Agonist anti-cd40 antibodies

Abstract

The present application relates generally to the identification of certain agonist anti-CD40 antibodies. Based on this, the present application provides novel agonist antibodies and their use in therapy.

Description

Technical Field

[0001] This application generally relates to the identification of certain agonist anti-CD40 antibodies. Based on this, the present invention provides novel agonist antibodies and their use in treatment. Background Technology

[0002] At certain points in their evolutionary process, tumors are recognized by the host's immune system. Despite evidence of recognition, some tumors grow rapidly, possibly due to a lack of danger signals that induce only a weak response, and possibly because they develop immune evasion strategies under increasing immune pressure.

[0003] Tumors treated in their early stages may respond better to immunotherapy alone. However, advanced cancers are more difficult to treat with all forms of therapy, including immunotherapy. The absence of warning signals in these cancers, coupled with the acquisition of increasingly sophisticated immune escape mechanisms, significantly hinders the host's 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 background with appropriate co-stimulatory markers, thereby eliciting a relevant T-cell response. Tumor cells often fail to present their own antigenic stimuli due to the low expression levels of essential co-stimulatory molecules (such as members of the B7 family) in their cells.

[0004] Since tumor antigens cannot be directly presented, cross-presentation may be the only natural antigen presentation method for tumor immunity. In this process, exogenous antigens derived from tumor cells (soluble antigens, apoptotic bodies, or live cancer cells) are taken up by dendritic cells (DCs). These exogenous antigens are not only processed and presented in MHC class II molecules via the classical pathway to elicit CD4+ T cell helper responses, but they are also internalized and displayed in MHC class I molecules for presentation to CD8+ T cells. However, unless the presenting DCs are also properly activated and presentation occurs against a suitable co-stimulatory background, this process will result in a weak response or tolerance. DCs in the tumor environment are often immature and can promote the expansion of regulatory T cells in the lymph nodes draining the tumor. Their function is also suppressed by infiltrating suppressor cells (such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) and cytokines within the tumor and draining lymph nodes. Therefore, effective immunotherapy by “helping” DCs to initiate antigen-specific T cell responses must overcome these obstacles and facilitate this cross-presentation process. Effective cross-initiation requires “licensed” dendritic cells (DCs) and high levels of antigen. The “licensing” or “regulation” of DCs is carried out by antigen-specific CD4+ helper T cells via cross-linking CD40. This linking alters the DC phenotype and function, inhibiting its potential to induce tolerance through autocrine signaling of cytokines such as IL-6 and IL-12, thereby enabling it to activate an effective cytotoxic T lymphocyte (CTL) response. Conversely, CTLs activated by unlicensed DCs are termed “unhelper,” resulting in T cell dysfunction or absence, and also inducing regulatory T cells. CD40, a cell surface molecule and a member of the tumor necrosis factor receptor superfamily, therefore broadly regulates immune activation and mediates tumor apoptosis.

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

[0006] CD40 is found in most melanomas and lung cancer, breast cancer, colon cancer, prostate cancer, pancreatic cancer, kidney cancer, ovarian cancer, head and neck cancer, as well as B-cell malignancies.

[0007] In mouse models of T-cell-mediated immunity, agonist anti-CD40 antibodies have been shown to replace T-cell helper antibodies provided by CD4+ lymphocytes. In tumor-bearing hosts, CD40 agonists trigger effective immune responses against tumor-associated antigens. For example, dendritic cells (DCs) can be "pretreated" with agonist anti-CD40 antibodies to upregulate their co-stimulatory markers, allowing them to activate CD8+ T cells upon encountering them. Thus, agonist anti-CD40 antibodies can replace CD4+ T-cell helper antibodies by inducing CD40 signaling on antigen-loaded DCs, leading to upregulation of B7 co-stimulatory molecules and release of IL-12, thereby enabling DCs to stimulate specific CTL responses.

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

[0009] Patients with CD40 or CD40L germline mutations exhibit significant immunosuppression, are susceptible to opportunistic infections, and have defects in 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, CD40 agonist antibodies have been shown to mimic CD40L signaling and substitute for the function of CD4+ lymphocytes. CD40 agonist antibodies can also overcome T-cell tolerance in tumor-bearing mice, elicit effective cytotoxic T-cell responses, and enhance the efficacy of anti-tumor vaccines.

[0011] These findings contradict the binding of CD40 on the surface of tumor cells, which in many cases mediates direct cytotoxic effects, leading to tumor regression through apoptosis and necrosis. Although the exact function of CD40 on tumor cells remains unclear, in vitro binding of CD40 inhibits the growth of solid tumor cells and high-grade B-cell lymphoma lineages. Furthermore, CD40-mediated tumor suppression has also been observed in vivo, including inhibition of breast cancer or B-cell lymphoma xenografts in immunocompromised mice.

[0012] These diverse roles of CD40 provide an opportunity that activation of CD40 in tumor-bearing animals can lead to: (i) direct cytotoxicity against tumors, and (ii) the delivery of tumor antigens to APCs that are simultaneously activated by CD40.

[0013] Agonistous monoclonal antibodies (mAbs) targeting CD40 have demonstrated therapeutic activity in a range of preclinical models. These findings, along with the dual function of CD40, make CD40 an attractive target for cancer therapy and provide a basis for the clinical development of agonist anti-CD40 antibodies.

[0014] However, there is still a need for other agonist monoclonal antibodies targeting human CD40. It is in this context that the present invention was developed. Invention Overview

[0016] The inventors have developed a novel agonist antiCD40 antibody suitable for use as an immunogenic active agent in the treatment of malignant tumors in patients.

[0017] The agonist anti-CD40 antibody of the present invention is suitable for use in a variety of activities, including but not limited to binding to: (a) CD40+ tumor blood vessels, allowing them to allow T cell transport; (b) CD40+ B cells in tumors, spleen, and lymph nodes, causing them to secrete elevated levels of autoantibodies against antigens expressed on tumor cells; and (c) CD40+ DCs and macrophages, causing them to upregulate co-stimulatory markers and release IL-12 to activate CD8+ T cells and stimulate specific CTL responses against cross-presented tumor antigens. Furthermore, when the antibody of the present invention is combined with other immune enhancers (such as local or systemic IL-2, TLR-7 agonists, and / or cytotoxic chemotherapy), effective anti-tumor CD8+ cytotoxic T lymphocyte responses can be observed. Therefore, the antibody of the present invention constitutes and provides a new universally applicable principle in the field of cancer therapy.

[0018] In a first aspect, the present invention comprises an isolated agonist anti-CD40 antibody or a fragment thereof, comprising (i) a VH chain containing three CDRs and (ii) a VL chain containing three CDRs, wherein one or more heavy chain complementarity-determining regions (CDRHs) are selected from:

[0019] a) A CDRH1 sequence containing SEQ ID NO:1;

[0020] b) A CDRH2 sequence containing SEQ ID NO:2;

[0021] c) A CDRH3 sequence containing SEQ ID NO:3; or

[0022] d) Contains any one of SEQ ID NO:1 to 3, with one or two amino acid substitutions, deletions, or insertions.

[0023] In one embodiment of the first aspect of the invention, the invention comprises an isolated agonist anti-CD40 antibody or a fragment thereof, comprising (i) a VH chain containing three CDRs and (ii) a VL chain containing three CDRs, wherein one or more heavy chain complementarity-determining regions (CDRHs) are selected from:

[0024] a) A CDRH1 sequence containing SEQ ID NO:1;

[0025] b) A CDRH2 sequence containing SEQ ID NO:2; or

[0026] c) Contains a CDRH2 sequence containing one or two amino acid substitutions, deletions, or insertions of SEQ ID NO:2.

[0027] In one embodiment of the first aspect of the invention, the isolated agonist antiCD40 antibody comprises the heavy chain variable region of SEQ ID NO:7.

[0028] In another embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody further comprises one or more light chain complementarity-determining regions (CDRLs) selected from:

[0029] a) A CDRL1 sequence containing SEQ ID NO:4;

[0030] b) A CDRL2 sequence containing SEQ ID NO:5; or

[0031] c) The CDRL3 sequence containing SEQ ID NO:6.

[0032] In a second aspect, the present invention comprises an isolated agonist anti-CD40 antibody or a fragment thereof, comprising (i) a VH chain containing three CDRs and (ii) a VL chain containing three CDRs, wherein one or more CDRLs are selected from:

[0033] a) A CDRL1 sequence containing SEQ ID NO:4;

[0034] b) A CDRL2 sequence containing SEQ ID NO:5;

[0035] c) A CDRL3 sequence containing SEQ ID NO:6; or

[0036] d) Contains any one of SEQ ID NO:4 to 6, with one or two amino acid substitutions, deletions, or insertions.

[0037] In one embodiment of the second aspect of the invention, the isolated agonist antiCD40 antibody comprises the light chain variable region of SEQ ID NO:8.

[0038] In another embodiment of the second aspect of the invention, the isolated agonist anti-CD40 antibody further comprises one or more CDRHs selected from the following:

[0039] a) A CDRH1 sequence containing SEQ ID NO:1;

[0040] b) A CDRH2 sequence containing SEQ ID NO:2; or

[0041] c) A CDRH3 sequence containing SEQ ID NO:3.

[0042] In a third aspect, the present invention is an isolated agonist anti-CD40 antibody comprising:

[0043] a) A CDRH1 sequence containing SEQ ID NO:1;

[0044] b) A CDRH2 sequence containing SEQ ID NO:2;

[0045] c) A CDRH3 sequence containing SEQ ID NO:3;

[0046] d) Contains a CDRL1 sequence of SEQ ID NO:4;

[0047] e) A CDRL2 sequence containing SEQ ID NO:5; and

[0048] f) Contains the CDRL3 sequence of SEQ ID NO:6.

[0049] In one embodiment of the third aspect of the invention, the isolated agonist antiCD40 antibody comprises the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8.

[0050] In one embodiment of the above aspects of the present invention, the agonist anti-CD40 antibody may be a mouse 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.

[0051] In one embodiment, the isolated agonist anti-CD40 antibody is a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, diabody, or single-chain antibody molecule.

[0052] In one implementation, the isolated agonist anti-CD40 antibody is a humanized antibody.

[0053] In one implementation, the isolated agonist anti-CD40 antibody is a monoclonal antibody.

[0054] In one embodiment, the isolated agonist anti-CD40 antibody is of type IgG1, IgG2a, IgG2b, IgG3, or IgG4. Preferably, the isolated agonist anti-CD40 antibody is of type IgG1.

[0055] In one embodiment, the isolated agonist anti-CD40 antibody is conjugated with a labeling group.

[0056] In one implementation, the isolated agonist anti-CD40 antibody enhances CD40 activity.

[0057] In one embodiment, the present invention comprises a nucleic acid molecule encoding the isolated agonist anti-CD40 antibody described herein.

[0058] In one embodiment, the present invention comprises a vector containing the nucleic acid molecule described herein.

[0059] In one embodiment, the invention comprises a host cell containing the nucleic acid molecules described herein.

[0060] In one embodiment, the present invention comprises an isolated agonist anti-CD40 antibody, which is adapted to achieve at least one or more of the following functions:

[0061] a. Promotes the secretion of autoantibodies by B cells against antigens expressed on tumor cells;

[0062] b. Upregulate co-stimulatory markers and release IL-12 to activate CD8+ T cells and stimulate specific cytotoxic T cell responses against cross-presented tumor antigens;

[0063] c. Increase antigen presentation by APCs (including macrophages, dendritic cells, and B cells);

[0064] d. Enhance the expression of MHC and immune co-stimulatory molecules (e.g., CD86, CD80, CD83, PD-L1, HLA-A, B, C, or HLA-DR);

[0065] e. Stimulates the production of pro-inflammatory cytokines (such as IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ); or

[0066] f. Inducing T cell activation;

[0067] g. Simulates CD40L signaling and replaces the function of CD4+ lymphocytes;

[0068] h. Overcoming T-cell tolerance in tumor-bearing individuals;

[0069] i. Induces an effective cytotoxic T cell response;

[0070] j. Enhance the effectiveness of anti-tumor vaccines; or

[0071] k. Makes the tumor vascular system more susceptible to immune infiltration.

[0072] In one embodiment, the present invention is a pharmaceutical composition comprising at least one isolated agonist antiCD40 antibody as described herein. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0073] In one embodiment, the pharmaceutical composition may further comprise an active agent, such as a radioisotope, a radionuclide, a toxin, or a therapeutic or chemotherapeutic group.

[0074] In another aspect, the present invention relates to a method for preparing the agonist anti-CD40 antibody described herein, comprising the steps of: preparing the agonist anti-CD40 antibody from a host cell that secretes the agonist anti-CD40 antibody.

[0075] In another aspect, the present invention relates to a method for treating or preventing malignant tumor-related conditions in a patient, comprising the steps of administering a therapeutically effective amount of at least one isolated agonist antiCD40 antibody disclosed herein to a patient in need of the antibody.

[0076] In one embodiment, the present invention includes a method for increasing antigen presentation of APCs (including macrophages, dendritic cells, and B cells) in an individual, comprising administering an effective amount of at least one isolated agonist antiCD40 antibody disclosed herein.

[0077] In one embodiment, the present invention includes a method for activating antigen-presenting cells in an individual, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0078] In one embodiment, the present invention includes a method for enhancing the expression of MHC and / or immune co-stimulatory molecules in an individual, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein. Preferably, the MHC and / or immune co-stimulatory molecule is selected from CD80, CD86, PD-L1, HLA-A, B, C, HLA-DR, and CD83.

[0079] In one embodiment, the invention includes a method for stimulating the production of pro-inflammatory cytokines in an individual, comprising administering an effective amount of at least one of the agonist anti-CD40 antibodies 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.

[0080] In one embodiment, the present invention includes a method for inducing T cell activation in an individual, comprising administering an effective amount of at least one of the agonist antiCD40 antibodies disclosed herein.

[0081] In one embodiment, the present invention includes a method for mimicking CD40L signaling and replacing CD4+ lymphocyte function in an individual, comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein.

[0082] In one embodiment, the invention includes a method for overcoming T cell tolerance in tumor-bearing animals or inducing an effective cytotoxic T cell response in an individual or enhancing the effectiveness of an anti-tumor vaccine, comprising administering an effective amount of at least one of the agonist anti-CD40 antibodies disclosed herein.

[0083] In one embodiment, the present invention includes a method for promoting the secretion of autoantibodies against antigens expressed on tumor cells by B cells in an individual, comprising administering an effective amount of the disclosed agonist antiCD40 antibody.

[0084] In one embodiment, the invention includes a method of upregulating a co-stimulatory marker and releasing IL-12 in an individual to activate CD8+ T cells and stimulate a specific cytotoxic T cell response against a cross-presented tumor antigen, comprising administering an effective amount of the disclosed agonist antiCD40 antibody.

[0085] The complex synergistic effects of events required for tumor eradication suggest that combination therapies can be beneficial in certain situations. Anti-CD40 agonist antibodies can be used in conjunction with other active agents that release antigens, promote cytokine release, enhance immune surveillance, and reduce inhibitory networks to enhance this effect.

[0086] Therefore, in one embodiment, the present invention includes a pharmaceutical formulation comprising: an effective amount of at least one disclosed agonist anti-CD40 antibody and one or more other immune enhancers. Such immune enhancers include, but are not limited to, IL-2, TLR-7 agonists, or systemic cytotoxic chemotherapeutic agents.

[0087] In another embodiment, the invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising administering to a patient in need an effective amount of at least one isolated agonist antiCD40 antibody disclosed herein and an effective amount of at least a second immune enhancer.

[0088] One treatment option is to alter the tumor microenvironment itself to promote the tumor as a source of stimulation for its own antigens. This can be achieved by introducing IL-2 and anti-CD40 antibodies into the tumor site. When administered directly in combination, this co-administration avoids the toxicities associated with systemic administration and successfully leads to regression of larger tumors as well as distant tumors, while maintaining long-term protective memory. Co-administration of IL-2 and CD40 agonists results in increased macrophage activity and B cell activation. Therefore, the combination of IL-2 and CD40 agonists can show significant benefits for a variety of cancers, where cancer regression is associated with a neutrophil-dominant inflammatory response.

[0089] Therefore, in one embodiment, the present invention comprises a pharmaceutical composition containing at least one agonist antiCD40 antibody and IL-2 as described herein.

[0090] In one embodiment, the invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising administering to a patient in need an effective amount of each of at least one of the isolated agonist antiCD40 antibody and IL-2 disclosed herein.

[0091] In one embodiment, the present invention includes a method for increasing antigen presentation of APCs (including macrophages, DCs, and B cells) in an individual, comprising administering an effective amount of each of at least one of the isolated agonist antiCD40 antibody and IL-2 disclosed herein.

[0092] In one embodiment, the present invention includes a method for activating antigen-presenting cells in an individual, comprising administering an effective amount of at least one isolated agonist antiCD40 antibody and IL-2 disclosed herein.

[0093] In one embodiment, the present invention includes a method for enhancing the expression of MHC and / or immune co-stimulatory molecules in an individual, comprising administering an effective amount of each of at least one of the agonist described herein, an anti-CD40 antibody, and IL-2. Preferably, the MHC and / or immune co-stimulatory molecule is selected from CD80, CD86, PD-L1, HLA-A, B, C, HLA-DR, and CD83.

[0094] In one embodiment, the invention includes a method for stimulating the production of pro-inflammatory cytokines in an individual, comprising administering an effective amount of each of at least one of the agonists described herein, an anti-CD40 antibody 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.

[0095] In one embodiment, the invention includes a method for inducing T cell activation in an individual, comprising administering an effective amount of each of at least one of the agonists described herein, namely, an anti-CD40 antibody and IL-2.

[0096] In one embodiment, the present invention includes a method for mimicking CD40L signaling and replacing CD4+ lymphocyte function in an individual, comprising administering an effective amount of at least one of the agonist anti-CD40 antibody and IL-2 described herein.

[0097] In one embodiment, the present invention includes a method for overcoming T cell tolerance in tumor-bearing animals or for inducing an effective cytotoxic T cell response or enhancing the effectiveness of an antitumor vaccine, comprising administering an effective amount of each of at least one of the agonists described herein, namely, an antiCD40 antibody and IL-2.

[0098] In one embodiment, the present invention includes a method for promoting the secretion of autoantibodies against antigens expressed on tumor cells by B cells in an individual, comprising administering an effective amount of the disclosed agonist antiCD40 antibody and IL-2.

[0099] In one embodiment, the present invention includes a method for upregulating a co-stimulatory marker and releasing IL-12 in an individual to activate CD8+ T cells and stimulate a specific cytotoxic T cell response against a cross-presented tumor antigen, comprising administering an effective amount of the disclosed agonist antiCD40 antibody and IL-2.

[0100] The most effective approach for treating or preventing malignant tumor-related conditions in patients may require combination therapy, in which treatment interventions are performed sequentially over time.

[0101] Therefore, in one embodiment, the present invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising sequentially administering, over time, an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein, along with other therapeutic interventions, to a patient in need. In some embodiments, the other therapeutic interventions are selected from surgery, radiotherapy, chemotherapy, thermotherapy, and immunotherapy.

[0102] One treatment option involves altering cells isolated from the patient and then transferring those cells back into the patient. This can be achieved by treating cells isolated from the patient with an anti-CD40 antibody. Cells treated with an anti-CD40 antibody can be treated with other active agents. In some embodiments, the active agent is selected from tumor-specific peptides, tumor cell lysates, cytokines, agonists, and mitogens.

[0103] Therefore, in one embodiment, the present invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising administering to a patient in need cells treated with an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein. In some embodiments, the cells have been isolated from the patient and are selected from dendritic cells (DCs), macrophages, B cells, myeloid cells, lymphoid cells, and hematopoietic stem cells.

[0104] Other objects, advantages, and novel features will be set forth in the description which follows, or will be apparent to those skilled in the art upon examination of the accompanying drawings and the detailed description of the following few non-limiting embodiments. Brief description of the attached diagram

[0106] This disclosure will be provided in detail in the following description of preferred embodiments with reference to the following figures, wherein:

[0107] Figure 1 The nucleotide sequence of the heavy chain of the humanized agonist anti-human CD40 antibody is shown.

[0108] Figure 2 The nucleotide sequence of the light chain of the humanized agonist anti-human CD40 antibody is shown.

[0109] Figure 3 The nucleotide and amino acid sequence of the heavy chain variable region (VH) of antibody SVX-3001 is shown.

[0110] Figure 4 The nucleotide and amino acid sequence of the light chain variable region (VL) of antibody SVX-3001 is shown.

[0111] Figure 5 The nucleotide and amino acid sequences of the gene sequence encoding the synthetic gene sequence of antibody SVX-3001 heavy chain are shown. This gene sequence was cloned into plasmid pcDNA3.1(+) to produce plasmid pcDNA3.1(+)_Selvax01HC.

[0112] Figure 6 The nucleotide and amino acid sequences of the gene sequence encoding the synthetic light chain of antibody SVX-3001 are shown. This gene sequence was cloned into plasmid pcDNA3.1(+) to produce plasmid pcDNA3.1(+)_Selvax01LC.

[0113] Figure 7 Displaying the plasmid map of pcDNA3.1(+)_Selvax01HC.

[0114] Figure 8 Displaying the plasmid map of pcDNA3.1(+)_Selvax01LC.

[0115] Figure 9The results of the ELISA assay show that the generated antibody is CD40-specific IgG.

[0116] Figure 10 The results of FACS analysis show the detection of SVX-3001 binding to CD40-expressing cells.

[0117] Figure 11 The results of a CFSE assay show the detection of cell division in human PBMCs in response to SVX-3001 stimulation.

[0118] Figure 12 The results of LEGENDplex assays show the detection of cytokines produced from human PBMCs in response to SVX-3001 stimulation (with and without IL-2).

[0119] Figure 13 The results of an FACS assay show the ability of SVX-3001 blocking antibodies B-B20 and LOB7 / 6 to bind to CD40.

[0120] Figure 14 A Biacore T200 sensor map showing epitope mapping of SVX-3001 relative to many different CD40 antibodies.

[0121] Figure 15 The results of FACS assays for SVX-3001-activated monocyte-derived dendritic cells (moDCs) are shown.

[0122] Figure 16 The results of a FACS assay show the dose-response of monocyte-derived dendritic cells (moDCs) to SVX-3001.

[0123] Figure 17 The nucleotide sequence of the gene encoding the synthetic gene of antibody SVX-3001 heavy chain was displayed and cloned into plasmid pcDNA3.4-TOPO to generate plasmid 20ACGJQC_Selvax01HC-pcDNA3.4-TOPO.

[0124] Figure 18 The nucleotide and amino acid sequences of the gene encoding the synthetic light chain of antibody SVX-3001 were displayed and cloned into plasmid pcDNA3.4-TOPO to generate plasmid 20ACGJRC_Selvax01LC-pcDNA3.4-TOPO.

[0125] Figure 19 Displaying the plasmid map of 20ACGJQC_Selvax01HC-pcDNA3.4-TOPO.

[0126] Figure 20Displaying the plasmid map of 20ACGJRC_Selvax01LC-pcDNA3.4-TOPO.

[0127] Brief description of sequence lists

[0128] Table 1: Sequence List

[0129]

[0130]

[0131] Invention Details

[0133] This invention relates to isolated agonist anti-human CD40 antibodies, which are particularly capable of increasing antigen presentation by APCs (including macrophages, dendritic cells, and B cells). In some embodiments, the antibody provides a method for enhancing the expression of MHC and / or immune co-stimulatory molecules. The antibody can also stimulate the production of pro-inflammatory cytokines that induce T cell activation. This is achieved by mimicking CD40L signaling and displacing the function of CD4+ lymphocytes. Therefore, the isolated agonist anti-CD40 antibodies described herein can improve T cell tolerance in tumor-bearing animals, induce effective cytotoxic T cell responses, and / or enhance the effectiveness of anti-tumor vaccines.

[0134] For convenience, the following sections provide an overview of the various meanings of the terms used herein. Following this, general aspects of agonist anti-CD40 antibodies are discussed, followed by specific examples demonstrating the properties and applications of various implementations of the antibodies.

[0135] definition

[0136] The scope of this invention is not limited to 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. Consistently, those skilled in the art will understand that variations and modifications can be readily made to the invention beyond the specific description herein. It should be understood that the invention includes all such variations and modifications. The invention also includes each step, feature, composition, and compound individually or collectively mentioned or indicated in the specification, and any and all combinations of such steps or features, or any combination of two or more.

[0137] In this application, unless otherwise expressly stated, the use of the singular includes the plural. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms such as "including" and "containing" is not restrictive. Furthermore, unless otherwise expressly stated, the terms "element" or "component" cover elements and components comprising one unit as well as elements and components comprising more than one subunit. Furthermore, the use of the term "part" can include a portion of a structural part or the entire structural part.

[0138] The term "antibody" refers to any intact immunoglobulin of the same type or a fragment thereof that can compete with intact antibodies for specific binding to a target antigen, and includes, for example, chimeric and bispecific antibodies. Intact antibodies typically contain at least two full-length heavy chains and two full-length light chains, but in some cases, they may contain fewer chains, such as naturally occurring antibodies in camels, which may contain only heavy chains. Antibodies can be derived from a single source or can be "chimeric," meaning that different parts of an antibody can be derived from two different antibodies, as described below. Antibodies or binding fragments can be generated in hybridomas, through recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise stated, the term "antibody" encompasses not only antibodies containing two full-length heavy chains and two full-length light chains, but also their derivatives, variants, fragments, and mutant proteins, as described below. Furthermore, unless explicitly excluded, antibodies encompass monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), chimeric antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some implementations, the term also covers peptide bodies.

[0139] Antibody heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined 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. In both the full-length light and heavy chains, the variable and constant regions are typically linked by “J” regions of about 12 or more amino acids, with the heavy chain also including “D” regions of about 10 or more amino acids. See Fundamental Immunology, Chapter 7 (edited by Paul, W., 2nd ed., Raven Press, NY (1989)) (introduced in its entirety for all purposes). The variable region of each light / heavy chain pair typically forms the antigen-binding site.

[0140] Variable regions typically exhibit the same general structure, consisting of three hypervariable regions (also known as complementarity-determining regions or CDRs) connecting relatively conserved framework regions (FRs). CDRs from each pair of chains are usually aligned through the framework regions, enabling the binding of specific epitopes. From the N-terminus to the C-terminus, the variable regions of both light and heavy chains typically contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Typically, amino acids are assigned to these domains according to the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).

[0141] In some embodiments, the antibody heavy chain binds to the epitope in the absence of the antibody light chain. In some embodiments, the antibody light chain binds to the epitope in the absence of the antibody heavy chain. In some embodiments, the antibody-binding region binds to the epitope in the absence of the antibody light chain. In some embodiments, the antibody-binding region binds to the epitope in the absence of the antibody heavy chain. In some embodiments, a single variable region specifically binds to the epitope in the absence of other variable regions.

[0142] In some embodiments, a definitive characterization of the CDR and identification of residues containing the antibody binding site are achieved by resolving the structure of the antibody and / or the antibody-ligand complex. In some embodiments, this can be achieved using any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, a variety of analytical methods can be employed to identify or approximately determine the CDR region. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the Contact definition.

[0143] By convention, the CDR regions in heavy chains are usually referred to as H1, H2, and H3, and numbered sequentially from the amino terminus to the carboxyl terminus. The CDR regions in light chains are usually referred to as L1, L2, and L3, and numbered sequentially from the amino terminus to the carboxyl terminus.

[0144] The term "light chain" includes full-length light chains and fragments thereof, which possess sufficient variable region sequences to confer binding specificity. Full-length light chains include a variable region domain (VL) and a constant region domain (CL). The variable region domain of the light chain is located at the amino terminus of the polypeptide. Light chains include κ chains and λ chains.

[0145] The term "heavy chain" includes full-length heavy chains and their fragments, which possess sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain (VH) and three constant region domains (CH1, CH2, and CH3). The VH domain is located at the amino terminus of the polypeptide, the CH domain at the carboxyl terminus, and CH3 is closest to the carboxyl terminus. Heavy chains can be any isoform, including IgG (including IgG1, IgG2, IgG3, and IgG4 isoforms), IgA (including IgA1 and IgA2 isoforms), IgM, and IgE.

[0146] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies with two distinct heavy / light chain pairs and two distinct binding sites. Bispecific antibodies can be produced by a variety of methods, including but not limited to hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0147] Each immunoglobulin chain typically consists of several "immunoglobulin domains," each containing approximately 90 to 110 amino acids and exhibiting a characteristic folding pattern. These domains are the basic units that make up antibody peptides. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The C-region of the heavy chain typically contains one or more domains responsible for effector functions. The number of constant region domains in the heavy chain will depend on the isotype. For example, the IgG heavy chain contains three C-region domains, referred to as CH1, CH2, and CH3. The antibodies provided can possess any of these isotypes and subtypes.

[0148] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chain of an antibody, typically comprising approximately 120 to 130 amino acids at the amino terminus of the heavy chain and approximately 100 to 110 amino acids at the amino terminus of the light chain. In some embodiments, the variable regions of different antibodies, and even antibodies from the same species, can differ significantly in their amino acid sequences. The variable region of an antibody often determines the specificity of a particular antibody for its target.

[0149] The term "epitaph" encompasses any determinant cluster capable of being bound by an antibody or T-cell receptor. An epitope is a region that binds to an antibody or T-cell receptor targeting that epitope; when 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 are located on proteins, but in some cases, they can be found on other types of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge characteristics. Typically, antibodies specific to a particular target epitope will preferentially recognize the epitope on the target in a complex mixture of proteins and / or macromolecules.

[0150] The term "polynucleotide" or "nucleic acid" includes single-stranded and double-stranded nucleotide polymers. The nucleotides in a polynucleotide can be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. These modifications include base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and internucleotide linkage modifications, such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphate, phosphoroanilothioate, phoshoraniladate, and phosphoroamidate.

[0151] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotides. In some embodiments, the oligonucleotide is 10 to 60 bases in length. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, for example, for constructing mutant genes. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides can include markers for detection assays, including radioactive markers, fluorescent markers, haptens, or antigenic markers. Oligonucleotides can be used as, for example, PCR primers, cloning primers, or hybridization probes.

[0152] "Isolated nucleic acid molecule" refers to a genome, mRNA, cDNA, or synthetically derived DNA or RNA, or a combination thereof, wherein the isolated polynucleotide is not linked to all or part of a naturally occurring polynucleotide to which it is linked, or is linked to a naturally occurring polynucleotide not linked to which it is linked. "Containing" an isolated nucleic acid molecule of the specified nucleic acid sequence may, in addition to the specified sequence, contain up to ten or even up to twenty other protein coding sequences or portions thereof, or may contain regulatory sequences that effectively link the expression of the coding region of the said nucleic acid sequence, and / or may contain a vector sequence.

[0153] Unless otherwise specified, the left end of any single-stranded polynucleotide sequence discussed herein is referred to as the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of nascent RNA transcripts is called the transcription direction; on a DNA strand with the same sequence as the RNA transcript, the 5' sequence region located at the 5' end of the RNA transcript is called the "upstream sequence"; on a DNA strand with the same sequence as the RNA transcript, the 3' sequence region located at the 3' end of the RNA transcript is called the "downstream sequence".

[0154] The term "control sequence" refers to a polynucleotide sequence that can influence the expression and processing of a coding sequence linked to it. The nature of such a control sequence can vary depending on the host organism. In specific embodiments, control sequences for prokaryotes may include promoters, ribosome binding sites, and transcription termination sequences. For example, control sequences for eukaryotes may include promoters containing one or more recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequences. A "control sequence" may include a leader sequence and / or a fusion partner sequence.

[0155] The term "vector" refers to any molecule or entity (such as nucleic acid, plasmid, bacteriophage, or virus) that can be used to transfer protein-coding information into a host cell.

[0156] The term "expression vector" or "expression construct" refers to a vector suitable for transforming host cells and containing (binding to the host cell) a nucleic acid sequence capable of directing and / or controlling the expression of one or more heterologous coding regions effectively linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control the transcription, translation, and, if introns are present, RNA splicing of the coding regions effectively linked thereto.

[0157] As used herein, “effective linkage” refers to a relationship in which the components to which the term is applied are in a position that allows them to perform their inherent functions under suitable conditions. For example, a control sequence in a vector that is “effectively linked” to a protein-coding sequence will be linked to the coding sequence in such a way that the protein-coding sequence can be expressed under conditions compatible with the transcriptional activity of the control sequence.

[0158] The term "host cell" refers to a cell that has been transformed by a nucleic acid sequence or is capable of being transformed by a nucleic acid sequence to express the target gene. This term includes the offspring of the parent cell, regardless of whether the offspring are identical to the original parent cell in morphology or genetic composition, as long as the target gene is present.

[0159] Unless otherwise specified, the term “MHC” as used herein includes MHC class I and MHC class II molecules.

[0160] The term "immune co-stimulatory molecules" includes cell surface molecules that function to amplify or counteract the initial activation signals provided to T cells by the T cell receptor (TCR) after interaction with an antigen or MHC. Examples of such molecules include CD86, CD80, CD83, PD-L1, HLA-A, B, C, and HLA-DR.

[0161] The term "transfection" refers to the uptake of foreign or exogenous DNA by cells, and the cells are considered "transfected" once the exogenous DNA has been introduced into the inner side of the cell membrane. Many 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, above; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more portions of exogenous DNA into a suitable host cell.

[0162] The term "transformation" refers to a change in the genetic characteristics of a cell. A cell is considered transformed when it has been modified to contain new DNA or RNA. For example, cells can be transformed by introducing new genetic material through transfection, transduction, or other techniques, causing the cells to undergo genetic modification from their original state. After transfection or transduction, the transformed DNA can recombine with the cell's DNA by physically integrating into the cell's chromosome, or it can be temporarily retained as an episome element without replication, or it can replicate independently as a plasmid. A cell is considered "stablely transformed" when the transformed DNA replicates with cell division.

[0163] The terms "peptide" or "protein" refer to a macromolecule having the amino acid sequence of a natural protein (i.e., a protein produced by naturally occurring and non-recombinant cells); or a molecule produced by genetically modified or recombinant cells, comprising a molecule having the amino acid sequence of a natural protein, or a molecule having one or more amino acids with a natural sequence that have been omitted, added, and / or substituted. The term also includes amino acid polymers, wherein one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and the polymer. The terms "peptide" and "protein" particularly include agonist anti-CD40 antibodies, or sequences having one or more amino acids with a deletion, addition, and / or substitution of an antigen-binding protein. The term "peptide fragment" refers to a peptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to a full-length natural protein. Such fragments may also contain modified amino acids compared to natural proteins. In some embodiments, the fragment is about 5 to 500 amino acids long. For example, the fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful peptide fragments include immunofunctional fragments of antibodies. In the case of agonist antiCD40 antibodies, useful fragments include, but are not limited to, CDR regions of the heavy and / or light chains, variable domains, a portion of an antibody chain including two CDRs, or only its variable regions.

[0164] The term "isolated protein" means that the protein: (1) is free from at least some of other proteins that are normally present with it; (2) is substantially free from other proteins of the same origin (e.g., of the same species); (3) is expressed by cells of a different species; (4) has been separated from at least about 50% of polynucleotides, lipids, carbohydrates or other substances that are bound to it in nature; (5) is effectively bound to polypeptides that are not bound to it in nature (through covalent or non-covalent interactions); or (6) is not present in nature. Typically, "isolated protein" constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Such isolated protein may be encoded by genomic DNA, cDNA, mRNA or other RNA of synthetic origin, or any combination thereof. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, preventative, research or other uses.

[0165] The term "amino acid" includes its normal meaning in this field.

[0166] "Variants" of peptides (such as antigen-binding proteins or antibodies) contain amino acid sequences in which one or more amino acid residues are inserted, deleted, and / or substituted relative to another peptide sequence. Variants include fusion proteins.

[0167] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by aligning and comparing sequences. "Percentage identity" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules, calculated based on the smallest molecule size among those compared. For these calculations, any vacancies in the alignment are preferably resolved using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in *Computational Molecular Biology* (edited by Lesk, AM), 1988, New York: Oxford University Press; *Biocomputing Informatics and Genome Projects* (edited by Smith, DW), 1993, New York: Academic Press; *Computer Analysis of Sequence Data, Part I* (edited by Griffin, AM, and Griffin, HG), 1994, New Jersey: Humana Press; *Von Heinje, G.*, 1987, *Sequence Analysis in Molecular Biology*, New York: Academic Press; *Sequence Analysis Primer* (edited by Gribskov, M., and Devereux, J.), 1991, New York: M. Stockton Press; and *Carillo et al.*, 1988, SIAM J. Applied Math. 48:1073.

[0168] When calculating percentage identity, the sequences being compared are typically aligned in a way that provides the maximum match between the sequences. An example of a computer program that can be used to determine percentage identity is the GCG package, which includes 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 compare two polypeptides or polynucleotides whose percentage sequence identity is to be determined. The sequences are aligned to achieve the best match for their respective amino acids or nucleotides (the “match span” determined by the algorithm). In conjunction with this algorithm, a vacancy opening penalty (calculated as 3 times the average diagonal, where the “average diagonal” is the average of the diagonals of the comparison matrix used; the “diagonal” is a fraction or number assigned to each perfect amino acid match by that specific comparison matrix) and a vacancy extension penalty (typically 1 / 10 of the vacancy opening penalty) are used, along with comparison matrices such as PAM 250 or BLOSUM 62. In some implementations, the algorithm also uses standard comparison matrices (PAM 250 comparison matrix see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; BLOSUM 62 comparison matrix see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0169] Examples of parameters that can be used when using the GAP procedure to determine the percentage identity of peptide or nucleotide sequences are as follows:

[0170] a. Algorithm: Needleman et al., 1970, J. Mol. Biol. 48: 443-453

[0171] b. Comparison matrix: from Henikoff et al., 1992, BLOSUM 62 cited above.

[0172] c. Penalty for open looks: 12 (but no penalty for open looks at the end of the game)

[0173] d. Penalty for open space: 4

[0174] e. Similarity threshold: 0

[0175] Some alignment schemes used to align two amino acid sequences can result in a match of only a short region in the two sequences, and this small aligned region can have very high sequence identity even if there is no significant relationship between the two full-length sequences. Therefore, if needed, the chosen alignment method (GAP procedure) can be modified to produce alignments spanning at least 50 or other numbers of consecutive amino acids across the target polypeptide.

[0176] In this document, the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations are used in accordance with conventional usage. See Immunology—A Synthesis (2nd edition, edited by ESGolub and DRGren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), non-natural amino acids such as -,-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of this invention. Examples of unconventional amino acids include: 4-hydroxyproline, -carboxyglutamic acid, -N,N,N-trimethyllysine, -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and -N-methylarginine, as well as other similar amino acids and imino acids (e.g., 4-hydroxyproline). According to standard usage and convention, in the peptide annotations used in this article, the left-hand direction is the direction of the amino terminus, and the right-hand direction is the direction of the carboxyl terminus.

[0177] 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 called the 5' direction. The direction of addition from 5' to 3' in a newly formed RNA transcript is called the transcription direction; on a DNA strand with the same sequence as the RNA, the 5' sequence region located at the 5' end of the RNA transcript is called the "upstream sequence"; on a DNA strand with the same sequence as the RNA, the 3' sequence region located at the 3' end of the RNA transcript is called the "downstream sequence".

[0178] Conserved amino acid substitutions can include non-naturally occurring amino acid residues that are typically incorporated through chemical peptide synthesis rather than synthesis within biological systems. These include peptide mimics and other inverted or reversed forms of the amino acid moiety.

[0179] The full disclosure of all publications cited herein (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) is incorporated herein by reference. No reference is acknowledged to constitute prior art or part of common general knowledge to those skilled in the art to which this invention pertains.

[0180] Naturally occurring residues can be classified into several categories based on their common side-chain properties:

[0181] a. Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0182] b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0183] c. Acids: Asp, Glu;

[0184] d. Alkaline: His, Lys, Arg;

[0185] e. Residues that affect chain orientation: Gly, Pro; and

[0186] f. Aromatics: Trp, Tyr, Phe.

[0187] For example, nonconservative substitution can involve exchanging a member of one class for a member of another class. Such substituted residues can be introduced, for example, into regions homologous to non-human antibodies in human antibodies, or into non-homologous regions of the molecule.

[0188] According to certain implementation schemes, the hydrophilicity index of the amino acids can be considered when modifying the agonist anti-CD40 antibody. Each amino acid has been assigned a hydrophilicity index based on its hydrophobic and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+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).

[0189] The importance of the hydrophilic amino acid index in conferring biological functions of protein-protein interactions is known in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be substituted with other amino acids having similar hydrophilic indices or fractions and still retaining similar biological activities. In some embodiments, when changes are made based on the hydrophilic index, substitutions of amino acids with a hydrophilic index within ±2 are included. In some embodiments, substitutions of amino acids with a hydrophilic index within ±1 are included, while in some embodiments, substitutions of amino acids with a hydrophilic index within ±0.5 are included.

[0190] It is also understood in the art that similar amino acid substitutions can be made effectively based on hydrophilicity, especially when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as is the case here. In some embodiments, the maximum local average hydrophilicity of a protein (determined by the hydrophilicity of its adjacent amino acids) is related to its immunogenicity and antigenicity, i.e., to the biological properties of the protein.

[0191] The following hydrophilicity values ​​were assigned to these amino acid residues: 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 modifications are made based on similar hydrophilicity values, in some embodiments, substitutions of amino acids with hydrophilicity values ​​within ±2 are included; in others, those within ±1 are included; and in still others, those within ±0.5 are included. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as “epitope core regions.”

[0192] Exemplary amino acid substitutions are listed in Table 2.

[0193] Table 2: Amino Acid Substitutions

[0194]

[0195]

[0196] The term "derivative" refers to a molecule that comprises chemical modifications rather than amino acid (or nucleic acid) insertions, deletions, or substitutions. In some embodiments, the derivative includes covalent modifications, including but not limited to chemical bonding with polymers, lipids, or other organic or inorganic moieties. In some embodiments, the chemically modified antigen-binding protein may have a longer cycling half-life than the unmodified antigen-binding protein. In some embodiments, the chemically modified antigen-binding protein may have improved targeting ability to desired cells, tissues, and / or organs. In some embodiments, the 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, for example, U.S. Patent Nos. 4640835, 4496689, 4301144, 4670417, 4791192, and 4179337. In some 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 homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers.

[0197] In some embodiments, the derivative is covalently modified with polyethylene glycol (PEG) subunits. In some embodiments, one or more water-soluble polymers are bonded to one or more specific sites on the derivative, such as the amino terminus. In some embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In some embodiments, PEG is used to improve the therapeutic capacity of antigen-binding proteins. In some embodiments, PEG is used to improve the therapeutic capacity of humanized antibodies. For example, certain such methods are discussed in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0198] Peptide analogues are commonly used in the pharmaceutical industry as non-peptide drugs that possess properties similar to a 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, 392 (1985); and Evans et al., J. Med. Chem., 30:1229 (1987), which are incorporated herein by reference for all purposes.) These compounds are typically developed using computerized molecular modeling. Peptide mimetics, structurally similar to therapeutic peptides, can be used to produce similar therapeutic or preventative effects. Typically, peptide mimics are structurally similar to exemplary polypeptides (i.e., polypeptides with biochemical properties or pharmacological activity), such as human antibodies, but one or more peptide bonds may optionally be replaced by methods known in the art with bonds selected from: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. In some embodiments, one or more amino acids of the common sequence may be systematically substituted with the same type of D-amino acid (e.g., replacing L-lysine with D-lysine) to produce a more stable peptide. Furthermore, constrained peptides containing a common sequence or substantially the same variation of the common sequence may be produced by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein by reference for all purposes); for example, by adding an internal cysteine ​​residue capable of forming an intramolecular disulfide bridge that cyclizes the peptide.

[0199] Throughout the instruction manual, the term "naturally occurring" used in conjunction with biological materials such as polypeptides, nucleic acids, and host cells refers to materials found in nature or material forms found in nature.

[0200] The term "agonist" refers to a compound that, upon binding to a receptor, elicits 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 modification of another compound, thereby enabling that other compound to bind directly to the receptor. An agonist may be referred to as an agonist of a specific receptor or receptor family (e.g., TNF or TNFR agonists).

[0201] The term "immunofunctional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy chain or light chain) refers to an antibody containing a portion of the antibody (regardless of how that portion is obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain, but is still specifically capable of functioning as an agonist of CD40. These bioactive fragments can be produced using recombinant DNA technology or by enzymatic or chemical cleavage of antigen-binding proteins (including intact antibodies). Immunofunctional immunoglobulin fragments include, but are not limited to, Fab, biantibodies (where the variable heavy chain domain and the variable light chain domain are located on the same polypeptide, linked by a short peptide linker that is too short to allow pairing between the two domains on the same chain), Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian source, including but not limited to humans, mice, rats, camels, or rabbits. It is also conceivable that the functional portions of the agonist anti-CD40 antibodies disclosed herein, such as one or more CDRs, can covalently bind to a second protein or small molecule to produce a therapeutic agent targeting a specific target in vivo, thereby possessing bifunctional therapeutic properties or having an extended serum half-life. Those skilled in the art will understand that agonist anti-CD40 antibodies may include non-protein components.

[0202] In some implementations, the polypeptide structure of the agonist antiCD40 antibody is based on the antibody, including but not limited to monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof.

[0203] The “Fc” region contains two heavy chain segments with antibody CH1 and CH2 domains. The two heavy chain segments are held together by two or more disulfide bonds and hydrophobic interactions with the CH3 domain.

[0204] The "Fab fragment" consists of a light chain and a heavy chain with a CH1 and variable region. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0205] The “Fab’ fragment” contains a light chain and a portion of a heavy chain. This portion includes the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, which allows interchain disulfide bonds to form between the two heavy chains of the two Fab’ fragments to form the F(ab’)2 molecule.

[0206] The “Fv region” contains variable regions from both heavy and light chains, but lacks constant regions.

[0207] A "single-chain antibody" is an Fv molecule in which the variable regions of the heavy and light chains 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 herein by reference.

[0208] A domain antibody is an immunoglobulin fragment that is functionally immunogenic, containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently linked to a peptide linker to produce a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0209] As used herein, "substantially pure" means that the described molecular species is the dominant species present, that is, on a molar basis, it is more abundant than any other molecular species in the same mixture. In some embodiments, substantially pure molecules are compositions in which the target molecule constitutes at least 50% (on a molar basis) of all present macromolecules. In other embodiments, substantially pure compositions comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecules present in the composition. In other embodiments, the target molecule is purified to substantially homogeneity, wherein contaminating molecules are undetectable in the composition by conventional detection methods, and thus the composition consists of a single detectable macromolecule.

[0210] The term "activator" is used in this document to refer to chemical compounds, mixtures of chemical compounds, biological macromolecules, or extracts made from biological materials.

[0211] As used herein, the terms “labeled” or “labeled” refer to the incorporation of a detectable label, such as the incorporation of a radiolabeled amino acid; or the attachment of a biotin moiety to a polypeptide, wherein the biotin moiety is detectable by a labeled avidin (e.g., streptavidin containing a fluorescent label); or the incorporation of an enzyme activity detectable by optical or colorimetric methods. In some embodiments, the label or labeling may also be therapeutic. Various methods for 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., radioactive isotopes or radionuclides). 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 1311) Fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinylate groups, and predefined peptide epitopes recognized by a second reporter molecule (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags). In some embodiments, the labels are linked by spacer arms of various lengths to reduce potential steric hindrance.

[0212] The term "therapeutic effective amount" refers to the amount of agonist antiCD40 antibody that produces a therapeutic response in mammals (preferably humans). This therapeutic effective amount can be readily determined by those skilled in the art.

[0213] As used herein, the term "pharmaceutical composition" (or active agent or drug) refers to a compound, composition, active agent or drug that, when properly administered to a patient, can induce a desired therapeutic effect. It does not necessarily require more than one ingredient.

[0214] Throughout this specification, unless the context otherwise requires, the word “comprising” or variations such as “including” or “containing” shall be understood to include the said integer or group of integers, but not exclude any other integer or group of integers.

[0215] Other definitions of the terms used herein can be found in the detailed description of the invention and apply 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 pertains.

[0216] The invention described herein may include one or more numerical ranges (e.g., dimensions, displacements, and field strengths). A numerical range is to be understood as including all values ​​within that range, including the value defining the range and values ​​adjacent to the range that result in the same or substantially the same result as the value immediately adjacent to the value defining the boundary of the range. For example, those skilled in the art will understand that a 10% variation in the upper or lower limit of the range is perfectly suitable and covered by the invention. More specifically, the variation in the upper or lower limit of the range is 5%, or a variation recognized in the art, whichever is greater.

[0217] Throughout this specification, relative language such as “about” and “approximately” is used. This language attempts to include at least 10% variability within a specified quantity or range. This variability can be +10% or -10% of the specified specific quantity.

[0218] Implementation Plan

[0219] A. Agonist antiCD40 antibody

[0220] When administered in an effective amount, the agonist anti-CD40 antibody of the present invention can modulate at least one or more of the following functions:

[0221] a. Increase antigen presentation by APCs (including macrophages, dendritic cells, and B cells); or

[0222] b. Enhance the expression of MHC and / or immune co-stimulatory molecules (such as CD86, CD80, CD83, PD-L1, HLA-A, B, C, or HLA-DR); or

[0223] c. Stimulates the production of pro-inflammatory cytokines (such as IL-12, IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-23, and IFN-γ); or

[0224] d. Inducing T cell activation; or

[0225] e. Mimic CD40L signaling and replace the function of CD4+ lymphocytes; or

[0226] f. Overcoming T-cell tolerance in tumor-bearing animals; or

[0227] g. Induces an effective cytotoxic T cell response; or

[0228] h. Enhance the effectiveness of anti-tumor vaccines; or

[0229] i. Makes the tumor vascular system more susceptible to immune infiltration.

[0230] In some embodiments, the provided agonist anti-CD40 antibody is a polypeptide comprising one or more CDRs as described herein. In some agonist anti-CD40 antibodies, the CDR is embedded in a "framework" region that orients the CDR, thereby achieving the appropriate binding properties of the CDR.

[0231] The antibodies disclosed herein have a variety of uses. As described herein, they can be used for a variety of therapeutic applications. For example, in some embodiments, agonist anti-CD40 antibodies can be used to treat malignant conditions, including but not limited to administration with other active agents such as IL-2. Other uses of agonist anti-CD40 antibodies include, for example, the diagnosis of disease states or conditions and screening assays to determine the presence or absence of CD40. Some agonist anti-CD40 antibodies described herein can also be used to treat consequences, symptoms, and / or pathologies associated with increased antigen presentation.

[0232] In one aspect, the present invention includes isolated agonist anti-CD40 antibodies or fragments thereof, comprising (i) a VH chain containing three CDRs and (ii) a VL chain containing three CDRs, wherein one or more heavy chain complementarity-determining regions (CDRHs) are selected from:

[0233] a) A CDRH1 sequence containing SEQ ID NO:1;

[0234] b) A CDRH2 sequence containing SEQ ID NO:2; or

[0235] c) Contains a CDRH2 sequence containing one or two amino acid substitutions, deletions, or insertions of SEQ ID NO:2.

[0236] In some embodiments, the provided agonist anti-CD40 antibody comprises one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs). In some embodiments, the agonist anti-CD40 antibody comprises (a) a polypeptide structure and (b) one or more CDRs inserted into and / or linked to that polypeptide structure. The polypeptide structure can take many different forms. For example, it can be or contain a framework of a naturally occurring antibody or a fragment or variant thereof, or it can be entirely synthetic. Examples of various polypeptide structures are further described below.

[0237] In some embodiments, the polypeptide structure of the agonist antiCD40 antibody is an antibody or antibody-derived, including but not limited to monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and portions or fragments of each of these. In some cases, the agonist antiCD40 antibody is an immunological fragment of an antibody (e.g., Fab, Fab', F(ab')2, or scFv). Various structures are further described and defined herein.

[0238] In one embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody further comprises:

[0239] a) A CDRH3 sequence containing SEQ ID NO:3;

[0240] b) A CDRL1 sequence containing SEQ ID NO:4;

[0241] c) A CDRL2 sequence containing SEQ ID NO:5; or

[0242] d) Contains the CDRL3 sequence of SEQ ID NO:6.

[0243] In another embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises:

[0244] A:

[0245] a) A CDRH1 sequence containing SEQ ID NO:1; or

[0246] b) A CDRH2 sequence containing SEQ ID NO:2; and

[0247] B:

[0248] c) A CDRH3 sequence containing SEQ ID NO:3;

[0249] d) Contains a CDRL1 sequence of SEQ ID NO:4;

[0250] e) A CDRL2 sequence containing SEQ ID NO:5; or

[0251] f) Contains the CDRL3 sequence of SEQ ID NO:6.

[0252] In another embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises:

[0253] a) A CDRH1 sequence containing SEQ ID NO:1;

[0254] b) A CDRH2 sequence containing SEQ ID NO:2;

[0255] c) A CDRH3 sequence containing SEQ ID NO:3;

[0256] d) Contains the CDRL1 sequence of SEQ ID NO:4;

[0257] e) A CDRL2 sequence containing SEQ ID NO:5; and

[0258] f) Contains the CDRL3 sequence of SEQ ID NO:6.

[0259] 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 another 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 another embodiment of the first aspect of the invention, the isolated agonist anti-CD40 antibody comprises both the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8.

[0260] SEQ ID NO:7 contains the CDRH1, CDRH2, and CDRH3 sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. SEQ ID NO:8 contains the CDRL1, CDRL2, and CDRL3 sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the antibody containing the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8 is referred to as SVX-3001.

[0261] In one embodiment, the isolated agonist anti-CD40 antibody is a monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, chimeric antibody, multispecific antibody, or antibody fragment thereof.

[0262] In one embodiment, the isolated agonist anti-CD40 antibody is a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, biantibody, or single-chain antibody molecule.

[0263] In one implementation, the isolated agonist anti-CD40 antibody is a humanized anti-human antibody.

[0264] In one implementation, the isolated agonist anti-CD40 antibody is a monoclonal antibody.

[0265] In one embodiment, the isolated agonist anti-CD40 antibody is of type IgG1, IgG2, IgG3, or IgG4. Preferably, the isolated agonist anti-CD40 antibody is of type IgG1.

[0266] In a particularly preferred embodiment, the isolated agonist antiCD40 antibody comprises the heavy chain sequence of SEQ ID NO:19 and / or the light chain sequence of SEQ ID NO:20.

[0267] In one embodiment, the isolated agonist anti-CD40 antibody is conjugated with a labeling group.

[0268] Other antibodies provided are variants of the aforementioned agonist anti-CD40 antibodies formed from combinations or sub-parts of the variable heavy and light chains shown in SEQ ID NO:7 and 8, comprising a variable light chain and / or a variable heavy chain having at least 50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or more than 99% identity with the amino acid sequences (whole sequences or sub-parts of sequences, such as one or more CDRs) in SEQ ID NO:7 and 8. In some cases, such antibodies comprise at least one heavy chain and one light chain, while in other cases, the variant forms comprise two identical light chains and two identical heavy chains (or sub-parts thereof).

[0269] In some embodiments, the agonist antiCD40 antibody comprises a heavy chain containing a variable region comprising at least 90% of the amino acid sequence identical to that of SEQ ID NO:7. In some embodiments, the agonist antiCD40 antibody comprises a heavy chain containing a variable region comprising at least 95% of the amino acid sequence identical to that of SEQ ID NO:7. In some embodiments, the agonist antiCD40 antibody comprises a heavy chain containing a variable region comprising at least 99% of the amino acid sequence identical to that of SEQ ID NO:7.

[0270] In some embodiments, the agonist antiCD40 antibody comprises a heavy chain containing at least 90% of the amino acid sequence identical to that of SEQ ID NO:19. In some embodiments, the agonist antiCD40 antibody comprises a heavy chain containing at least 95% of the amino acid sequence identical to that of SEQ ID NO:19. In some embodiments, the agonist antiCD40 antibody comprises a heavy chain containing at least 99% of the amino acid sequence identical to that of SEQ ID NO:19.

[0271] In some embodiments, the agonist anti-CD40 antibody comprises a sequence having at least 90%, 90-95%, and / or 95-99% identity with one or more CDRs, wherein said one or more CDRs are derived from at least one sequence of SEQ ID NO: 1 to 3 and 4 to 6. In some embodiments, there are 1, 2, 3, 4, 5, or 6 CDRs (each having at least 90%, 90-95%, and / or 95-99% identity with the aforementioned sequence).

[0272] In some embodiments, the agonist antiCD40 antibody comprises a light chain containing a variable region comprising at least 90% of the same amino acid sequence as SEQ ID NO:8. In some embodiments, the agonist antiCD40 antibody comprises a light chain containing a variable region comprising at least 95% of the same amino acid sequence as SEQ ID NO:8. In some embodiments, the agonist antiCD40 antibody comprises a light chain containing a variable region comprising at least 99% of the same amino acid sequence as SEQ ID NO:8.

[0273] In some embodiments, the agonist antiCD40 antibody comprises a light chain containing at least 90% of the same amino acid sequence as SEQ ID NO:20. In some embodiments, the agonist antiCD40 antibody comprises a light chain containing at least 95% of the same amino acid sequence as SEQ ID NO:20. In some embodiments, the agonist antiCD40 antibody comprises a light chain containing at least 99% of the same amino acid sequence as SEQ ID NO:20.

[0274] In one implementation, the isolated agonist anti-CD40 antibody enhances CD40 activity.

[0275] B. Preparation of agonist anti-CD40 antibodies

[0276] In one embodiment, the present invention includes a method for preparing the agonist anti-CD40 antibody described herein, comprising the step of preparing the agonist anti-CD40 antibody from a host cell that secretes the agonist anti-CD40 antibody.

[0277] Typically, a fully monoclonal agonist antibody against CD40 can be generated as follows: Mice containing an immunoglobulin gene are immunized with the target CD40, and antibody-expressing lymphocytes (such as B cells) are obtained from the mice. These recovered cells are fused with a myeloid cell line to prepare an immortalized hybridoma cell line, and these hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the target antigen. In some embodiments, the preparation of hybridoma cell lines that produce CD40-specific agonist antibodies is provided.

[0278] In some embodiments, phage display technology is used to generate monoclonal antibodies. In some embodiments, this technology generates monoclonal antibodies. In some embodiments, a polynucleotide encoding a single Fab or Fv antibody fragment is expressed on the surface of the phage particle. See, for example, 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 some embodiments, phages are “screened” to identify antibody fragments that have affinity for the target. Thus, some methods mimic immune selection by displaying a library of antibody fragments on the surface of filamentous phages and subsequently selecting phages by their binding to the target. In some methods, high-affinity functional antibody fragments are isolated. In some such embodiments, a complete antibody gene library is created by cloning a naturally rearranged V gene from peripheral blood lymphocytes. See, for example, Mullinax et al., Proc Natl Acad Sci (USA), 87:8095-8099 (1990).

[0279] According to certain embodiments, the antibodies of the present invention are prepared using transgenic mice having an inserted, substantial portion of an antibody-producing genome, but which are deficient in endogenous mouse antibody production. Therefore, such mice are capable of producing immunoglobulin molecules and antibodies, but lack the production of mouse immunoglobulins and antibodies. Techniques for achieving this result are disclosed in the patents, applications, and references cited herein. In some embodiments, methods such as those disclosed in PCT Publication No. WO 98 / 24493 or Mendez et al., Nature Genetics, 15:146-156 (1997) may be employed, which are incorporated herein by reference for all purposes.

[0280] In some implementations, CD40-specific agonist antibodies are generated by exposing spleen cells (B or T cells) to an antigen in vitro and then reconstituted the exposed cells in immunocompromised mice (e.g., SCID or nod / SCID). See, for example, Brams et al., J. Immunol. 160:2051-2058 (1998); Carballido et al., Nat. Med., 6:103-106 (2000). In some of these methods, implantation of fetal tissue into SCID mice (SCID-hu) results in long-term hematopoiesis and human T cell development. See, for example, McCune et al., Science, 241:1532-1639 (1988); Ifversen et al., Sem. Immunol., 8:243-248 (1996). In some cases, humoral immune responses in such chimeric mice depend on the co-development of T cells in the animal. See, for example, Martensson et al., Immunol., 83:1271-179 (1994). In some methods, peripheral blood lymphocytes are transplanted into SCID mice. See, for example, Mosier et al., Nature, 335:256-259 (1988). In some such embodiments, higher levels of B cell production are detected when the transplanted cells are treated with an initiator such as staphylococcal enterotoxin A (SEA). See, for example, Martensson et al., Immunol., 84:224-230 (1995); Murphy et al., Blood, 86:1946-1953 (1995).

[0281] As will be understood, antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding specific antibodies can be used to transform suitable mammalian host cells. Transformation can be performed by any known method for introducing polynucleotides into host cells, including, for example, packaging the polynucleotides in a virus (or viral vector) and transducing the host cells with the virus (or vector), or by transfection methods known in the art, such as those exemplified in 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 method 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 the cell nucleus.

[0282] Mammalian cell lines suitable as expression hosts 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, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and many other cell lines. Particularly preferred cell lines are selected by identifying which cell lines exhibit high expression levels and produce antibodies against CD40-specific agonists.

[0283] In some embodiments, the agonist CD40 antibody comprises an immunoglobulin molecule of at least one of the isotypes IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM. In some embodiments, the agonist CD40 antibody comprises a human κ light chain and / or a human heavy chain. In some embodiments, the heavy chain is an isotype of IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM. In some embodiments, the clonal agonist CD40 antibody is used for expression in mammalian cells. In some embodiments, the agonist CD40 antibody comprises a constant region other than any constant region of the isotypes IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM.

[0284] In some embodiments, the agonist CD40 antibody comprises a human λ light chain and a human IgG2 heavy chain. In some embodiments, the agonist CD40 antibody comprises a human λ light chain and a human IgG4 heavy chain. In some embodiments, the agonist CD40 antibody comprises a human λ light chain and a human IgG1 heavy chain. In some embodiments, the agonist CD40 antibody comprises a human λ light chain and a human IgG3, IgE, IgA, IgD, or IgM heavy chain. In other embodiments, the agonist CD40 antibody comprises a human κ light chain and a human IgG2 heavy chain. In some embodiments, the agonist CD40 antibody comprises a human κ light chain and a human IgG4 heavy chain. In some embodiments, the agonist CD40 antibody comprises a human κ light chain and a human IgG1 heavy chain. In some embodiments, the agonist CD40 antibody comprises a human κ light chain and a human IgG3, IgE, IgA, IgD, or IgM heavy chain. In some embodiments, the agonist CD40 antibody includes an antibody variable region linked to a constant region, wherein said constant region is neither a constant region of the IgG2 isotype nor a constant region of the IgG4 isotype. Preferably, the agonist CD40 antibody comprises a human IgG1 heavy chain and a light chain. In some embodiments, the clonal agonist CD40 antibody is used for expression in mammalian cells.

[0285] In some embodiments, conserved modification of the heavy and light chains of an antibody from at least one of the hybridoma lines described herein will produce an agonist CD40 antibody having similar function and chemical properties to the antibody from the hybridoma line. Conversely, in some embodiments, a substantial alteration of the function and / or chemical properties of the agonist CD40 antibody can be achieved by selective substitution in the amino acid sequences of the heavy and light chains, wherein said substitution has significantly different effects on maintaining (a) the structure of the molecular backbone (e.g., sheet or helical conformation) in the substituted region, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chains.

[0286] For example, “conservative amino acid substitution” can involve replacing a native amino acid residue with a non-native residue such that the polarity or charge of the amino acid residue at that position has little or no effect. Alternatively, any native residue in a polypeptide can be replaced with alanine, as previously described for “alanine scanning mutagenesis”.

[0287] The desired amino acid substitution (whether conserved or non-conservative) can be determined by those skilled in the art when such substitution is required. In some embodiments, amino acid substitution can be used to identify important residues of agonist CD40 antibodies, or to increase or decrease the agonist activity of agonist CD40 antibodies, as described herein.

[0288] In some embodiments, the agonist CD40 antibody comprises one or more polypeptides. In some embodiments, any of a variety of expression vector / host systems may be used to express a polynucleotide molecule encoding a polypeptide 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 phage, plasmid, or copious DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculoviruses); 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.

[0289] In some embodiments, a polypeptide comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself is recombinantly expressed in yeast. Some of these embodiments use commercially available expression systems, such as the Pichia pastoris expression system (Invitrogen, San Diego, CA), performed according to the manufacturer's instructions. In some embodiments, this system relies on a pre-pro-alpha sequence to guide secretion. In some embodiments, transcription of the insert fragment is driven by an alcohol oxidase (AOX1) promoter upon methanol induction.

[0290] In some 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 some embodiments, the method for purifying polypeptides from yeast growth medium is the same as the method for purifying polypeptides from bacterial and mammalian cell supernatants.

[0291] In some embodiments, a nucleic acid encoding a polypeptide comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself is cloned into a baculovirus expression vector such as pVL1393 (PharMingen, San Diego, CA). In some embodiments, this vector can be used, according to the manufacturer's instructions (PharMingen), to infect fall armyworm (Spodoptera frugiperda) cells in sF9 protein-free medium and produce recombinant polypeptides. In some embodiments, the polypeptide is purified and concentrated from this medium using a heparin-agarose column (Pharmacia).

[0292] In some 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. Certain insect systems used for polypeptide expression are well known to those skilled in the art. In one such system, the alfalfa silver-striped armyworm (Autographa californica) nucleopolyhedrovirus (AcNPV) is used as a vector to express a foreign gene in fall armyworm cells or Trichoplusia larvae. In some embodiments, the nucleic acid molecule encoding the polypeptide can be inserted into a non-essential gene of the virus, such as a polyhedrosis protein gene, and placed under the control of the gene's promoter. In some embodiments, successful insertion of the nucleic acid molecule will inactivate the non-essential gene. In some embodiments, this inactivation results in a detectable characteristic. For example, inactivation of the polyhedrosis protein gene results in the production of a virus lacking a capsid protein.

[0293] In some implementations, the recombinant virus can be used to infect fall armyworm cells or larvae of the genus *Spodoptera litura*. See, for example, Smith et al., J. Virol., 46:584 (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA), 91:3224-7 (1994).

[0294] In some embodiments, a polypeptide comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself is prepared in bacterial cells and produced in bacteria as an insoluble inclusion body. In some embodiments, host cells containing such inclusion bodies are collected by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, and 1 mM EDTA; and treated with 0.1 mg / ml lysozyme (Sigma, St. Louis, MO) at room temperature for 15 minutes. In some embodiments, the lysate is clarified by sonication, and cell debris is precipitated by centrifugation at 12000X g for 10 minutes. In some embodiments, the precipitate containing the polypeptide is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA; placed on 50% glycerol; and centrifuged at 6000X g for 30 minutes. In some embodiments, the precipitate may be resuspended in a Mg-free solution. ++ and Ca ++ The peptide is prepared in standard phosphate-buffered saline (PBS). In some embodiments, the peptide is further purified by fractionation of the resuspended precipitate in a denaturing SDS-polyacrylamide gel (see, for example, Sambrook et al., cited above). In some embodiments, the gel may be soaked in 0.4 M KCl to visualize the protein, which can then be cleaved and electrolyzed in an SDS-free gel running buffer. According to some embodiments, a glutathione-S-transferase (GST) fusion protein is produced in bacteria as a soluble protein. In some embodiments, this GST fusion protein is purified using a GST purification module (Pharmacia).

[0295] In some embodiments, it is desirable to “refold” certain peptides, such as peptides containing one or more agonist anti-CD40 antibody components or agonist anti-CD40 antibodies themselves. In some embodiments, such peptides are generated using certain recombinant systems discussed herein. In some embodiments, peptides are “refolded” and / or oxidized to form a desired tertiary structure and / or generate disulfide bonds. In some embodiments, this structure and / or linkage is related to certain biological activities of the peptide. In some embodiments, refolding is achieved using any of a number of methods known in the art. Exemplary methods include, but are not limited to, exposing a dissolved peptide active agent to a pH typically above 7 in the presence of a dissociative agent. An exemplary dissociative agent is guanidine. In some embodiments, the refolding / oxidation solution also contains a reducing agent and an oxidized form of that reducing agent. In some embodiments, the reducing agent and its oxidized form are present in a proportion that will produce a specific redox potential that allows disulfide bond shuffling to occur. In some embodiments, this shuffling allows the formation of cysteine ​​bridges. Exemplary redox pairs include, but are not limited to, cysteine / cystamine, glutathione / dithiodiGSH, cuprous ion / chloride ion, dithiothreitol DTT / oxidized DTT (dithiane), and 2-mercaptoethanol (bME) / dithiobME. In some embodiments, a co-solvent is used to improve the efficiency of refolding. Exemplary co-solvents include, but are not limited to, glycerol, polyethylene glycol of various molecular weights, and arginine.

[0296] In some embodiments, the peptide is substantially purified to contain one or more agonist anti-CD40 antibody fractions or the agonist anti-CD40 antibody itself. Certain protein purification techniques are known to those skilled in the art. In some embodiments, protein purification involves separating the peptide fraction from a non-peptide fraction. In some embodiments, the peptide is purified using chromatography and / or electrophoresis techniques. 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-agarose), ion exchange chromatography, size exclusion chromatography, and reversed-phase chromatography; gel filtration; hydroxyapatite chromatography; isoelectric focusing; polyacrylamide gel electrophoresis; and combinations of these and other techniques. In some embodiments, the peptide is purified by rapid protein liquid chromatography or high-performance liquid chromatography (HPLC). In some embodiments, the purification steps may be modified or certain steps may be omitted, and a suitable method for preparing a substantially purified peptide may still be produced.

[0297] In some embodiments, the degree of purification of the peptide preparation is quantified. Certain methods for quantifying the degree of purification are known to those skilled in the art. Some exemplary methods include, but are not limited to, determining the specific binding activity of the preparation and assessing the amount of peptide in the preparation by SDS / PAGE analysis. Some exemplary methods for assessing the amount of purified peptide preparation include calculating the binding activity of the preparation and comparing it to the binding activity of the initial extract. In some embodiments, the result of such calculation is expressed as a "purification fold". The unit used to express the amount of binding activity depends on the specific assay performed.

[0298] In some embodiments, partial purification involves peptides comprising one or more agonist anti-CD40 antibody components or the agonist anti-CD40 antibody itself. In some embodiments, partial purification can be achieved by using fewer purification steps or by using a different form of the same universal purification protocol. For example, in some embodiments, cation exchange column chromatography performed using an HPLC device typically produces a greater “purification fold” than the same technique using a low-pressure chromatography system. In some embodiments, methods resulting in a lower degree of purification may have advantages in terms of the overall recovery of the peptide or in maintaining the binding activity of the peptide.

[0299] In some cases, the electrophoretic migration of peptides can vary, sometimes significantly, with different SDS / PAGE conditions. See, for example, Capaldi et al., Biochem. Biophys. Res. Comm., 76:425 (1977). It should be understood that the apparent molecular weight of purified or partially purified peptides can differ under different electrophoretic conditions.

[0300] C. Nucleic acid molecules encoding anti-CD40 agonist antibodies

[0301] In one embodiment, the invention includes a nucleic acid molecule encoding the isolated agonist anti-CD40 antibody disclosed herein.

[0302] Those skilled in the art will recognize that the above discussion can be used for the identification, evaluation, and / or generation of agonist anti-CD40 antibodies, and also for nucleic acid sequences encoding such antibodies. Therefore, nucleic acid sequences encoding such antibodies are considered. For example, the antibody may have at least 80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater identity with at least one nucleic acid sequence described in SEQ ID NO: 9 or 10 or with at least one to six CDRs (and various combinations thereof) encoded by the nucleic acid sequence of SEQ ID NO: 9 or 10.

[0303] In some embodiments, antibodies (or nucleic acid sequences encoding them) are contemplated within the scope of this invention, wherein the nucleic acid sequence encoding the particular antibody (or the nucleic acid sequence itself) is capable of selectively hybridizing under stringent conditions with any nucleic acid sequence encoding proteins SEQ ID NO:7 and 8. In one embodiment, suitable moderately stringent conditions include pre-washing in a solution of 5×SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); overnight hybridization at 50–65°C in 5×SSC or, in the case of cross-species homology, at 45°C in 0.5×SSC; followed by washing twice at 65°C for 20 minutes each time with 2×, 0.5×, and 0.2×SSC containing 0.1% SDS. Such hybridization DNA sequences are also within the scope of this invention, as are the nucleotide sequences encoding antibody polypeptides encoded by the hybridization DNA sequences and the amino acid sequences encoded by these nucleic acid sequences, based on codon degeneracy. In some embodiments, variants of the CDR include nucleic acid sequences hybridizing with one or more CDRs within the above-described sequences and the amino acid sequences encoded by these sequences.

[0304] The phrase "selective hybridization" as used herein refers to detectable selective binding. The polynucleotides, oligonucleotides, and fragments thereof of the present invention can selectively hybridize with nucleic acid chains under hybridization and washing conditions that minimize detectable binding to nonspecific nucleic acids. Highly stringent conditions can be used to achieve selective hybridization conditions known in the art and discussed herein. Typically, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the present invention and the target nucleic acid sequence will be at least 80%, more typically having a preferred increasing homology of at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is partial or complete identity between them. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are compared for maximum matching. Vacancies are allowed (in either of the two matching sequences) to maximize the match; the vacancy length is preferably 5 or less, more preferably 2 or less. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them) are homologous, as used herein, if the alignment score is greater than 5 (in standard deviations) using the ALIGN procedure, a mutation data matrix, and a vacancy penalty of 6 or greater. See Dayhoff, MO, in Atlas of Protein Sequence and Structure, pp. 101–110 (Vol. 5, National Biomedical Research Foundation (1972)) and Appendix 2 of this volume, pp. 1–10. More preferably, two sequences or portions thereof are homologous if, after optimal alignment using the ALIGN procedure, the amino acids are greater than or equal to 50%. Hereinafter, the term “corresponding” is used to mean that a polynucleotide sequence is wholly or partially homologous (i.e., identical, not strictly evolutionarily related) to a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. Conversely, in this paper, the term "complementary" is used to refer to a complementary sequence that is wholly or partially homologous to a reference polynucleotide sequence. For example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATA" and is complementary to the reference sequence "GTATA".

[0305] In one embodiment, the present invention includes a vector comprising the nucleic acid molecule described herein.

[0306] In one embodiment, the invention includes a host cell containing the nucleic acid molecules described herein.

[0307] D. A composition containing at least one agonist anti-CD40 antibody.

[0308] In one embodiment, the present invention comprises a composition containing at least one agonist anti-CD40 antibody as described herein. Preferably, the composition is a pharmaceutical composition. Thus, in a preferred embodiment of the invention, pharmaceutical compositions comprising at least one isolated agonist anti-CD40 antibody as described herein and a pharmaceutically acceptable excipient are included.

[0309] In one alternative embodiment, the present invention provides a pharmaceutical composition comprising an agonist anti-CD40 antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0310] In some embodiments, the present invention provides a pharmaceutical composition comprising an agonist anti-CD40 antibody and a therapeutically effective amount of at least one other therapeutic agent, as well as a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0311] When the composition contains at least one other therapeutic agent, the therapeutic agent is preferably selected from radioisotopes, radionuclides, toxins, or therapeutic and chemotherapeutic groups. Therefore, in a preferred form, the present invention includes a pharmaceutical formulation comprising: an effective amount of at least one agonist anti-CD40 antibody disclosed herein and at least one second immune enhancer. Therapeutic agents include, but are not limited to, chemical compositions synthesized in vitro, antibodies, antigen-binding regions, and combinations and conjugates thereof. In some embodiments, the therapeutic agent may act as an agonist, antagonist, allosteric modulator, or toxin. In some embodiments, the therapeutic agent may function to inhibit or stimulate its target, thereby promoting an immune response against malignant tumors. Such immune enhancers include, but are not limited to, IL-2, TLR-7 agonists, or systemic cytotoxic chemotherapeutic agents.

[0312] In some embodiments, the acceptable formulation material is preferably non-toxic to the receptor at the dose and concentration used. In some embodiments, the formulation material is for subcutaneous injection and / or intratumoral administration. In some embodiments, the pharmaceutical composition may comprise formulation materials for modifying, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); swelling agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other sugars (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; and hydrophilic agents. Polymers (e.g., polyvinylpyrrolidone); low molecular weight peptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, dehydrated sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton); stability enhancers (e.g., sucrose or sorbitol); tension enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery carriers; diluents; excipients and / or adjuvants. Remington's Pharmaceutical Sciences, 18th edition, edited by ARGennaro, 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.

[0313] In some embodiments, agonist anti-CD40 antibodies and / or therapeutic molecules are linked to half-life-extending vectors known in the art. Such vectors include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of ABP), and dextran. For example, such vectors are described in U.S. Application Serial No. 09 / 428082 (now U.S. Patent No. 6660843) and published PCT Application No. WO 99 / 25044, which are incorporated herein by reference for all purposes.

[0314] In some embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art based on, for example, the intended route of administration, delivery method, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, cited above. In some embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antibodies of the present invention.

[0315] In some embodiments, the primary solvent or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, in some embodiments, suitable solvents or carriers may be water for injection, physiological saline solutions, or artificial cerebrospinal fluid, possibly supplemented with other materials common in parenteral administration compositions. In some embodiments, the saline solution comprises isotonic phosphate buffered saline. In some embodiments, neutral buffered saline solutions or saline solutions mixed with serum albumin are other exemplary solvents. In some embodiments, the pharmaceutical composition comprises Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, and therefore may further comprise sorbitol or suitable alternatives. In some embodiments, a composition containing an agonist antiCD40 antibody (with or without at least one other therapeutic agent) for storage can be prepared by mixing the selected composition having the desired purity with an optional formulation (Remington's Pharmaceutical Sciences, cited above) in the form of a lyophilized cake or aqueous solution. In addition, in some embodiments, compositions containing an agonist anti-CD40 antibody (with or without at least one other therapeutic agent) may be formulated into lyophilized products using suitable excipients such as sucrose.

[0316] In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. The preparation of such a pharmaceutically acceptable composition is within the capabilities of those skilled in the art.

[0317] In some embodiments, the formulation component is present at a concentration acceptable at the application site. In some embodiments, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8.

[0318] In some embodiments, when considering parenteral (preferably intratumoral or peritumoral) administration, the therapeutic composition may be in the form of a pyrogen-free, parenterically acceptable aqueous solution containing the desired agonist anti-CD40 antibody (with or without other therapeutic agents) in a pharmaceutically acceptable solvent. In some embodiments, the solvent for parenteral injection is sterile distilled water, in which the agonist anti-CD40 antibody, with or without at least one other therapeutic agent, is formulated into a sterile isotonic solution and appropriately stored. In some embodiments, the preparation may involve formulating the desired molecule with substances such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), microspheres, or liposomes, which can provide controlled or sustained release of the product, which can then be delivered via depot injection.

[0319] In some embodiments, the pharmaceutical composition may involve mixing an effective amount of an agonist anti-CD40 antibody, with or without at least one other therapeutic agent, with a non-toxic excipient suitable for preparing tablets. In some embodiments, a unit-dose solution can be prepared by dissolving the tablet in sterile water or another suitable solvent. In some embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or gum arabic; or lubricants such as magnesium stearate, stearic acid, or talc.

[0320] Other pharmaceutical compositions will be apparent to those skilled in the art, including those involving agonist anti-CD40 antibodies, formulations with or without at least one other therapeutic agent, and formulations in sustained or controlled delivery formulations. In some embodiments, techniques for formulating various other sustained or controlled delivery methods are also known to those skilled in the art, such as liposome carriers, biodegradable microparticles or porous microspheres, and reservoir-type injections. See, for example, PCT application number PCT / US93 / 00829, which describes the controlled release of porous polymeric microparticles for delivering pharmaceutical compositions. In some embodiments, sustained-release formulations may comprise a semi-permeable polymeric matrix in the form of a shaped article, such as a film or microcapsule. Sustained-release matrices may include polyesters, hydrogels, polylactic acid (US3,773,919 and EP 058481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., above), or poly-D(-)-3-hydroxybutyric acid (EP 133988). In some embodiments, the sustained-release composition may also include liposomes, which may be prepared by any of several methods known in the art. See, for example, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); EP 036, 676; EP 088, 046 and EP 143, 949.

[0321] Pharmaceutical compositions intended for internal administration are typically sterile. In some embodiments, this can be achieved through filtration using a sterile filter membrane. In some embodiments, sterilization using this method may be performed before or after lyophilization and reconstitution when the composition is lyophilized. In some embodiments, compositions intended for parenteral administration may be stored in lyophilized or solution form. In some embodiments, parenteral compositions are typically placed in containers with sterile inlets and outlets, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0322] In some embodiments, once the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In some embodiments, such formulations can be stored in ready-to-use form or in a form reconstituted before administration (e.g., lyophilized form).

[0323] In some embodiments, a cartridge is provided for producing a single-dose administration unit. In some embodiments, the cartridge may include a first container containing a dried protein and a second container containing an aqueous formulation. In some embodiments, the cartridge includes a single-chamber and multi-chamber pre-filled syringe (e.g., a liquid syringe and lyosyringe).

[0324] In some embodiments, the effective amount of the pharmaceutical composition comprising an agonist anti-CD40 antibody (with or without at least one other therapeutic agent) for treatment will depend, for example, on the treatment context and objective. Those skilled in the art will recognize that, according to some embodiments, the appropriate dose level for treatment will therefore depend in part on the molecule delivered, the indication for using the agonist anti-CD40 antibody (with or without at least one other therapeutic agent), the route of administration, the individual's body size (weight, body surface area, tumor size, or organ size), and / or condition (age and general health). In some embodiments, clinicians may titrate the dose and modify the route of administration to obtain optimal therapeutic effect. In some embodiments, depending on the factors described above, the typical dose may range from about 0.1 μg / kg to at most about 100 mg / kg or higher. In some embodiments, the dose may range from 0.1 μg / kg to about 100 mg / kg, or 1 μg / kg to about 100 mg / kg, or 5 μg / kg to about 100 mg / kg. For example, the effective dose is preferably between 20 μg / kg and 200 μg / kg. Therefore, when patients receive 6 doses, the corresponding total dose over 2 weeks is between 120 μg / kg and 1.2 mg / kg.

[0325] In some embodiments, the dosing frequency will take into account the pharmacokinetic parameters of the agonist anti-CD40 antibody and / or any other therapeutic agent in the formulation used. In some embodiments, the clinician will administer the composition until a dose is reached to achieve the desired effect. Therefore, in some embodiments, the composition may 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 a period of time, or as a continuous infusion via an implantable device or catheter. Further refinement of the appropriate dose is routinely performed by those skilled in the art and falls within their usual scope of work. In some embodiments, the appropriate dose can be determined by using appropriate dose-response data.

[0326] In some embodiments, the route of administration of the pharmaceutical composition conforms to known methods. Intratumoral or peritumoral injection is preferred; however, other routes of administration may be suitable when the composition is formulated for site-specific delivery. Such routes of administration may include intravenous, intraperitoneal, intramuscular, subcutaneous, intraarterial, portal venous, or intralesional routes; via a continuous release system or via an implanted device. In some embodiments, the composition may be administered by bolus or continuous infusion or via an implanted device.

[0327] In some embodiments, the composition can be administered topically by implanting a membrane, sponge, or other suitable material that has absorbed or encapsulated the desired molecule. In some embodiments using an implantable device, the device can be implanted into any suitable tissue or organ, and the delivery of the desired molecule can be achieved by diffusion, timed release of a bolus, or continuous administration.

[0328] E. Methods used to treat or prevent diseases

[0329] In one embodiment, the invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising administering an effective amount of at least one isolated agonist antiCD40 antibody disclosed herein to a patient in need.

[0330] In one embodiment, the present invention includes a method for increasing antigen presentation of APCs (including macrophages, dendritic cells, and B cells) in an individual, comprising administering an effective amount of at least one isolated agonist antiCD40 antibody disclosed herein.

[0331] In one embodiment, the present invention includes a method for activating antigen-presenting cells in an individual, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein.

[0332] Activation of antigen-presenting cells in an individual can be measured using assays known in the art. For example, antigen presentation from activated antigen-presenting cells in the individual can be measured using an antigen recall assay, in which PBMCs from a human donor previously exposed to Epstein-Barr virus (EBV) are attacked with EBV antigen in the presence of an agonist anti-CD40 antibody.

[0333] In one embodiment, the invention includes a method for activating dendritic cells in an individual, comprising administering an effective amount of at least one isolated agonist antiCD40 antibody disclosed herein.

[0334] Activation of dendritic cells in an individual can be measured using assays known in the art. For example, the stimulatory effect of activated dendritic cells from that individual on T cells can be measured using a mixed lymphocyte reaction.

[0335] In one embodiment, the present invention includes a method for enhancing the expression of MHC and / or immune co-stimulatory molecules in an individual, comprising administering an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein. Preferably, the MHC and / or immune co-stimulatory molecule is selected from CD80, CD86, PD-L1, HLA-A, B, C, HLA-DR, and CD83.

[0336] The expression of MHC and / or immune costimulatory molecules in an individual can be measured using assays known in the art. For example, the expression of MHC and / or immune costimulatory molecules in cells derived from that individual can be measured using a fluorescence-activated cell sorting assay.

[0337] In one embodiment, the invention includes a method for stimulating the production of pro-inflammatory cytokines in an individual, comprising administering an effective amount of at least the 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.

[0338] The expression of pro-inflammatory cytokines in an individual can be measured using assays known in the art. For example, the expression of pro-inflammatory cytokines in cells from that individual can be measured using ELISA or LEGENDplex assays.

[0339] In one embodiment, the present invention includes a method for inducing T cell activation in an individual, comprising administering an effective amount of at least one of the agonist antiCD40 antibodies disclosed herein.

[0340] In one embodiment, the present invention includes a method for mimicking CD40L signaling and replacing CD4+ lymphocyte function in an individual, comprising administering an effective amount of at least one agonist anti-CD40 antibody disclosed herein.

[0341] In one embodiment, the invention includes a method for overcoming T cell tolerance in tumor-bearing animals or inducing an effective cytotoxic T cell response or enhancing the effectiveness of an antitumor vaccine in an individual, comprising administering an effective amount of at least one of the agonist antiCD40 antibodies disclosed herein.

[0342] In one embodiment, the present invention includes a method for promoting the secretion of autoantibodies against antigens expressed on tumor cells by B cells in an individual, comprising administering an effective amount of the disclosed agonist antiCD40 antibody.

[0343] In one embodiment, the invention includes a method for upregulating co-stimulatory markers and releasing IL-12 in an individual to activate CD8+ T cells and stimulating a specific cytotoxic T cell response against a cross-presented tumor antigen, comprising administering an effective amount of the disclosed agonist antiCD40 antibody.

[0344] The complex synergistic relationships among events required for tumor eradication suggest that combination therapy can be beneficial in certain situations. Anti-CD40 agonist antibodies can be administered in combination with adjunctive drugs that release antigens, promote cytokine release, enhance immune surveillance, and reduce the inhibitory network to enhance this effect.

[0345] In another embodiment, the invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising administering to a patient in need an effective amount of at least one isolated agonist antiCD40 antibody disclosed herein and an effective amount of at least a second immune enhancer.

[0346] Co-administration of IL-2 and CD40 agonists

[0347] One treatment option is to alter the tumor microenvironment itself, promoting the tumor as a source of stimulation for its own antigens. This can be achieved by introducing IL-2 in combination with anti-CD40 antibodies into or near the tumor site. When administered directly, this co-administration avoids the toxicities associated with systemic administration and successfully leads to regression of larger tumors as well as distant tumors, while maintaining long-term protective memory. Co-administration of IL-2 and CD40 agonists results in increased macrophage activity and B cell activation. The combination of IL-2 and CD40 agonists has shown significant benefits for a variety of cancers, with cancer regression associated with a co-dominant neutrophil- and T-cell inflammatory response.

[0348] Therefore, in one embodiment, the present invention includes a pharmaceutical composition comprising at least one agonist antiCD40 antibody and IL-2 as described herein.

[0349] In one embodiment, the invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising administering to a patient in need an effective amount of at least one of the agonists described herein, an anti-CD40 antibody and IL-2.

[0350] In one embodiment, the present invention includes a method for increasing antigen presentation of APCs (including macrophages, DCs, and B cells) in an individual, comprising administering an effective amount of at least one of the agonists described herein, an anti-CD40 antibody, and IL-2.

[0351] In one embodiment, the present invention includes a method for activating antigen-presenting cells in an individual, comprising administering an effective amount of at least one of the agonist anti-CD40 antibody and IL-2 disclosed herein.

[0352] Activation of antigen-presenting cells in an individual can be measured by assays known in the art. For example, antigen presentation from activated antigen-presenting cells in the individual can be measured using a viral antigen recall assay in which PBMCs from a human donor previously exposed to Epstein-Barr virus (EBV) are attacked with EBV antigen in the presence of an agonist anti-CD40 antibody.

[0353] In one embodiment, the invention includes a method for activating dendritic cells in an individual, comprising administering an effective amount of at least one isolated agonist antiCD40 antibody and IL-2 disclosed herein.

[0354] Activation of dendritic cells in an individual can be measured using assays known in the art. For example, the stimulatory effect of activated dendritic cells from that individual on T cells can be measured using a mixed lymphocyte reaction.

[0355] In one embodiment, the present invention includes a method for enhancing the expression of MHC and / or immune co-stimulatory molecules in an individual, comprising administering an effective amount of each of at least one of the agonist described herein, an anti-CD40 antibody, and IL-2. Preferably, the MHC and / or immune co-stimulatory molecule is selected from CD80, CD86, PD-L1, HLA-A, B, C, HLA-DR, and CD83.

[0356] The expression of MHC and / or immune costimulatory molecules in an individual can be measured using assays known in the art. For example, the expression of MHC and / or immune costimulatory molecules in cells derived from that individual can be measured using a fluorescence-activated cell sorting assay.

[0357] In one embodiment, the invention includes a method for stimulating the production of pro-inflammatory cytokines in an individual, comprising administering an effective amount of each of at least one of the agonists described herein, an anti-CD40 antibody 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.

[0358] The expression of pro-inflammatory cytokines in an individual can be measured using assays known in the art. For example, the expression of pro-inflammatory cytokines in cells from that individual can be measured using ELISA or LEGENDplex assays.

[0359] In one embodiment, the invention includes a method for inducing T cell activation in an individual, comprising administering an effective amount of each of at least one of the agonists described herein, namely, an anti-CD40 antibody and IL-2.

[0360] In one embodiment, the present invention includes a method for mimicking CD40L signaling and replacing CD4+ lymphocyte function in an individual, comprising administering an effective amount of at least one of the agonist anti-CD40 antibody and IL-2 described herein.

[0361] In one embodiment, the invention includes a method for overcoming T-cell tolerance in tumor-bearing animals or inducing an effective cytotoxic T-cell response or enhancing the effectiveness of an anti-tumor vaccine in an individual, comprising administering an effective amount of each of at least one of the agonists described herein, namely, an anti-CD40 antibody and IL-2.

[0362] In one embodiment, the present invention includes a method for promoting the secretion of autoantibodies against antigens expressed on tumor cells by B cells in an individual, comprising administering an effective amount of the disclosed agonist antiCD40 antibody and IL-2.

[0363] In one embodiment, the invention includes a method for upregulating co-stimulatory markers and releasing IL-12 in an individual to activate CD8+ T cells and stimulating a specific cytotoxic T cell response against a cross-presented tumor antigen, comprising administering an effective amount of the disclosed agonist antiCD40 antibody and IL-2.

[0364] The most effective approach for treating or preventing malignant tumor-related conditions in patients may require combination therapy, in which treatment interventions are performed sequentially over time.

[0365] Therefore, in one embodiment, the present invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising sequentially administering, over time, an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein, along with other therapeutic interventions, to a patient in need. In some embodiments, the other therapeutic interventions are selected from surgery, radiotherapy, chemotherapy, hyperthermia, and immunotherapy.

[0366] One treatment option involves altering cells isolated from the patient and then transferring those cells back into the patient. This can be achieved by treating cells isolated from the patient with an anti-CD40 antibody. Cells treated with an anti-CD40 antibody can be treated with other active agents. In some embodiments, the active agent is selected from tumor-specific peptides, tumor cell lysates, cytokines, agonists, and mitogens.

[0367] Therefore, in one embodiment, the present invention includes a method for treating or preventing malignant tumor-related conditions in a patient, comprising administering to a patient in need cells treated with an effective amount of at least one isolated agonist anti-CD40 antibody disclosed herein. In some embodiments, the cells have been isolated from the patient and are selected from dendritic cells (DCs), macrophages, B cells, myeloid cells, lymphoid cells, and hematopoietic stem cells.

[0368] Other features of the invention are described more fully in the following embodiments. However, it should be understood that this detailed description is included merely for illustrative purposes and should not be construed in any way as limiting the extensive description of the invention above. Example

[0369] Example 1 - Production of SVX-3001 antibody

[0370] Sequence data

[0371] The heavy chain of the humanized agonist anti-human CD40 antibody (SEQ ID NO 13, Figure 1 ) and the light chain of the humanized agonist anti-human CD40 antibody (SEQ ID NO 14, Figure 2 The nucleotide sequence of was obtained from North Coast Biologics. Figure 1 and Figure 2 In the diagram, HindIII and XbaI sites are shown in bold, and the coding sequences of the heavy and light chains are underlined. This humanized agonist anti-human CD40 antibody is named SVX-3001. The heavy chain variable regions of SVX-3001 (SEQ ID NO 9 and SEQ ID NO 7) are identified. Figure 3 ) and the light chain variable region of SVX-3001 (SEQ ID NO 10 and SEQ ID NO 8, Figure 4 The coding sequence and amino acid sequence of ), as well as the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3. Figure 3 and Figure 4 In the figure, the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) are shown in bold.

[0372] Gene synthesis and cloning

[0373] The heavy chain encoding SVX-3001 (SEQ ID NO 15) contains HindIII and XbaI restriction enzyme sites for cloning. Figure 5 ) and SVX-3001 light chain (SEQ ID NO 17, Figure 6The DNA sequence of ) was synthesized using GenScript HK and cloned into the vector pcDNA3.1(+) to generate the plasmid pcDNA3.1(+)_Selvax01HC( Figure 7 ) and pcDNA3.1(+)_Selvax01LC( Figure 8 Genscript HK provides 100 μg of transfection-grade plasmids (≥90% supercoiled, ≤0.01 EU / ug endotoxin) in TE buffer. Figure 5 and Figure 6 In the diagram, HindIII and XbaI sites are shown in bold, and 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 κ light chain constant region is shown underlined.

[0374] Production of supernatant containing SVX-3001 antibody

[0375] The day before transfection, HEK 293T cells were placed in DMEM (Gibco) containing 10% FCS (Hyclone) at a concentration of 4 x 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 10 cells / well in 6-well tissue culture plates (Corning Falcon). Three hours before transfection, the culture medium was removed and replaced with 1.5 ml / well of DMEM + 1% FCS. A transfection mixture was prepared by adding 1.5 μg pcDNA3.1(+)_Selvax01HC DNA and 1.5 μg pcDNA3.1(+)_Selvax01LC DNA to a sterile tube containing 300 μl of additive-free DMEM, followed by 6 μl of 1 μg / ml 25kD linear polyethyleneimine (PEI) (Polysciences) and vortexing. The transfection mixture was incubated at room temperature for 10 minutes, followed by cell addition. Cells were incubated at 37°C for 3 hours, followed by the addition of 1.5 ml / well of DMEM + 10% FCS. Cells were then returned to a 37°C, 5% CO2 culture environment.

[0376] Supernatant containing SVX-3001 antibody was collected from transiently transfected cells 18 hours and 2 days post-transfection and replaced with DMEM + 10% FCS. A final collection of culture supernatant was conducted 7 days post-transfection. All culture supernatant was centrifuged to remove cell debris and stored at 4°C.

[0377] Production of purified SVX-3001 antibody

[0378] One day before transfection, HEK 293T cells were placed in DMEM (Gibco) containing 10% ultra-low IgG FCS (Gibco) at a concentration of 4 x 10⁻⁶ cells / mL. 5Cells were seeded at a density of 10 cells / well in 6-well tissue culture plates (Corning Falcon). Three hours before transfection, the culture medium was removed and replaced with 1.5 ml / well of DMEM + 1% ultra-low IgG FCS. A transfection mixture was prepared by adding 1.5 μg pcDNA3.1(+)_Selvax01HC and 1.5 μg p pcDNA3.1(+)_Selvax01LC to a sterile tube containing 300 μl of additive-free DMEM, followed by 6 μl of 1 μg / ml 25kD linear polyethyleneimine (PEI) (Polysciences) and vortexing. The transfection mixture was incubated at room temperature for 10 minutes, followed by cell addition. Cells were incubated at 37°C for 3 hours, followed by the addition of 1.5 ml / well of DMEM + 10% ultra-low IgG FCS. Cells were then returned to 37°C, 5% CO2 culture.

[0379] Supernatants containing SVX-3001 antibody were collected from transiently transfected cells at 2 and 5 days post-transfection and replaced with DMEM + 10% ultra-low IgG FCS. A final collection of culture supernatants was conducted at 7 days post-transfection. The culture supernatants from different time points were combined, filtered through a 0.22 μm filter, and stored at 4°C. At the Harry Perkins Institute for Medical Research Monoclonal Antibody Laboratory, SVX-3001 antibody was purified from the combined supernatants using Protein G. The purified SVX-3001 antibody was administered in PBS at a concentration of 1 mg / ml.

[0380] Example 2 - ELISA detection of human IgG specific for human CD40

[0381] Perform ELISA using the Invitrogen IgG (Total) Human ELISA Kit with plates. Coat each well of the ELISA plate (Corning Costar) 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 (Novoprotein) with a C-terminal 6His tag. Incubate the plates overnight at 4°C. Wash the wells twice with 400 μl / well of wash buffer, then add 250 μl / well of blocking buffer. Incubate the plates at room temperature for 2 hours. Wash wells twice with 400 μl / well of wash buffer, then add 100 μl / well of recombinant human IgG standard serially diluted from 100 ng / ml to 1.56 ng / ml, control anti-CD40 antibody Lob 7 / 4 IgG1 (University of Southampton) serially diluted from approximately 100 ng / ml to 1.56 ng / ml, and supernatant from HEK 293T cells transiently transfected with plasmids pcDNA3.1(+)_Selvax01HC and pcDNA3.1(+)_Selvax01LC serially diluted from 1 / 10 to 1 / 10000. Incubate plates at room temperature for 2 hours on a microplate shaker set to 400 rpm. Wash wells four times with 400 μl / well of wash buffer, then add 100 μl / well of substrate solution containing tetramethylbenzidine (TMB). Incubate plates at room temperature for 15 minutes. Add 100 μl of stop solution (1 M H3PO4) to each well. The assay included a dilution control only (no antibody), recombinant human IgG standard (25 ng / ml), SVX-3001 antibody (estimated 27.3 ng / ml), and Lob 7 / 4 IgG1 antibody (estimated 42.2 ng / ml). Captured antibodies were detected using HRP anti-human IgG antibody. Absorbance at 450 nm was measured using an EnSpire multimode microplate reader (Perkin Elmer).

[0382] Results: Recombinant human IgG standard was detected in wells coated with purified anti-human IgG monoclonal capture antibody, but not in wells coated with recombinant CD40 protein. Figure 9 Anti-CD40 antibody SVX-3001 and Lob 7 / 4 IgG1 could be detected in wells coated with purified anti-human IgG monoclonal capture antibody and wells coated with recombinant CD40 protein. Figure 9 ).

[0383] Conclusion: The supernatant from HEK 293T cells transiently transfected with plasmids pcDNA3.1(+)_Selvax01HC and pcDNA3.1(+)_Selvax01LC was confirmed to contain human IgG protein (i.e. antibody SVX-3001) that is specific to human CD40 protein.

[0384] Example 3 - FACS analysis for detecting antibody binding to antigens on cells

[0385] 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 PE-labeled anti-human CD19 antibody. SVX-3001 antibody bound to the cell surface was detected using BV421-labeled anti-human IgG antibody. Gated lymphocytes were analyzed based on FSC-A and SSC-A signals.

[0386] Methods: Human erythrocyte sedimentation rate (ESR) tannins were obtained from the Australian Red Cross Blood Services (ethical approval: RDHS-243-15, Office of Human Research Ethics, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from the ESR tannins samples by density centrifugation using a Ficoll Paque PLUS (GE Healthcare LifeSciences).

[0387] FACS staining was performed on 96-well U-bottom plates (Corning Falcon). All incubations were performed on ice in the dark. The FACS buffer used for washing 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 supernatant containing SVX-3001 antibody, human IgG1 isotype control (BioLegend), BV421 anti-mouse Ig (BD Biosciences), and PE anti-human CD19 (BioLegends).

[0388] 10 6FACS staining was performed on individual PBMCs / wells. Cell pellets were resuspended in 20 μl of supernatant containing SVX-3001 antibody and incubated for 30 min. 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 min. 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 min. Cells were washed once with FACS buffer, once with PBS, and resuspended in 100 μl of PBS containing 1% formaldehyde and incubated for 20 min. Cells were washed twice with FACS buffer and resuspended in 200 μl of FACS buffer for analysis.

[0389] FACS staining controls included an unstained control, PE anti-human CD19 and BV421 mouse anti-human Ig monostained controls, and a human IgG1 isotype control. Cell staining was analyzed using a FACS Canto II flow cytometer (BD). The PMT voltage and gate used for analysis were set using the FACS staining controls.

[0390] Results: The supernatant containing SVX-3001 antibody bound to CD40 on the surface of CD19+ peripheral blood lymphocytes. Figure 10 ). Figure 10 (A) Shows the PE anti-CD19 control, which shows PE fluorescence due to staining of cells expressing CD19 on their cell surface. Figure 10 (B) shows the BV421 anti-human IgG control, which shows background BV421 fluorescence due to the binding of the anti-human IgG antibody to the CD19+ cell subset expressing IgG on the cell surface. Figure 10 (C) Staining of human IgG1 isotype control. Figure 10 (D) shows the test staining of the supernatant containing SVX-3001 antibody, which shows BV421 fluorescence due to the presence of SVX-3001 antibody that binds to CD40 on the surface of B cells.

[0391] Conclusion: The SVX-3001 antibody is specific for the native CD40 protein expressed on the surface of human immune cells.

[0392] Example 4 - CFSE assay for detecting cell division in response to stimuli

[0393] The ability of SVX-3001 to stimulate cell division rate in PMBCs was measured using CFSE. CFSE-labeled human PBMCs were incubated for 7 days with 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, and 0.001 μg / ml of SVX-3001 antibody or human IgG1 isotype control antibody, followed by staining with Zombie Aqua and PE anti-CD19.

[0394] Methods: Human erythrocyte sedimentation rate (ESR) tannins were obtained from the Australian Red Cross Blood Services (ethical approval: RDHS-243-15, Office of Human Research Ethics, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from the ESR tannins samples by density centrifugation using a Ficoll Paque PLUS (GE Healthcare LifeSciences).

[0395] PBMC of human beings at 2x 10 7 Cells were suspended in PBS at 100 μM / ml and stained with CFSE (Life Technologies Australia) using 25 μl of 100 μM CFSE per 1 ml of cells. The cells were mixed with CFSE by gently inverting for 10 minutes, and then 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.

[0396] Cells were cultured in 96-well plates (Nunc) at a density of 5 x 10⁻⁶ cells / well. 5 Cells / well were cultured in RPMI + 10% FCS to a final volume of 200 μl / well. Cells were stimulated with supernatant containing SVX-3001 antibody 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 and 5% CO2 for 7 days, and then harvested for FACS staining.

[0397] FACS staining was performed on 96-well U-bottom plates (Corning Falcon). All incubations were performed on ice in the dark. The FACS buffer used for washing 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 (BioLegends).

[0398] Cell pellets for FACS staining were resuspended in 20 μl of PE anti-human CD19 diluted in FACS buffer and incubated for 30 min. Cells were washed twice with PBS and resuspended in 100 μl of Zombie Aqua diluted in PBS and incubated for 15 min. Cells were washed once with FACS buffer, once with PBS, and resuspended in 100 μl of PBS containing 1% formaldehyde and incubated for 20 min. Cells were washed twice with FACS buffer and resuspended in 200 μl of FACS buffer for analysis.

[0399] FACS staining controls include unstained control, monostained control, and FMO control. Cell staining was analyzed using a FACS Canto II flow cytometer (BD). The PMT voltage and gate were set using the FACS staining controls for analysis.

[0400] Results: At concentrations above 0.01 mg / ml, cell division stimulated by SVX-3001 antibody in human PBMCs was higher than the background level of cell division observed in human PBMCs stimulated with human IgG1 isotype control antibody. Figure 11 ). Figure 11 (A) Shows the use of PBMC gating with FSC-A and SSC-A to exclude fragments. Figure 11 (B) shows the use of single-cell gating with FSC-A and FSC-H to exclude cell clusters. Figure 11 (C) shows the use of live cell gating with Zombie Aqua and SSC-A to exclude dead cells. Figure 11 (D) shows that dividing cells were identified by comparing CFSE with PE-anti-CD19, based on the reduction of CFSE fluorescence. Figure 11 (E) shows the percentage of dividing cells in PBMC cultures stimulated with different concentrations of human IgG1 antibody.

[0401] Conclusion: The SVX-3001 antibody has stimulatory (agonist) activity that can lead to increased cell division in human immune cells.

[0402] Example 5 - LEGENDplex assay for detecting PMBC cytokine responses to SVX-3001 stimulation

[0403] LEGENDplex assays were performed to determine the levels of cytokines produced in response to SVX-3001 stimulation of PMBCs.

[0404] Methods: Human blood samples were obtained from healthy volunteers (ethical approval: HRE2017-0767, Office of Human Research Ethics, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from human blood samples by density centrifugation using a 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) + 10% FCS and 5% FCS to allow cells to recover from freezing. Cells were labeled using the Cell Trace Violet proliferation kit (Life Technologies Australia).

[0405] Cells were cultured in 96-well plates (Nunc) at a density of 5 x 10⁻⁶ cells / well. 5 Cells / well were cultured in RPMI + 10% FCS to a final volume of 200 μl / well. Cells were cultured under irritation-free conditions with 1 μg / ml purified SVX-3001 antibody and 10 ng / ml recombinant human IL-2 (Peprotech) or 1 μg / ml purified SVX-3001 antibody and 10 ng / ml recombinant human IL-2. Cells were cultured at 37°C and 5% CO2 for 3 days, after which the supernatant was harvested. Soluble analytes in the supernatant were determined using a LEGENDplex bead-based immunoassay (BioLegend). LEGENDplex data were acquired using an LSR Tortessa Cell Analyzer (BD).

[0406] Results: Stimulation of human PBMCs with purified SVX-3001 antibody resulted in increased levels of IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, and IL-23 in the culture medium after 3 days of culture, and decreased levels of IL-13 and TNF-β. Figure 12 Stimulation of human PBMCs with a combination of SVX-3001 and IL-2, instead of using SVX-3001 or IL-2 alone, resulted in the release of IFN-γ into the culture medium after 3 days of culture. Figure 12 The cytokine concentrations measured in the culture medium control (RPMI + 10% FCS + 10 ng / ml IL-2) were... Figure 12 The middle part is indicated by a dashed line.

[0407] Conclusion: The SVX-3001 antibody has stimulatory (agonist) activity that leads to differential release of cytokines from human immune cells.

[0408] Example 6 - FACS Antibody Competition Study

[0409] FACS assays were performed to determine the binding ability of SVX-3001 blocking 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 SVX-3001 antibody. Binding of mouse anti-human CD40 antibodies to human PBMCs was detected using BV421-labeled anti-mouse Ig antibody.

[0410] Methods: Human erythrocyte sedimentation rate (ESR) tannins were obtained from the Australian Red Cross Blood Services (ethical approval: RDHS-243-15, Office of Human Research Ethics, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from the ESR tannins samples by density centrifugation using a Ficoll Paque PLUS (GE Healthcare LifeSciences).

[0411] FACS staining was performed on 96-well U-bottom plates (Corning Falcon). All incubations were performed on ice in the dark. The FACS buffer used for washing 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 the supernatant containing SVX-3001 antibody. The staining reagents were anti-CD40 antibody LOB7 / 6 (LSBio), anti-CD40 antibody B-B20 (Abcam), and BV421 anti-mouse Ig (BDBiosciences).

[0412] 5×10 for each hole 5 FACS staining was performed on individual PBMCs. Cell pellets were resuspended in 200 μl of supernatant containing 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, or 0.001 μg / ml SVX-3001 antibody and incubated for 30 min. Cell pellets (without washing) were then resuspended in 20 μl of LOB7 / 6 diluted in FACS buffer or B-B20 diluted in FACS buffer and incubated for 30 min. 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 min. Cells were washed twice with PBS and resuspended in 100 μl of PBS containing 1% formaldehyde and incubated for 20 min. Cells were washed twice with FACS buffer and resuspended in 200 μl of FACS buffer for analysis.

[0413] FACS staining controls include an unstained control, a non-blocking control, and a non-primary antibody control. Cell staining was analyzed using a FACS Canto II flow cytometer (BD). The PMT voltage and gate were set using the FACS staining controls.

[0414] Results: Staining of human PBMCs by anti-CD40 antibody B-B20 was blocked by SVX-3001, while staining by anti-CD40 antibody LOB7 / 6 was not blocked by SVX-3001. Figure 13 (A) Unstained control. (B) LOB7 / 6 staining. (C) LOB7 / 6 staining in the presence of 1 μg / ml SVX-3001. (D) B-B20 staining. (E) B-B20 staining in the presence of 1 μg / ml SVX-3001.

[0415] 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.

[0416] Example 7 - Study on surface plasmon resonance antibody competition

[0417] The ability of SVX-3001 to compete with a range of anti-CD40 antibodies was measured using surface plasmon resonance.

[0418] Methods: Pair binding assays were performed using a Biacore T200 (GE Healthcare) via surface plasmon resonance (SPR). HBS-EP+ buffer was always used as the run buffer.

[0419] Recombinant human CD40 extracellular domains (CD40-Fc; BioLegend) containing a C-terminal human IgG1 Fc domain and a 6His tag were immobilized onto the surface of the S-series sensor chip CM5 (GE Healthcar) using an amine conjugation kit (GE Healthcare). CD40 Fc was diluted to 25 μg / ml in 10 mM sodium acetate buffer at pH 5.0 for immobilization. Target immobilization level: 1000 RU. Washing solution: ethanolamine.

[0420] The reagents used in the competition study were purified SVX-3001 antibody, recombinant CD40L (BioLegend), and purified anti-CD40 antibodies Lob 7 / 4 IgG1 (University of Southampton), CP-870893 IgG1 (University of Southampton), S2C6 (Mabtech), G28.5 (BioXCell), and LOB7 / 6 (LSBio).

[0421] After injecting at 10 μl / min for 180 seconds, the first sample (Sample 1) was bound to a concentration sufficient to saturate the surface-fixed CD40-Fc for pairwise binding analysis. This concentration ranged from 12.5 to 200 μg / ml for the reagents tested. The second sample (Sample 2) was then injected at 10 μl / min for 180 seconds. Regeneration was performed using glycine-HCl buffer at pH 1.5, followed by injection at 10 μl / min for 30 seconds, stabilization for 5 seconds, and then testing the next pair. Saturation was confirmed and a baseline reaction was established using the same pairing method for Sample 1 and Sample 2.

[0422] result: Figure 14 The image shows the Biacore T200 sensor map plotted for epitope mapping via paired binding of SVX-3001, Lob 7 / 4IgG1, and LOB7 / 6. Table 3 summarizes the results for all test pairs.

[0423] Table 3: Blocking effect of sample 1 on sample 2 when using a fixed CD40-Fc

[0424]

[0425] Conclusions: SVX-3001 and antibodies Lob 7 / 4 IgG1, CP-870893 IgG1, S2C6, and G28.5 bind to the same or overlapping epitopes on human CD40. SVX-3001 binds to different epitopes that do not overlap with antibody LOB7 / 6. The epitopes of SVX-3001 do not overlap with the binding sites of CD40L.

[0426] It has been reported that antibodies Lob 7 / 4, SGN40 (derived from antibody S2C6), and CP-870893 all bind to epitopes in the CRD1 region of human CD40 (Cancer Cell 33, 664-675, April 9, 2018). Based on SPR data, it is inferred that SVX-3001 also binds to epitopes in the CRD1 region (P25942; Cys26-Cys59) of human CD40.

[0427] Example 8 - Study on surface plasmon resonance antibody affinity

[0428] The affinity of SVX-3001 for the extracellular domain of CD40 was measured using surface plasmon resonance.

[0429] Methods: Kinetic analysis was performed using a Biacore T200 (GE Healthcare) via surface plasmon resonance (SPR). HBS-EP+ buffer was always used as the run buffer.

[0430] Purified SVX-3001 was immobilized onto the surface of the S-Series sensor chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare). SVX-3001 was diluted to 25 μg / ml in 10 mM sodium acetate buffer at pH 5.0 for immobilization. Target immobilization level: 600 RU. Wash solution: ethanolamine. The reference surface used for analysis was either untreated or had a human IgG1 isotype control (BioLegend) immobilized onto the surface using the same method used for SVX-3001.

[0431] The reagents used for kinetic studies are the recombinant human CD40 extracellular domain with a C-terminal 6His tag (CD40-6His; Novoprotein) and the recombinant human CD40 extracellular domain with a C-terminal human IgG1 Fc domain and a 6His tag (CD40-Fc; BioLegend).

[0432] The injection parameters for the sample were: contact time: 120 seconds, flow rate: 30 μl / min, and dissociation time: 300 seconds. The injection parameters for regeneration were: contact time: 30 seconds, flow rate: 30 μl / min, and stabilization time: 0 seconds. The regeneration solution was either a 10 mM glycine-HCl buffer solution at 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. Curve fitting was performed using a 1:1 binding model.

[0433] Results: Table 4 shows the results of three experiments using CD40-6His as the analyte and the results using CD40-F. C Kinetic and affinity constants of SVX-3001 in three tests as an analyte.

[0434] Table 4: Kinetic and Affinity Constants of SVX-3001

[0435]

[0436] Conclusion: The equilibrium dissociation constant (Ki) of SVX-3001 calculated using CD40-6His as the analyte is... D The value is 6.111 x 10. -9 M (6.111 nM), K of SVX-3001 calculated using CD40-Fc as the analyte D It is 5.826 x 10 -10 M (0.5826 nM). Binding rate constant (k) calculated using two analytes. a Similar to K, therefore K D The difference appears to be due to the dissociation rate constants (K0) of the two analytes.d This is due to the difference in ).

[0437] Example 9 - Activation of monocyte-derived dendritic cells using anti-CD40 antibody

[0438] The stimulation of monocyte-derived dendritic cells (moDCs) by IFN-γ in the presence of SVX-3001, CD40L, or LPS was measured by referencing the expression of co-stimulatory molecules.

[0439] Methods: Human erythrocyte sedimentation rate (ESR) amber layer samples were obtained from the Australian Red Cross Blood Service (ethical approval: RDHS-243-15, Office of Human Research Ethics, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from the human ESR amber layer samples by density centrifugation using a Ficoll Paque PLUS (GE Healthcare LifeSciences). Human PBMCs were then in RPMI (Gibco) + 10% FCS (Hyclone) at 5 x 10⁻⁶ ppm. 6 Cells were seeded at a density of 6-well plates (CorningFalcon) and incubated at 37°C for 2 hours to allow monocytes to adhere to the plastic. The culture medium containing unadhered cells was then removed.

[0440] Adherent monocytes were cultured for 7 days in RPMI + 10% FCS containing 80 ng / ml recombinant human GM-CSF (Shenandah Biotechnology), 10 ng / ml recombinant human IL-4 (Shenandah Biotechnology), and 10 μg / ml polymyxin B (Sigma-Aldrich) to differentiate into monocyte-derived dendritic cells (moDCs). The medium was replaced with a solution containing GM-CSF, IL-4, and polymyxin B on day 4.

[0441] On day 7, cells were stimulated in a medium containing GM-CSF and IL-4 but without polymyxin B. Stimulation conditions included a medium control (no stimulation), 1 μg / ml SVX-3001, 1 μg / ml human IgG1 isotype control (BioLegend), 0.67 mg / ml recombinant CD40L (BioLegends), and 1 μg / ml LPS (Sigma-Aldrich) with 20 ng / ml recombinant human IFN-γ (Shenandoah Biotechnology). On day 9, cells were treated with 1X Brefeldin A solution (BioLegend) for 4 hours, then harvested and stained for FACS analysis (48-hour stimulation).

[0442] FACS staining was performed on 96-well U-bottom plates (Corning Falcon). The FACS buffer used for washing and diluting the antibody consisted of PBS containing 1% BSA (Sigma), 1% FCS (Hyclone), and 0.01% w / v sodium azide (Sigma). The 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 (BioLegent).

[0443] Each stained sample contained one moDC from one well of a 6-well plate used for cell culture and stimulation. Cells were washed twice with phosphate-buffered saline (PBS), resuspended in 100 μl of Zombie UV diluted in PBS, and incubated for 15 min. Cells were washed twice with FACS buffer, resuspended in 100 μl of staining mixture (containing 50 μl of Brilliant Stain buffer (BD) and 50 μl of anti-cell surface labeled antibody diluted in FACS buffer), and incubated for 30 min. Cells were washed twice with PBS, resuspended in 100 μl of fixation / permeabilization solution (BD), and incubated for 20 min. Cells were washed twice with Perm / Wash buffer (BD), resuspended in 100 μl of staining mixture (containing 50 μl of Brilliant Stain buffer and 50 μl of anti-intracellular labeled antibody diluted in Perm / Wash buffer). Cells were washed twice with Perm / Wash buffer, resuspended in 200 μl of FACS buffer, and analyzed.

[0444] FACS staining controls included unstained and monostained controls. Cell staining was analyzed using an LSL Ortessa Cell Analyzer (BD). PMT voltage and compensation values ​​were set using FACS staining controls. Gating for moDC analysis was size (FSC-A vs. SSC-A), single cell count (FSC-A vs. FSC-H), viable cells (Zombie UV negative), and CD3 count. - CD14 -Cells (CD3 vs. CD14) and CD11b + CD11c + Cells (CD11b vs. CD11c). The median fluorescence intensity (MFI) of cell surface markers was determined for the entire moDC population.

[0445] Results: Stimulation of moDCs with SVX-3001, CD40L, or LPS in combination with IFN-γ for 48 hours led to upregulation of cell surface markers HLA-A, B, C, HLA-DR, CD80, CD83, CD86, and PD-L1, as well as increased IL-12 expression. Figure 15 As shown. These markers were not upregulated in unstimulated moDCs or moDCs stimulated with human IgG1 isotype control antibody.

[0446] Conclusion: The anti-CD40 antibody SVX-3001 exhibits agonistic activity against human monocyte-derived dendritic cells (moDCs) (an example of antigen-presenting cells (APCs),) leading to increased expression of co-stimulatory molecules B7-1 (CD80), B7-2 (CD86), and PD-L1 (CD274), as well as HLA-A, B, C (MHC class I), HLA-DR (MHC class II), CD83, and the pro-inflammatory cytokine IL-12. This agonistic activity is dependent on the antibody's antigen-binding domain, as no such response was observed with human IgG1 isotype control antibodies possessing the same Fc domain.

[0447] Example 10 - Activation of monocyte-derived dendritic cells with anti-CD40 antibody - dose-response

[0448] The stimulation of monocyte-derived dendritic cells (moDCs) in the presence of SVX-3001 was measured by referencing the expression of co-stimulatory molecules. The dose-response effect of SVX-3001 stimulation was also measured.

[0449] Methods: Human erythrocyte sedimentation rate (ESR) amber layer samples were obtained from the Australian Red Cross Blood Service (ethical approval: RDHS-243-15, Office of Human Research Ethics, Curtin University). Peripheral blood mononuclear cells (PBMCs) were isolated from the human ESR amber layer samples by density centrifugation using a Ficoll Paque PLUS (GE Healthcare LifeSciences). Human PBMCs were then in RPMI (Gibco) + 10% FCS (Hyclone) at 5 x 10⁻⁶ ppm. 6 Cells were seeded at a density of 6-well plates (CorningFalcon) and incubated at 37°C for 2 hours to allow monocytes to adhere to the plastic. The culture medium containing unadhered cells was then removed.

[0450] Adherent monocytes were cultured for 7 days in RPMI + 10% FCS containing 80 ng / ml recombinant human GM-CSF (Shenandah Biotechnology), 10 ng / ml recombinant human IL-4 (Shenandah Biotechnology), and 10 μg / ml polymyxin B (Sigma-Aldrich) to differentiate into monocyte-derived dendritic cells (moDCs). The medium was replaced with a solution containing GM-CSF, IL-4, and polymyxin B on day 4.

[0451] On day 7, cells were stimulated in a 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 anti-CD40 antibody SVX-3001. Controls included unstimulated medium alone, 1 μg / ml human IgG1 isotype control (BioLegend), and 1 μg / ml LPS (SigmaAldrich) with 20 ng / ml recombinant human IFN-γ (Shenandoah Biotechnology). On day 9, cells were treated with 1X Brefeldin A solution (BioLegend) for 4 hours, then harvested and stained for FACS analysis (48-hour stimulation).

[0452] FACS staining was performed on 96-well U-bottom plates (Corning Falcon). The FACS buffer used for washing and diluting the antibody consisted of PBS containing 1% BSA (Sigma), 1% FCS (Hyclone), and 0.01% w / v sodium azide (Sigma). The 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 (BioLegent).

[0453] Each stained sample contained one moDC from one well of a 6-well plate used for cell culture and stimulation. Cells were washed twice with phosphate-buffered saline (PBS), resuspended in 100 μl of Zombie UV diluted in PBS, and incubated for 15 min. Cells were washed twice with FACS buffer, resuspended in 100 μl of staining mixture (containing 50 μl of Brilliant Stain buffer (BD) and 50 μl of anti-cell surface labeled antibody diluted in FACS buffer), and incubated for 30 min. Cells were washed twice with PBS, resuspended in 100 μl of fixation / permeabilization solution (BD), and incubated for 20 min. Cells were washed twice with Perm / Wash buffer (BD), resuspended in 100 μl of staining mixture (containing 50 μl of Brilliant Stain buffer and 50 μl of anti-intracellular labeled antibody diluted in Perm / Wash buffer). Cells were washed twice with Perm / Wash buffer, resuspended in 200 μl of FACS buffer, and analyzed.

[0454] FACS staining controls included unstained and monostained controls. Cell staining was analyzed using an LSR Tortessa cell analyzer (BD). PMT voltage and compensation values ​​were set using FACS staining controls. Gating for moDC analysis was size (FSC-A vs. SSC-A), single cell count (FSC-A vs. FSC-H), viable cell count (Zombie UV vs. SSC-A), and CD3 count. - CD14 - Cells (CD3 vs. CD14), CD11b + CD11c + Cells (CD11b vs. CD11c) and CD1a + Cells (CD1a vs. SSC-A). The median fluorescence intensity (MFI) of cell surface markers was determined for the entire moDC population.

[0455] Results: Stimulation of human moDCs with anti-CD40 antibodies SVX-3001, APX005M and CP-870893 for 48 hours 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 The mean fluorescence intensity (MFI) of FACS staining of moDCs stimulated with SVX-3001 for 48 hours at different concentrations is shown. The MFI of unstimulated moDCs is shown as dashed lines.

[0456] Conclusion: The anti-CD40 antibody SVX-3001 provides dose-dependent agonist signals to human moDCs, leading to the upregulation of cell surface markers HLA-A, B, C, HLA-DR, CD80, CD83, CD86, and PD-L1, as well as the cytokine IL-12.

[0457] Example 11 - Gene Synthesis of SVX-3001 Codon Optimization Sequence

[0458] Use GeneArt, Thermo Fisher Scientific's GeneOptimizer TM For expression in humans, the DNA sequences encoding the SVX-3001 heavy and light chains were optimized, resulting in the gene sequence 20ACGJQC_Selvax01HC (SEQ ID NO 21). Figure 17 ) and 20ACGJRC_Selvax01LC(SEQ ID NO 22, Figure 18 ).exist Figure 17 and 18 In the diagram, the Kozak sequence is shown in bold, and the coding sequences of the heavy and light chains are indicated by underscores.

[0459] The gene sequences 20ACGJQC_Selvax01HC and 20ACGJRC_Selvax01LC were synthesized by GeneArt, Thermo Fisher Scientific, and inserted into pcDNA3.4-TOPO to produce the plasmid 20ACGJQC_Selvax01HC-pcDNA3.4-TOPO. Figure 19 ) and 20ACGJRC_Selvax01LC-pcDNA3.4-TOPO( Figure 20 ). sequence list <110> Selvax Pty Ltd <120> Agonist antiCD40 antibody <130> 288168 <160> twenty two <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDRH1 <400> 1 Gly Tyr Ser Ile Thr Thr Asn Tyr Tyr Trp Asn 1 5 10 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDRH2 <400> 2 Tyr Ile Arg Tyr Asp Gly Thr Thr Tyr Tyr Tyr Ala Pro Ser Leu Lys Gly 1 5 10 15 <210> 3 <211> 3 <212> PRT <213> Artificial sequence <220> 81 <223> CDRH3 <400> 3 Leu Asp Tyr 1 <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDRL1 <400> 4 Arg Ser Ser Gln Ser Leu Glu Asn Ser Asn Gly Asn Thr Phe Leu Asn 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDRL2 <400> 5 Arg Val Ser Asn Arg Phe Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDRL3 <400> 6 Leu Gln Val Thr His Val Pro Tyr Thr 82 1 5 <210> 7 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region sequence <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Tyr Ile Arg Tyr Asp Gly Thr Thr Tyr Tyr Tyr Ala Pro Ser Leu 50 55 60 Lys Gly Arg Phe Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Thr Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 8 <211> 113 <212> PRT <213> Artificial sequence <220> 83 <223> Light chain variable region sequence <400> 8 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Asp Arg Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser 20 25 30 Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Glu Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 9 <211> 336 <212> DNA <213> Artificial sequence <220> <223> Heavy chain variable region nucleotide sequence <400> 9 caggtgcaac tgcagcagag cggccccggg ctggtgaagc ctagccagtc actgtccctc 60 84 acctgcgccg ttagcggcta tagcattacc accaactact actggaattg gatccggcag 120 gcccccggta agggcctgga gtgggtcggg tacatccggt atgacggcac aacctactat 180 gccccctctt tgaaaggcag attcagtatc acccgggaca ctagcaagaa ccaattcttt 240 ctgcagctga cctctgtgac tccagaggac acagctactt actactgcgc acggcttgat 300 tattggggac agggaacgct ggtgacagtc tcgagt 336 <210> 10 <211> 339 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of light chain variable region <400> 10 gacatcgtga tgacccagag ccccctgagc ctgtcagtta gcctggggga tagggccagc 60 atcagttgcc ggtcttcaca aagtctggaa aacagcaacg gcaatacctt tcttaactgg 120 ttccagcaga agcctggcca gtctccccag ctgctgattt acagagtgtc caatcggttt 180 tccggcgtgc ccgaccggtt ctccgggagc ggctctggta ccgactttac actcaaaatc 240 agccgcgtcg aggccgagga tgaaggcgtg tacttctgct tgcaggtgac ccacgtgcca 300 tatactttcg gaggaggcac caagctggag atcaagcgt 339 <210> 11 <211> 277 <212> PRT <213> Homo sapiens <400> 11 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30 85 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr 100 105 110 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly 115 120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu 180 185 190 Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200 205 Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn 210 215 220 86 Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235 240 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His 245 250 255 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser 260 265 270 Val Gln Glu Arg Gln 275 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Polyhistidine tag <400> 12 His His His His His His 1 5 <210> 13 <211> 1417 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain nucleotide sequence <400> 13 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gacaggtgca actgcagcag agcggccccg ggctggtgaa gcctagccag 120 tcactgtccc tcacctgcgc cgttagcggc tatagcatta ccaccaacta ctactggaat 180 87 tggatccggc aggcccccgg taagggcctg gagtgggtcg ggtacatccg gtatgacggc 240 acaacctact atgccccctc tttgaaaggc agattcagta tcacccggga cactagcaag 300 aaccaattct ttctgcagct gacctctgtg actccagagg acacagctac ttactactgc 360 gcacggcttg attattgggg acagggaacg ctggtgacag tctcgagtgc tagcaccaag 420 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 480 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1080 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1380 tccctgtctc cgggtaaatg agtcctagct gtctaga 1417 <210> 14 <211> 741 <212> DNA <213> Artificial sequence 88 <220> <223> Light chain nucleotide sequence <400> 14 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtgacatcgt gatgacccag agccccctga gcctgtcagt tagcctgggg 120 gatagggcca gcatcagttg ccggtcttca caaagtctgg aaaacagcaa cggcaatacc 180 tttcttaact ggttccagca gaagcctggc cagtctcccc agctgctgat ttacagagtg 240 tccaatcggt tttccggcgt gcccgaccgg ttctccggga gcggctctgg taccgacttt 300 acactcaaaa tcagccgcgt cgaggccgag gatgaaggcg tgtacttctg cttgcaggtg 360 acccacgtgc catatacttt cggaggaggc accaagctgg agatcaagcg tacggtagcg 420 gccccatctg tcttcatctt cccgccatct gatgagcagt tgaaatctgg aactgcctct 480 gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg gaaggtggat 540 aacgccctcc aatcgggtaa ctcccaggag agtgtcacag agcaggacag caaggacagc 600 acctacagcc tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc 660 tacgcctgcg aagtcaccca tcagggcctg agctcgcccg tcacaaagag cttcaacagg 720 ggagagtgtt agtaatctag a 741 <210> 15 <211> 1410 <212> DNA <213> Artificial sequence <220> <223> Selvax01HC nucleotide sequence <400> 15 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gacaggtgca actgcagcag agcggccccg ggctggtgaa gcctagccag 120 tcactgtccc tcacctgcgc cgttagcggc tatagcatta ccaccaacta ctactggaat 180 89 tggatccggc aggcccccgg taagggcctg gagtgggtcg ggtacatccg gtatgacggc 240 acaacctact atgccccctc tttgaaaggc agattcagta tcacccggga cactagcaag 300 aaccaattct ttctgcagct gacctctgtg actccagagg acacagctac ttactactgc 360 gcacggcttg attattgggg acagggaacg ctggtgacag tctcgagtgc tagcaccaag 420 ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 480 ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 540 gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 600 ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660 gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 720 aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1080 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1380 tccctgtctc cgggtaaatg ataatctaga 1410 <210> 16 <211> 462 <212> PRT <213> Artificial Sequence 90 <220> <223> Amino acid sequence of Selvax01HC <400> 16 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val 20 25 30 Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser 35 40 45 Ile Thr Thr Asn Tyr Tyr Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Gly Tyr Ile Arg Tyr Asp Gly Thr Thr Tyr Tyr 65 70 75 80 Ala Pro Ser Leu Lys Gly Arg Phe Ser Ile Thr Arg Asp Thr Ser Lys 85 90 95 Asn Gln Phe Phe Leu Gln Leu Thr Ser Val Thr Pro Glu Asp Thr Ala 100 105 110 Thr Tyr Tyr Cys Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 115 120 125 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 130 135 140 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 145 150 155 160 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 91 165 170 175 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 180 185 190 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 195 200 205 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 210 215 220 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 92 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355 360 365 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 17 <211> 741 <212> DNA <213> Artificial sequence <220> <223> Selvax01LC nucleotide sequence <400> 17 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtgacatcgt gatgacccag agccccctga gcctgtcagt tagcctgggg 120 gatagggcca gcatcagttg ccggtcttca caaagtctgg aaaacagcaa cggcaatacc 180 tttcttaact ggttccagca gaagcctggc cagtctcccc agctgctgat ttacagagtg 240 tccaatcggt tttccggcgt gcccgaccgg ttctccggga gcggctctgg taccgacttt 300 93 acactcaaaa tcagccgcgt cgaggccgag gatgaaggcg tgtacttctg cttgcaggtg 360 acccacgtgc catatacttt cggaggaggc accaagctgg agatcaagcg tacggtagcg 420 gccccatctg tcttcatctt cccgccatct gatgagcagt tgaaatctgg aactgcctct 480 gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg gaaggtggat 540 aacgccctcc aatcgggtaa ctcccaggag agtgtcacag agcaggacag caaggacagc 600 acctacagcc tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc 660 tacgcctgcg aagtcaccca tcagggcctg agctcgcccg tcacaaagag cttcaacagg 720 ggagagtgtt agtaatctag a 741 <210> 18 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Selvax01LC <400> 18 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser 20 25 30 Val Ser Leu Gly Asp Arg Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Glu Asn Ser Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe 65 70 75 80 94 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Glu Gly Val Tyr Phe 100 105 110 Cys Leu Gln Val Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 19 <211> 442 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain amino acid sequence 95 <400> 19 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Tyr Ile Arg Tyr Asp Gly Thr Thr Tyr Tyr Ala Pro Ser Leu 50 55 60 Lys Gly Arg Phe Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Thr Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 96 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 97 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 20 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Light chain amino acid sequence <400> 20 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Asp Arg Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser 20 25 30 Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 98 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Glu Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 21 <211> 1410 <212> DNA <213> Artificial Sequence <220> <223> 20ACGJQC_Selvax01HC Nucleotide Sequence 99 <400> 21 aagcttgcca ccatggaaac ccctgctcag ctgctgtttc tgctgctgct gtggctgcct 60 gatacaacag gacaggtcca gctccagcag tctggccctg gacttgtgaa gcctagccag 120 agcctgtctc tgacatgtgc cgtgtctggc tacagcatca ccaccaacta ctactggaac 180 tggatccggc aggcccctgg caaaggactg gaatgggtcg gatatatcag atacgacggc 240 accacctact acgcccctag cctgaagggc agattctcca tcaccagaga caccagcaag 300 aaccagttct tcctgcagct gaccagcgtg acccctgagg ataccgccac ctattactgc 360 gccagactgg attattgggg ccagggcaca ctggtcacag tgtctagcgc ctctacaaag 420 ggccctagcg ttttcccact ggctcctagc agcaagagca catctggtgg aacagccgct 480 ctgggctgcc tggtcaagga ttactttcct gagcctgtga ccgtgtcctg gaattctggt 540 gctctgacaa gcggcgtgca cacctttcca gccgtgctgc aaagcagcgg cctgtactct 600 ctgtctagcg tcgtgacagt gcctagcagc tctctgggca cccagaccta catctgcaac 660 gtgaaccaca agcctagcaa caccaaggtg gacaagaagg tggaacccaa gagctgcgac 720 aagacccaca cctgtcctcc atgtcctgct ccagaactgc tcggcggacc ctccgttttc 780 ctgtttccac ctaagcctaa ggacaccctg atgatcagca gaacccctga agtgacctgc 840 gtggtggtgg atgtgtccca cgaggatccc gaagtgaagt tcaattggta cgtggacggc 900 gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtacaacag cacctacaga 960 gtggtgtccg tgctgacagt gctgcaccag gactggctga acggcaaaga gtacaagtgc 1020 aaggtgtcca acaaggccct gcctgctcct atcgagaaaa ccatcagcaa ggccaagggc 1080 cagccaagag aaccccaggt ttacaccctg cctccaagcc gggaagagat gaccaagaat 1140 caggtgtccc tgacctgcct cgtgaagggc ttctaccctt ccgatatcgc cgtggaatgg 1200 gagagcaatg gccagcctga gaacaactac aagacaaccc ctcctgtgct ggacagcgac 1260 ggctcattct tcctgtacag caagctgacc gtggacaagt ctaggtggca gcagggcaac 1320 gtgttcagct gttctgtgat gcacgaggcc ctgcacaacc actacaccca gaaatctctg 1380 100 agtctgagcc ccggcaagtg atgatctaga 1410 <210> 22 <211> 741 <212> DNA <213> Artificial sequence <220> <223> 20ACGJRC_Selvax01LC Nucleotide sequence <400> 22 aagcttgcca ccatggaaac ccctgctcag ctgctgtttc tgctgctgct gtggctgcct 60 gataccaccg gcgatatcgt gatgacacag agccctctga gcctgtccgt gtctctgggc 120 gatagagcca gcatcagctg tagaagcagc cagagcctgg aaaacagcaa cggcaacacc 180 ttcctgaact gttccagca gaagcccgga cagtctcccc agctgctgat ctacagagtg 240 tccacagat tcagcgggt gcccgataga ttttctgca gcggctctgg caccgacttc 300 accctgaaga ttagcagt ggaagccgag gacgaggcg tgtactctg tctgcaagtg 360 acccacgtgc catacacctt tggcggaggc accaagctgg aaatcaagg aacagtggcc 420 gctccgagcg tgttcatctt tccaccaagc gacgagcagc tgaaaagcgg cacagcctct 480 gtcgtgtgcc tgctgaacaa cttctacccc agagaagcca aggtgcagtg gaaggtggac 540 aatgccctgc agagcggcaa tagccagag agcgtgaccg aggaggc agaggacc 600 acatacagcc tgagcagcac cctgacactg agcaagccg actacgagaa gcacaagtg 660 tacgcctgcg aagtgacaca ccaggcctg tctagccctg tgaccagag cttcaaccgg 720 ggcgagtgct gatgatctag a 741

Claims

1. An isolated agonist anti-CD40 antibody or its antigen-binding fragment, comprising (i) a VH chain containing three CDRs and (ii) a VL chain containing three CDRs, wherein: (a) The CDRH1 sequence consists of SEQ ID NO:1; (b) The CDRH2 sequence consists of SEQ ID NO:2; (c) The CDRH3 sequence consists of SEQ ID NO:3; (d) The CDRL1 sequence consists of SEQ ID NO:4; (e) The CDRL2 sequence consists of SEQ ID NO:5; and (f) The CDRL3 sequence consists of SEQ ID NO:

6.

2. The isolated agonist antiCD40 antibody according to claim 1, comprising the heavy chain variable region of SEQ ID NO:

7.

3. The isolated agonist antiCD40 antibody according to claim 1, comprising the light chain variable region of SEQ ID NO:

8.

4. The isolated agonist antiCD40 antibody according to claim 1, wherein the antibody comprises the heavy chain of SEQ ID NO:19 or the light chain of SEQ ID NO:

20.

5. The isolated agonist anti-CD40 antibody according to claim 1, comprising the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:

8.

6. The isolated agonist antiCD40 antibody according to any one of claims 1-5, wherein the isolated agonist antiCD40 antibody is a humanized antibody against human CD40.

7. The isolated agonist antiCD40 antibody according to any one of claims 1-5, wherein the isolated agonist antiCD40 antibody is a monoclonal antibody.

8. The isolated agonist antiCD40 antibody according to any one of claims 1-5, wherein the antibody is an IgG antibody.

9. The isolated agonist antiCD40 antibody according to any one of claims 1-5, wherein the antibody comprises the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO:

20.

10. A pharmaceutical composition comprising at least one agonist antiCD40 antibody according to any one of claims 1 to 9.

11. A pharmaceutical composition comprising at least one isolated agonist antiCD40 antibody of any one of claims 1 to 9 and a pharmaceutically acceptable excipient.

12. The pharmaceutical composition of claim 10 or 11, wherein the composition further comprises other active agents selected from radioisotopes, radionuclides, toxins, or therapeutic and chemotherapeutic groups.

13. A method for preparing an agonist anti-CD40 antibody according to any one of claims 1 to 9, comprising the step of preparing the agonist anti-CD40 antibody from a host cell that secretes the agonist anti-CD40 antibody.

14. Use of at least one isolated agonist antiCD40 antibody according to any one of claims 1 to 9 in the preparation of a composition for increasing antigen presentation of APC in vitro.

15. The use according to claim 14, wherein the APC is a macrophage, a dendritic cell (DC), or a B cell.

16. A method for increasing antigen presentation of APCs in vitro, comprising treating APCs isolated from a patient with at least one agonist antiCD40 antibody of any one of claims 1 to 9.

17. The method of claim 16, wherein the APC is a macrophage, a dendritic cell (DC), or a B cell.

18. Use of at least one isolated agonist antiCD40 antibody according to any one of claims 1 to 9 in the preparation of a composition for activating antigen-presenting cells in vitro.

19. The use according to claim 18, wherein the antigen-presenting cell is a macrophage, a dendritic cell (DC), or a B cell.

20. A method for activating antigen-presenting cells in vitro, comprising treating APCs isolated from a patient with at least one agonist antiCD40 antibody of any one of claims 1 to 9.

21. The method of claim 20, wherein the APC is a macrophage, a dendritic cell (DC), or a B cell.

22. A pharmaceutical composition comprising an effective amount of at least one agonist antiCD40 antibody of any one of claims 1 to 9 and at least a second immune enhancer.

23. The pharmaceutical composition of claim 22, wherein the immune enhancer is selected from IL-2, TLR-7 agonists or systemic cytotoxic chemotherapeutic agents.

24. A pharmaceutical composition comprising at least one agonist antiCD40 antibody of any one of claims 1 to 9 and IL-2.

25. Use of the agonist antiCD40 antibody and IL-2 of any one of claims 1 to 9 in the preparation of a composition for increasing antigen presentation of APC in vitro.

26. The use of claim 25, wherein the APC is a macrophage, a dendritic cell (DC), or a B cell.

27. A method for increasing antigen presentation of APCs in vitro, comprising treating APCs isolated from a patient with an agonist antiCD40 antibody and IL-2 according to any one of claims 1 to 9.

28. The method of claim 27, wherein the APC is a macrophage, a dendritic cell (DC), or a B cell.

29. Use of at least one isolated agonist antiCD40 antibody and IL-2 of any one of claims 1 to 9 in the preparation of a composition for activating antigen-presenting cells in vitro.

30. The use of claim 29, wherein the APC is a macrophage, a dendritic cell (DC), or a B cell.

31. A method for activating antigen-presenting cells in vitro, comprising treating APCs isolated from a patient with at least one isolated agonist antiCD40 antibody and IL-2 according to any one of claims 1 to 9.

32. The method of claim 31, wherein the APC is a macrophage, a dendritic cell (DC), or a B cell.

33. A nucleic acid molecule encoding an agonist anti-CD40 antibody according to any one of claims 1 to 9.

34. The nucleic acid molecule of claim 33, wherein the molecule comprises the sequence of SEQ ID NO: 9 encoding the heavy chain variable region of the agonist antiCD40 antibody.

35. The nucleic acid molecule of claim 33, wherein the molecule comprises a sequence having at least 80, 80-85, 85-90, 90-95, 95-97, 97-99 or greater identity with SEQ ID NO:

9.

36. The nucleic acid molecule of any one of claims 33 to 35, further comprising the sequence of SEQ ID NO: 10 encoding the light chain variable region of the said agonist anti-CD40 antibody.

37. The nucleic acid molecule of claim 33, wherein the molecule comprises the sequence of SEQ ID NO: 21 encoding the heavy chain of the agonist antiCD40 antibody.

38. The nucleic acid molecule of claim 33, wherein the molecule comprises a sequence having at least 80, 80-85, 85-90, 90-95, 95-97, 97-99 or greater identity with SEQ ID NO:

21.

39. The nucleic acid molecule of any one of claims 37 to 38, further comprising the sequence of SEQ ID NO: 22 encoding the light chain of the said agonist antiCD40 antibody.

Images