Agonist anti CD40 antibody

Abstract

To provide an agonist anti CD40 antibody.SOLUTION: The application generally relates to identification of a specific agonist anti CD40 antibody. Based on the identification, the invention provides a novel agonist antibody, and use of the agonist antibody in therapy. The agonist anti CD40 antibody is suitable for use in various activities including coupling to CD40+DC and macrophage however not limited thereto. The antibody is combined with other immunopotentiators (for example, topical or systemic IL-2, TLR-7 agonists and / or cytotoxicity chemotherapy), therefore occurrence of strong anti-tumor CD8+ cytotoxicity T lymphocyte response can be detected.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field This application relates generally to the identification of specific agonistic anti-CD40 antibodies. Based thereon, the present invention provides novel agonistic antibodies and their use in therapy. [Background technology]

[0002] Background technology At some point during their evolution, tumors are recognized by the host immune system, but some tumors grow despite evidence of recognition, possibly because they may lack danger signals and therefore induce only a weak response, and perhaps because they develop immune evasion strategies under increasing immune pressure.

[0003] Tumors treated at an early stage of development are more likely to respond better to monoimmunotherapy. However, advanced cancers are more resistant to all forms of treatment, including immunotherapy. The lack of their danger signals, coupled with the acquisition of increasingly complex immune evasion mechanisms, poses significant obstacles to the host adaptive immune response, which relies on antigen-presenting cells (APCs) such as dendritic cells (DCs) to sample, process, and present tumor-derived antigens in the correct context with appropriate costimulatory markers to trigger relevant T cell responses. Tumor cells often do not present their own antigenic stimuli due to low expression levels of essential costimulatory molecules, such as B7 family members.

[0004] Because tumor antigens cannot be presented directly, cross-presentation may be the only natural mode of antigen presentation for tumor immunity. During this process, exogenous antigens derived from tumor cells (soluble antigens, apoptotic bodies, or live cancer cells) are taken up by DCs and, rather than simply following the classical pathway of processing and presentation in the context of MHC class II molecules to elicit CD4+ T cell help, the exogenous antigens are also internalized and presented in the context of MHC class I molecules for presentation to CD8+ T cells. However, unless the presenting DCs are also properly activated and presentation occurs in the context of appropriate costimulation, this process results in a weak response or resistance. DCs within the tumor environment are often immature and may promote the proliferation of regulatory T cells in tumor-draining lymph nodes. Their function can also be suppressed by infiltrating suppressor cells, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), as well as cytokines within the tumor and draining lymph nodes. Therefore, effective immunotherapy that "helps" DCs prime antigen-specific T cell responses must overcome these obstacles and support the cross-presentation process. Effective cross-priming requires "licensed" DCs and high levels of antigen. Licensing or "conditioning" of DCs is achieved by antigen-specific CD4+ T helper cells via CD40 cross-linking. This ligation alters DC phenotype and function, inhibiting their tolerogenic potential through autocrine signaling by cytokines such as IL-6 and IL-12, thus enabling them to activate potent cytotoxic T lymphocyte (CTL) responses. In contrast, CTLs activated by unlicensed DCs have been termed "helpless," resulting in T cell anergy or deletion and the induction of regulatory T cells. Thus, the cell surface molecule CD40, a member of the tumor necrosis factor receptor superfamily, broadly regulates immune activation and mediates tumor apoptosis.

[0005] CD40 is a transmembrane protein that is a member of the TNF receptor superfamily. CD40 is expressed by APCs, and binding of its natural ligand (CD154 or CD40L) on T helper cells and platelets activates APCs, including DCs, macrophages, and B cells.

[0006] CD40 is found on the majority of melanomas and carcinomas of the lung, breast, colon, prostate, pancreas, kidney, ovary and head and neck, as well as B-cell malignancies.

[0007] Agonistic anti-CD40 antibodies have been shown to replace the T cell help provided by CD4+ lymphocytes in a mouse model of T cell-mediated immunity. In tumor-bearing hosts, CD40 agonists induce effective immune responses against tumor-associated antigens. For example, DCs can be "preconditioned" with agonistic anti-CD40 antibodies to upregulate costimulatory markers, enabling them to activate CD8+ T cells upon encountering them. Thus, agonistic anti-CD40 antibodies can replace CD4+ T cell help by signaling CD40 on antigen-loaded DCs, leading to the upregulation of B7 costimulatory molecules and the release of IL-12, thereby endowing them with the ability to stimulate specific CTL responses.

[0008] Thus, ligation of CD40 on the surface of APCs enhances the expression of MHC and costimulatory molecules such as CD86, CD80, CD83, PD-L1, HLA-A, B, C, or HLA-DR, stimulates the production of pro-inflammatory cytokines such as IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ, and induces T cell activation, all of which are essential for cell-mediated immune responses.

[0009] Patients with germline mutations in either CD40 or CD40L are severely immunosuppressed, susceptible to opportunistic infections, and have deficient T cell-dependent immune responses, including IgG production, germinal center formation, and memory B cell induction.

[0010] In mouse models of T cell-mediated immunity, agonistic CD40 antibodies have been shown to mimic CD40L signaling and substitute for the function of CD4+ lymphocytes. Agonistic CD40 antibodies can also overcome T cell tolerance in tumor-bearing mice, elicit effective cytotoxic T cell responses, and enhance the efficacy of antitumor vaccines.

[0011] These findings contrast with CD40 ligation on the surface of tumor cells, which mediates a direct cytotoxic effect, often resulting in tumor regression via apoptosis and necrosis. Although the exact function of CD40 on tumor cells is unknown, CD40 engagement in vitro inhibits the growth of solid tumor cells and aggressive B-cell lymphoma lines. Furthermore, CD40-mediated tumor inhibition has also been observed in vivo, including the inhibition of breast carcinoma or B-cell lymphoma xenografts in immunocompromised mice.

[0012] These diverse roles for CD40 provide an opportunity for CD40 activation in tumor-bearing animals to potentially have (i) a direct cytotoxic effect on the tumor and (ii) presentation of tumor antigens to APCs that are simultaneously activated by CD40.

[0013] Agonistic monoclonal antibodies (mAbs) against CD40 have already demonstrated therapeutic activity in various preclinical models. These findings, together with the dual functionality of CD40, make it an attractive target for cancer therapy and form the rationale for the clinical development of agonistic anti-CD40 antibodies.

[0014] However, there remains a need for further agonistic monoclonal antibodies directed against human CD40. It is against this background that the present invention was developed. Summary of the Invention

[0015] Summary of the Invention The present inventors have developed novel agonistic anti-CD40 antibodies suitable for use as immunogenic agents capable of treating malignancies in patients.

[0016] The agonist anti-CD40 antibodies of the present invention (a) inhibit CD40 receptors that allow T cell trafficking; + (b) CD40 in tumors, spleens, and lymph nodes, which secrete increased levels of autoantibodies against antigens expressed on tumor cells. + B cells; (c) upregulate costimulatory markers, release IL-12, and promote CD8 + CD40, which activates T cells and stimulates specific CTL responses against cross-presented tumor antigens + The antibodies of the present invention are suitable for use in a variety of activities, including, but not limited to, binding to DCs and macrophages. Furthermore, when combined with other immune enhancing agents (e.g., local or systemic IL-2, TLR-7 agonists and / or cytotoxic chemotherapy), they can produce potent anti-tumor CD8 + The generation of a cytotoxic T lymphocyte response can be observed. Antibodies therefore represent and offer a new principle of general application in the field of neoplastic therapy.

[0017] In one aspect, the invention provides a V H and (ii) a V containing three CDRs. L and an isolated agonist anti-CD40 antibody or fragment thereof comprising a heavy chain, wherein one or more heavy chain complementarity determining regions (CDRHs) are selected from the following: a) a CDRH1 sequence comprising SEQ ID NO:1; b) a CDRH2 sequence comprising SEQ ID NO:2; c) a CDRH3 sequence comprising SEQ ID NO: 3; or d) any one of SEQ ID NOs: 1 to 3 containing one or two amino acid substitutions, deletions or insertions; is selected from the group consisting of:

[0018] In one embodiment of the first aspect of the invention, the present invention provides a V H chain and (ii) a V containing three CDRsL and an isolated agonist anti-CD40 antibody or fragment thereof comprising a heavy chain, wherein one or more heavy chain complementarity determining regions (CDRHs) are selected from the following: (a) a CDRH1 sequence comprising SEQ ID NO: 1; (b) a CDRH2 sequence comprising SEQ ID NO:2; or (c) a CDRH2 sequence comprising SEQ ID NO: 2 containing one or two amino acid substitutions, deletions, or insertions; is selected from the group consisting of:

[0019] In one embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises the heavy chain variable region of SEQ ID NO:7.

[0020] In another embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody is: a) a CDRL1 sequence comprising SEQ ID NO:4; b) a CDRL2 sequence comprising SEQ ID NO:5; or c) a CDRL3 sequence comprising SEQ ID NO: 6 and further comprising one or more light chain complementarity determining regions (CDRLs) selected from the group consisting of:

[0021] In a second aspect, the present invention provides a V H chain and (ii) a V containing three CDRs L and an isolated agonist anti-CD40 antibody or fragment thereof comprising a chain, wherein one or more CDRLs are selected from the following: a) a CDRL1 sequence comprising SEQ ID NO:4; b) a CDRL2 sequence comprising SEQ ID NO:5; or c) a CDRL3 sequence comprising SEQ ID NO: 6 d) any one of SEQ ID NOs: 4 to 6 containing one or two amino acid substitutions, deletions, or insertions; is selected from the group consisting of:

[0022] In one embodiment of the second aspect of the invention, the isolated agonist anti-CD40 antibody comprises the light chain variable region of SEQ ID NO:8.

[0023] In another embodiment of the second aspect of the invention, the isolated agonist anti-CD40 antibody is: a) a CDRH1 sequence comprising SEQ ID NO:1; b) a CDRH2 sequence comprising SEQ ID NO:2; or c) a CDRH3 sequence comprising SEQ ID NO:3 and further comprising one or more CDRHs selected from the group consisting of:

[0024] In a third aspect, the present invention provides a method for manufacturing a semiconductor device comprising: a) a CDRH1 sequence comprising SEQ ID NO:1; b) a CDRH2 sequence comprising SEQ ID NO:2; c) a CDRH3 sequence comprising SEQ ID NO:3; d) a CDRL1 sequence comprising SEQ ID NO:4; e) a CDRL2 sequence comprising SEQ ID NO:5; and f) a CDRL3 sequence comprising SEQ ID NO: 6 and an isolated agonist anti-CD40 antibody comprising:

[0025] In one embodiment of the third aspect of the invention, the isolated agonist anti-CD40 antibody comprises the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8.

[0026] In one embodiment of the above aspects of the invention, the agonist anti-CD40 antibody can be a murine antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment thereof.

[0027] In one embodiment, the isolated agonist anti-CD40 antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single chain antibody molecule.

[0028] In one embodiment, the isolated agonist anti-CD40 antibody is a humanized antibody.

[0029] In one embodiment, the isolated agonist anti-CD40 antibody is a monoclonal antibody.

[0030] In one embodiment, the isolated agonist anti-CD40 antibody is of the IgG1, IgG2a, IgG2b, IgG3, or IgG4 type. Preferably, the isolated agonist anti-CD40 antibody is of the IgG1 type.

[0031] In one embodiment, the isolated agonist anti-CD40 antibody is coupled to a labeling group.

[0032] In one embodiment, the isolated agonist anti-CD40 antibody enhances CD40 activity.

[0033] In one embodiment, the invention includes a nucleic acid molecule encoding an isolated agonist anti-CD40 antibody described herein.

[0034] In one embodiment, the invention includes a vector comprising a nucleic acid molecule described herein.

[0035] In one embodiment, the invention includes a host cell comprising a nucleic acid molecule described herein.

[0036] In one embodiment, the invention includes an isolated agonist anti-CD40 antibody suitable at least to accomplish at least one or more of the following functions: a) Promoting the secretion of autoantibodies by B cells against antigens expressed on tumor cells; b. Upregulating costimulatory markers and releasing IL-12 to activate CD8+ T cells and stimulate specific cytotoxic T cell responses against cross-presented tumor antigens; c.Increased antigen presentation by APCs (including macrophages, DCs, and B cells); d. enhancing the expression of MHC and immune co-stimulatory molecules (e.g., CD86, CD80, CD83, PD-L1, HLA-A, B, C, or HLA-DR); e. Stimulation of the production of pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ); or fInduction of T cell activation; g. Mimicking the signal of CD40L and substituting the function of CD4+ lymphocytes; h. Overcoming T cell tolerance in tumor-bearing subjects; i. eliciting an effective cytotoxic T cell response; j. enhancing the efficacy of anti-tumor vaccines; or k. Making tumor vasculature more permissive to immune infiltration.

[0037] In one embodiment, the invention is a pharmaceutical composition comprising at least one isolated agonist anti-CD40 antibody described herein. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0038] In one embodiment, the pharmaceutical composition may further comprise an active agent such as a radioisotope, radionuclide, toxin, therapeutic group or chemotherapeutic group.

[0039] In another aspect, the invention resides in a method of making an agonist anti-CD40 antibody described herein, comprising preparing the agonist anti-CD40 antibody from a host cell that secretes the agonist anti-CD40 antibody.

[0040] In yet another aspect, the invention resides in a method for treating or preventing symptoms associated with a malignancy in a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0041] In one embodiment, the present invention includes a method for increasing antigen presentation by APCs (including macrophages, DCs, and B cells) in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0042] In one embodiment, the present invention includes a method of activating antigen-presenting cells in a subject comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0043] In one embodiment, the invention includes a method of enhancing expression of MHC and / or immune costimulatory molecules in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein. Preferably, the MHC and / or immune costimulatory molecules are selected from CD80, CD86, PD-L1, HLA-A, B, C, HLA-DR, and CD83.

[0044] In one embodiment, the invention includes a method of stimulating the production of pro-inflammatory cytokines in a subject, comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein. Preferably, the pro-inflammatory cytokines are selected from the list of IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ.

[0045] In one embodiment, the invention includes a method of inducing T cell activation in a subject comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein.

[0046] In one embodiment, the invention includes a method of mimicking CD40L signaling and substituting for CD4+ lymphocyte function in a subject, comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein.

[0047] In one embodiment, the present invention includes a method for overcoming T cell tolerance in a tumor-bearing animal, or inducing an effective cytotoxic T cell response, or enhancing the effectiveness of an anti-tumor vaccine in a subject, comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein.

[0048] In one embodiment, the invention includes a method of promoting the secretion of autoantibodies by B cells against antigens expressed on tumor cells in a subject, comprising administering an effective amount of an agonist anti-CD40 antibody disclosed herein.

[0049] In one embodiment, the invention includes a method of upregulating costimulatory markers, releasing IL-12 to activate CD8+ T cells, and stimulating a specific cytotoxic T cell response against cross-presented tumor antigens in a subject, comprising administering an effective amount of an agonist anti-CD40 antibody disclosed herein.

[0050] The complex coordination of events required for tumor eradication suggests that combination therapeutic approaches may also be beneficial in some situations. Agonistic anti-CD40 antibodies could be used with additional arms to release antigens, promote cytokine release, increase immune surveillance, and decrease inhibitory networks to enhance this effect.

[0051] Thus, in one embodiment, the present invention includes a pharmaceutical formulation comprising an effective amount of at least one agonist anti-CD40 antibody disclosed herein together with one or more additional immune enhancing agents, including, but not limited to, IL-2, a TLR-7 agonist, or a systemic cytotoxic chemotherapeutic agent.

[0052] In another embodiment, the present invention includes a method for treating or preventing symptoms associated with a malignancy in a patient, comprising administering to a patient in need thereof an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein and an effective amount of at least a second immune enhancing agent.

[0053] One therapeutic option is to alter the tumor microenvironment itself, encouraging the tumor to act as its own source of antigenic stimulation. This can be achieved by introducing IL-2 with an anti-CD40 antibody into the tumor site. When co-injected directly, this co-administration can successfully induce regression of larger tumors as well as distant tumors while avoiding the toxicity associated with systemic administration and maintaining long-term protective memory. Co-administration of IL-2 and a CD40 agonist can result in increased macrophage activity and B cell activation. Therefore, the combination of IL-2 and a CD40 agonist can demonstrate significant benefits against a variety of cancers whose regression is associated with a neutrophil-dominated inflammatory response.

[0054] Thus, in one embodiment, the present invention includes a pharmaceutical composition comprising at least one agonist anti-CD40 antibody described herein and IL-2.

[0055] In one embodiment, the present invention comprises a method for treating or preventing symptoms associated with a malignancy in a patient, comprising administering to a patient in need thereof an effective amount of each of at least one agonist anti-CD40 antibody described herein and IL-2.

[0056] In one embodiment, the present invention includes a method for increasing antigen presentation by APCs (including macrophages, DCs, and B cells) in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody disclosed herein and IL-2.

[0057] In one embodiment, the present invention includes a method of activating antigen-presenting cells in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein and IL-2.

[0058] In one embodiment, the invention includes a method of enhancing expression of MHC and / or immune costimulatory molecules in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody disclosed herein and IL-2. Preferably, the MHC and / or immune costimulatory molecules are selected from CD80, CD86, PD-L1, HLA-A, B, C, HLA-DR, and CD83.

[0059] In one embodiment, the present invention includes a method of stimulating the production of pro-inflammatory cytokines in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody disclosed herein and IL-2. Preferably, the pro-inflammatory cytokines are selected from the list of IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ.

[0060] In one embodiment, the invention includes a method of inducing T cell activation in a subject comprising administering an effective amount of each of at least one agonist anti-CD40 antibody described herein and IL-2.

[0061] In one embodiment, the invention includes a method of mimicking CD40L signaling and substituting for CD4+ lymphocyte function in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody described herein and IL-2.

[0062] In one embodiment, the invention includes a method for overcoming T cell tolerance in a tumor-bearing animal, or inducing an effective cytotoxic T cell response, or enhancing the effectiveness of an anti-tumor vaccine in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody described herein and IL-2.

[0063] In one embodiment, the invention includes a method of promoting the secretion of autoantibodies by B cells against antigens expressed on tumor cells in a subject, comprising administering an effective amount of an agonist anti-CD40 antibody disclosed herein and IL-2.

[0064] In one embodiment, the invention includes a method of upregulating costimulatory markers, releasing IL-12 to activate CD8+ T cells, and stimulating a specific cytotoxic T cell response against cross-presented tumor antigens in a subject, comprising administering an effective amount of an agonist anti-CD40 antibody disclosed herein and IL-2.

[0065] The most effective method for treating or preventing symptoms associated with a malignancy in a patient may require a combination therapeutic approach in which therapeutic interventions are administered sequentially over time.

[0066] Thus, in one embodiment, the present invention includes a method for treating or preventing symptoms associated with a malignancy in a patient, comprising sequentially administering to a patient in need thereof an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein with an additional therapeutic intervention, which in some embodiments is selected from the group consisting of surgery, radiation therapy, chemotherapy, hyperthermia, and immunotherapy.

[0067] One treatment option is to modify cells isolated from a patient and then return these cells to the patient. This can be achieved by treating the cells isolated from the patient with an anti-CD40 antibody. The cells treated with the anti-CD40 antibody can be treated with an additional agent. In some embodiments, the agent is selected from the group consisting of tumor-specific peptides, tumor cell lysates, cytokines, agonists, and mitogens.

[0068] Thus, in one embodiment, the present invention comprises a method for treating or preventing symptoms associated with a malignancy in a patient, comprising administering to a patient in need thereof an effective amount of cells treated with at least one isolated agonist anti-CD40 antibody disclosed herein. In some embodiments, the cells are isolated from the patient and are selected from the group consisting of DCs, macrophages, B cells, myeloid cells, lymphoid cells, and hematopoietic stem cells.

[0069] Additional objects, advantages and novel features will be described in the following description or will become apparent to those skilled in the art upon examination of the drawings and the following detailed description of several non-limiting embodiments.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS The present disclosure provides details in the following description of preferred embodiments with reference to the following drawings: [Brief explanation of the drawings]

[0071] [Figure 1] FIG. 1 shows the nucleotide sequence of the heavy chain of a humanized agonistic anti-human CD40 antibody.

[0072] [Figure 2] FIG. 2 shows the nucleotide sequence of the light chain of a humanized agonistic anti-human CD40 antibody.

[0073] [Figure 3] FIG. 3 shows the nucleotide and amino acid sequences of the heavy chain variable region (VH) of antibody SVX-3001.

[0074] [Figure 4] FIG. 4 shows the nucleotide and amino acid sequences of the light chain variable region (VL) of antibody SVX-3001.

[0075] [Figure 5]FIG. 5 shows the nucleotide and amino acid sequences of the synthetic gene sequence encoding the heavy chain of antibody SVX-3001 that was cloned into the plasmid pcDNA3.1(+) to produce the plasmid pcDNA3.1(+)_Selvax01HC.

[0076] [Figure 6] FIG. 6 shows the nucleotide and amino acid sequences of the synthetic gene sequence encoding the light chain of antibody SVX-3001 that was cloned into the plasmid pcDNA3.1(+) to produce the plasmid pcDNA3.1(+)_Selvax01LC.

[0077] [Figure 7] FIG. 7 shows the plasmid map of pcDNA3.1(+)_Selvax01HC.

[0078] [Figure 8] FIG. 8 shows the plasmid map of pcDNA3.1(+)_Selvax01LC.

[0079] [Figure 9] FIG. 9 shows the results of an ELISA assay demonstrating that the antibodies produced were CD40-specific IgG.

[0080] [Figure 10-1] FIG. 10 shows the results of a FACS analysis detecting the binding of SVX-3001 on cells expressing CD40. [Figure 10-2] FIG. 10 shows the results of a FACS analysis detecting the binding of SVX-3001 on cells expressing CD40. [Figure 10-3] FIG. 10 shows the results of a FACS analysis detecting the binding of SVX-3001 on cells expressing CD40. [Figure 10-4] FIG. 10 shows the results of a FACS analysis detecting the binding of SVX-3001 on cells expressing CD40.

[0081] [Figure 11-1]FIG. 11 shows the results of a CFSE assay to detect cell division in human PBMCs in response to stimulation with SVX-3001. [Figure 11-2] FIG. 11 shows the results of a CFSE assay to detect cell division in human PBMCs in response to stimulation with SVX-3001. [Figure 11-3] FIG. 11 shows the results of a CFSE assay to detect cell division in human PBMCs in response to stimulation with SVX-3001. [Figure 11-4] FIG. 11 shows the results of a CFSE assay to detect cell division in human PBMCs in response to stimulation with SVX-3001. [Figure 11-5] FIG. 11 shows the results of a CFSE assay to detect cell division in human PBMCs in response to stimulation with SVX-3001.

[0082] [Figure 12-1] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2. [Figure 12-2] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2. [Figure 12-3] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2. [Figure 12-4] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2. [Figure 12-5] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2. [Figure 12-6] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2. [Figure 12-7] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2. [Figure 12-8] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2. [Figure 12-9] FIG. 12 shows the results of a LEGENDplex assay to detect cytokine production from human PBMCs in response to stimulation with SVX-3001 with or without IL-2.

[0083] [Figure 13-1] FIG. 13 shows the results of a FACS assay determining the ability of SVX-3001 to block the binding of antibodies B-B20 and LOB7 / 6 to CD40. [Figure 13-2] FIG. 13 shows the results of a FACS assay determining the ability of SVX-3001 to block the binding of antibodies B-B20 and LOB7 / 6 to CD40. [Figure 13-3] FIG. 13 shows the results of a FACS assay determining the ability of SVX-3001 to block the binding of antibodies B-B20 and LOB7 / 6 to CD40. [Figure 13-4] FIG. 13 shows the results of a FACS assay determining the ability of SVX-3001 to block the binding of antibodies B-B20 and LOB7 / 6 to CD40. [Figure 13-5] FIG. 13 shows the results of a FACS assay determining the ability of SVX-3001 to block the binding of antibodies B-B20 and LOB7 / 6 to CD40.

[0084] [Figure 14-1]FIG. 14 shows the Biacore T200 sensogram for epitope mapping of SVX-3001 with several different CD40 antibodies. [Figure 14-2] FIG. 14 shows the Biacore T200 sensogram for epitope mapping of SVX-3001 with several different CD40 antibodies. [Figure 14-3] FIG. 14 shows the Biacore T200 sensogram for epitope mapping of SVX-3001 with several different CD40 antibodies. [Figure 14-4] FIG. 14 shows the Biacore T200 sensogram for epitope mapping of SVX-3001 with several different CD40 antibodies. [Figure 14-5] FIG. 14 shows the Biacore T200 sensogram for epitope mapping of SVX-3001 with several different CD40 antibodies. [Figure 14-6] FIG. 14 shows the Biacore T200 sensogram for epitope mapping of SVX-3001 with several different CD40 antibodies.

[0085] [Figure 15-1] FIG. 15 shows the results of a FACS assay determining activation of monocyte-derived dendritic cells (moDCs) by SVX-3001. [Figure 15-2] FIG. 15 shows the results of a FACS assay determining activation of monocyte-derived dendritic cells (moDCs) by SVX-3001.

[0086] [Figure 16-1] FIG. 16 shows the results of a FACS assay determining the dose response of monocyte-derived dendritic cells (moDCs) to SVX-3001. [Figure 16-2] FIG. 16 shows the results of a FACS assay determining the dose response of monocyte-derived dendritic cells (moDCs) to SVX-3001. [Figure 16-3]FIG. 16 shows the results of a FACS assay determining the dose response of monocyte-derived dendritic cells (moDCs) to SVX-3001. [Figure 16-4] FIG. 16 shows the results of a FACS assay determining the dose response of monocyte-derived dendritic cells (moDCs) to SVX-3001.

[0087] [Figure 17] FIG. 17 shows the nucleotide sequence of the synthetic gene sequence encoding the heavy chain of antibody SVX-3001 that was cloned into the plasmid pcDNA3.4-TOPO to produce the plasmid 20ACGJQC_Selvax01HC-pcDNA3.4-TOPO.

[0088] [Figure 18] FIG. 18 shows the nucleotide and amino acid sequences of the synthetic gene sequence encoding the light chain of antibody SVX-3001 that was cloned into the plasmid pcDNA3.4-TOPO to produce the plasmid 20ACGJRC_Selvax01LC-pcDNA3.4-TOPO.

[0089] [Figure 19] FIG. 19 shows the plasmid map of 20ACGJQC_Selvax01HC-pcDNA3.4-TOPO.

[0090] [Figure 20] Figure 20 shows the plasmid map of 20ACGJRC_Selvax01LC-pcDNA3.4-TOPO. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] DETAILED DESCRIPTION OF THE INVENTION

[0091] Detailed Description of the Invention The present invention relates, inter alia, to isolated agonistic anti-human CD40 antibodies that can increase antigen presentation by APCs (including macrophages, DCs, and B cells). In certain embodiments, the antibodies provide a method for enhancing the expression of MHC and / or immune costimulatory molecules. They can also stimulate the production of pro-inflammatory cytokines and induce T cell activation. They do so by mimicking the signaling of CD40L and substituting for the function of CD4+ lymphocytes. In doing so, the isolated agonistic anti-CD40 antibodies described herein can improve T cell tolerance in tumor-bearing animals, induce effective cytotoxic T cell responses, and / or enhance the efficacy of anti-tumor vaccines.

[0092] For convenience, the following section generally outlines the various meanings of terms used herein. Following this discussion, general aspects relating to agonistic anti-CD40 antibodies are discussed, followed by specific examples illustrating the properties of various embodiments of the antibodies and how they may be used. definition

[0093] The present invention is not limited in scope by the following specific embodiments. This detailed description is for illustrative purposes only. Functionally equivalent products, compositions, and methods are within the scope of the invention described herein. Consistent with this position, those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations or any two or more steps or features.

[0094] In this application, the use of the singular includes the plural unless otherwise stated. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limiting. Also, terms such as "element" or "component" encompass both elements and components that contain one unit and elements and components that contain more than one subunit, unless otherwise stated. Also, the use of the term "portion" can include a part of a moiety or the entire portion.

[0095] The term "antibody" refers to an intact immunoglobulin of any isotype or a fragment thereof capable of competing with the intact antibody for specific binding to a target antigen, including, for example, chimeric antibodies and bispecific antibodies. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, but in some instances may include naturally occurring antibodies in camelids, which may contain fewer chains, such as only heavy chains. Antibodies may be derived from a single source or may be "chimeric," i.e., different portions of the antibody may be derived from two different antibodies, as described further below. Antibodies or binding fragments may be produced in hybridomas, by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below. Additionally, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0096] Antibody heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses, including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically connected by a "J" region of about 12 or more amino acids, with heavy chains also containing a "D" region of about 10 additional amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form the antigen-binding site.

[0097] Variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. From the N-terminus to the C-terminus, both light chain and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is typically based on the Kabat Sequences of Proteins of Immunological Interest definition (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or according to Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).

[0098] In certain embodiments, an antibody heavy chain binds to an epitope in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an epitope in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an epitope in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an epitope in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an epitope in the absence of other variable regions.

[0099] In certain embodiments, the delineation of CDRs and the identification of residues comprising the antibody binding site are achieved by solving the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be used to identify or approximate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0100] By convention, the CDR regions in the heavy chain are typically referred to as H1, H2, and H3, numbered consecutively from the amino terminus to the carboxy terminus, and the CDR regions in the light chain are typically referred to as L1, L2, and L3, numbered consecutively from the amino terminus to the carboxy terminus.

[0101] The term "light chain" includes full-length light chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length light chain contains the variable region domain V L and constant region domain C L The variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0102] The term "heavy chain" includes full-length heavy chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length heavy chain contains the variable region domain V H, as well as three constant region domains C H 1. C H 2 and C H Includes 3. V H The domain is located at the amino terminus of the polypeptide and is C H The domain is located at the carboxyl terminus and is C H 3 is closest to the carboxy terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0103] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including, but not limited to, hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0104] Individual immunoglobulin chains typically consist of several "immunoglobulin domains," each consisting of approximately 90-110 amino acids and with a characteristic folding pattern. These domains are the basic units from which antibody polypeptides are constructed. In humans, IgA and IgD isotypes contain four heavy chains and four light chains; IgG and IgE isotypes contain two heavy chains and two light chains; and IgM isotypes contain five heavy chains and five light chains. The heavy chain C region typically contains one or more domains that can be responsible for effector functions. The number of heavy chain constant region domains depends on the isotype. IgG heavy chains, for example, contain C H 1. C H 2 and C H 3. The antibodies provided can have any of these isotypes and subtypes.

[0105] The term "variable region" or "variable domain" refers to a portion of an antibody's light and / or heavy chain, typically comprising approximately the amino-terminal 120-130 amino acids of the heavy chain and about 100-110 amino-terminal amino acids of the light chain. In certain embodiments, the variable regions of different antibodies vary widely in amino acid sequence, even among antibodies of the same species. The variable regions of an antibody typically determine the specificity of a particular antibody for its target.

[0106] The term "epitope" includes any determinant capable of binding by an antibody or a T-cell receptor. An epitope is a region that engages with an antibody or the T-cell receptor that targets the epitope; if the epitope is part of a protein, it includes specific amino acids that directly contact the antibody or T-cell receptor. In most cases, epitopes reside on proteins, but in some instances, they can reside on other types of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural and / or charge characteristics. Generally, antibodies specific for a particular target epitope preferentially recognize that epitope on the target in a complex mixture of proteins and / or macromolecules.

[0107] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate.

[0108] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotides. In some embodiments, oligonucleotides are 10-60 bases in length. In other embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, for example, for use in constructing mutant genes. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides can contain labels, including radioactive labels, fluorescent labels, hapten labels, or antigen labels, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers, or hybridization probes.

[0109] By "isolated nucleic acid molecule" is meant DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or any combination thereof, where the isolated polynucleotide is not related to all or a portion of a polynucleotide found in nature or is linked to a polynucleotide with which it is not linked in nature. An isolated nucleic acid molecule "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include coding sequences for up to 10, or even up to 20, other proteins or portions thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0110] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; the region of the sequence on the DNA strand that has the same sequence as an RNA transcript that is 5'-terminal to the 5'-end of the RNA transcript is referred to as the "upstream sequence"; and the region of the sequence on the DNA strand that has the same sequence as an RNA transcript that is 3'-terminal to the 3'-end of the RNA transcript is referred to as the "downstream sequence."

[0111] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend on the host organism. In certain embodiments, prokaryotic control sequences can include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, eukaryotic control sequences can include a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence, and a transcription termination sequence. A "control sequence" can include a leader sequence and / or a fusion partner sequence.

[0112] The term "vector" refers to any molecule or entity (eg, nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell.

[0113] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that (in conjunction with the host cell) direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when introns are present, affect RNA splicing of the coding region operably linked thereto.

[0114] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under appropriate conditions. For example, a control sequence in a vector "operably linked" to a protein-coding sequence is ligated such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0115] The term "host cell" means a cell that has been transformed or is capable of being transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present.

[0116] As used herein, the term "MHC" includes reference to both MHC class I and MHC class II molecules unless otherwise specified.

[0117] The term "immune costimulatory molecule" includes cell surface molecules that act to amplify or counteract the initial activation signal provided by the T cell receptor (TCR) to T cells after interaction with antigen or MHC. Examples of such molecules include CD86, CD80, CD83, PD-L1, HLA-A, B, C, and HLA-DR.

[0118] The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell; a cell is "transfected" when exogenous DNA is introduced inside the cell membrane. Several transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al. See, e.g., Chu et al., 1981, Gene 13:197; Chu et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Using such techniques, one or more exogenous DNA moieties can be introduced into a suitable host cell.

[0119] The term "transformation" refers to a change in the genetic characteristics of a cell; a cell is transformed when it has been modified to contain new DNA or RNA. For example, a cell is genetically modified and transformed from its native state by introducing new genetic material by transfection, transduction, or other techniques. After transfection or transduction, the transforming DNA can recombine with the cell's DNA by being physically integrated into the cell's chromosomes, or it can be maintained transiently as an episomal element without replication, or it can replicate independently as a plasmid. A cell is considered "stably transformed" if the transforming DNA is replicated as the cell divides.

[0120] The terms "polypeptide" and "protein" refer to polymers having the amino acid sequence of a native protein, i.e., a protein produced by naturally occurring non-recombinant cells; or molecules produced by genetically engineered or recombinant cells and having the amino acid sequence of a native protein, or molecules having one or more amino acid deletions, additions, and / or substitutions of the native sequence. The terms also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. The terms "polypeptide" and "protein" specifically encompass sequences of agonist anti-CD40 antibodies or antigen-binding proteins having one or more amino acid deletions, additions, and / or substitutions. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. In certain embodiments, fragments are between about 5 and 500 amino acids in length. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies. In the case of agonist anti-CD40 antibodies, useful fragments include, but are not limited to, CDR regions, heavy and / or light chain variable domains, portions of antibody chains containing two CDRs, or only the variable regions thereof, etc.

[0121] The term "isolated protein" means that the protein of interest (1) is free from at least some other proteins with which it is normally found; (2) is essentially free from other proteins from the same source, e.g., the same species; (3) is expressed by cells from a different species; (4) is separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) is operatively associated (by covalent or noncovalent interactions) with polypeptides with which it is not naturally associated; or (6) does not exist in nature. Typically, an "isolated protein" comprises at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof, can encode such an isolated protein. Preferably, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other uses.

[0122] The term "amino acid" includes its ordinary meaning in the art.

[0123] A "variant" of a polypeptide (e.g., an antigen-binding protein, or an antibody) comprises an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted into the amino acid sequence compared to another polypeptide sequence. Variants include fusion proteins.

[0124] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: Stockton Press; and Examples include those described in Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0125] When calculating percent identity, the sequences to be compared are typically aligned to maximize the sequence identity. One example of a computer program that can be used to determine percent identity is the GCG program package, including GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acids or nucleotides (the "matched span" determined by the algorithm). A gap opening penalty (calculated as 3 times the average diagonal, where "average diagonal" is the average of the diagonal lines of the comparison matrix used; "diagonal" is the score or number assigned to each perfect amino acid match by a specific comparison matrix) and a gap extension penalty (usually 1 / 10 times the gap opening penalty), and a comparison matrix such as PAM 250 or BLOSUM 62 are used with the algorithm. In certain embodiments, standard comparison matrices (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919) are also used by the algorithm.

[0126] Examples of parameters that can be used in determining percent identity of a polypeptide or nucleotide sequence using the GAP program are as follows: a. Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453 b. Comparison matrix: BLOSUM 62 from Henikoff et al., 1992 (ibid.) c. Gap penalty: 12 (but no penalty for end gaps) d. Gap length penalty: 4 e. Similarity threshold: 0

[0127] Certain alignment schemes for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this small aligned region may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Thus, the selected alignment method (GAP program) can be adjusted, if desired, to result in alignment over at least 50 or other numbers of consecutive amino acids of the target polypeptide.

[0128] As used herein, the 20 conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids, such as -, -disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include 4-hydroxyproline, -carboxyglutamate, -N,N,N-trimethyllysine, -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0129] Similarly, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction; the region of the sequence on the DNA strand that has the same sequence as the RNA and is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence"; and the region of the sequence on the DNA strand that has the same sequence as the RNA and is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0130] Conservative amino acid substitutions can include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

[0131] The entire disclosures of all publications cited herein (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) are incorporated herein by reference. No reference is admitted to constitute prior art or to be part of the common general knowledge of those working in the field to which this invention pertains.

[0132] Naturally occurring residues can be divided into classes based on common side chain properties: a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues that influence chain orientation: Gly, Pro; and f. Aromatics: Trp, Tyr, Phe.

[0133] For example, non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class. Such substituted residues can be introduced, for example, into regions of the human antibody that are homologous with the non-human antibody, or into the non-homologous regions of the molecule.

[0134] According to certain embodiments, modifications to agonist anti-CD40 antibodies can be made taking into account the hydropathic index of amino acids. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0135] The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is understood in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be substituted for other amino acids with similar hydropathic indices or scores and still retain similar biological activity. In certain embodiments, modifications based on hydropathic index include substitutions of amino acids with hydropathic indices within ±2. In certain embodiments, those within ±1 are included, and in certain embodiments, those within ±0.5 are included.

[0136] It is also understood in the art that substitutions of like amino acids can be made effectively on the basis of hydrophilicity, particularly when the biologically functional proteins or peptides thereby generated are intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0137] These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5), and tryptophan (-3.4). When making changes based on similar hydrophilicity values, certain embodiments include substitutions of amino acids whose hydrophilicity values ​​are within ±2, certain embodiments include those within ±1, and certain embodiments include those within ±0.5. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also called "epitope core regions."

[0138] Exemplary amino acid substitutions are shown in Table 2. [Table 2]

[0139] The term "derivative" refers to a molecule that contains a chemical modification other than an amino acid (or nucleic acid) insertion, deletion, or substitution. In certain embodiments, a derivative includes a covalent modification, including, but not limited to, chemical conjugation with a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, a chemically modified antigen-binding protein may have a greater circulating half-life than an antigen-binding protein that is not chemically modified. In certain embodiments, a chemically modified antigen-binding protein may have improved targeting ability to desired cells, tissues, and / or organs. In some embodiments, a derivative antigen-binding protein is covalently modified to include one or more water-soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In certain embodiments, the derivative antigen binding protein comprises one or more polymers including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, and mixtures of such polymers.

[0140] In certain embodiments, the derivative is covalently modified with polyethylene glycol (PEG) subunits. In certain embodiments, one or more water-soluble polymers are attached to one or more specific positions of the derivative, for example, the amino terminus. In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In certain embodiments, PEG is used to improve the therapeutic potential of an antigen-binding protein. In certain embodiments, PEG is used to improve the therapeutic potential of a humanized antibody. Certain such methods are described, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0141] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics." Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber & Freidinger, TINS, p. 392 (1985); and Evans et al., J. Med. Chem., 30:1229 (1987) (incorporated herein by reference for any purpose). Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce similar therapeutic or prophylactic effects. In general, peptidomimetics are structurally similar to paradigmatic polypeptides (i.e., polypeptides with biochemical properties or pharmacological activity), such as human antibodies, but have one or more peptide bonds optionally replaced by bonds selected from --CH2NH--, --CH2S--, --CH2-CH2--, --CH=CH-(cis and trans), --COCH2--, --CH(OH)CH2--, and --CH2SO--, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used in certain embodiments to generate more stable peptides. Furthermore, constrained peptides containing a consensus sequence or substantially identical consensus sequence variants can be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein by reference for any purpose); for example, by adding internal cysteine ​​residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[0142] The term "naturally occurring" as used throughout this specification in reference to biological material, such as a polypeptide, nucleic acid, host cell, etc., refers to a material that is found in nature or a form of a material that is found in nature.

[0143] The term "agonist" refers to a compound that can combine with a receptor to produce a cellular response. An agonist can be a ligand that binds directly to the receptor. Alternatively, an agonist can bind indirectly to the receptor, for example, by (a) forming a complex with another molecule that binds directly to the receptor, or (b) otherwise causing the modification of another compound so that the other compound binds directly to the receptor. Agonists are sometimes referred to as agonists of a particular receptor or receptor family (e.g., TNF or TNFR agonists).

[0144] The term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antibody refers to a species of antibody that contains a portion of the antibody (regardless of how the portion is obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically functioning as an agonist of CD40. These biologically active fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of antigen-binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, diabodies (a heavy chain variable domain on the same polypeptide connected with a light chain variable domain via a short peptide linker that is too short to allow pairing between the two domains on the same chain), Fab', F(ab'), Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian source, including, but not limited to, human, mouse, rat, camelid, or rabbit. It is further contemplated that a functional portion of the agonist anti-CD40 antibodies disclosed herein, e.g., one or more CDRs, can be covalently linked to a second protein or small molecule to create a therapeutic agent with bifunctional therapeutic properties directed against a specific target in the body or with a long serum half-life. As will be understood by those skilled in the art, agonist anti-CD40 antibodies can include non-proteinaceous components.

[0145] In certain embodiments, the polypeptide structure of the agonist anti-CD40 antibody is based on an antibody, including, but not limited to, a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, a synthetic antibody (sometimes referred to herein as an "antibody mimetic"), a chimeric antibody, a humanized antibody, an antibody fusion (sometimes referred to herein as an "antibody conjugate"), and a fragment thereof, respectively.

[0146] The "Fc" region is the C H 1 and C H The two heavy chain fragments contain two or more disulfide bonds and a C H The three domains are held together by hydrophobic interactions.

[0147] A "Fab fragment" is a fragment of one light chain and one heavy chain. H 1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0148] A "Fab' fragment" contains one light chain and a VH domain and C H 1 domain and C H 1 Domain and C H and a portion of one heavy chain, including the region between the two domains, such that interchain disulfide bonds can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0149] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0150] A "single-chain antibody" is an Fv molecule in which the heavy and light chain variable regions are linked by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0151] A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V H The two V domains of a bivalent domain antibody are covalently linked with a peptide linker to create a bivalent domain antibody. H The regions can target the same or different antigens.

[0152] As used herein, "substantially pure" means that the recited molecular species is the predominant species present, i.e., more abundant on a molar basis than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the target species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition comprises at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, the target species is purified to essential homogeneity, such that contaminating species cannot be detected in the composition by conventional detection methods, and thus the composition consists of a single detectable macromolecular species.

[0153] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.

[0154] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or by attachment of a biotin moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In certain embodiments, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g.,3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In certain embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0155] The term "therapeutically effective amount" refers to the amount of an agonist anti-CD40 antibody determined to produce a therapeutic response in a mammal, preferably a human. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.

[0156] As used herein, the term "pharmaceutical composition" (or agent or drug) refers to a chemical compound, composition, agent, or drug that is capable of inducing a desired therapeutic effect when properly administered to a patient, without necessarily requiring more than one component.

[0157] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0158] Other definitions of selected terms used herein may be found in the detailed description of the invention and may be applied throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0159] The inventions described herein may include one or more ranges of values ​​(e.g., size, displacement, field strength, etc.). A range of values ​​is understood to include all values ​​within the range, including the values ​​defining the range and values ​​immediately adjacent to the range that produce the same or substantially the same results as the values ​​defining the boundaries of the range. For example, one skilled in the art will understand that a 10% variation of the upper or lower limit of a range may be entirely appropriate and is encompassed by the present invention. More specifically, a variation of the upper or lower limit of a range may be 5%, or whichever is greater, as generally recognized in the art.

[0160] Throughout this specification, relative terms such as the words "about" and "approximately" are used. This language seeks to incorporate a variability of at least 10% for a specified number or range. The variance may be plus or minus 10% of the particular number specified. Embodiment A. Agonistic anti-CD40 antibodies

[0161] Agonist anti-CD40 antibodies of the invention, when administered in an effective amount, can modulate at least one or more of the following functions: a. Increased antigen presentation by APCs (including macrophages, DCs, and B cells); or b. enhancing the expression of MHC and immune co-stimulatory molecules (e.g., CD86, CD80, CD83, PD-L1, HLA-A, B, C, or HLA-DR); or c. Stimulation of the production of pro-inflammatory cytokines (e.g., IL-12, IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ); or Induction of dT cell activation; or e. Mimicking the signaling of CD40L and substituting for the function of CD4+ lymphocytes; or f. Overcoming T cell tolerance in tumor-bearing animals; or g. Inducing an effective cytotoxic T cell response; or h. enhancing the efficacy of anti-tumor vaccines; or i. Making the tumor vasculature more permissive to immune infiltration.

[0162] In some embodiments, the agonist anti-CD40 antibodies provided are polypeptides comprising one or more CDRs described herein. In some agonist anti-CD40 antibodies, the CDRs are embedded within "framework" regions that orient the CDRs to achieve the appropriate binding properties of the CDRs.

[0163] The antibodies of the present invention disclosed herein have a variety of utilities. They can be used in a variety of therapeutic applications, as described herein. For example, in some embodiments, agonistic anti-CD40 antibodies are useful for treating malignant conditions, including, but not limited to, when administered in conjunction with other agents, such as IL-2. Other uses for agonistic anti-CD40 antibodies include, for example, diagnosing disease states or symptoms and screening assays for determining the presence or absence of CD40. Some of the agonistic anti-CD40 antibodies described herein are also useful in treating consequences, symptoms, and / or pathologies associated with increased antigen presentation.

[0164] In one aspect, the invention includes an isolated agonist anti-CD40 antibody or fragment thereof comprising: (i) a VH chain comprising three CDRs; and (ii) a VL chain comprising three CDRs, wherein one or more heavy chain complementarity determining regions (CDRHs) are selected from the following: a) a CDRH1 sequence comprising SEQ ID NO:1; b) a CDRH2 sequence comprising SEQ ID NO:2; or c) a CDRH2 sequence comprising SEQ ID NO:2 containing one or two amino acid substitutions, deletions or insertions.

[0165] In some embodiments, the provided agonist anti-CD40 antibodies comprise one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs). In some embodiments, the agonist anti-CD40 antibodies comprise (a) a polypeptide structure and (b) one or more CDRs inserted into and / or linked to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, it can be or comprise a naturally occurring antibody framework or a fragment or variant thereof, or it can be completely synthetic in nature. Examples of various polypeptide structures are further described below.

[0166] In certain embodiments, the polypeptide structure of the agonist anti-CD40 antibody is or is derived from an antibody, including, but not limited to, a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, a synthetic antibody (sometimes referred to herein as an "antibody mimetic"), a chimeric antibody, a humanized antibody, an antibody fusion (sometimes referred to herein as an "antibody conjugate"), and portions or fragments of each. In some examples, the agonist anti-CD40 antibody is an immunological fragment of an antibody (e.g., a Fab, a Fab', a F(ab')2, or a scFv). The various structures are further described and defined herein.

[0167] In one embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises: a) a CDRH3 sequence comprising SEQ ID NO:3; b) a CDRL1 sequence comprising SEQ ID NO:4; c) a CDRL2 sequence comprising SEQ ID NO:5; or d) a CDRL3 sequence comprising SEQ ID NO:6 Further includes:

[0168] In a further embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises: A: a) a CDRH1 sequence comprising SEQ ID NO: 1; or b) a CDRH2 sequence comprising SEQ ID NO:2; and B: c) a CDRH3 sequence comprising SEQ ID NO:3; d) a CDRL1 sequence comprising SEQ ID NO:4; e) a CDRL2 sequence comprising SEQ ID NO:5; or f) a CDRL3 sequence comprising SEQ ID NO: 6 Includes:

[0169] In another embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises: a) a CDRH1 sequence comprising SEQ ID NO:1; b) a CDRH2 sequence comprising SEQ ID NO:2; c) a CDRH3 sequence comprising SEQ ID NO:3; d) a CDRL1 sequence comprising SEQ ID NO:4; e) a CDRL2 sequence comprising SEQ ID NO:5; and f) a CDRL3 sequence comprising SEQ ID NO: 6 Includes:

[0170] In another embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises the heavy chain variable region of SEQ ID NO: 7. In a further embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises the light chain variable region of SEQ ID NO: 8. In a further embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8.

[0171] SEQ ID NO:7 comprises the CDRH1, CDRH2, and CDRH3 sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. SEQ ID NO:8 comprises the CDRL1, CDRL2, and CDRL3 sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, an antibody comprising the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8 is known as SVX-3001.

[0172] In one embodiment, the isolated agonist anti-CD40 antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment thereof.

[0173] In one embodiment, the isolated agonist anti-CD40 antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single chain antibody molecule.

[0174] In one embodiment, the isolated agonist anti-CD40 antibody is a humanized anti-human antibody.

[0175] In one embodiment, the isolated agonist anti-CD40 antibody is a monoclonal antibody.

[0176] In one embodiment, the isolated agonist anti-CD40 antibody is of the IgG1, IgG2, IgG3, or IgG4 type. Preferably, the isolated agonist anti-CD40 antibody is of the IgG1 type.

[0177] In a particularly preferred embodiment, the isolated agonist anti-CD40 antibody comprises the heavy chain sequence of SEQ ID NO:19 and / or the light chain sequence of SEQ ID NO:20.

[0178] In one embodiment, the isolated agonist anti-CD40 antibody is coupled to a labeling group.

[0179] Other antibodies provided are variants of the above-described agonist anti-CD40 antibodies formed by combinations or subportions of variable heavy and variable light chains set forth in SEQ ID NOs: 7 and 8, each comprising a variable light chain and / or variable heavy chain (either the entire sequence or a subportion of the sequence, e.g., one or more CDRs) that have at least 50%, 50-60, 60-70, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater than 99% identity to the amino acid sequences of the sequences in SEQ ID NOs: 7 and 8. In some examples, such antibodies comprise at least one heavy chain and one light chain, while in other examples, the variants comprise two identical light chains and two identical heavy chains (or subportions thereof).

[0180] In certain embodiments, the agonist anti-CD40 antibody comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the agonist anti-CD40 antibody comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the agonist anti-CD40 antibody comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 7.

[0181] In certain embodiments, the agonist anti-CD40 antibody comprises a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the agonist anti-CD40 antibody comprises a heavy chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the agonist anti-CD40 antibody comprises a heavy chain comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 19.

[0182] In some embodiments, the agonist anti-CD40 antibody comprises a sequence at least 90%, 90-95%, and / or 95-99% identical to one or more CDRs from at least one of the sequences set forth in SEQ ID NOs: 1-3 and 4-6. In some embodiments, there are 1, 2, 3, 4, 5, or 6 CDRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0183] In certain embodiments, the agonist anti-CD40 antibody comprises a light chain comprising a variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 8. In certain embodiments, the agonist anti-CD40 antibody comprises a light chain comprising a variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO: 8. In certain embodiments, the agonist anti-CD40 antibody comprises a light chain comprising a variable region comprising an amino acid sequence at least 99% identical to SEQ ID NO: 8.

[0184] In certain embodiments, the agonist anti-CD40 antibody comprises a light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 20. In certain embodiments, the agonist anti-CD40 antibody comprises a light chain comprising an amino acid sequence at least 95% identical to SEQ ID NO: 20. In certain embodiments, the agonist anti-CD40 antibody comprises a light chain comprising an amino acid sequence at least 99% identical to SEQ ID NO: 20.

[0185] In one embodiment, the isolated agonist anti-CD40 antibody enhances CD40 activity. B. Preparation of Agonistic Anti-CD40 Antibodies

[0186] In one embodiment, the invention includes a method of making an agonist anti-CD40 antibody described herein, comprising preparing the agonist anti-CD40 antibody from a host cell that secretes the agonist anti-CD40 antibody.

[0187] Generally, a complete monoclonal agonistic antibody against CD40 can be produced as follows: Mice containing immunoglobulin genes are immunized with the CD40 of interest, and lymphocytes (such as B cells) from the mice that express the antibody are obtained. These collected cells are fused with a myeloid cell line to prepare immortal hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. In a specific embodiment, the production of hybridoma cell lines that produce agonistic antibodies specific to CD40 is provided.

[0188] In certain embodiments, monoclonal antibodies are generated using phage display technology. In certain embodiments, such technology produces monoclonal antibodies. In certain embodiments, polynucleotides encoding single Fab or Fv antibody fragments are expressed on the surface of phage particles. See, e.g., Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991); U.S. Patent No. 5,885,793. In certain embodiments, phage are "screened" to identify antibody fragments with affinity for a target. Thus, certain such processes mimic immune selection by displaying antibody fragment repertoires on the surface of filamentous bacteriophage, followed by selection of phage by target binding. In certain such procedures, high-affinity functional antibody fragments are isolated. In certain such embodiments, a complete repertoire of antibody genes is generated by cloning naturally rearranged V genes from peripheral blood lymphocytes. For example, Mullinax et al., Proc Natl Acad See Sci (USA), 87:8095-8099 (1990).

[0189] According to certain embodiments, the antibodies of the present invention are prepared by utilizing transgenic mice that have a significant portion of their genome that produces antibodies, but that are deficient in endogenous mouse antibody production. Such mice are capable of producing immunoglobulin molecules and antibodies, but are deficient in the production of mouse immunoglobulin molecules and antibodies. Techniques utilized to achieve this result are disclosed in the patents, applications, and references disclosed herein. In certain embodiments, methods such as those disclosed in PCT Published Application No. WO 98 / 24893 or Mendez et al., Nature Genetics, 15:146-156 (1997) can be used, which are incorporated herein by reference for any purpose.

[0190] In certain embodiments, agonistic antibodies specific for CD40 are produced by exposing splenocytes (B cells or T cells) to antigen in vitro and then reconstituting the exposed cells in immunocompromised mice, such as SCID or nod / SCID. See, e.g., Brams et al., J. Immunol. 160:2051-2058 (1998); Carballido et al., Nat. Med., 6:103-106 (2000). In certain such approaches, engraftment of fetal tissue into SCID mice (SCID-hu) results in long-term hematopoiesis and human T cell development. See, e.g., McCune et al., Science, 241:1532-1639 (1988); Ifversen et al., Sem. Immunol., 8:243-248 (1996). In certain instances, humoral immune responses in such chimeric mice depend on the co-generation of T cells in the animal. See, e.g., Martensson et al., Immunol., 83:1271-179 (1994). In a particular approach, peripheral blood lymphocytes are transplanted into SCID mice. See, e.g., Mosier et al., Nature, 335:256-259 (1988). In certain such embodiments, higher levels of B cell production are detected when such transplanted cells are treated with a priming agent, such as staphylococcal enterotoxin A (SEA). See, e.g., Martensson et al., Immunol., 84:224-230 (1995); Murphy et al., Blood, 86:1946-1953 (1995).

[0191] As will be understood, antibodies can be expressed in cell lines other than hybridoma cell lines. A sequence encoding a particular antibody can be used to transform a suitable mammalian host cell. Transformation can be by any known method for introducing a polynucleotide into a host cell, including, for example, packaging the polynucleotide into a virus (or viral vector) and transducing the virus (or vector) into the host cell, or by transfection procedures known in the art, such as those exemplified by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which are incorporated herein by reference). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into nuclei.

[0192] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and several other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce agonistic antibodies specific to CD40.

[0193] In certain embodiments, the agonistic CD40 antibody comprises at least one immunoglobulin molecule of an IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, the agonistic CD40 antibody comprises a human kappa light chain and / or a human heavy chain. In certain embodiments, the heavy chain is an IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, the agonistic CD40 antibody is cloned for expression in mammalian cells. In certain embodiments, the agonistic CD40 antibody comprises a constant region other than any of the constant regions of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotypes.

[0194] In certain embodiments, the agonistic CD40 antibody comprises a human lambda light chain and a human IgG2 heavy chain. In certain embodiments, the agonistic CD40 antibody comprises a human lambda light chain and a human IgG4 heavy chain. In certain embodiments, the agonistic CD40 antibody comprises a human lambda light chain and a human IgG1 heavy chain. In certain embodiments, the agonistic CD40 antibody comprises a human lambda light chain and a human IgG3, IgE, IgA, IgD, or IgM heavy chain. In other embodiments, the agonistic CD40 antibody comprises a human kappa light chain and a human IgG2 heavy chain. In certain embodiments, the agonistic CD40 antibody comprises a human kappa light chain and a human IgG4 heavy chain. In certain embodiments, the agonistic CD40 antibody comprises a human kappa light chain and a human IgG1 heavy chain. In certain embodiments, the agonistic CD40 antibody comprises a human kappa light chain and a human IgG3, IgE, IgA, IgD, or IgM heavy chain. In certain embodiments, the agonistic CD40 antibody comprises an antibody variable region linked to a constant region that is neither an IgG2 isotype nor an IgG4 isotype. Preferably, the agonistic CD40 antibody comprises human IgG1 heavy and light chains. In certain embodiments, the agonistic CD40 antibody is cloned for expression in mammalian cells.

[0195] In certain embodiments, conservative modifications to the heavy and light chains of an antibody derived from at least one hybridoma line described herein produce an agonist CD40 antibody with functional and chemical characteristics similar to those of the antibody derived from the hybridoma line. In contrast, in certain embodiments, substantial alterations in the functional and / or chemical characteristics of an agonist CD40 antibody can be achieved by selecting substitutions in the amino acid sequences of the heavy and light chains that have significantly different effects on (a) the structure of the molecular backbone, e.g., sheet or helix structure, in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining side chain bulk.

[0196] For example, a "conservative amino acid substitution" can involve substituting a native amino acid residue with a non-native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Additionally, any native residue in a polypeptide can be substituted with alanine, as previously described for "alanine scanning mutagenesis."

[0197] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify critical residues of an agonist CD40 antibody, or to increase or decrease the agonist activity of an agonist CD40 antibody described herein.

[0198] In certain embodiments, the agonist CD40 antibody comprises one or more polypeptides. In certain embodiments, any of a variety of expression vector / host systems can be utilized to express polynucleotide molecules encoding polypeptides comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself. Such systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0199] In certain embodiments, polypeptides comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself are recombinantly expressed in yeast. Certain such embodiments use commercially available expression systems, such as the Pichia expression system (Invitrogen, San Diego, CA), according to the manufacturer's instructions. In certain embodiments, such systems rely on a pre-pro-alpha sequence to direct secretion. In certain embodiments, transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction with methanol.

[0200] In certain embodiments, secreted polypeptides comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself are purified from yeast growth medium. In certain embodiments, the methods used to purify polypeptides from yeast growth medium are the same as those used to purify polypeptides from bacterial and mammalian cell supernatants.

[0201] In certain embodiments, nucleic acids encoding polypeptides comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself are cloned into a baculovirus expression vector, such as pVL1393 (PharMingen, San Diego, CA). In certain embodiments, such vectors can be used according to the manufacturer's instructions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free medium to produce recombinant polypeptides. In certain embodiments, polypeptides are purified and concentrated from such medium using a heparin-Sepharose column (Pharmacia).

[0202] In certain embodiments, a polypeptide comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself is expressed in an insect system. Specific insect systems for polypeptide expression are well known to those skilled in the art. One such system uses Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector for expressing foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. In certain embodiments, a nucleic acid molecule encoding a polypeptide can be inserted into a non-essential gene of the virus, such as the polyhedrin gene, and placed under the control of that gene's promoter. In certain embodiments, successful insertion of the nucleic acid molecule renders the non-essential gene inactive. In certain embodiments, its inactivation results in a detectable characteristic. For example, inactivation of the polyhedrin gene results in the production of a virus lacking coat protein.

[0203] In certain embodiments, the recombinant virus can be used to infect S. frugiperda cells or Trichoplusia larvae. See, e.g., Smith et al. See, e.g., Engelhard et al., J. Virol., 46:584 (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA), 91:3224-7 (1994).

[0204] In certain embodiments, polypeptides comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself produced in bacterial cells are produced as insoluble inclusion bodies within the bacteria. In certain embodiments, host cells containing such inclusion bodies are harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris (pH 8), 1 mM EDTA; and treated with 0.1 mg / ml lysozyme (Sigma, St. Louis, MO) for 15 minutes at room temperature. In certain embodiments, the lysate is clarified by sonication, and cell debris is pelleted by centrifugation at 12,000 x g for 10 minutes. In certain embodiments, the polypeptide-containing pellet is resuspended in 50 mM Tris (pH 8) and 10 mM EDTA; layered on 50% glycerol; and centrifuged at 6000 x g for 30 minutes. In certain embodiments, the pellet is purified by centrifugation with 0.1 mg / ml lysozyme (Sigma, St. Louis, MO). ++ and Ca ++ The polypeptide can be resuspended in standard phosphate-buffered saline (PBS) without SDS. In certain embodiments, the polypeptide is further purified by fractionating the resuspended pellet on a denaturing SDS-polyacrylamide gel (see, e.g., Sambrook et al., supra). In certain embodiments, such gels can be soaked in 0.4 M KCl to visualize the protein, which can be excised and electroeluted in a gel running buffer lacking SDS. According to certain embodiments, glutathione-S-transferase (GST) fusion proteins are produced in bacteria as soluble proteins. In certain embodiments, such GST fusion proteins are purified using a GST purification module (Pharmacia).

[0205] In certain embodiments, it is desirable to "refold" a particular polypeptide, e.g., a polypeptide comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself. In certain embodiments, such polypeptides are produced using certain recombinant systems discussed herein. In certain embodiments, the polypeptide is "refolded" and / or oxidized to form a desired tertiary structure and / or generate disulfide bonds. In certain embodiments, such structures and / or linkages are associated with a particular biological activity of the polypeptide. In certain embodiments, refolding is achieved using any of several procedures known in the art. Exemplary methods include, but are not limited to, exposing the solubilized polypeptide agent to a pH typically greater than 7 in the presence of a chaotropic agent. An exemplary chaotropic agent is guanidine. In certain embodiments, the refolding / oxidizing solution also includes a reducing agent and an oxidized form of the reducing agent. In certain embodiments, the reducing agent and its oxidized form are present in a ratio that generates a particular redox potential that allows disulfide shuffling to occur. In certain embodiments, such shuffling allows the formation of cysteine ​​bridges. Exemplary redox pairs include, but are not limited to, cysteine / cystamine, glutathione / dithiobis(GSH), cupric chloride, dithiothreitol / dithiane-DTT, and 2-mercaptoethanol (bME) / dithio-bME. In certain embodiments, cosolvents are used to increase the efficiency of refolding. Exemplary cosolvents include, but are not limited to, glycerol, polyethylene glycols of various molecular weights, and arginine.

[0206] In certain embodiments, polypeptides comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself are substantially purified. Certain protein purification techniques are known to those skilled in the art. In certain embodiments, protein purification involves crude fractionation of the polypeptide fraction from non-polypeptide fractions. In certain embodiments, chromatographic and / or electrophoretic techniques are used to purify the polypeptide. Exemplary purification methods include, but are not limited to, ammonium sulfate precipitation; PEG precipitation; immunoprecipitation; centrifugation after heat denaturation; chromatography, including but not limited to affinity chromatography (e.g., protein A-Sepharose), ion exchange chromatography, exclusion chromatography, and reverse-phase chromatography; gel filtration; hydroxyapatite chromatography; isoelectric focusing; polyacrylamide gel electrophoresis; and combinations of such techniques with other techniques. In certain embodiments, polypeptides are purified by fast protein liquid chromatography or high-pressure liquid chromatography (HPLC). In certain embodiments, purification steps can be modified or certain steps can be omitted, still resulting in a suitable method for preparing a substantially purified polypeptide.

[0207] In certain embodiments, the degree of purification of a polypeptide preparation is quantified. Specific methods for quantifying the degree of purification are known to those skilled in the art. Specific exemplary methods include, but are not limited to, determining the specific binding activity of the preparation and assessing the amount of polypeptide in the preparation by SDS / PAGE analysis. Specific exemplary methods for assessing the amount of purification of a polypeptide preparation include calculating the binding activity of the preparation and comparing it with the binding activity of the initial extract. In certain embodiments, the result of such calculation is expressed as "fold purification." The units used to express the amount of binding activity depend on the specific assay being performed.

[0208] In certain embodiments, a polypeptide comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself is partially purified. In certain embodiments, partial purification can be achieved by using fewer purification steps or by utilizing different forms of the same general purification scheme. For example, in certain embodiments, cation exchange column chromatography performed using an HPLC instrument generally results in a greater "purification factor" than the same technique using a low-pressure chromatography system. In certain embodiments, methods resulting in a lower degree of purification may have advantages in overall recovery of the polypeptide or in maintaining the binding activity of the polypeptide.

[0209] In certain cases, the electrophoretic migration of a polypeptide can vary, sometimes significantly, under different conditions of SDS / PAGE. See, e.g., Capaldi et al., Biochem. Biophys. Res. Comm., 76:425 (1977). It will be understood that the apparent molecular weight of a purified or partially purified polypeptide may vary under different electrophoretic conditions. C. Nucleic Acid Molecules Encoding Agonist Anti-CD40 Antibodies

[0210] In one embodiment, the invention includes a nucleic acid molecule encoding an isolated agonist anti-CD40 antibody disclosed herein.

[0211] Those skilled in the art will understand that the above discussion can be used to identify, evaluate, and / or generate agonist anti-CD40 antibodies, as well as nucleic acid sequences that can encode those antibodies. Accordingly, nucleic acid sequences encoding those antibodies are contemplated. For example, the antibody can have at least 80, 80-85, 85-90, 90-95, 95-97, 97-99% or more identity to at least one nucleic acid sequence set forth in SEQ ID NO: 9 or 10, or at least one to six CDRs encoded by the nucleic acid sequence of SEQ ID NO: 9 or 10 (and various combinations thereof).

[0212] In some embodiments, an antibody (or the nucleic acid sequence encoding it) is contemplated within the present invention if the nucleic acid sequence encoding the particular antibody (or the nucleic acid sequence itself) can selectively hybridize under stringent conditions to either of the nucleic acid sequences encoding the proteins of SEQ ID NOs: 7 and 8. In one embodiment, suitable moderately stringent conditions include a prewash in a solution of 5xSSC; 0.5% SDS, 1.0 mM EDTA (pH 8:0); hybridization at 50°C, -65°C, 5xSSC overnight, or, for cross-species homology, 45°C, 0.5xSSC; followed by two washes at 65°C for 20 minutes each in 2x, 0.5x, and 0.2xSSC containing 0.1% SDS. Such hybridizing DNA sequences are also within the scope of the present invention, as are nucleotide sequences encoding the antibody polypeptides encoded by the hybridizing DNA sequences and the amino acid sequences encoded by these nucleic acid sequences, due to coding degeneracy. In some embodiments, CDR variants include one or more CDR-hybridizing nucleic acid sequences within the above sequences and the amino acid sequences encoded by those sequences.

[0213] The phrase "selectively hybridize" in this context means detectably and selectively bind. The polynucleotides, oligonucleotides, and fragments thereof according to the present invention selectively hybridize to nucleic acid strands under hybridization and washing conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions, known in the art and discussed herein, can be used to achieve selective hybridization conditions. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the present invention and the nucleic acid sequence of interest is at least 80%, with more typically increases of at least 85%, 90%, 95%, 99%, and 100% homology being preferred. Two amino acid sequences are homologous if there is partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred, with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences at least 30 amino acids in length derived therefrom) are homologous, as this term is used herein, if they have an alignment score of greater than 5 (in standard deviation units) using the program ALIGN with a mutation data matrix and a gap penalty of 6 or more. See Dayhoff, MO, in Atlas of Protein Sequence and Structure, pp. 101-110 (Vol. 5, National Biomedical Research Foundation (1972)) and pages 1-10 of Supplement 2 to this volume. Two sequences, or portions thereof, are more preferably homologous if their amino acids are 50% or more identical when optimally aligned using the ALIGN program.The term "corresponding" is used herein to mean that a polynucleotide sequence is homologous (i.e., identical, but not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term "complementary" is used herein to mean that a complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For purposes of illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0214] In one embodiment, the invention includes a vector comprising a nucleic acid molecule described herein.

[0215] In one embodiment, the invention includes a host cell comprising a nucleic acid molecule described herein. D. Compositions Comprising at Least One Agonist Anti-CD40 Antibody

[0216] In one embodiment, the present invention includes a composition comprising at least one agonist anti-CD40 antibody described herein. Preferably, the composition is a pharmaceutical composition. Accordingly, a preferred embodiment of the present invention includes a pharmaceutical composition comprising at least one isolated agonist anti-CD40 antibody described herein and a pharmaceutically acceptable excipient.

[0217] In an alternative embodiment, the present invention provides a pharmaceutical composition comprising an agonist anti-CD40 antibody together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0218] In certain embodiments, the present invention provides pharmaceutical compositions comprising an agonist anti-CD40 antibody and a therapeutically effective amount of at least one additional therapeutic agent, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0219] When the composition includes at least one additional therapeutic agent, the agent is preferably selected from the group consisting of a radioisotope, a radionuclide, a toxin, or a therapeutic and a chemotherapy. Thus, in a preferred embodiment, the present invention includes a pharmaceutical formulation comprising an effective amount of at least one agonist anti-CD40 antibody disclosed herein together with at least a second immune-enhancing agent. Agents include, but are not limited to, in vitro synthetically prepared chemical compositions, antibodies, antigen-binding regions, and combinations and conjugates thereof. In certain embodiments, the agent may act as an agonist, antagonist, allosteric modulator, or toxin. In certain embodiments, the agent may act to inhibit or stimulate its target, thereby promoting an immune response against malignant tumors. Such immune-enhancing agents include, but are not limited to, IL-2, TLR-7 agonists, or systemic cytotoxic chemotherapeutic agents.

[0220] In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations used. In some embodiments, the formulation materials are for sc and / or intratumoral administration. In certain embodiments, pharmaceutical compositions can include formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition. In certain embodiments, suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (mannitol, glycine, etc.); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming agents. ions (such as sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates, such as polysorbate 20, polysorbate 80, Triton); stability enhancing agents (e.g., sucrose or sorbitol); tonicity agents (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (Remington's Pharmaceuticals, Inc., New York, NY, USA). Sciences, 18 th Edition, A.R. Gennaro, ed., Mack Publishing Company (1995). In some embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% sucrose.

[0221] In certain embodiments, the agonist anti-CD40 antibody and / or therapeutic molecule is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylated ABP) and dextran. Such vehicles are described, for example, in U.S. Patent Application No. 09 / 428,082, now U.S. Patent No. 6,660,843, and published PCT application No. WO 99 / 25044, which are incorporated herein by reference for any purpose.

[0222] In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery mode, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibodies of the invention.

[0223] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier may be water for injection, saline, or artificial cerebrospinal fluid, supplemented with other materials common in compositions for parenteral administration. In some embodiments, saline includes isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. In certain embodiments, the pharmaceutical composition comprises Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In certain embodiments, compositions containing an agonist anti-CD40 antibody, with or without at least one additional therapeutic agent, can be prepared for storage by mixing the selected composition having the desired purity with any formulation agent in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, supra). Additionally, in certain embodiments, compositions comprising an agonist anti-CD40 antibody, with or without at least one additional therapeutic agent, can be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0224] In certain embodiments, pharmaceutical compositions can be selected for parenteral delivery. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0225] In certain embodiments, formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0226] In certain embodiments, when parenteral administration (preferably intratumoral or peritumoral administration) is contemplated, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired agonist anti-CD40 antibody, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water in which the agonist anti-CD40 antibody, with or without at least one additional therapeutic agent, has been formulated as a properly preserved, sterile, isotonic solution. In certain embodiments, preparations may include formulating the desired molecule in agents such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can provide controlled or sustained release of the product, allowing delivery via depot injection.

[0227] In certain embodiments, the pharmaceutical composition may comprise an effective amount of an agonist anti-CD40 antibody, with or without at least one additional therapeutic agent, in a mixture with non-toxic excipients suitable for tablet manufacture. In certain embodiments, the tablet may be dissolved in sterile water or another suitable vehicle to prepare a solution in unit dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0228] Additional pharmaceutical compositions, including formulations comprising agonist anti-CD40 antibodies, with or without at least one additional therapeutic agent, in sustained- or controlled-delivery formulations will be apparent to those skilled in the art. In certain embodiments, techniques for formulating various other sustained- or controlled-delivery means, such as liposomes alone, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT / US 93 / 00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can comprise semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (US 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al. al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra), or poly-D(-)-3-hydroxybutyric acid (EP 133,988). In certain embodiments, sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046, and EP 143,949.

[0229] The pharmaceutical composition used for in vivo administration is typically sterile.In certain embodiments, this can be achieved by filtration through sterile filtration membrane.In certain embodiments, when the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution.In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in solution.In certain embodiments, parenteral compositions are generally placed into a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0230] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) prior to administration.

[0231] In certain embodiments, the kit is provided for producing single-dose units.In certain embodiments, the kit can comprise both a first container with dry protein and a second container with aqueous formulation.In certain embodiments, the kit comprises single-chamber and multi-chamber pre-filled syringe (for example, liquid syringe and lyosyringe).

[0232] In certain embodiments, the effective amount of a pharmaceutical composition comprising an agonist anti-CD40 antibody, with or without at least one additional therapeutic agent, used therapeutically will depend, for example, on the context and purpose of the treatment. Therefore, those skilled in the art will understand that appropriate dosage levels for treatment will vary depending, in part, on the molecule being delivered, the indication for which the agonist anti-CD40 antibody, with or without at least one additional therapeutic agent, is being used, the route of administration, and the subject's size (body weight, surface tumor size, or organ size) and / or condition (age and general health). In certain embodiments, clinicians can titrate the dosage and modify the route of administration to achieve optimal therapeutic efficacy. In certain embodiments, typical dosages can range from about 0.1 μg / kg to up to about 100 mg / kg or more, depending on the factors described above. In certain embodiments, dosages can range from 0.1 μg / kg to up to about 100 mg / kg; or 1 μg / kg to up to about 100 mg / kg; or 5 μg / kg to up to about 100 mg / kg. For example, the effective dose is preferably 20 μg / kg to 200 μg / kg. Thus, if a patient receives 6 doses, the corresponding total dose over 2 weeks is 120 μg / kg to 1.2 mg / kg.

[0233] In certain embodiments, the dosing frequency takes into account the pharmacokinetic parameters of the agonist anti-CD40 antibody and / or any additional therapeutic agents in the formulation used. In certain embodiments, the clinician administers the composition until a dosage that achieves the desired effect is reached. Thus, in certain embodiments, the composition can be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routine and within the scope of routine practice by those skilled in the art. In certain embodiments, the appropriate dosage can be confirmed through the use of appropriate dose-response data.

[0234] In certain embodiments, the administration route of pharmaceutical compositions follows known methods.Preferably, intratumoral or peritumoral injection is used, but if the composition is formulated for site-specific delivery, other administration routes may be suitable.Such administration routes may include intravenous, intraperitoneal, intramuscular, subcutaneous, intraarterial, intraportal, or intralesional routes; by sustained release system or by implantation device.In certain embodiments, the composition can be administered by bolus injection, or continuously by infusion, or by implantation device.

[0235] In certain embodiments, the compositions can be administered locally via implantation of a membrane, sponge, or another suitable material into which the desired molecule has been absorbed or encapsulated. In certain embodiments, when an implantation device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, timed-release bolus, or continuous administration. E. Methods for Treating or Preventing Conditions

[0236] In one embodiment, the present invention comprises a method for treating or preventing symptoms associated with a malignancy in a patient, comprising administering to a patient in need thereof an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0237] In one embodiment, the present invention includes a method for increasing antigen presentation by APCs (including macrophages, DCs, and B cells) in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0238] In one embodiment, the present invention includes a method of activating antigen-presenting cells in a subject comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0239] The activation of antigen-presenting cells in a subject can be measured by assays known in the art. For example, antigen presentation by activated antigen-presenting cells from a subject can be measured using a viral antigen recall assay, in which PBMCs from a human donor previously exposed to Epstein-Barr virus (EBV) are challenged with EBV antigen in the presence of an agonist anti-CD40 antibody.

[0240] In one embodiment, the present invention includes a method of activating dendritic cells in a subject comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0241] Activation of dendritic cells in a subject can be measured by assays known in the art. For example, stimulation of T cells by activated dendritic cells from a subject can be measured using a mixed lymphocyte reaction.

[0242] In one embodiment, the invention includes a method of enhancing expression of MHC and / or immune costimulatory molecules in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein. Preferably, the MHC and / or immune costimulatory molecules are selected from CD80, CD86, PD-L1, HLA-A, B, C, HLA-DR, and CD83.

[0243] The expression of MHC and / or immune co-stimulatory molecules in a subject can be measured by assays known in the art. For example, the expression of MHC and / or immune co-stimulatory molecules in cells from a subject can be measured using a fluorescence-activated cell sorting assay.

[0244] In one embodiment, the invention includes a method of stimulating the production of pro-inflammatory cytokines in a subject, comprising administering an effective amount of at least an agonist anti-CD40 antibody disclosed herein. Preferably, the pro-inflammatory cytokines are selected from IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ.

[0245] The expression of pro-inflammatory cytokines in a subject can be measured by assays known in the art. For example, the expression of pro-inflammatory cytokines in cells derived from a subject can be measured using ELISA or LEGENDplex assay.

[0246] In one embodiment, the invention includes a method of inducing T cell activation in a subject comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein.

[0247] In one embodiment, the invention includes a method of mimicking CD40L signaling and substituting for CD4+ lymphocyte function in a subject, comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein.

[0248] In one embodiment, the present invention includes a method for overcoming T cell tolerance in a tumor-bearing animal, or inducing an effective cytotoxic T cell response, or enhancing the effectiveness of an anti-tumor vaccine in a subject, comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein.

[0249] In one embodiment, the invention includes a method of promoting the secretion of autoantibodies by B cells against antigens expressed on tumor cells in a subject, comprising administering an effective amount of an agonist anti-CD40 antibody disclosed herein.

[0250] In one embodiment, the invention includes a method of upregulating costimulatory markers, releasing IL-12 to activate CD8+ T cells, and stimulating a specific cytotoxic T cell response against cross-presented tumor antigens in a subject, comprising administering an effective amount of an agonist anti-CD40 antibody disclosed herein.

[0251] The complex coordination of events required for tumor eradication suggests that combination therapeutic approaches may also be beneficial in some situations. Agonistic anti-CD40 antibodies could be administered with additional arms to release antigens, promote cytokine release, increase immune surveillance, and decrease inhibitory networks to enhance this effect.

[0252] In another embodiment, the present invention includes a method for treating or preventing symptoms associated with a malignancy in a patient, comprising administering to a patient in need thereof an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein and an effective amount of at least a second immune enhancing agent. Coadministration of IL-2 and CD40 agonists

[0253] One therapeutic option is to alter the tumor microenvironment itself, encouraging the tumor to act as its own source of antigenic stimulation. This can be achieved by introducing IL-2 with anti-CD40 antibodies into or near the tumor site. When co-injected directly, this co-administration successfully induces regression of larger tumors as well as distant tumors, avoiding the toxicity associated with systemic administration and maintaining long-term protective memory. Co-administration of IL-2 and CD40 agonists can result in increased macrophage activity and activation of T and B cells. The combination of IL-2 and CD40 agonists can demonstrate significant benefit against a variety of cancers whose regression is associated with a neutrophil- and T-cell-dominated inflammatory response.

[0254] Thus, in one embodiment, the present invention includes a pharmaceutical composition comprising at least one agonist anti-CD40 antibody described herein and IL-2.

[0255] In one embodiment, the present invention comprises a method for treating or preventing symptoms associated with a malignancy in a patient, comprising administering to a patient in need thereof an effective amount of each of at least one agonist anti-CD40 antibody described herein and IL-2.

[0256] In one embodiment, the present invention includes a method for increasing antigen presentation by APCs (including macrophages, DCs, and B cells) in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody disclosed herein and IL-2.

[0257] In one embodiment, the present invention includes a method of activating antigen-presenting cells in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein and IL-2.

[0258] The activation of antigen-presenting cells in a subject can be measured by assays known in the art. For example, antigen presentation by activated antigen-presenting cells from a subject can be measured using a viral antigen recall assay, in which PBMCs from a human donor previously exposed to Epstein-Barr virus (EBV) are challenged with EBV antigen in the presence of an agonist anti-CD40 antibody.

[0259] In one embodiment, the present invention includes a method of activating dendritic cells in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein and IL-2.

[0260] Activation of dendritic cells in a subject can be measured by assays known in the art. For example, stimulation of T cells by activated dendritic cells from a subject can be measured using a mixed lymphocyte reaction.

[0261] In one embodiment, the invention includes a method of enhancing expression of MHC and / or immune costimulatory molecules in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody disclosed herein and IL-2. Preferably, the MHC and / or immune costimulatory molecules are selected from CD80, CD86, PD-L1, HLA-A, B, C, HLA-DR, and CD83.

[0262] The expression of MHC and / or immune co-stimulatory molecules in a subject can be measured by assays known in the art. For example, the expression of MHC and / or immune co-stimulatory molecules in cells from a subject can be measured using a fluorescence-activated cell sorting assay.

[0263] In one embodiment, the invention includes a method of stimulating the production of pro-inflammatory cytokines in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody disclosed herein and IL-2. Preferably, the pro-inflammatory cytokines are selected from IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ.

[0264] The expression of pro-inflammatory cytokines in a subject can be measured by assays known in the art. For example, the expression of pro-inflammatory cytokines in cells derived from a subject can be measured using ELISA or LEGENDplex assay.

[0265] In one embodiment, the invention includes a method of inducing T cell activation in a subject comprising administering an effective amount of each of at least one agonist anti-CD40 antibody described herein and IL-2.

[0266] In one embodiment, the invention includes a method of mimicking CD40L signaling and substituting for CD4+ lymphocyte function in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody described herein and IL-2.

[0267] In one embodiment, the invention includes a method for overcoming T cell tolerance in a tumor-bearing animal, or inducing an effective cytotoxic T cell response, or enhancing the effectiveness of an anti-tumor vaccine in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody described herein and IL-2.

[0268] In one embodiment, the invention includes a method of promoting the secretion of autoantibodies by B cells against antigens expressed on tumor cells in a subject, comprising administering an effective amount of an agonist anti-CD40 antibody disclosed herein and IL-2.

[0269] In one embodiment, the invention includes a method of upregulating costimulatory markers, releasing IL-12 to activate CD8+ T cells, and stimulating a specific cytotoxic T cell response against cross-presented tumor antigens in a subject, comprising administering an effective amount of an agonist anti-CD40 antibody disclosed herein and IL-2.

[0270] The most effective method for treating or preventing symptoms associated with a malignancy in a patient may require a combination therapeutic approach in which therapeutic interventions are administered sequentially over time.

[0271] Thus, in one embodiment, the present invention includes a method for treating or preventing symptoms associated with a malignancy in a patient, comprising sequentially administering to a patient in need thereof an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein with an additional therapeutic intervention, which in some embodiments is selected from the group consisting of surgery, radiation therapy, chemotherapy, hyperthermia, and immunotherapy.

[0272] One treatment option is to modify cells isolated from a patient and then return these cells to the patient. This can be achieved by treating the cells isolated from the patient with an anti-CD40 antibody. The cells treated with the anti-CD40 antibody can be treated with an additional agent. In some embodiments, the agent is selected from the group consisting of tumor-specific peptides, tumor cell lysates, cytokines, agonists, and mitogens.

[0273] Thus, in one embodiment, the present invention comprises a method for treating or preventing symptoms associated with a malignancy in a patient, comprising administering to a patient in need thereof an effective amount of cells treated with at least one isolated agonist anti-CD40 antibody disclosed herein. In some embodiments, the cells are isolated from the patient and are selected from the group consisting of DCs, macrophages, B cells, myeloid cells, lymphoid cells, and hematopoietic stem cells.

[0274] Further features of the present invention are more fully described in the following examples, however, it should be understood that this detailed description is included solely for the purpose of illustrating the invention and should in no way be construed as a limitation on the broad description of the invention set forth above. [Example]

[0275] Example 1 - Production of SVX-3001 antibody Sequence data The nucleotide sequences of the humanized agonistic anti-human CD40 antibody heavy chain (SEQ ID NO: 13, Figure 1) and the humanized agonistic anti-human CD40 antibody light chain (SEQ ID NO: 14, Figure 2) were obtained from North Coast Biologics. In Figures 1 and 2, the HindIII and XbaI sites are shown in bold, and the coding sequences of the heavy and light chains are underlined. The humanized agonistic anti-human CD40 antibody was designated SVX-3001. The coding and amino acid sequences of the SVX-3001 heavy chain variable region (SEQ ID NO: 9 and SEQ ID NO: 7, Figure 3) and the SVX-3001 light chain variable region (SEQ ID NO: 10 and SEQ ID NO: 8, Figure 4), along with the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3), were identified. In Figures 3 and 4, the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) are shown in bold. Gene synthesis and cloning

[0276] DNA sequences encoding the SVX-3001 heavy chain (SEQ ID NO: 15, Figure 5) and SVX-3001 light chain (SEQ ID NO: 17, Figure 6) with HindIII and XbaI restriction enzyme sites for cloning were synthesized by GenScript HK and cloned into the vector pcDNA3.1(+) to generate the plasmids pcDNA3.1(+)_Selvax01HC (Figure 7) and pcDNA3.1(+)_Selvax01LC (Figure 8), respectively. Genscript HK provided 100 μg of each plasmid, transfection grade (≥90% supercoiled, ≤0.01 EU / μg endotoxin) in TE buffer. In Figures 5 and 6, the HindIII and XbaI sites are shown in bold and the coding sequences are underlined. The amino acid sequences of the heavy / light chain variable regions are shown in plain text, the signal peptide sequences are shown in bold, and the human IgG1 heavy chain constant region / human kappa light chain constant region are underlined. Production of SVX-3001 antibody-containing supernatant

[0277] One day before transfection, HEK 293T cells were cultured at 4 × 10 in DMEM (Gibco) containing 10% FCS (Hyclone). 5 Cells were seeded into 6-well tissue culture plates (Corning-Falcon) at a density of 100 cells / well. The medium was removed 3 hours before transfection and replaced with 1.5 ml / well of DMEM + 1% FCS. The transfection mix was created by adding 1.5 μg of pcDNA3.1(+)_Selvax01HC DNA and 1.5 μg of pcDNA3.1(+)_Selvax01LC DNA to a sterile tube containing 300 μl of DMEM (no additives), followed by 6 μl of polyethyleneimine (PEI), 25 kD, linear (Polysciences) at 1 μg / ml and mixing by vortexing. The transfection mix was incubated at room temperature for 10 minutes before being added to the cells. The cells were incubated at 37°C for 3 hours, after which 1.5 ml / well of DMEM + 10% FCS was added. The cells were then returned to culture at 37°C in 5% CO2.

[0278] SVX-3001 antibody-containing supernatants from transiently transfected cells were collected 18 hours and 2 days after transfection and replaced with DMEM + 10% FCS. Culture supernatants were finally harvested 7 days after transfection. All culture supernatants were centrifuged to remove cell debris and stored at 4°C. Production of purified SVX-3001 antibody

[0279] One day before transfection, HEK 293T cells were cultured at 4 × 10 in DMEM (Gibco) containing 10% ultra-low IgG FCS (Gibco). 5 Cells were seeded into 6-well tissue culture plates (Corning-Falcon) at a density of 100 cells / well. The medium was removed 3 hours before transfection and replaced with 1.5 ml / well of DMEM + 1% ultra-low IgG FCS. The transfection mix was created by adding 1.5 μg of pcDNA3.1(+)_Selvax01HC and 1.5 μg of pcDNA3.1(+)_Selvax01LC to a sterile tube containing 300 μl of DMEM (no additives), followed by 6 μl of polyethyleneimine (PEI), 25 kD, linear (Polysciences) at 1 μg / ml and vortexing. The transfection mix was incubated at room temperature for 10 minutes before being added to the cells. The cells were incubated at 37°C for 3 hours, after which 1.5 ml / well of DMEM + 10% ultra-low IgG FCS was added. The cells were then returned to culture at 37°C in 5% CO2.

[0280] SVX-3001 antibody-containing supernatants from transiently transfected cells were collected 2 days and 5 days after transfection and replaced with DMEM + 10% ultra-low IgG FCS. Culture supernatants were finally collected on day 7 after transfection. Culture supernatants from different time points were pooled, filtered through a 0.22 μm filter, and stored at 4°C. SVX-3001 antibody was purified from the pooled supernatants using Protein G at the Harry Perkins Institute of Medical Research Monoclonal Antibody Facility. Purified SVX-3001 antibody was supplied at 1 mg / ml in PBS. Example 2 - ELISA to detect human IgG with specificity for human CD40

[0281] ELISA was performed using the IgG (Total) Human ELISA Kit with Plates (Invitrogen). Wells of an ELISA plate (Corning-Costar) were coated with 100 μl / well of purified anti-human IgG monoclonal capture antibody at the recommended concentration or 1 μg / ml of recombinant human CD40 extracellular domain with a C-terminal 6His tag (Novoprotein). The plate was incubated overnight at 4°C. After washing the wells twice with 400 μl / well of wash buffer, 250 μl / well of blocking buffer was added. The plate was incubated for 2 hours at room temperature. After washing the wells twice with 400 μl / well of wash buffer, 100 μl / well of serial 1 / 2 dilutions of recombinant human IgG standard from 100 ng / ml to 1.56 ng / ml and control anti-CD40 antibody Lob 7 / 4 from approximately 100 ng / ml to 1.56 ng / ml were added. Serial 1 / 2 dilutions of IgG1 (University of Southampton) and serial 1 / 10 dilutions of supernatant from HEK 293T cells transiently transfected with the plasmids pcDNA3.1(+)_Selvax01HC and pcDNA3.1(+)_Selvax01LC from 1 / 10 to 1 / 10,000 were added. The plate was incubated for 2 hours at room temperature on a microplate shaker set at 400 rpm. After washing the wells four times with 400 μl / well of wash buffer, 100 μl / well of substrate solution containing tetramethylbenzidine (TMB) was added. The plate was incubated for 15 minutes at room temperature. 100 μl of stop solution (1 M H3PO4) was added to each well. Tests included a diluent-only control (no antibody), recombinant human IgG standard (25 ng / ml), SVX-3001 antibody (estimated at 27.3 ng / ml), and Lob 7 / 4 IgG1 antibody (estimated at 42.2 ng / ml). Captured antibody was detected using an HRP anti-human IgG antibody. Absorbance at 450 nm was measured using an EnSpire multimode plate reader (Perkin Elmer).

[0282] Results: Recombinant human IgG standard was detectable in wells coated with purified anti-human IgG monoclonal capture antibody but not in wells coated with recombinant CD40 protein (Figure 9). Anti-CD40 antibodies SVX-3001 and Lob 7 / 4 IgG1 were detectable in wells coated with purified anti-human IgG monoclonal capture antibody and in wells coated with recombinant CD40 protein (Figure 9).

[0283] Conclusion: Supernatants from HEK 293T cells transiently transfected with the plasmids pcDNA3.1(+)_Selvax01HC and pcDNA3.1(+)_Selvax01LC were confirmed to contain human IgG protein (i.e., antibody SVX-3001) with specificity for human CD40 protein. Example 3 - FACS analysis to detect binding of antibodies to antigens on cells

[0284] FACS analysis was performed to determine whether SVX-3001 binds to CD40 expressed on the surface of human peripheral blood mononuclear cells (PBMCs). PBMCs were stained with a PE-labeled anti-human CD19 antibody. Cell surface-bound SVX-3001 antibody was detected using a BV421-labeled anti-human IgG antibody. Lymphocytes were gated based on FSC-A and SSC-A signals.

[0285] Methods: Human buffy coat samples were obtained from the Australian Red Cross Blood Services (ethics approval: RDHS-243-15, Human Research Ethics Office, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from the human buffy coat samples by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences).

[0286] FACS staining was performed in 96-well U-bottom plates (Corning-Falcon). All incubations were performed on ice in the dark. The FACS buffer used for washing steps and antibody dilution consisted of PBS containing 1% BSA (Sigma), 1% FCS (Hyclone), and 0.01% w / v sodium azide (Sigma). The staining reagents were SVX-3001 antibody-containing supernatant, human IgG1 isotype control (BioLegend), BV421 anti-mouse Ig (BD Biosciences), and PE anti-human CD19 (BioLegend).

[0287] FACS staining was performed using 10 wells 6This assay was performed on 100 human PBMCs. Cells were pelleted and resuspended in 20 μl of SVX-3001 antibody-containing supernatant and incubated for 30 minutes. Cells were washed twice with FACS buffer and resuspended in 20 μl of BV421 anti-mouse Ig diluted in FACS buffer and incubated for 30 minutes. Cells were washed twice with FACS buffer and resuspended in 20 μl of PE anti-human CD19 diluted in FACS buffer and incubated for 30 minutes. Cells were washed once with FACS buffer and once with PBS, resuspended in 100 μl of 1% formaldehyde in PBS and incubated for 20 minutes. Cells were washed twice with FACS buffer and resuspended in 200 μl of FACS buffer for analysis.

[0288] FACS staining controls included an unstained control, PE anti-human CD19 and BV421 mouse anti-human Ig single-stained controls, and a human IgG1 isotype control. Cell staining was analyzed using a FACS Canto II flow cytometer (BD). PMT voltages and gates for analysis were set using the FACS staining controls.

[0289] Results: The supernatant containing the SVX-3001 antibody inhibited CD19 + It bound to CD40 on the surface of peripheral blood lymphocytes (Figure 10). Figure 10(A) shows a PE-anti-CD19 control showing PE fluorescence staining of cells expressing CD19 on the cell surface. Figure 10(B) shows PE-anti-CD19 control showing PE fluorescence staining of cells expressing CD19 on the cell surface. + Figure 10(C) shows a BV421 anti-human IgG control, which shows background BV421 fluorescence due to binding of anti-human IgG antibodies to a subset of cells. Figure 10(D) shows test staining with SVX-3001 antibody-containing supernatant, showing BV421 fluorescence due to the presence of SVX-3001 antibodies bound to CD40 on the surface of B cells.

[0290] Conclusion: The SVX-3001 antibody has specificity for the native CD40 protein expressed on the surface of human immune cells. Example 4 - CFSE assay to detect cell division in response to stimuli

[0291] The ability of SVX-3001 to stimulate the cell division rate of PBMCs was measured using CFSE. CFSE-labeled human PBMCs were cultured with SVX-3001 antibody or a human IgG1 isotype control antibody at 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, and 0.001 μg / ml for 7 days and then stained with Zombie Aqua and PE anti-CD19.

[0292] Methods: Human buffy coat samples were obtained from the Australian Red Cross Blood Services (ethics approval: RDHS-243-15, Human Research Ethics Office, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from the human buffy coat samples by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences).

[0293] 2x10 human PBMCs in PBS 7 Cells were suspended at 100 μM per ml and stained with CFSE (Life Technologies Australia) using 25 μl of 100 μM CFSE for every ml of cells. Cells were mixed with CFSE by gentle inversion for 10 minutes, after which at least 4 volumes of RPMI (Gibco) + 10% FCS (Hyclone) were added. Cells were washed twice with RPMI + 10% FCS and then resuspended in RPMI + 10% FCS for culture.

[0294] Cells were plated at 5x10 in a final volume of 200µl / well of RPMI + 10% FCS. 5 Cells were cultured in 96-well plates (Nunc) at 1000 cells / well. Cells were stimulated with SVX-3001 antibody-containing supernatant or human IgG1 isotype control antibody (BioLegend) at antibody concentrations of 1 mg / ml, 0.1 mg / ml, 0.01 mg / ml, and 0.01 mg / ml. Cells were cultured at 37°C in 5% CO for 7 days and then harvested for FACS staining.

[0295] FACS staining was performed in 96-well U-bottom plates (Corning-Falcon). All incubations were performed on ice in the dark. The FACS buffer used for washing steps and antibody dilution consisted of PBS containing 1% BSA (Sigma), 1% FCS (Hyclone), and 0.01% w / v sodium azide (Sigma). The staining reagents were Zombie Aqua (BioLegend) and PE anti-human CD19 (BioLegend).

[0296] Cells for FACS staining were pelleted and resuspended in 20 μl of PE anti-human CD19 diluted in FACS buffer and incubated for 30 minutes. Cells were washed twice with PBS, resuspended in 100 μl of Zombie Aqua diluted in PBS, and incubated for 15 minutes. Cells were washed once with FACS buffer and once with PBS, resuspended in 100 μl of 1% formaldehyde in PBS, and incubated for 20 minutes. Cells were washed twice with FACS buffer and resuspended in 200 μl of FACS buffer for analysis.

[0297] FACS staining controls included unstained controls, single-stained controls, and FMO controls. Cell staining was analyzed using a FACS Canto II flow cytometer (BD). PMT voltages and gates for analysis were set using the FACS staining controls.

[0298] Results: At concentrations above 0.01 mg / ml, the SVX-3001 antibody stimulated human PBMC cell division above the background level seen in human PBMCs stimulated with a human IgG1 isotype control antibody (Figure 11). Figure 11(A) shows that a PBMC gate on FSC-A vs. SSC-A was used to exclude debris. Figure 11(B) shows that a single cell gate on FSC-A vs. FSC-H was used to exclude cell clumps. Figure 11(C) shows that a live cell gate on Zombie Aqua vs. SSC-A was used to exclude dead cells. Figure 11(D) shows that dividing cells were identified based on a decrease in CFSE fluorescence with CFSE vs. PE-anti-CD19. Figure 11(E) shows a graph of the percent dividing cells in PBMC cultures stimulated with different concentrations of human IgG1 antibody.

[0299] Conclusion: The SVX-3001 antibody has agonistic activity that leads to an increase in cell-dividing human immune cells. Example 5 - LEGENDplex assay to detect cytokine responses to stimulation of PMBCs with SVX-3001

[0300] A LEGENDplex assay was performed to determine the level of cytokine production in response to stimulation of PMBCs with SVX-3001.

[0301] Methods: Human blood samples were obtained from healthy volunteers (ethics approval: HRE 2017-0767, Human Research Ethics Office, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from human blood samples by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences) and stored at -80°C in FCS (Hyclone) containing 10% DMSO (Sigma). Human PBMCs were thawed and cultured overnight at 37°C in RPMI (Gibco) containing 5% FCS + 10% FCS to recover cells from freezing. Cells were labeled using the Cell Trace Violet Proliferation Kit (Life Technologies Australia).

[0302] Cells were plated at 5x10 in a final volume of 200µl / well of RPMI + 10% FCS. 5 Cells were cultured in 96-well plates (Nunc) at 100 cells / well. Cells were cultured unstimulated, with 1 μg / ml purified SVX-3001 antibody, 10 ng / ml recombinant human IL-2 (Peprotech), or with 1 μg / ml purified SVX-3001 antibody and 10 ng / ml recombinant human IL-2. Cells were cultured at 37°C with 5% CO for 3 days, and then the supernatant was harvested. Supernatants were assayed for soluble analytes using LEGENDplex bead-based immunoassays (BioLegend). LEGENDplex data were acquired using an LSRFortessa Cell Analyser (BD).

[0303] Results: Stimulation of human PBMCs with purified SVX-3001 antibody resulted in increases in IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, and IL-23 and decreases in IL-13 and TNFβ in the culture medium after 3 days of culture (Figure 12). Stimulation of human PBMCs with a combination of SVX-3001 and IL-2, but not SVX-3001 or IL-2 alone, released IFNγ into the culture medium after 3 days of culture (Figure 12). Cytokine concentrations measured in the culture medium control (RPMI + 10% FCS + 10 ng / ml IL-2) are shown as dotted lines in Figure 12.

[0304] Conclusion: The SVX-3001 antibody has agonistic activity that leads to differential release of cytokines from human immune cells. Example 6 - Antibody competition studies by FACS

[0305] A FACS assay was performed to determine the ability of SVX-3001 to block the binding of antibodies B-B20 and LOB7 / 6 to CD40. Human PBMCs were stained with mouse anti-human CD40 antibodies B-B20 and LOB7 / 6 in the presence or absence of 1 μg / ml of SVX-3001 antibody. Binding of mouse anti-human CD40 antibodies to human PBMCs was detected using an anti-mouse Ig antibody labeled with BV421.

[0306] Methods: Human buffy coat samples were obtained from the Australian Red Cross Blood Services (ethics approval: RDHS-243-15, Human Research Ethics Office, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from the human buffy coat samples by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences).

[0307] FACS staining was performed in 96-well U-bottom plates (Corning-Falcon). All incubations were performed on ice in the dark. The FACS buffer used for washing steps and antibody dilution consisted of PBS containing 1% BSA (Sigma), 1% FCS (Hyclone), and 0.01% w / v sodium azide (Sigma). The blocking reagent was SVX-3001 antibody-containing supernatant. The staining reagents were anti-CD40 antibody LOB7 / 6 (LSBio), anti-CD40 antibody B-B20 (Abcam), and BV421 anti-mouse Ig (BD Biosciences).

[0308] FACS staining, 5x10 per well 5 Assays were performed on human PBMCs. Cells were pelleted and resuspended in 200 μl of SVX-3001 antibody-containing supernatant at 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, or 0.001 μg / ml and incubated for 30 minutes. Cells were pelleted (without washing) and resuspended in 20 μl of LOB7 / 6 diluted in FACS buffer or B-B20 diluted in FACS buffer and incubated for 30 minutes. Cells were washed twice with FACS buffer, resuspended in 20 μl of BV421 anti-mouse Ig diluted in FACS buffer, and incubated for 30 minutes. Cells were washed twice with PBS, resuspended in 100 μl of 1% formaldehyde in PBS, and incubated for 20 minutes. Cells were washed twice with FACS buffer and resuspended in 200 μl of FACS buffer for analysis.

[0309] FACS staining controls included unstained controls, no blocking controls, and no primary antibody controls. Cell staining was analyzed using a FACS Canto II flow cytometer (BD). PMT voltages and gates for analysis were set using the FACS staining controls.

[0310] Results: Staining of human PBMCs with anti-CD40 antibody B-B20 was blocked by SVX-3001, whereas staining with anti-CD40 antibody LOB7 / 6 was not blocked by SVX-3001 (Figure 13). (A) Unstained control. (B) Staining with LOB7 / 6. (C) Staining with LOB7 / 6 in the presence of 1 μg / ml SVX-3001. (D) Staining with B-B20. (E) Staining with B-B20 in the presence of 1 μg / ml SVX-3001.

[0311] Conclusion: Antibodies SVX-3001 and B-B20 bind to the same or overlapping epitopes on the human CD40 molecule. Antibodies SVX-3001 and LOB7 / 6 bind to different epitopes on the human CD40 molecule. Example 7 - Antibody competition studies by surface plasmon resonance

[0312] The ability of SVX-3001 to compete with a range of anti-CD40 antibodies was measured by surface plasmon resonance.

[0313] Methods: Pairwise binding analysis by surface plasmon resonance (SPR) was performed using a Biacore T200 (GE Healthcare). HBS-EP+ buffer was used as the running buffer throughout.

[0314] Recombinant human CD40 extracellular domain (CD40-Fc; BioLegend) with a C-terminal human IgG1 Fc domain and a 6His tag was immobilized on the surface of a Series S Sensor Chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare). For immobilization, CD40-Fc was diluted to 25 μg / ml in 10 mM sodium acetate buffer (pH 5.0). Target immobilization level: 1000 RU. Wash solution: ethanolamine.

[0315] The reagents used in the competition studies were purified SVX-3001 antibody, recombinant CD40L (BioLegend), and purified anti-CD40 antibodies Lob 7 / 4 IgG1 (University of Southampton), CP-870,893 IgG1 (University of Southampton), S2C6 (Mabtech), G28.5 (BioXCell), and LOB7 / 6 (LSBio).

[0316] Pairwise binding analysis was performed by binding the first sample (Sample 1) at a concentration sufficient to saturate the immobilized CD40-Fc on the surface after 180 seconds of injection at 10 μl / min. This concentration ranged from 12.5 to 200 μg / ml for the reagent being tested. The second sample (Sample 2) was then injected at 10 μl / min for 180 seconds. Regeneration was performed using glycine-HCl buffer, pH 1.5, for 30 seconds at 10 μl / min with a 5-second stabilization period before testing the next pair. Pairs in which Sample 1 and Sample 2 were identical were used to confirm saturation and establish a baseline response.

[0317] Results: Biacore T200 sensograms for epitope mapping by pairwise binding for SVX-3001, Lob 7 / 4 IgG1 and LOB7 / 6 are shown in Figure 14. Results for all pairs tested are summarized in Table 3. [Table 3-1] [Table 3-2]

[0318] Conclusions: SVX-3001 binds to an epitope on human CD40 that is identical to or overlaps with the epitopes of antibodies Lob 7 / 4 IgG1, CP-870,893 IgG1, S2C6, and G28.5. SVX-3001 binds to a distinct epitope that does not overlap with the epitope of antibody LOB7 / 6. The epitope of SVX-3001 does not overlap with the binding site of CD40 L.

[0319] The antibodies Lob 7 / 4, SGN40 (derived from antibody S2C6), and CP-870,893 have all been reported to bind to epitopes within the CRD1 region of human CD40 (Cancer Cell 33, 664-675, April 9, 2018). Based on SPR data, SVX-3001 also binds to an epitope within the CRD1 region of human CD40 (P25942; Cys26-Cys59). Example 8 - Antibody affinity testing by surface plasmon resonance

[0320] The affinity of SVX-3001 for the extracellular domain of CD40 was measured using surface plasmon referencing.

[0321] Methods: Kinetic analysis by surface plasmon resonance (SPR) was performed using a Biacore T200 (GE Healthcare). HBS-EP+ buffer was used as the running buffer throughout.

[0322] Purified SVX-3001 was immobilized on the surface of a Series S Sensor Chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare). For immobilization, SVX-3001 was diluted to 25 μg / ml in 10 mM sodium acetate buffer (pH 5.0). Target immobilization level: 600 RU. Wash solution: ethanolamine. Reference surfaces for analysis were either untreated or had a human IgG1 isotype reference (BioLegend) immobilized on the surface by the same process used for SVX-3001.

[0323] The reagents used in the kinetic study were recombinant human CD40 extracellular domain with a C-terminal 6His tag (CD40-6His; Novoprotein) and C-terminal human IgG1 The recombinant human CD40 extracellular domain had an Fc domain and a 6His tag (CD40-Fc; BioLegend).

[0324] The sample injection parameters were: contact time: 120 s, flow rate: 30 μl / min, dissociation time: 300 s. The injection parameters for regeneration were: contact time: 30 s, flow rate: 30 μl / min, stabilization period: 0 s. The regeneration solution was either 10 mM glycine-HCl buffer pH 1.5 or 50 mM NaOH. The concentrations of CD40-His and CD40-Fc used for kinetic analysis were 2 mM, 4 mM, 8 mM (in duplicate), 16 mM, and 32 mM. A 1:1 binding model was used for curve fitting.

[0325] Results: The kinetic and affinity constants of SVX-3001 from three experiments with CD40-Fc as the analyte and three experiments with CD40-Fc as the analyte are shown in Table 4. [Table 4]

[0326] Conclusion: The calculated equilibrium dissociation constant (K D ) is 6.111x10 -9 M (6.111 nM) and the calculated K of SVX-3001 using CD40-Fc as the analyte. D is 5.826x10 -10 The association rate constant (k) calculated using both analytes was M (0.5826 nM). a ) were similar, so K D The difference between the dissociation rate constants (k d ) is likely due to the difference. Example 9 - Activation of monocyte-derived dendritic cells by anti-CD40 antibodies

[0327] Stimulation of monocyte-derived dendritic cells (moDCs) in the presence of SVX-3001, CD40L, or LPS with IFN-γ was measured with reference to the expression of costimulatory molecules.

[0328] Methods: Human buffy coat samples were obtained from the Australian Red Cross Blood Service (ethics approval: RDHS-243-15, Human Research Ethics Office, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coat samples by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences). Human PBMCs were cultured at 5x10 in RPMI (Gibco) + 10% FCS (Hyclone). 6 Monocytes were seeded into 6-well plates (Corning-Falcon) at a density of 100 cells / well and incubated at 37°C for 2 hours to allow adherence of the monocytes to the plastic. The medium containing non-adherent cells was then removed.

[0329] Adherent monocytes were differentiated into monocyte-derived dendritic cells (moDCs) by culturing for 7 days in RPMI + 10% FCS containing 80 ng / ml recombinant human GM-CSF (Shenandoah Biotechnology), 10 ng / ml recombinant human IL-4 (Shenandoah Biotechnology), and 10 μg / ml polymyxin B (Sigma-Aldrich). The medium containing GM-CSF, IL-4, and polymyxin B was replaced on day 4.

[0330] Cells were stimulated on day 7 in medium containing GM-CSF and IL-4 but without polymyxin B. Stimulation conditions included medium control (no stimulation), 1 μg / ml SVX-3001, 1 μg / ml human IgG1 isotype control (BioLegend), 0.67 mg / ml recombinant CD40L (BioLegend), and 1 μg / ml LPS (Sigma-Aldrich) containing 20 ng / ml recombinant human IFN-γ (Shenandoah Biotechnology). On day 9, cells were treated with 1x Brefeldin A solution (BioLegend) for 4 hours, after which they were harvested and stained for FACS analysis (48 hours of stimulation).

[0331] FACS staining was performed in 96-well U-bottom plates (Corning-Falcon). The FACS buffer used for washing steps and antibody dilution consisted of PBS containing 1% BSA (Sigma), 1% FCS (Hyclone), and 0.01% w / v sodium azide (Sigma). Staining reagents included BUV805 anti-CD3 (BD), Alexafluor700 anti-CD14 (BioLegend), BV605 anti-CD11b (BioLegend), BV711 anti-CD11c (BioLegend), APC anti-CD1a (BioLegend), PerCP-Cy5.5 anti-HLA-A, B, C (BioLegend), APC-H7 anti-HLA-DR (BD), FITC anti-CD80 (BioLegend), PE-Cy7 anti-CD83 (BioLegend), BUV395 anti-CD86 (BD), BV510 anti-PD-L1 (BioLegend), BV421 anti-IL-12 (BD), and Zombie UV (BioLegend).

[0332] Each staining sample contained moDCs from one well of a 6-well plate used for cell culture and stimulation. Cells were washed twice with PBS and resuspended in 100 μl of Zombie UV diluted in phosphate-buffered saline (PBS) and incubated for 15 minutes. Cells were washed twice with FACS buffer and resuspended in 100 μl of staining mixture containing 50 μl of Brilliant Stain Buffer (BD) and 50 μl of antibodies against cell surface markers diluted in FACS buffer and incubated for 30 minutes. Cells were washed twice with PBS and resuspended in 100 μl of Fixation / Permeabilization Solution (BD) and incubated for 20 minutes. Cells were washed twice with Perm / Wash Buffer (BD) and resuspended in 100 μl of staining mixture containing 50 μl of Brilliant Stain Buffer and 50 μl of antibodies against intracellular markers diluted in Perm / Wash Buffer. Cells were washed twice with Perm / Wash buffer and resuspended in 200 μl of FACS buffer for analysis.

[0333] FACS staining controls included unstained and single-stained controls. Cell staining was analyzed using an LSR Fortessa Cell Analyser (BD). PMT voltage and compensation values ​​were set using the FACS staining controls. Gates for analysis of moDCs were determined by size (FSC-A vs. SSC-A), singlets (FSC-A vs. FSC-H), live (Zombie UV negative), and CD3 - CD14 - cells (CD3 vs. CD14) and CD11b + CD11c + The median fluorescence intensity (MFI) of cell surface markers was determined for the entire moDC population.

[0334] Results: As shown in Figure 15, stimulation of moDCs with SVX-3001, CD40L, or LPS containing IFN-γ for 48 hours resulted in upregulation of cell surface markers HLA-A, B, C, HLA-DR, CD80, CD83, CD86, and PD-L1 and increased expression of IL-12. These markers were not upregulated in unstimulated moDCs or moDCs stimulated with a human IgG1 isotype control antibody.

[0335] Conclusion: The anti-CD40 antibody SVX-3001 has agonistic activity on human monocyte-derived dendritic cells (moDCs), an example of antigen-presenting cells (APCs), resulting in increased expression of the costimulatory molecules B7-1 (CD80), B7-2 (CD86), and PD-L1 (CD274), along with HLA-A, B, and C (MHC class I), HLA-DR (MHC class II), CD83, and the pro-inflammatory cytokine IL-12. This agonistic activity is dependent on the antigen-binding domain of the antibody, as this response is not observed with a human IgG1 isotype control antibody that shares the same Fc domain. Example 10 - Activation of Monocyte-Derived Dendritic Cells by Anti-CD40 Antibodies - Dose Response

[0336] Stimulation of monocyte-derived dendritic cells (moDCs) in the presence of SVX-3001 was measured with reference to the expression of costimulatory molecules. The dose response of the stimulatory effect of SVX-3001 was also measured.

[0337] Methods: Human buffy coat samples were obtained from the Australian Red Cross Blood Service (ethics approval: RDHS-243-15, Human Research Ethics Office, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coat samples by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences). Human PBMCs were cultured at 5x10 in RPMI (Gibco) + 10% FCS (Hyclone). 6 Monocytes were seeded into 6-well plates (Corning-Falcon) at a density of 100 cells / well and incubated at 37°C for 2 hours to allow adherence of the monocytes to the plastic. The medium containing non-adherent cells was then removed.

[0338] Adherent monocytes were differentiated into monocyte-derived dendritic cells (moDCs) by culturing for 7 days in RPMI + 10% FCS containing 80 ng / ml recombinant human GM-CSF (Shenandoah Biotechnology), 10 ng / ml recombinant human IL-4 (Shenandoah Biotechnology), and 10 μg / ml polymyxin B (Sigma-Aldrich). The medium containing GM-CSF, IL-4, and polymyxin B was replaced on day 4.

[0339] Cells were stimulated on day 7 in medium containing GM-CSF and IL-4 but without polymyxin B. Stimulation conditions included 1 μg / ml, 0.316 μg / ml, 0.1 μg / ml, 0.0316 μg / ml, and 0.01 μg / ml of the anti-CD40 antibody SVX-3001. Controls included medium alone (no stimulation), 1 μg / ml of human IgG1 isotype control (BioLegend), and 1 μg / ml of LPS (Sigma-Aldrich) containing 20 ng / ml of recombinant human IFN-γ (Shenandoah Biotechnology). On day 9, cells were treated with 1x Brefeldin A solution (BioLegend) for 4 hours, after which they were harvested and stained for FACS analysis (48 hours of stimulation).

[0340] FACS staining was performed in 96-well U-bottom plates (Corning-Falcon). The FACS buffer used for washing steps and antibody dilution consisted of PBS containing 1% BSA (Sigma), 1% FCS (Hyclone), and 0.01% w / v sodium azide (Sigma). Staining reagents included BUV805 anti-CD3 (BD), Alexafluor700 anti-CD14 (BioLegend), BV605 anti-CD11b (BioLegend), BV711 anti-CD11c (BioLegend), APC anti-CD1a (BioLegend), PerCP-Cy5.5 anti-HLA-A, B, C (BioLegend), APC-H7 anti-HLA-DR (BD), FITC anti-CD80 (BioLegend), PE-Cy7 anti-CD83 (BioLegend), BUV395 anti-CD86 (BD), BV510 anti-PD-L1 (BioLegend), BV421 anti-IL-12 (BD), and Zombie UV (BioLegend).

[0341] Each staining sample contained moDCs from one well of a 6-well plate used for cell culture and stimulation. Cells were washed twice with PBS and resuspended in 100 μl of Zombie UV diluted in phosphate-buffered saline (PBS) and incubated for 15 minutes. Cells were washed twice with FACS buffer and resuspended in 100 μl of staining mixture containing 50 μl of Brilliant Stain Buffer (BD) and 50 μl of antibodies against cell surface markers diluted in FACS buffer and incubated for 30 minutes. Cells were washed twice with PBS and resuspended in 100 μl of Fixation / Permeabilization Solution (BD) and incubated for 20 minutes. Cells were washed twice with Perm / Wash Buffer (BD) and resuspended in 100 μl of staining mixture containing 50 μl of Brilliant Stain Buffer and 50 μl of antibodies against intracellular markers diluted in Perm / Wash Buffer. Cells were washed twice with Perm / Wash buffer and resuspended in 200 μl of FACS buffer for analysis.

[0342] FACS staining controls included unstained and single-stained controls. Cell staining was analyzed using an LSR Fortessa Cell Analyser (BD). PMT voltage and compensation values ​​were set using the FACS staining controls. Gates for analysis of moDCs were size (FSC-A vs. SSC-A), singlet (FSC-A vs. FSC-H), live (Zombie UV vs. SSC-A), CD3 - CD14 - cells (CD3 vs. CD14), CD11b + CD11c + The median fluorescence intensity (MFI) of cell surface markers was determined for the entire moDC population.

[0343] Results: Stimulation of human moDCs for 48 hours with anti-CD40 antibodies SVX-3001, APX005 M, and CP-870,893 resulted in dose-dependent upregulation of cell surface markers HLA-A, B, C (MHC class I), HLA-DR (MHC class II), CD80, CD83, CD86, and PD-L1, as well as the cytokine IL-12. Figure 16 shows the mean fluorescence intensity (MFI) of FACS staining of moDCs stimulated with SVX-3001 at different concentrations for 48 hours. The MFI of unstimulated moDCs is indicated by the dotted line.

[0344] Conclusions: The anti-CD40 antibody SVX-3001 provides a dose-dependent agonistic signal to human moDCs that results in upregulation of cell surface markers HLA-A, B, C, HLA-DR, CD80, CD83, CD86, and PD-L1, as well as the cytokine IL-12. Example 11 - Gene synthesis of codon-optimized sequence of SVX-3001

[0345] The DNA sequences encoding the SVX-3001 heavy chain and the SVX-3001 light chain were optimized for expression in Homo sapiens using GeneOptimizer™ by GeneArt, Thermo Fisher Scientific, resulting in the gene sequences 20ACGJQC_Selvax01HC (SEQ ID NO: 21, Figure 17) and 20ACGJRC_Selvax01LC (SEQ ID NO: 22, Figure 18). In Figures 17 and 18, the Kozak sequences are shown in bold and the coding sequences for the heavy and light chains are underlined.

[0346] The gene sequences 20ACGJQC_SelvaxO1HC and 20ACGJRC_SelvaxO1LC were synthesized by GeneArt, Thermo Fisher Scientific, and inserted into pcDNA3.4-TOPO to generate 20ACGJQC_SelvaxO1HC-pcDNA3.4-TOPO (Figure 19) and 20ACGJRC_SelvaxO1LC-pcDNA3.4-TOPO (Figure 20). The present invention provides, for example, the following items. (Item 1) (i) V containing three CDRs H chain and (ii) a V containing three CDRs L and wherein one or more heavy chain complementarity determining regions (CDRHs) are selected from the following: (d) a CDRH1 sequence comprising SEQ ID NO:1; (e) a CDRH2 sequence comprising SEQ ID NO:2; or (f) a CDRH2 sequence comprising SEQ ID NO: 2 containing one or two amino acid substitutions, deletions, or insertions; 1. An isolated agonist anti-CD40 antibody or fragment thereof selected from the group consisting of: (Item 2) (a) a CDRH3 sequence comprising SEQ ID NO:3; (b) a CDRL1 sequence comprising SEQ ID NO:4; (c) a CDRL2 sequence comprising SEQ ID NO:5; or (d) a CDRL3 sequence comprising SEQ ID NO: 6 2. The isolated agonist anti-CD40 antibody of item 1, further comprising: (Item 3) A: (a) a CDRH1 sequence comprising SEQ ID NO: 1; or (b) a CDRH2 sequence comprising SEQ ID NO:2; and B: (c) a CDRH3 sequence comprising SEQ ID NO:3; (d) a CDRL1 sequence comprising SEQ ID NO:4; (e) a CDRL2 sequence comprising SEQ ID NO:5; or (f) a CDRL3 sequence comprising SEQ ID NO: 6 3. The isolated agonist anti-CD40 antibody of item 1 or 2, comprising: (Item 4) (a) a CDRH1 sequence comprising SEQ ID NO: 1; (b) a CDRH2 sequence comprising SEQ ID NO:2; (c) a CDRH3 sequence comprising SEQ ID NO:3; (d) a CDRL1 sequence comprising SEQ ID NO:4; (e) a CDRL2 sequence comprising SEQ ID NO:5; and (f) a CDRL3 sequence comprising SEQ ID NO: 6 4. The isolated agonist anti-CD40 antibody according to items 1 to 3, comprising: (Item 5) 7. The isolated agonist anti-CD40 antibody of any of the preceding items, comprising a heavy chain variable region of SEQ ID NO:7. (Item 6) 8. The isolated agonist anti-CD40 antibody of any of the preceding items, comprising a light chain variable region of SEQ ID NO:8. (Item 7) 19. The isolated agonist anti-CD40 antibody of any of the preceding items, comprising a heavy chain of SEQ ID NO: 19 or a light chain of SEQ ID NO: 20. (Item 8) 7. The isolated agonist anti-CD40 antibody of any of the preceding items, comprising a heavy chain variable region of SEQ ID NO:7 and a light chain variable region of SEQ ID NO:8. (Item 9) 8. The isolated agonistic anti-CD40 antibody of any of the preceding items, which is a humanized antibody directed against human CD40. (Item 10) 9. The isolated agonistic anti-CD40 antibody of item 8, which is a monoclonal antibody. (Item 11) The isolated agonist anti-CD40 antibody of any of the preceding items, which is an IgG antibody. (Item 12) 19. The isolated agonist anti-CD40 antibody of any of the preceding items, comprising a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 20. (Item 13) 13. A pharmaceutical composition comprising at least one agonist anti-CD40 antibody according to any one of items 1 to 12. (Item 14) 13. A pharmaceutical composition comprising at least one isolated agonist anti-CD40 antibody according to any one of items 1 to 12 and a pharmaceutically acceptable excipient. (Item 15) 15. The pharmaceutical composition according to item 13 or 14, further comprising an additional active agent. (Item 16) 16. The pharmaceutical composition of item 15, wherein the additional active agent is selected from the group consisting of a radioisotope, a radionuclide, a toxin, or a therapeutic and a chemotherapeutic group. (Item 17) 13. A method for producing the agonist anti-CD40 antibody according to any one of items 1 to 12, comprising the step of preparing the agonist anti-CD40 antibody from a host cell that secretes the agonist anti-CD40 antibody. (Item 18) 13. A method for treating or preventing symptoms associated with a malignant tumor in a patient, comprising administering to a patient in need thereof an effective amount of at least one isolated agonist anti-CD40 antibody according to any one of paragraphs 1 to 12. (Item 19) 13. A method for increasing antigen presentation by APCs (including macrophages, DCs, and B cells) in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody of any one of items 1 to 12. (Item 20) 13. A method for activating antigen-presenting cells in a subject, the method comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody according to any one of items 1 to 12. (Item 21) 13. A method for enhancing expression of MHC and / or immune co-stimulatory molecules in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody of any one of paragraphs 1 to 12. (Item 22) 13. A method for stimulating the production of pro-inflammatory cytokines in a subject, the method comprising administering an effective amount of at least an agonistic anti-CD40 antibody according to any one of items 1 to 12. (Item 23) Item 24. A method for inducing T cell activation in a subject, comprising administering an effective amount of at least one agonist anti-CD40 antibody described in any one of Items 1 to 12. 13. A method for mimicking CD40L signaling and substituting for the function of CD4+ lymphocytes in a subject, the method comprising administering an effective amount of at least one agonistic anti-CD40 antibody according to any one of items 1 to 12. (Item 25) 13. A method for overcoming T cell tolerance in a subject bearing a tumor, or inducing an effective cytotoxic T cell response, or enhancing the effectiveness of an anti-tumor vaccine, comprising administering to a subject an effective amount of at least one agonistic anti-CD40 antibody according to any one of items 1 to 12. (Item 26) 13. A method for promoting secretion of autoantibodies by B cells against antigens expressed on tumor cells in a subject, the method comprising administering an effective amount of the agonist anti-CD40 antibody of any one of items 1 to 12. (Item 27) 13. A method of upregulating costimulatory markers, releasing IL-12 to activate CD8+ T cells, and stimulating specific cytotoxic T cell responses against cross-presented tumor antigens in a subject, comprising administering an effective amount of the agonist anti-CD40 antibody of any one of paragraphs 1 to 12. (Item 28) 13. A pharmaceutical composition comprising an effective amount of at least one agonist anti-CD40 antibody according to any one of items 1 to 12 together with at least a second immune enhancing agent. (Item 29) 29. The pharmaceutical composition of item 28, wherein the immune enhancing agent is selected from IL-2, a TLR-7 agonist, or a systemic cytotoxic chemotherapeutic agent. (Item 30) 13. A method for treating or preventing symptoms associated with a malignant tumor in a patient, comprising administering to a patient in need thereof an effective amount of at least one isolated agonist anti-CD40 antibody according to any one of paragraphs 1 to 12 and an effective amount of at least a second immune enhancing agent. (Item 31) 13. A pharmaceutical composition comprising at least one agonistic anti-CD40 antibody according to any one of items 1 to 12 and IL-2. (Item 32) 13. A method for treating or preventing symptoms associated with a malignant tumor in a patient, comprising administering to a patient in need thereof an effective amount of each of at least one agonistic anti-CD40 antibody according to any one of paragraphs 1 to 12 and IL-2. (Item 33) A method for increasing antigen presentation by APCs (including macrophages, DCs, and B cells) in a subject, comprising administering an effective amount of an agonist according to any one of items 1 to 12. The method comprises administering an anti-CD40 antibody and IL-2. (Item 34) 13. A method of activating antigen-presenting cells in a subject, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody of any one of items 1 to 12 and IL-2. (Item 35) 13. A method for promoting secretion of autoantibodies by B cells against antigens expressed on tumor cells in a subject, the method comprising administering effective amounts of the agonistic anti-CD40 antibody of any one of items 1 to 12 and IL-2. (Item 36) 13. A method of upregulating costimulatory markers, releasing IL-12 to activate CD8+ T cells, and stimulating a specific cytotoxic T cell response against cross-presented tumor antigens in a subject, comprising administering an effective amount of the agonist anti-CD40 antibody of any one of paragraphs 1 to 12 and IL-2. (Item 37) Item 38. A method for increasing antigen presentation by APCs (including macrophages, DCs, and B cells) in a subject, comprising administering effective amounts of at least one agonist anti-CD40 antibody described in any one of Items 1 to 12 and IL-2. 13. A method for enhancing expression of MHC and / or immune co-stimulatory molecules in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody of any one of paragraphs 1 to 12 and IL-2. (Item 39) 13. A method of stimulating the production of a pro-inflammatory cytokine (e.g., IL-12) in a subject, comprising administering an effective amount of each of at least one agonist anti-CD40 antibody of any one of paragraphs 1 to 12 and IL-2. (Item 40) 13. A method for inducing T cell activation in a subject, the method comprising administering an effective amount of each of at least one agonistic anti-CD40 antibody according to any one of items 1 to 12 and IL-2. (Item 41) 13. A method for mimicking CD40L signaling and substituting for the function of CD4+ lymphocytes in a subject, the method comprising administering an effective amount of each of at least one agonistic anti-CD40 antibody according to any one of items 1 to 12 and IL-2. (Item 42) 13. A method for overcoming T cell tolerance in a tumor-bearing subject, or inducing an effective cytotoxic T cell response, or enhancing the effectiveness of an anti-tumor vaccine, comprising administering to a subject an effective amount of each of at least one agonistic anti-CD40 antibody according to any one of items 1 to 12 and IL-2. (Item 43) A nucleic acid molecule encoding the agonist anti-CD40 antibody according to any one of Items 1 to 12. (Item 44) A nucleic acid molecule encoding the heavy chain of an agonist anti-CD40 antibody comprising SEQ ID NO:9. (Item 45) 44. The nucleic acid molecule of item 43, wherein the molecule has at least 80, 80-85, 85-90, 90-95, 95-97, 97-99 or more identities to SEQ ID NO:9. (Item 46) 46. ​​The nucleic acid molecule of any one of items 43 to 45, further comprising a light chain of an agonist anti-CD40 antibody comprising SEQ ID NO: 10. (Item 47) A nucleic acid molecule encoding the heavy chain of an agonist anti-CD40 antibody comprising SEQ ID NO:21. (Item 48) 44. The nucleic acid molecule of item 43, wherein the molecule has at least 80, 80-85, 85-90, 90-95, 95-97, 97-99 or more sequences identical to SEQ ID NO: 21. (Item 49) 49. The nucleic acid molecule of any one of items 46 to 48, further comprising a light chain of an agonist anti-CD40 antibody comprising SEQ ID NO: 22.

Claims

[Claim 1] The invention described in the present specification.

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