 109 1B5-NK-VL CDR1 (KABAT) RASQGVRKYLA 110 1B5-NK-VL CDR1 (CHOTIA) RASQGVRKYLA 111 1B5-NK-VL CDR2 (KABAT) AASTLQS 112 1B5-NK-VL CDR2 (CHOTIA) AASTLQS FROM 1B5-NK-VL CDR3 (KABAT) QKYFSAPYT 114 1B5-NK-VL CDR3 (CHOTIA) QKYFSAPYT 115 3G5 VH with underlined leader sequence Atggagtttgggctgacctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagttggtggaatct cagttatctggtctgatggaagtaataa Attctatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctatatctgcaaatga 116 3G5 VL with underlined leader sequence Atggaagccccagcgcagcttctcttcctcctgctactctctggctcccagatagcactggagaaatagtgatgacgcag

- 67 042934

Tctccagccaccctgtctgtgtctccagggggaaagagccaccctctcctgcagggccagtcagagtgttagaagtaac ctgaagattttgcagt ttattactgtcagcagcataataagtggatcaccttcggccaagggacacgactggagattaaa 117 3C3 VH with underlined leader sequence Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgg cagttatatggtatgatggaagttataaatactat Gcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctg 118 3C3 VL with underlined leader sequence Atggacatgagggtccctgctcagctcctgggactcctgctgctctggctcccagataccagatgtgacatccagatgac ctgcatccactttgcaatcaggggtccc Atctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaactta ttactgtcaaaagtataagagtgccccattcactttcggccctgggaccaaagtggatatcaaa 119 3B6 VH with underlined leader sequence Atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgtgaggtgcagctgttggagtctgggg Gaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagct tataactggtactggtggtagcacatactacgcag Actccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatgtgcaaatgaacagcctgagagc 120 3B6 VL with underlined leader sequence Atgaggctccctgctcagctcctggggctgctaatgctctgggtctctggatccagtggggatattgtgatgactcagtctc Cactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgcatagtactggata atcagcagagtggaggctgaggattttgg ggtttattactgcatgcaagctctacaaactccgtggacgttcggccacgggaccaaggtggaaatcaaa 121 6H6 VH with underlined leader sequence Atggagtttgggctgagctgggtattcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggg

- 68 042934

Gaggcgtggtccagcctgggaggtccctgagattctcctgtgcagcgtctggattcaccctcagtagtagctatggcatgcactg tctgcaaatgaacagcctgagagccgagg Acacggctgtctattactgtgcgagagcggggggttcgggggaggtattataactactttgactactggggccagggaaccct ggtcaccgtctcctca 122 6H6 VL with underlined leader sequence Atggaagccccagcgcagcttctcttcctcctgctactctctggctcccagataccactggagaaatagtgatgacgcagtctcc gccactggtatcccagccaggttcag 123 1B4 VH with underlined leader sequence Atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccaatgtgaggtgcagctgttggaatctggggga ggtattactggtagtggtgctaacacattctacacagactccgtga Agggccggttcaccatttccagagacaattccaataattcgctgtatctgcaaatgaacagcctgagagccgatgacacggc Cgtatactactgtgcgaaaagaaatggtgggagttactactactactactacggcatggacgtctggggccaagggaccacggtc accgtgtcct ca 124 1B4 VL with underlined leader sequence Atgaggctccctgctcagctcctggggctgctaatgctctctgggtctctggatccagtggggatattgtgatgactcagtctccac tgatctatttgggttctaatcgggcctccggggtccctgac Aggttcagtggcagtggatcaggcacacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactg catgcaagctctacaaattccgtggacgttcggccaagggaccaaggtggaaatcaaa 125 2E1.2 VH with underlined leader sequence AtggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtCcag cctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaAggggct ggagtgggtggcagttatatgggatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagAcaattccaag aacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagcgggaagttcggggaggtattataact actttgactactggggccagggaaccctggtcaccgtctcctca 126 2E1.2 VL2 with underlined leader sequence Atggaagccccagcgcagcttctcttcctcctgctactctctggctcccagataccactggagaaaatagtgatgacgcagtctccagcca

- 69 042934

ccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttaggagcaacttagcctggtatcagcaga aacctggccaggctcccaggctcctcatctatggtgcatccaccaggggccactggtatcccagacaggttcagtggcagtgggtctg ggacagagttcactctcaccatcagcagcctgcagtct gaagattttgcagtttatcactgtcagcagtataataagtggctcattttc ggcggagggaccaaggtggagatcaaa 127 1B5 VH with underlined leader sequence Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtccagc Ctgggaggtccctgagactctcctgtgcagcgtctcctgtgcagcgtctggattcaccttcagtagctttggcatgcactgggtccgccaggctccaggcaaggggctgg aGtgggtgacacttatggtttgatggaagttctaaatactatgcagactccgtgaagggccgattcaccatctccagagacaactccaacaac acGctgtatctgcaaatgaac agcctgagagccgaggacacggctgtatattactgtgtgagaggttttgcagcagtggctgggtggtacttcga tttctggggccgtggcaccctggtcactgtctcctca 128 1B5 VL with underlined leader sequence Atggacatgagggtccctgctcagctcctgggactcctgctgctctggctcccagataccagatgtgacatccagatgacccagtctccatcctcc cTgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcgttagaaagtatttagcctggtatcagcagaaaccaggga aAgttcctaagctcctgatctatgctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcac caTcagcagcctgca gcctgaagatgttgcaacttattactgtcaaaagtatttcagtgccccgtacacttttggccaggggaccaaactggaga tcaaa 129 3B6-NS VH with underlined leader sequence Atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgtgaggtgcagctgttggagtctgggggaggctt ggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggct ccagggaaggggctggagtgggtct caggtataactggtactggtggtagcacatactacgcagactccgtgaagggccggttcac catctccagacaattccaagaacacgctgtatgtgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcga a a agggctggtgggagcttcta eta eta eta cggta tggacgtctggggcca aggga ccatea egg cc gtctcctca 130 3B6-NS VL with underlined leader sequence atgaggctccctgctcagctcctggggctgctaatgctctgggtctctggatccagtggggatattgtgatgactcagtctccactctcc ctgcccgtcacccctggagagccggcctccatctcctcctgcaggtctagtcagagcctcctgcatagtactggatacaactatttggattg gtacctgcagaagccagggcagtctccacacagctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggc agtggatcaggcacagattttttaacactgaaaat cagcagagtggaggctgaggattttggggtttattactgcatgcaagctctacaa a ctccgtgga cgttcggccacgggaccaaggtgga a atca a a 131 1B5-NK VH with underlined leader sequence atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgt

- 70 042934

ggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctttggcatgcactgggtccgccaggctc caggcaaggggctggagtgggtgacacttatggtttgatggaagttctaaatactatgcagactccgtgaagggccgattcacca tctccagagacaactccaagaacacgctgtatctg caaatgaacagcctgagagccgaggacaccggctgtatattactgtgtgaga ggttttgcagcagtggctgggtggtacttcgatttctggggccgtggcaccctggtcactgtctcctca 132 1B5-NK VL with underlined leader sequence Atggacatgagggtccctgctcagctcctgggactcctgctgctctggctcccagataccagatgtgacatccagatgacccagtctc catcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcgttagaaagtatttagcctggtatca gcagaaaccagggaaagttcctaagctcctgatctatgctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtgga tctgggacagatttcactctcaccatcagcagcct gcagcctgaagatgttgcaacttattactgtcaaaagtatttcagtgccccgta cacttttggccaggggaccaaactggagatcaaa 133 Extracellular domain of human CD40 EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCH QHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIA TGVSDTICEPCP VGFF SNVS S AFEKCHPWTSCETKDLVVQQ AGTNKTD VVCGPQDRL R 134 Immunoglobulin 2 heavy gamma chain constant region (IgHG2) (Uniprot P01859) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSN KGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 135 3C3 heavy chain with variable region in italics and constant domain in bold QVQLVESGGGWQPGRSLRLSCAGSGFIFSRYGMYWVRQAPGKGLEWVAVIWYDGSYKYY ADSVKGRFTISRDNSKNTLYLQШSLRAEDTAVYYCARESPWYYFDYWGQGTLVTVSS^Sτ KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

- 71 042934

SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQ PREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 136 3C3 light chain with variable region in italics and constant domain in bold DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQQKPGKVPKLLIYAASTLQSGVPSRFS GSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGTKVDIKRJVAAPSVF#¥PSDEQ LKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 137 3C3 heavy chain with leader sequence in underline, variable region in italics and constant domain in bold MEFGLSWVFLNALLRGNOCOVOLVESGGGWOPGRSLRLSCAGSGFIFSRYGAYYWFRQ APGKGLEWVA VIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARES /Wr/7WWG7/J7FASASTKGPSVFPLAPC^RSTSESTA SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVERKCCVECPPCPAPPVAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG 138 3C3 light chain with leader sequence in underline, variable region in italics and constant domain in bold MGWSCIIEFLNAAAAGVGSDIOMTQSPSSLSASVGDRVTITCRASOGISNYLAWYOOKPG KVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGTKV D/ARTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNS OESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGEC

CLAIM

Claims (26)

1. An agonist isolated monoclonal antibody that binds to human CD40, said antibody comprising heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 19, 21, 23, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO: 25, 27, 29, respectively. 2. An antibody according to claim 1, which additionally exhibits at least one of the following properties: (a) induces cellular apoptosis; (b) enhances the activity of the cell to stimulate T cells, as measured by an increase in the expression of IL-12p40; (c) enhances B cell activation as measured by increased expression of at least one cell surface marker selected from the group consisting of HLA-DR V450, CD54-FE, CD86-APC and CD83 BV510, CD19 V500, CD54-FE , HLA-DR V450, CD23 PerCP-Cy5.5, CD69APC, CD86-APC, CD38 PerCP-Cy5.5 and CD71-PE; (d) binds to human CD40 with an equilibrium dissociation constant Kd of ^10-10 M or less; (e) cross-reacts with CD40 cynomolgus monkeys; and/or (f) binds to human CD40 resulting in cell activation as measured by an NFkB-driven reporter cell line. 3. An antibody according to claim 1 which contains an IgG2 heavy chain constant region. 4. An isolated antibody that binds to human CD40 and contains CDR sequences from the heavy and light chain variable regions, respectively, containing the amino acid sequence shown in SEQ ID NOs: 17 and 18. 5. An antibody according to any one of claims 1 to 4, characterized in that said antibody is a human antibody. - 72 042934 6. An antibody according to any one of claims 1 to 4, characterized in that said antibody contains a human constant region. 7. An antibody according to any one of claims 1 to 4, characterized in that said antibody is an antigen binding fragment, a Fab, Fab', (Fab') ₂ , Fv or scFv fragment. 8. A molecular conjugate comprising an antibody according to any one of claims 1 to 7 coupled to a tumor antigen, self antigen or pathogen component. 9. A bispecific molecule for inducing or enhancing an immune response to a cancer cell, comprising an antibody according to any one of claims 1 to 7 coupled to a second molecule, wherein the second molecule binds to a T cell receptor, an NK cell receptor, or another tumor necrosis factor receptor . 10. An isolated nucleic acid encoding the heavy chain variable region of an antibody according to any one of claims 1-7. 11. An isolated nucleic acid encoding a light chain variable region of an antibody according to any one of claims 1-7. 12. An isolated nucleic acid encoding both the heavy chain variable region and the light chain variable region of an antibody according to any one of claims 1-7. 13. An expression vector containing a nucleic acid molecule according to any one of claims 10-12. 14. Host cell transformed with the expression vector of claim 13. 15. A composition for inducing or enhancing an immune response to a cancer cell, comprising said antibody according to any one of claims 1 to 7, a molecular conjugate according to claim 8 or a bispecific molecule according to claim 9, and a carrier. 16. Composition according to claim 15, additionally containing an adjuvant. 17. The composition of claim 15, further comprising one or more other antibodies. 18. The composition of claim 17 wherein said one or more other antibodies bind to CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, galectin-9, CEACAM1, BTLA, CD69, galectin-1 , TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-4, B7-1, B7-2, CD28, 4-1BB (CD137 ), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, DR3 or CD28H. 19. A method of inducing or enhancing an immune response against an antigen in a subject, comprising administering to said subject an antibody according to any one of claims 1-7, a molecular conjugate according to claim 8, a composition according to any one of claims 15-18, or a bispecific molecule according to claim 9 in an amount effective to induce or enhance an immune response against the antigen. 20. The method of claim 19, further comprising the step of administering an antigen, wherein said antigen is administered simultaneously, separately, or sequentially with respect to administration of said antibody, composition, or bispecific molecule. 21. A method for inhibiting the growth of CD40-expressing cells, comprising bringing said cells into contact with an antibody according to any one of claims 1-7, a composition according to any one of claims 15-18, or a bispecific molecule according to claim 9 in an amount effective to inhibit reproduction CD40 expressing cells. 22. A method of inducing or enhancing an immune response to a cancer cell in a subject, comprising administering to said subject an antibody according to any one of claims 1 to 7, a composition according to any one of claims 15 to 18, or a bispecific molecule according to claim 9 in an effective amount, wherein the cancer selected from the group consisting of chronic lymphoblastic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and B-cell marginal cell lymphoma. 23. The method of any one of claims 19-22, further comprising administering to the subject one or more therapeutic agents, wherein said therapeutic agent is another antibody selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-PD-L1 antibody. CTLA-4. 24. The method of claim 23, wherein said first and second antibodies are administered simultaneously or sequentially. 25. Use of an antibody according to any one of claims 1-7 to induce or enhance an immune response against an antigen in a subject. 26. The use of an antibody according to any one of claims 1 to 7 in the manufacture of a medicament for inducing or enhancing an immune response to a cancer cell.