Antibodies that bind to CD40 and uses thereof

Abstract

An isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human CD40 is provided. Nucleic acid molecules encoding the antibody or antigen-binding portion thereof, expression vectors, host cells, and methods for expressing the antibody or antigen-binding portion thereof are also provided. Immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibody or antigen-binding portion thereof, as well as methods of treatment using the same, are also provided.

Description

[Technical Field]

[0001] Related Applications and References This application claims priority to U.S. Provisional Patent Application No. 63 / 001,612, filed March 30, 2020.

[0002] The above-referenced applications, and all documents cited therein or during the prosecution thereof ("Application Citations"), and all documents cited or referenced herein (including, but not limited to, all documents, patents, and published patent applications cited herein) ("Documents Cited Herein"), and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products described herein or in any document incorporated herein by reference, are hereby incorporated by reference and may be used in the practice of the present invention. More particularly, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences described in this disclosure are incorporated by reference, along with the Genbank sequence as of the earliest effective filing date of this disclosure.

[0003] The present disclosure generally relates to isolated monoclonal antibodies, particularly murine, chimeric, or humanized monoclonal antibodies, or antigen-binding portions thereof, that specifically bind to human CD40 with high affinity and functionality. Nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, expression vectors, host cells, and methods for expressing the antibodies or antigen-binding portions thereof are also provided. The present disclosure further provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof, as well as methods of treatment using the anti-CD40 antibodies or antigen-binding portions thereof of the present disclosure. [Background technology]

[0004] B lymphocyte activation requires antigen receptor-mediated stimulation and costimulation, and CD40 is one of the costimulatory molecules involved in the activation process (Jodi L. Karnell et al., (2019) Advanced Drug Delivery Review 141:92-103).

[0005] CD40, a type I transmembrane protein, is a member of the TNF receptor superfamily. It was first characterized on B lymphocytes, where it is constitutively expressed and signals to promote B cell activation and proliferation. Later, it was found on dendritic cells (DCs), monocytes, macrophages, and non-hematopoietic cells. The primary ligand for CD40 is CD40L, which is expressed primarily by activated T cells and B cells and platelets, and is also found on mononuclear cells, natural killer cells, and basophils under inflammatory conditions (Jodi L. Karnell et al., (2019), supra). The widespread distribution of this costimulatory pair indicates their central role in immune processes. For example, binding of CD40 to CD40L on DCs promotes cytokine production and the induction of costimulatory molecules, leading to T cell activation and differentiation (Quezada SA et al., (2004) Annu Rev Immunol. 22:307-328).

[0006] CD40 is also expressed on tumors, including B-cell malignancies, lung cancer, bladder cancer, gastric cancer, breast cancer, and ovarian cancer, and has been reported to be involved in the pathology of several inflammatory diseases, including autoimmune diseases, atherothrombosis, cancer, and respiratory diseases (Costello et al., (1999) Immunol Today 20(11):488-493; Tong et al., (2003) Cancer Gene Ther 10(1):1-13; Lee et al., (2014) Curr Cancer Drug Targets 14(7):610-620; Ara A et al., (2018), supra; Lee et al., (1999) Proc Natl Acad Sci USA 96:9136-9141; Stamenkovic et al., (1989) EMBO J. 8:1403-1410). On the one hand, CD40-mediated signaling causes tumor cell growth inhibition and cell death in several B cell-derived tumor lines (Grafton et al., (1997) Cell. Immunol. 182:45-56), and on the other hand, in certain other B cell malignancies, it induces increased expression of multiple factors that protect tumor cells from apoptosis (Lee et al., (1999) Proc Natl Acad Sci USA 96:9136-9141).

[0007] Agonist anti-CD40 antibodies, which activate or induce CD40 signaling upon binding to CD40, and antagonist anti-CD40 antibodies, which block or inhibit CD40 signaling that can be induced by CD40L binding, have been developed for disease treatment. The agonist anti-CD40 antibody celicrelumab (Pfizer and VLST) has shown clinical efficacy in some settings in patients with advanced cancer (Vonderheide et al., (2013) Clin Cancer Res. 19(5):1035-1043). Dacetuzumab (Settte Geneticscs), a weaker CD40 agonist than cericlerumab, has shown antitumor activity in diffuse large B-cell lymphoma, multiple myeloma, and CLL, and is being tested in combination with rituximab and gemcitabine in the treatment of relapsed or refractory DLBCL (Advani R et al., (2009) J Clin Oncol. 27:4371-4377; Furman RR et al., (2010) Leuk Lymphoma. 51:228-235; Forero-Torres A et al., (2012) Leuk Lymphoma 54(2):277-283). The antagonist anti-CD40 antibody lucatumumab (Novartis) is being tested in clinical trials to treat multiple myeloma and chronic lymphocytic leukemia (Hassan SB et al., (2014) Immunopharmacol immunotoxicol 36(2):96-104). Furthermore, biologics that stimulate CD40 signaling have shown efficacy in treating infectious and autoimmune diseases, including HIV-1 / AIDS, tuberculosis, and malaria (Elizabeth A Thompson, et al., (2015) J Immunol. 195(3):1015-1024). Summary of the Invention [Problem to be solved by the invention]

[0008] There is a continuing need for better anti-CD40 antibodies with improved pharmaceutical characteristics.

[0009] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. [Means for solving the problem]

[0010] The present disclosure provides isolated monoclonal antibodies, e.g., murine, human, chimeric, or humanized monoclonal antibodies, or antigen-binding portions thereof, that bind to CD40 (e.g., human CD40 and monkey CD40) and have binding affinity to CD40 that is equivalent, if not greater, and activity to activate CD40 signaling that is equivalent, if not greater, than prior art anti-CD40 antibodies, such as dacetuzumab and celicrelumab.

[0011] The antibodies, or antigen-binding portions thereof, of the present disclosure can be used in a variety of applications, including the detection of CD40 protein and the treatment and prevention of CD40-associated diseases, such as cancer, infectious diseases, and autoimmune diseases.

[0012] Thus, in one aspect, the disclosure provides a method for detecting a nucleotide sequence comprising: i) a heavy chain variable region that may comprise a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to (1) SEQ ID NOs: 1, 4, and 7, respectively; (2) SEQ ID NOs: 2, 5, and 8, respectively; or (3) SEQ ID NOs: 3, 6, and 9, respectively; and / or ii) a light chain variable region that may comprise a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to (1) SEQ ID NOs: 1, 4, and 7, respectively; (2) SEQ ID NOs: 2, 5, and 8, respectively; or (3) SEQ ID NOs: 3, 6, and 9, respectively. The present invention relates to an isolated monoclonal antibody (e.g., a murine, chimeric, or humanized antibody) that binds to CD40, or an antigen-binding portion thereof, having a light chain variable region whose CDR3 region can comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to (1) each of SEQ ID NOs: 10, 13, and 16; (2) each of SEQ ID NOs: 11, 14, and 17; or (3) each of SEQ ID NOs: 12, 15, and 18.

[0013] An antibody, or antigen-binding portion thereof, of the disclosure may comprise a heavy chain variable region, which may comprise a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, and a light chain variable region, which may comprise a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, the VH CDR3 region, the VL CDR1 region, the VL CDR2 region, and the VL The CDR3 region can comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) each of SEQ ID NOs: 1, 4, 7, 10, 13 and 16; (2) each of SEQ ID NOs: 2, 5, 8, 11, 14 and 17; or (3) each of SEQ ID NOs: 3, 6, 9, 12, 15 and 18, wherein the antibody or antigen-binding portion thereof binds to CD40.

[0014] The heavy chain variable region of an antibody, or antigen-binding portion thereof, of the present disclosure may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 19, 20 (X1=A or S), 21, or 22, wherein the antibody, or antigen-binding portion thereof, binds to CD40. The amino acid sequence of SEQ ID NO: 19 may be encoded by the nucleotide sequence of SEQ ID NO: 31 or 32, and the amino acid sequence of SEQ ID NO: 20 (X1=S) may be encoded by the nucleotide sequence of SEQ ID NO: 33.

[0015] The light chain variable region of an antibody, or antigen-binding portion thereof, of the present disclosure may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 23, 24 (X1=K, X2=F; or X1=Y, X2=Y), 25, or 26, wherein the antibody, or antigen-binding portion thereof, binds to CD40. The amino acid sequence of SEQ ID NO: 23 may be encoded by the nucleotide sequence of SEQ ID NO: 34 or 35. The amino acid sequence of SEQ ID NO: 24 (X1=K, X2=F) may be encoded by the nucleotide sequence of SEQ ID NO: 36.

[0016] The antibodies or antigen-binding portions thereof of the present disclosure may be: (1) SEQ ID NOs: 19 and 23, respectively; (2) SEQ ID NOs: 20 (X1=A) and 24 (X1=K, X2=F), respectively; (3) SEQ ID NOs: 20 (X1=S) and 24 (X1=K, X2=F), respectively; (4) SEQ ID NOs: 20 (X1=A) and 24 (X1=Y, X2=Y), respectively; (5) SEQ ID NOs: 20 (X1=S) and 24 (X1=Y, (5) a heavy chain variable region and a light chain variable region having an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of SEQ ID NOs: 21 and 25; or (6) a heavy chain variable region and a light chain variable region having an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of SEQ ID NOs: 22 and 26, respectively, and which binds to CD40.

[0017] The isolated monoclonal antibody of the present disclosure, or an antigen-binding portion thereof, may comprise a heavy chain and a light chain linked by a disulfide bond, the heavy chain may comprise a heavy chain variable region and a heavy chain constant region, and the light chain may comprise a light chain variable region and a light chain constant region, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region, the heavy chain variable region and the light chain variable region may comprise the amino acid sequences described above, and the antibody, or an antigen-binding portion thereof, binds to CD40. The heavy chain constant region may be, for example, a human IgG2 constant region having the amino acid sequence set forth in SEQ ID NO: 28, or a human IgG1 constant region having, for example, the amino acid sequence set forth in SEQ ID NO: 27, and the light chain constant region may be, for example, a human kappa constant region having the amino acid sequence set forth in SEQ ID NO: 29. The heavy chain constant region, e.g., the Fc fragment, may be modified to have reduced or enhanced FcR binding affinity. The amino acid sequences of SEQ ID NOs: 27, 28 and 29 can be encoded by the nucleotide sequences of SEQ ID NOs: 37, 38 and 39, respectively.

[0018] In certain embodiments, antibodies of the present disclosure may comprise or consist of two heavy chains and two light chains, each heavy chain may comprise a heavy chain constant region, heavy chain variable region, or CDR sequence described above, and each light chain may comprise a light chain constant region, light chain variable region, or CDR sequence described above, wherein the antibody binds to CD40. Antibodies of the present disclosure may be, for example, full-length antibodies of the IgG1, IgG2, or IgG4 isotype. In other embodiments, antibodies or antigen-binding portions thereof of the present disclosure may be single-chain variable fragment (scFv) antibodies, or antibody fragments, such as Fab or F(ab')2 fragments.

[0019] The present disclosure also provides bispecific molecules that may include an antibody of the present disclosure, or an antigen-binding portion thereof, linked to a second functional group (e.g., a secondary antibody) having a different binding specificity than the antibody or antigen-binding portion thereof. The present disclosure also provides immunoconjugates, e.g., antibody-drug conjugates, that may include an antibody of the present disclosure, or an antigen-binding portion thereof, linked to a therapeutic agent, e.g., a cytotoxin. In another embodiment, the antibody of the present disclosure, or an antigen-binding portion thereof, may be engineered into part of a chimeric antigen receptor (CAR). Additionally, immune cells, e.g., T cells and NK cells, that may include the chimeric antigen receptor are provided. Furthermore, the antibody of the present disclosure, or an antigen-binding portion thereof, may be encoded by or used in conjunction with an oncolytic virus.

[0020] Nucleic acid molecules encoding the antibodies, or antigen-binding portions thereof, of the present disclosure are also encompassed by the present disclosure, as well as expression vectors that may contain such nucleic acids and host cells that may contain such expression vectors. Also provided are methods for preparing anti-CD40 antibodies, or antigen-binding portions thereof, of the present disclosure using host cells, which may include (i) expressing the antibody in the host cell and (ii) isolating the antibody from the host cell or a cell culture thereof.

[0021] Also provided are compositions that can include an antibody, or antigen-binding portion thereof, immunoconjugate, bispecific molecule, oncolytic virus, CAR, CAR-T cell, nucleic acid molecule, expression vector, or host cell of the present disclosure, and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition can further include a therapeutic agent, such as an anti-cancer agent.

[0022] In yet another aspect, the present disclosure provides a method of modulating an immune response in a subject, the method comprising administering to the subject an antibody, or antigen-binding portion thereof, of this disclosure, such that the immune response in the subject is modulated. Preferably, the antibody, or antigen-binding portion thereof, of this disclosure augments, stimulates, or increases the immune response in the subject. In some embodiments, the method comprises administering a bispecific molecule, immunoconjugate, CAR-T cell, or oncolytic virus encoding or carrying the antibody of this disclosure, or alternatively, a nucleic acid molecule capable of expressing them in the subject.

[0023] In a further aspect, the present disclosure provides a method of inhibiting tumor growth in a subject in need thereof, comprising administering a therapeutically effective amount of a composition of the present disclosure to the subject. The tumor may be a solid or non-solid tumor, including, but not limited to, B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastric intestine cancer, prostate cancer, bladder cancer, renal cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal carcinoma. In some embodiments, at least one additional anti-cancer antibody may be administered together with the antibody, or antigen-binding portion thereof, of the present disclosure, including, for example, an anti-VISTA antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-STAT3 antibody, and / or an anti-ROR1 antibody. In yet another embodiment, an antibody of the present disclosure, or an antigen-binding portion thereof, is administered with a cytokine (e.g., IL-2, IL-21, GM-CSF, and / or IL-4), or a costimulatory antibody (e.g., anti-CD137 and / or anti-GITR antibody). In another embodiment, an antibody of the present disclosure, or an antigen-binding portion thereof, is administered with a chemotherapeutic agent, which may be a cytotoxic drug, e.g., epirubicin, oxaliplatin, and / or 5-fluorouracil (5-FU). An antibody of the present disclosure, or an antigen-binding portion thereof, may be, for example, murine, human, chimeric, or humanized.

[0024] In another aspect, the present disclosure provides a method for treating or alleviating an infectious disease in a subject in need thereof, comprising administering a therapeutically effective amount of a composition of the present disclosure to the subject. The infectious disease can be a disease caused by a viral, bacterial, fungal, or mycoplasmal infection. In certain embodiments, the infectious disease is AIDS, tuberculosis, or malaria. In certain embodiments, the subject can further be administered at least one anti-infective agent, such as an antiviral agent, an antibacterial agent, an antifungal agent, or an antimycoplasmal agent.

[0025] In another aspect, the present disclosure provides a method of treating or alleviating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure. In certain embodiments, the subject may further be administered at least one anti-inflammatory agent.

[0026] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0027] Accordingly, it is not the purpose of the present invention to encompass any previously known products, methods of making the products, or methods of using the products, and therefore, the applicant reserves the right, and hereby discloses, a disclaimer of any previously known products, processes, or methods. It is further noted that the present invention does not intend to encompass within its scope any products, processes, or methods of making the products or methods of using the products that do not satisfy the USPTO's written description and enablement requirements (35 U.S.C. § 112, first paragraph) or the EPO (European Patent Convention (EPC) Article 83), and therefore, the applicant reserves the right, and hereby discloses, a disclaimer of any previously described products, methods of making the products, or methods of using the products. Compliance with Article 53(c) EPC and Rules 28(b) and (c) EPC may be advantageous in the practice of the present invention. All rights are expressly reserved to expressly disclaim any embodiment that is the subject of any issued patent of the applicant in this application line or in any other line or in any prior application of any third party. Nothing stated herein should be construed as a warranty.

[0028] It is noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have the meaning ascribed to them by U.S. patent law; e.g., they may mean "includes," "included," "including," and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them by U.S. patent law, e.g., they may permit elements not expressly specified, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention.

[0029] The following detailed description, presented by way of example and not intended to limit the invention to only the particular embodiments described, can be best understood in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0030] [Figure 1A] 1 shows the binding ability of mouse antibodies 1A3 and 1D1 to human CD40 in a capture ELISA. [Figure 1B] Figure 1 shows the binding ability of mouse antibody C1H1 to human CD40 in a capture ELISA. [Figure 2A] 1 shows the binding ability of mouse antibodies 1A3 and 1D1 to 293T cells expressing human CD40 in a cell-based binding FACS assay. [Figure 2B] 1 shows the binding ability of mouse antibody C1H1 to 293T cells expressing human CD40 in a cell-based coupled FACS assay. [Figure 3A] 1 shows the blocking ability of murine antibodies 1A3 and 1D1 against human CD40-CD40L binding in a competitive ELISA. [Figure 3B] Figure 1 shows the blocking ability of mouse antibody C1H1 against human CD40-CD40L binding in a competitive ELISA. [Figure 4A] Figure 1 shows the ability of murine antibodies 1A3 and 1D1 to block benchmark human CD40 binding in a competitive ELISA. [Figure 4B] Figure 1 shows the ability of murine antibody C1H1 to block benchmark-human CD40 binding in a competitive ELISA. [Figure 5A] 1 shows the activity of murine antibodies 1A3 and 1D1 to activate CD40 signaling in a cell-based reporter assay. [Figure 5B] Figure 1 shows the activity of mouse antibody C1H1 to activate CD40 signaling in a cell-based reporter assay. [Figure 6A] 1 shows the binding ability of chimeric antibody 1A3 to human CD40 in a capture ELISA. [Figure 6B] 1 shows the binding ability of chimeric antibody 1D1 to human CD40 in a capture ELISA. [Figure 6C]1 shows the binding ability of chimeric antibody C1H1 to human CD40 in a capture ELISA. [Figure 7A] 1 shows the activity of chimeric antibody 1A3 to activate CD40 signaling in a cell-based reporter assay. [Figure 7B] 1 shows the activity of chimeric antibody 1D1 to activate CD40 signaling in a cell-based reporter assay. [Figure 7C] 1 shows the activity of chimeric antibody C1H1 to activate CD40 signaling in a cell-based reporter assay. [Figure 8] 1 shows the binding ability of humanized antibodies huC1H1-V1 and huC1H1-V2 to human CD40 in capture ELISA. [Figure 9] This shows the binding ability of humanized antibodies huC1H1-V1 and huC1H1-V2 to 293T cells expressing human CD40 in cell-based binding FACS. [Figure 10] 1 shows the blocking ability of humanized antibodies huC1H1-V1 and huC1H1-V2 against human CD40-CD40L binding in a competitive ELISA. [Figure 11] Figure 1 shows the ability of humanized antibodies huC1H1-V1 and huC1H1-V2 to block benchmark human CD40 binding in a competitive ELISA. [Figure 12] 1 shows the activity of humanized antibodies huC1H1-V1 and huC1H1-V2 to activate CD40 signaling in a cell-based reporter assay. DETAILED DESCRIPTION OF THE INVENTION

[0031] To ensure that this disclosure may be more readily understood, some terms are first defined. Further definitions are set forth throughout the detailed description.

[0032] The term "CD40" refers to tumor necrosis factor receptor superfamily member 5 (TNFR5). The term "CD40" includes variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific for human CD40 protein may, in some cases, cross-react with CD40 protein from species other than humans, such as monkeys. In other embodiments, an antibody specific for human CD40 protein may be completely specific for human CD40 protein and may not exhibit other species or other types of cross-reactivity, or may cross-react with CD40 from certain other species but not all other species.

[0033] The term "human CD40" refers to a CD40 protein having an amino acid sequence derived from a human, such as the amino acid sequence of human CD40 having Genbank accession number NP_001241.1 (Amini M et al., (2020) Life Sci 254:117774). The terms "monkey or rhesus CD40" and "mouse CD40" refer to monkey and mouse CD40 sequences, respectively, such as those having the amino acid sequences with Genbank accession numbers NP_001252791.1 and NP_035741.2, respectively.

[0034] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by such cells or the liver that result in selective damage to, destruction of, or elimination from the human body of invading pathogens, pathogen-infected cells or tissues, cancerous cells, or, in cases of autoimmune or pathological inflammation, normal human cells or tissues.

[0035] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as CD40, through at least one antigen-binding site, where the antigen-binding site is usually located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-chain Fv (scFv) antibodies, heavy-chain antibodies (HCAbs), light-chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, fusion proteins containing the antigen-binding site of an antibody, and any other modified immunoglobulin molecule containing an antigen-binding site (e.g., dual variable domain immunoglobulin molecules), as long as the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies may be any of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules, including but not limited to toxins and radioisotopes. Unless expressly indicated otherwise, the term "antibody," as used herein, includes the "antigen-binding portion" of an intact antibody. IgG is a glycoprotein that may comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H The heavy chain constant region can be composed of three domains: C H1 , C H2 and C H3 Each light chain can be composed of a light chain variable region (referred to herein as V L The light chain constant region can consist of one domain, C L V H and VL The regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0036] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD40 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) Fab fragments, V L , V H , C L and C H1 (ii) a F(ab')2 fragment, a bivalent fragment that may contain two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H1 (iv) a V fragment of a single arm of an antibody; L and V H Fv fragment consisting of domains, (v) V H (vi) isolated complementarity-determining regions (CDRs); and (viii) nanobodies, heavy chain variable regions containing a single variable domain and two constant domains. In addition, Fv fragments contain two domains, V and V. L and V Hare encoded by separate genes, but they are grouped together as V L and V H These regions can be joined using recombinant methods by synthetic linkers that allow them to be produced as a single protein chain (known as a single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the regions pair to form a monovalent molecule. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0037] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CD40 protein is substantially free of antibodies that specifically bind to antigens other than CD40 protein). However, an isolated antibody that specifically binds to human CD40 protein may have cross-reactivity to other antigens, such as CD40 proteins from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0038] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0039] The term "murine antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences. The murine antibodies of the present disclosure may include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "murine antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto murine framework sequences.

[0040] The term "chimeric antibody" refers to an antibody made by combining genetic material from a non-human source with genetic material from a human, or more generally, a chimeric antibody is an antibody that has genetic material from a particular species along with genetic material from another species.

[0041] The term "humanized antibody" as used herein refers to antibodies derived from non-human species in which the protein sequences have been modified to increase their similarity to antibody forms naturally produced in humans.

[0042] The phrases "antibody that recognizes an antigen" and "antibody that is specific for an antigen" are used herein synonymously with the term "antibody that specifically binds to an antigen."

[0043] As used herein, an antibody or antigen-binding portion thereof that "specifically binds to human CD40" is intended to refer to an antibody that binds to human CD40 protein (and optionally, one or more non-human derived CD40 proteins) but does not substantially bind to non-CD40 proteins. Preferably, the antibody has a "high affinity," i.e., a binding affinity of 5.0 x 10 -8 M or less, more preferably 1.0 × 10 -8 K below M DIt binds to the human CD40 protein.

[0044] As used herein, the term "does not substantially bind" to proteins or cells means that the antibody does not bind to proteins or cells or does not bind with high affinity, i.e., binds with less than 1.0 x 10 -6 M or more, more preferably 1.0 × 10 -5 M or more, more preferably 1.0 × 10 -4 M or more, more preferably 1.0 × 10 -3 M or more, and even more preferably 1.0 x 10 -2 K over M D This means that the molecule binds to a protein or cell.

[0045] The term "high affinity" for an IgG antibody refers to an affinity of 1.0 × 10 for the target antigen. -6 M or less, more preferably 9.0 × 10 -9 M or less, more preferably 5.0 × 10 -9 M or less, and even more preferably 1.0 x 10 -9 M or less, and even more preferably 5.0 x 10 -10 K below M D However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is defined as 10 -6 M or less, more preferably 10 -7 M or less, and even more preferably, 10 -8 K below M D It refers to an antibody having the following structure:

[0046] As used herein, "K" assoc " or "K a While the term "K" is intended to refer to the binding rate of a particular antibody-antigen interaction, as used herein, dis " or "K d The term "K" as used herein is intended to refer to the off-rate of a particular antibody-antigen interaction. DThe term "dissociation constant" is intended to refer to the dissociation constant, which is K d Against K a The ratio of (i.e., K d / K a ) and expressed as molar concentration (M). D The K value can be determined using methods well established in the art. D A preferred method for determining is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore™ system.

[0047] The half-maximal effective concentration (EC) is also known as 50 The term "antibody concentration" refers to the concentration of antibody that induces a response halfway between baseline and maximum after a particular exposure time.

[0048] The half maximal inhibitory concentration (IC 50 The term "antibody concentration" refers to a concentration of antibody that inhibits a specific biological or biochemical function by 50% compared to the absence of the antibody.

[0049] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals such as non-human primates, sheep, dogs, cats, cows, and horses are preferred.

[0050] The term "therapeutically effective amount" refers to an amount of an antibody, or antigen-binding portion thereof, of the present disclosure sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as cancer) and / or reduce the severity of the disease or condition. A therapeutically effective amount is understood in relation to the condition being treated, and the actual effective amount is readily understood by one of ordinary skill in the art.

[0051] The term "agonist CD40 antibody" or "agonist anti-CD40 antibody" refers to an anti-CD40 antibody that binds to CD40 and activates or induces CD40 signaling, thereby promoting, for example, immune cell activation and proliferation and cytokine and chemokine production. On the other hand, the term "antagonist CD40 antibody" refers to an anti-CD40 antibody that blocks or inhibits CD40 signaling that can be induced by CD40L binding. Agonist CD40 antibodies can promote the innate and adaptive immune responses of tumor-bearing subjects against tumors through, for example, enhanced antigen-presenting capacity of APCs, activation of tumor-specific CD4+ and CD8+ T cells, secretion of cytokines and chemokines by lymphocytes and monocytes, and enhanced tumor cell death by cytotoxic lymphocytes and NK cells.

[0052] Percent "identity," as used herein in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence (introducing gaps, if necessary), are the same or have a specified percentage of nucleotides or amino acid residues that are the same, with or without considering conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity when compared and aligned as measured using a sequence comparison algorithm or by visual inspection for maximum correspondence.

[0053] Various aspects of the disclosure are described in further detail in the following subsections.

[0054] The antibodies, or antigen-binding portions thereof, of the present disclosure specifically bind to human CD40 with binding affinity comparable to, if not greater than, the aforementioned anti-CD40 antibodies, such as dacetuzumab and celicrelumab.

[0055] Further functional properties include the ability to block CD40-CD40L binding and activate CD40 signaling.

[0056] A preferred antibody of the present disclosure is a humanized monoclonal antibody. Additionally or alternatively, the antibody can be, for example, a chimeric monoclonal antibody.

[0057] [Table 1]

[0058] Exemplary antibodies, or antigen-binding portions thereof, of the present disclosure are structurally and chemically characterized as described below and in the Examples below. The amino acid sequence ID numbers of the heavy and light chain variable regions of the antibodies are summarized in Table 1 above, and some antibodies have the same V H or V L The heavy chain constant region in the antibody can be, for example, a human IgG1 or IgG2 heavy chain constant region having the amino acid sequences shown in SEQ ID NOs: 27 and 28, respectively, and the light chain constant region in the antibody can be, for example, a human κ constant region having the amino acid sequence shown in SEQ ID NO: 29. These antibodies can also have a mouse IgG1 or IgG2 heavy chain constant region, and / or a mouse κ constant region.

[0059] The heavy and light chain variable region CDRs in Table 1 are defined by the Kabat numbering system, however, as is well known in the art, CDR regions can also be determined by other systems, such as Chothia, IMGT, AbM, or Contact numbering systems / methods, based on the heavy / light chain variable region sequences.

[0060] V of other anti-CD40 antibodies that bind to human CD40 H and V L The sequences (or CDR sequences) of the anti-CD40 antibodies of the present disclosure are H and V L Preferably, V H and V L When chains (or CDRs within such chains) are mixed and matched, a particular V H / V L V from involution H The sequence is structurally similar to V H Similarly, preferably, a particular V H / V L V from involution L The sequence is structurally similar to V L It is replaced by an array.

[0061] Thus, in one embodiment, an antibody of the present disclosure, or an antigen-binding portion thereof, (a) a heavy chain variable region comprising the amino acid sequence listed above in Table 1; and (b) a light chain variable region comprising an amino acid sequence listed above in Table 1, or the V of another anti-CD40 antibody L wherein the antibody specifically binds to human CD40.

[0062] In another embodiment, an antibody, or antigen-binding portion thereof, of the present disclosure: (a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable region listed above in Table 1; and (b) comprises the CDR1, CDR2, and CDR3 regions of a light chain variable region listed above in Table 1 or the CDRs of another anti-CD40 antibody, wherein the antibody specifically binds to human CD40.

[0063] In yet another embodiment, the antibody, or antigen-binding portion thereof, comprises the CDRs of another antibody that binds human CD40, e.g., the heavy chain variable CDR2 region of an anti-CD40 antibody combined with CDR1 and / or CDR3 from the heavy chain variable region, and / or CDR1, CDR2, and / or CDR3 from the light chain variable region of a different anti-CD40 antibody.

[0064] Furthermore, it is well known in the art that the CDR3 domain alone, independent of the CDR1 and / or CDR2 domains, can determine the binding specificity of an antibody to a cognate antigen, and that multiple antibodies can be predictably generated with the same binding specificity based on a common CDR3 sequence. For example, Klimka et al.,British J.of Cancer 83(2):252-260(2000);Beiboer et al.,J.Mol.Biol.296:833-849(2000);Rader et al.,Proc.Natl.Acad.Sci.USA95:8910-8915(1998);Barbas et al. al.,J.Am.Chem.Soc.116:2161-2162(1994);Barbas et al.,Proc.Natl.Acad.Sci.USA92:2529-2533(1995);Ditzel et al.,J.Immunol.157:739-749(1996);Berezov et al.,BIAjournal 8:Scientific Review 8(2001);Igarashi et al. See, e.g., Bourgeois et al., J. Virol 72:807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994), and Xu and Davis, Immunity 13:37-45 (2000). See also U.S. Patent Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905; and 5,760,185, each of which is incorporated herein by reference in its entirety.

[0065] Thus, in another embodiment, an antibody of the present disclosure comprises CDR2 of the heavy chain variable region of an anti-CD40 antibody and at least CDR3 of the heavy and / or light chain variable region of an anti-CD40 antibody, or CDR3 of the heavy and / or light chain variable region of another anti-CD40 antibody, wherein the antibody is capable of specifically binding to human CD40. These antibodies preferably (a) compete for binding to CD40; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have similar binding affinity as the anti-CD40 antibody of the present disclosure. In yet another embodiment, the antibody may further comprise CDR2 of the light chain variable region of an anti-CD40 antibody, or CDR2 of the light chain variable region of another anti-CD40 antibody, wherein the antibody is capable of specifically binding to human CD40. In another embodiment, an antibody of the present disclosure may further comprise CDR1 of the heavy and / or light chain variable region of an anti-CD40 antibody, or CDR1 of the heavy and / or light chain variable region of another anti-CD40 antibody, wherein the antibody is capable of specifically binding to human CD40.

[0066] In another embodiment, an antibody of the present disclosure comprises heavy and / or light chain variable region sequences of CDR1, CDR2, and CDR3 sequences that differ from those of an anti-CD40 antibody of the present disclosure by one or more conservative modifications. It is understood in the art that certain conservative sequence modifications can be made without eliminating antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0067] Thus, in one embodiment, the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: (a) the heavy chain variable region CDR1 sequence comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (b) the heavy chain variable region CDR2 sequence comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (c) the heavy chain variable region CDR3 sequence comprises a sequence listed in Table 1 above, and / or a conservative modification thereof; and / or (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 sequences comprise the sequences listed in Table 1 above; and / or conservative modifications thereof; (e) The antibody specifically binds to human CD40.

[0068] The antibodies of the present disclosure have one or more of the following functional properties described above, such as high affinity binding to human CD40 and the ability to activate CD40 signaling on CD40-expressing cells.

[0069] In various embodiments, the antibody can be, for example, a murine, human, humanized, or chimeric antibody.

[0070] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not substantially affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the present disclosure can be substituted with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function (i.e., the above-mentioned functions) using the functional assays described herein.

[0071] The antibodies of the present disclosure may be prepared by using the V of an anti-CD40 antibody of the present disclosure as starting material to engineer modified antibodies. H / V L The antibody may be prepared using antibodies having one or more of the following sequences: H and / or V L ), for example, by modifying one or more residues in one or more CDR regions and / or one or more framework regions. Additionally or alternatively, antibodies may be engineered by modifying residues in the constant region(s), for example, to alter the effector functions of the antibody.

[0072] In certain embodiments, CDR grafting techniques can be used to engineer the variable regions of antibodies. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular natural antibody by constructing expression vectors containing CDR sequences from that particular natural antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad. USA 86:10029-10033; see also U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0073] Accordingly, another embodiment of the present disclosure relates to isolated monoclonal antibodies, or antigen-binding portions thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above, and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above. H and V L While comprising CDR sequences, they may comprise different framework sequences.

[0074] Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as Kabat et al., (1991) (cited above); Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836 (the contents of each of which are expressly incorporated herein by reference). As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available at the attached Genbank accession numbers 1-69 (NG--0010109, NT--024637 and BC070333), 3-33 (NG--0010109 and NT--024637) and 3-7 (NG--0010109 & NT--024637). As another example, the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available under the attached Genbank accession numbers: 1-69 (NG--0010109, NT--024637, and BC070333), 5-51 (NG--0010109 and NT--024637), 4-34 (NG--0010109 and NT--024637), 3-30.3 (CAJ556644), and 3-23 (AJ406678).

[0075] The antibody protein sequence is compared against compiled protein sequence databases using one of the sequence similarity search methods known to those skilled in the art, called Gapped BLAST (Altschul et al., (1997), supra).

[0076] Preferred framework sequences for use in the antibodies of this disclosure are structurally similar to the framework sequences used by the antibodies of this disclosure. H The CDR1, CDR2, and CDR3 sequences can be grafted into framework regions having the same sequence as found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted into framework regions that contain one or more mutations compared to the germline sequence. For example, in some cases, it has been found to be beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0077] Another type of variable region modification is the V H and / or V L Amino acid residues within the CDR1, CDR2, and / or CDR3 regions are mutated to improve one or more binding characteristics (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays known in the art. Preferably, conservative modifications (as known in the art) are introduced. The mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Furthermore, typically, no more than one, two, three, four, or five residues within the CDR regions are altered.

[0078] Thus, in another embodiment, the present disclosure provides a V that comprises (a) a sequence of the present disclosure or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions. H (b) a V region comprising an amino acid sequence of the disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; H(c) a V comprising an amino acid sequence of the present disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; H (d) a V comprising an amino acid sequence of the present disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; L (e) a V comprising an amino acid sequence of the present disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; L and (f) a V comprising an amino acid sequence of the disclosure or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions. L An isolated anti-CD40 monoclonal antibody, or an antigen-binding portion thereof, is provided, comprising a heavy chain variable region including a CDR3 region.

[0079] The engineered antibodies of the disclosure may be modified, e.g., to improve the properties of the antibody. H and / or V L These framework modifications include those made to framework residues within the following: (a) nucleotide sequence (SEQ ID NO: 1); (b) nucleotide sequence (SEQ ID NO: 2); (c) nucleotide sequence (SEQ ID NO: 3); (d) nucleotide sequence (SEQ ID NO: 4); (e) nucleotide sequence (SEQ ID NO: 5); (f) nucleotide sequence (SEQ ID NO: 6); (g) nucleotide sequence (SEQ ID NO: 7); (h) nucleotide sequence (SEQ ID NO: 8); (i) nucleotide sequence (SEQ ID NO: 9); (j) nucleotide sequence (SEQ ID NO: 10); (j) nucleotide sequence (SEQ ID NO: 11); (j) nucleotide sequence (SEQ ID NO: 12); (j) nucleotide sequence (SEQ ID NO: 13); (j) nucleotide sequence (SEQ ID NO: 14); (j) nucleotide sequence (SEQ ID NO: 15); (j) nucleotide sequence (SEQ ID NO: 16); (j) nucleotide sequence (SEQ ID NO: 17); (j) nucleotide sequence (SEQ ID NO: 18); (j) nucleotide sequence (SEQ ID NO: 19); (k) nucleotide sequence (SEQ ID NO: 20); (k) nucleotide sequence (SEQ ID NO: 21); (k) nucleotide sequence (SEQ ID NO: 22); (k) nucleotide sequence (SEQ ID NO: 23); (k) nucleotide sequence (SEQ ID NO: 24); (k) nucleotide sequence (SEQ ID NO: 25); (k) nucleotide sequence (SEQ ID NO: 26); (k) nucleotide sequence (SEQ ID NO: 27); (k) nucleotide sequence (SEQ ID NO: 28); (k) nucleotide sequence (SEQ ID NO: 29); (k) nucleotide sequence (SEQ ID NO: 30); (k) nucleotide sequence (SEQ ID NO: 31); (k) nucleotide sequence (SEQ ID

[0080] Another type of framework modification involves mutating one or more residues within the framework regions or one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach, also known as "deimmunization," is described in further detail in U.S. Patent Application Publication No. 20030153043.

[0081] In addition to, or instead of, modifications made within the framework or CDR regions, antibodies of the disclosure can be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, antibodies of the disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) to similarly alter one or more functional properties of the antibody or to alter its glycosylation.

[0082] In one embodiment, the hinge region between CH1 and CH2 is modified such that the number of cysteine ​​residues in the hinge region is changed, e.g., increased or decreased. This technique is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0083] In another embodiment, the Fc-hinge region of the antibody is mutated to increase or decrease the biological half-life of the antibody. More particularly, one or more amino acid mutations are made to the C of the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding compared to native Fc-hinge region SpA binding. H2 -C H3 This technique is described in more detail in U.S. Patent No. 6,165,745.

[0084] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such glycosylation can increase the affinity of the antibody for an antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861.

[0085] Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present disclosure, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose in their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Application Publication No. 2004 / 0110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22). As another example, European Patent No. 1,176,195 describes cell lines in which the FUT8 gene, encoding a fucosyltransferase, has been functionally disrupted, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating α-1,6 bond-related enzymes. Furthermore, European Patent No. 1,176,195 describes cell lines that bind to the Fc region of antibodies or have no enzymatic activity, and have low enzymatic activity for the addition of fucose to N-acetylglucosamine, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662).WO 03 / 035835 describes Lec13 cells, a mutant CHO cell line that reduces the ability to attach fucose to Asn(297)-linked carbohydrates and also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with modified glycosylation profiles can also be produced in chicken eggs, as described in WO 06 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, such as Lemna. Methods for producing antibodies in plant systems are disclosed in U.S. patent application corresponding to Alston & Bird LLP Attorney Docket No. 040989 / 314911, filed August 11, 2006. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit a bisecting GlcNac structure, resulting in increased ADCC activity of the antibody (see also Umana et al., (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of antibodies can be cleaved using a fucosidase enzyme; for example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14:5516-23).

[0086] Another modification of the antibodies herein contemplated by the present disclosure is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or a fragment thereof, is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono(C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See, e.g., EP 154 316 and EP 0 401 384.

[0087] The antibodies of the present disclosure can be characterized by their various physical properties in order to detect and / or distinguish between different classes thereof.

[0088] For example, an antibody may contain one or more glycosylation sites in either the light chain or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity or altered pK of the antibody, resulting in altered antigen binding (Marshall et al., (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al., (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al., (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing the NXS / T sequence. In some cases, it is preferable to have an anti-CD40 antibody that does not contain variable region glycosylation, which can be achieved by selecting an antibody that does not contain glycosylation motifs in the variable region or by mutating residues within the glycosylated region.

[0089] In a preferred embodiment, the antibody does not contain an asparagine isomerism site. Deamidation of asparagine can occur at NG or DG sequences, resulting in the generation of isoaspartic acid residues that introduce bonds into the polypeptide chain and reduce its stability (isoaspartic acid effect).

[0090] Each antibody has a unique isoelectric point (pI), generally within the pH range of 6 to 9.5. The pI of an IgG1 antibody is typically within the pH range of 7 to 9.5, and the pI of an IgG4 antibody is typically within the pH range of 6 to 8. It has been speculated that antibodies with a pI outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferable to have an anti-CD40 antibody with a pI value within the normal range. This can be achieved by selecting an antibody with a pI within the normal range or by mutating charged surface residues.

[0091] In another aspect, the present disclosure provides nucleic acid molecules encoding the heavy and / or light chain variable regions, or CDRs, of the antibodies of the present disclosure. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acids of the present disclosure can be, for example, DNA or RNA, and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0092] Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

[0093] A preferred nucleic acid molecule of the present disclosure is the V H and V L Once V H and V L Once the DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L or V HThe DNA fragment encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0094] V H The isolated DNA encoding the region is V H The DNA encoding the heavy chain constant region (C H1 , C H2 and C H3 The heavy chain constant region can be converted into a full-length heavy chain gene by operably linking it to another DNA molecule encoding the V. The sequences of human heavy chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG2 constant region. For a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain C H1 It may be operably linked to another DNA molecule encoding only the constant region.

[0095] V L The isolated DNA encoding the region is V L The DNA encoding the light chain constant region, C L The light chain constant region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking it to another DNA molecule encoding the same. The sequences of human light chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0096] To generate the scFv gene, H and V LThe sequences are linked by a flexible linker L and V H V can be expressed as a continuous single-chain protein with H and V L The DNA fragment encoding the nucleotide sequence (Gly4-Ser) is operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0097] The monoclonal antibodies (mAbs) of the present disclosure can be produced using the well-known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display technology. Chimeric or humanized antibodies are also well known in the art. See, e.g., U.S. Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370, the contents of which are specifically incorporated herein by reference in their entireties.

[0098] Antibodies of the present disclosure can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods, as are well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0099] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from viral elements that direct high-level protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), and adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or β-globin promoter, can be used. Furthermore, regulatory elements are composed of sequences derived from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

[0100] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. H The segment is located at C H operatively connected to the V L The segment is located at C L The variable regions are used to generate full-length antibody genes of any antibody isotype by inserting them operably linked to an expression vector already encoding heavy and light chain constant regions of the desired isotype. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0101] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0102] For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. While it is theoretically possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, most preferably mammalian host cells, is most preferred, because such cells are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies.

[0103] Preferred mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells (including, e.g., dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, as described in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium that the host cells are grown in. The antibody can be recovered from the culture medium using standard protein purification methods.

[0104] The antibodies of the present disclosure can be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; WO 02 / 096910; WO 07 / 038,658; WO 07 / 051,081; WO 07 / 059,404; WO 08 / 083,312; and WO 08 / 103,693; U.S. Patent Application Publication Nos. 2006 / 0024317; 2006 / 0004081; and 2006 / 0247295, the disclosures of which are incorporated herein by reference.

[0105] In another aspect, the present disclosure features bispecific molecules comprising one or more antibodies of this disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, "bispecific molecule" includes molecules with three or more specificities.

[0106] In one embodiment, the bispecific molecule has a third specificity in addition to the anti-Fc binding specificity and the anti-CD40 binding specificity. The third specificity can be for an anti-enhancement factor (EF), e.g., a molecule that binds to a surface protein involved in cytotoxic activity, thereby increasing the immune response against the target cell. For example, an anti-enhancement factor can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) or other immune cells, resulting in an increased immune response against the target cell.

[0107] Bispecific molecules can be in many different formats and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody format, except that instead of having two binding arms of the same specificity, they have two binding arms with different specificities. At the other end are bispecific molecules consisting of two single-chain antibody fragments (scFvs) linked by a peptide chain, the so-called Bs(scFv)2 construct. Intermediate-sized bispecific molecules contain two different F(ab) fragments linked by a peptidyl linker. These and other formats of bispecific molecules can be prepared by genetic recombination, somatic cell hybridization, or chemical methods. See, for example, Kufer et al. (cited above); Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000), and the references cited therein.

[0108] Also provided herein are oncolytic viruses that preferentially infect and kill cancer cells. The antibodies of the present disclosure can be used together with the oncolytic viruses. Alternatively, oncolytic viruses encoding the antibodies of the present disclosure can be introduced into the human body.

[0109] Chimeric Antigen Receptor Also provided herein is a chimeric antigen receptor (CAR) comprising an anti-CD40 scFv, which comprises the CDRs and heavy / light chain variable regions described herein.

[0110] An anti-CD40 CAR can comprise: (a) an extracellular antigen-binding domain comprising an anti-CD40 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0111] CARs may contain a signal peptide at the N-terminus of the extracellular antigen-binding domain, which directs the nascent receptor to the endoplasmic reticulum, and a hinge peptide at the N-terminus of the extracellular antigen-binding domain, which makes the receptor more accessible for binding. CARs preferably contain a major intracellular signaling domain and one or more costimulatory signaling domains in the intracellular signaling domain. The most commonly used and most effective major intracellular signaling domain is the ITAM-containing CD3-zeta cytoplasmic domain, whose phosphorylation leads to T cell activation. The costimulatory signaling domain may be derived from costimulatory proteins such as CD28, CD137, and OX40.

[0112] CARs may further include factors that promote T cell proliferation, persistence, and anti-tumor activity, such as cytokines and costimulatory ligands.

[0113] Also provided are engineered immune effector cells comprising the CARs provided herein. In some embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the immune effector cells are T cells.

[0114] In another aspect, the present disclosure provides pharmaceutical compositions that may include one or more antibodies or antigen-binding portions thereof, bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors, or host cells of the present disclosure formulated with a pharmaceutically acceptable carrier. The antibodies or antigen-binding portions thereof, bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors, or host cells may be administered separately when the composition includes two or more antibodies (or antigen-binding portions thereof, bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors, or host cells). The compositions may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug, e.g., an anti-tumor drug.

[0115] Pharmaceutical compositions can contain any excipient. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003) (the disclosure of which is incorporated herein by reference).

[0116] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the phrase "parenteral administration" refers to methods of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion. Alternatively, the pharmaceutical composition of the present disclosure may be administered by a non-parenteral route, for example, a topical, epidermal, or mucosal route, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

[0117] The pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They may also be formulated as microemulsions, liposomes, or other ordered structures suitable to high drug concentration.

[0118] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration, but will generally be that amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.01% to about 99% of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0119] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect, together with the necessary pharmaceutical carrier. Alternatively, antibodies can be administered as sustained-release formulations when less frequent administration is required.

[0120] For administration of antibodies, the dosage may range from about 0.0001 to 100 mg / kg.

[0121] A "therapeutically effective dose" of an anti-CD40 antibody, or antigen-binding portion thereof, of the present disclosure preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of impairment or disability resulting from disease affliction. For example, for the treatment of a subject with a tumor, a "therapeutically effective dose" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared to an untreated subject. A therapeutically effective amount of a therapeutic antibody can reduce tumor size or ameliorate symptoms in a subject, which may typically be a human or another mammal.

[0122] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used.See, for example, "Sustained and Controlled Release Drug Delivery Systems," JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0123] Therapeutic compositions can be administered by medical devices, such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0124] In certain embodiments, monoclonal antibodies of the present disclosure may be formulated to ensure proper distribution in vivo. For example, to ensure that therapeutic antibodies of the present disclosure cross the blood-brain barrier, they may be formulated in liposomes, which may further contain targeting molecules to facilitate selective delivery to specific cells or organs. For example, US Pat. No. 4,522,811; US ​​Pat. No. 5,374,548; US Pat. No. 5,416,016; and US Pat. No. 5,399,331; VVRanade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al. al.,(1988)Biochem.Biophys.Res.Commun.153:1038;Bloeman et al.,(1995)FEBS Lett.357:140;M.Owais et al.,(1995)Antimicrob.Agents Chemother.39:180;Briscoe et al. al.,(1995)Am.J.Physiol.1233:134;Schreier et al.,(1994)J.Biol.Chem.269:9090;Keinanen See, e.g., and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

[0125] The pharmaceutical compositions of the present disclosure have numerous in vitro and in vivo utilities, including, for example, the treatment and / or prevention of cancer, or more generally, enhancing immune responses in patients with cancer. The pharmaceutical compositions can be administered to a human subject in vivo, for example, to inhibit tumor growth or treat or alleviate an infectious or autoimmune disease.

[0126] Given the ability of the pharmaceutical composition of the present disclosure to inhibit the proliferation and survival of cancer cells, the present disclosure provides a method for inhibiting the proliferation of tumor cells in a subject in need thereof, comprising administering the pharmaceutical composition of the present disclosure to the subject so that tumor growth is inhibited in the subject.Non-limiting examples of tumors that can be treated with the pharmaceutical composition of the present disclosure include, but are not limited to, B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, renal cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal carcinoma.In addition, the pharmaceutical composition of the present disclosure can also be applied to refractory or recurrent malignant tumors whose growth can be inhibited by the composition of the present disclosure.

[0127] These and other methods of the present disclosure are discussed in further detail below.

[0128] In another aspect, the present disclosure provides a method of combination therapy in which the pharmaceutical composition of the present disclosure is co-administered with one or more additional antibodies or non-antibody drugs that are effective in inhibiting tumor growth in a subject. In one embodiment, the present disclosure provides a method for inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition of the present disclosure and one or more additional antibodies, such as an anti-TIM3 antibody, an anti-PD-L1 antibody, and an anti-PD-1 antibody and / or an anti-CTLA-4 antibody. In certain embodiments, the subject is human. In certain embodiments, the pharmaceutical composition of the present disclosure can be further combined with standard cancer treatments. For example, CD40 signaling activation by the pharmaceutical composition of the present disclosure can be combined with a chemotherapy regimen in addition to blocking CTLA-4 and / or PD-1. For example, the chemotherapeutic agent can be administered together with the pharmaceutical composition of the present disclosure and can be a cytotoxic drug. For example, epitubicin, oxaliplatin, and 5-FU are administered to a patient undergoing anti-CD40 therapy. Other treatments that may be combined with anti-CD40 therapy include, but are not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone deprivation.

[0129] The combination of therapeutic agents described herein can be administered simultaneously in a single composition in a pharmaceutically acceptable carrier or as separate compositions containing each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0130] Furthermore, when two or more doses of a combination therapy are administered sequentially, the order of sequential administration may be reversed or maintained in the same order at each time of administration, sequential administration may be combined with simultaneous administration, or any combination thereof.

[0131] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are hereby expressly incorporated by reference. [Example]

[0132] Example 1: Generation of murine anti-CD40 monoclonal antibodies using hybridoma technology immunization Mice were immunized according to the method described in E. Harlow and D. Lane, "Antibody: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998. Recombinant human CD40 protein (AA region 21-193 of Uniprot number P25942, amino acid residues 21-193 of SEQ ID NO: 30) with a C-terminal human IgG1 Fc tag (SEQ ID NO: 27) was used as the immunogen. Human CD40-his protein (Acro biosystems, Cat# CD0-H5228) was used to determine antiserum titers and to screen hybridomas secreting antigen-specific antibodies.

[0133] The immunization dose contained 25 μg of human CD40-Fc protein per mouse per injection for both the primary and boost immunizations. To enhance immune responses, Freund's complete and incomplete adjuvants (Sigma, St. Louis, Mo., USA) were used for the primary and boost immunizations, respectively. Briefly, the adjuvant-antigen mixture was first prepared by gently mixing the adjuvant in a vial using a vortex mixer. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL microcentrifuge tube. Antigen was prepared in PBS or saline with a concentration ranging from 0.5 to 1.0 mg / mL. Next, the calculated amount of antigen was added to the microcentrifuge tube along with the adjuvant, and the resulting mixture was mixed by gentle vortexing for 2 minutes to create a water-in-oil emulsion. The adjuvant-antigen emulsion was then drawn up into an appropriate syringe for injection into animals. A total of 25 μg of antigen was injected in a volume of 50–100 μl. Each animal was immunized and then boosted 2–3 times depending on the antiserum titer. Animals with good titers were given a final booster by intraperitoneal injection prior to fusion.

[0134] Hybridoma fusion and screening Cells of a mouse myeloma cell line (SP2 / 0-Ag14, ATCC #CRL-1581) were cultured to reach logarithmic phase just prior to fusion. Spleen cells from immunized mice were sterilely prepared and fused with myeloma cells according to the method described by Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256:495-497 (1975). The fused "hybrid cells" were then distributed into 96-well plates in DMEM / 20% FCS / HAT medium. Viable hybridoma colonies were observed microscopically 7 to 10 days after fusion. After two weeks, supernatants from each well were subjected to ELISA-based screening using recombinant human CD40-his protein and cell-based binding FACS using 293T-CD40 cells expressing human CD40 protein (uniprot#P25942-1, SEQ ID NO: 30) on their cell membranes. Hybridoma-secreting antibodies that bound to human CD40-his protein and showed high specificity to 293T-CD40 cells, namely clones 1A3, 1D1, and C1H1, were subcloned by limiting dilution to confirm the clonality of the cell line and then purified as monoclonal antibodies. Briefly, a Protein A Sepharose column (Bestchrom (Shanghai) Biosciences, Cat#AA0273) was washed with PBS buffer for 5 to 10 column volumes. The cell supernatant was passed through the column, and the column was then washed with PBS buffer until the protein absorbance reached baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7) and immediately collected in a 1.5 ml tube containing neutralization buffer (1 M Tris-HCl, pH 9.0). Fractions containing immunoglobulins were pooled and dialyzed in PBS overnight at 4°C. The in vitro functional activity of the purified monoclonal antibodies was then characterized as follows.

[0135] Example 2 Affinity Determination of Murine Anti-CD40 Monoclonal Antibodies Using BIACORE Surface Plasmon Resonance Technology The purified anti-CD40 mouse monoclonal antibody (mAb) produced in Example 1 was characterized for affinity and binding kinetics by a Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[0136] Briefly, goat anti-mouse IgG (GE Healthcare, Cat# BR100838, Mouse Antibody Capture Kit) was covalently coupled via primary amines to a CM5 chip (GE Healthcare, carboxymethyl dextran-coated chip #BR100530) using a standard amine coupling kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA), and a Protein G chip (GE Healthcare, Cat# 29-1793-15) was used for benchmark affinity determination. Unreacted moieties on the biosensor surface were blocked with ethanolamine. Next, the purified anti-CD40 antibody of the present disclosure and two benchmark antibodies, BM1 (dacetuzumab, Genentech Inc., also referred to as CD40-BM1, produced in-house using heavy and light chain amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively) and BM2 (celicrelumab, Abgenix Inc., also referred to as CD40-BM2, produced in-house using heavy and light chain amino acid sequences set forth in SEQ ID NOs: 42 and 43, respectively), were each flowed over the chip at a concentration of 66.7 nM at a flow rate of 10 μL / min. Next, HBS-EP (provided by Biacore) was added. +Serially diluted recombinant human CD40-his (Acro biosystems, Cat# CD0-H5228, starting at 80 nM in two-fold serial dilutions) or cynomolgus monkey CD40-his protein (Acro biosystems, Cat# CD0-C52H6, starting at 80 nM in two-fold serial dilutions) in buffer were flowed over the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were followed for 2 minutes, and dissociation kinetics were followed for 10 minutes. Binding and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore evaluation software. D , K. a and K. d The values ​​were determined and are summarized in Table 2 below.

[0137] [Table 2]

[0138] The murine antibody C1H1 of the present disclosure specifically bound to human and monkey CD40 with higher binding affinity than BM1 and BM2.

[0139] Example 3 Binding activity of mouse anti-CD40 monoclonal antibodies The binding activity of the mouse anti-CD40 antibodies was measured by capture ELISA and flow cytometry (FACS).

[0140] For the capture ELISA, 96-well microplates were coated with 100 μl / well of 2 μg / ml AffiniPure goat anti-mouse IgG F(ab')2 fragment specific (Jackson Immuno Research, Cat#115-005-072) in PBS and incubated overnight at 4° C. Plates were washed once with wash buffer (PBS+0.05% v / v Tween®-20, PBST) and then blocked with 200 μl / well of blocking buffer (5% w / v non-fat milk in PBST) for 2 hours at 37° C. Plates were washed four times and incubated with 100 μl / well of serially diluted anti-CD40 antibodies of the present disclosure, BM1, BM2, and negative control hIgG (Human Immunoglobulin for Intravenous Injection (pH 4), Hualan Biological Engineering Inc.) (5-fold serial dilutions in 2.5% w / v nonfat milk in PBST, starting at 66.7 nM) for 40 minutes at 37° C., then washed again four times. Plates containing the capture anti-CD40 antibody were incubated with 100 μl of biotin-labeled human CD40-Fc protein (amino acid residues 21-193 of SEQ ID NO: 30 linked to the N-terminal amino acid residues 99-330 of SEQ ID NO: 28, 1.15 nM in 2.5% w / v nonfat milk in PBST) for 40 minutes at 37° C., washed four times, and incubated with streptavidin-conjugated HRP (1:10,000 dilution in PBST, Jackson Immuno Research, Cat# 016-030-084, 100 μl / well) for 40 minutes at 37° C. After the final wash, plates were incubated with 100 μl / well of ELISA substrate TMB (Innoreagents, Cat# TMB-S-002) at room temperature. The reaction was stopped with 50 μl / well of 1 M H2SO4 for 3-10 min, and the absorbance of each well was read on a microplate reader using dual wavelength mode with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values ​​were then plotted against antibody concentration. Data were analyzed using Graphpad Prism software, and EC 50 The values ​​were reported.

[0141] For the binding of anti-CD40 antibodies to 293T-CD40 cells tested by flow cytometry (FACS), Biosion's in-house prepared 293T-CD40 cells stably expressing full-length human CD40 (uniprot #P25942-1, SEQ ID NO: 30) on the cell membrane were used. 293T-CD40 cells were prepared by transfecting 293T cells with the pCMV-TP plasmid, which contained the CD40 coding sequence between the EcoRI and XbaI sites, according to the Lipofectamine 3000 transfection reagent (Thermo Fisher) instructions. Specifically, 293T-CD40 cells were harvested from cell culture flasks, washed twice, and resuspended in phosphate-buffered saline (PBS) containing 2% v / v fetal bovine serum (FACS buffer). 2 × 10 cells were cultured per well in a 96-well plate. 5 Cells were incubated with 100 μl of the disclosed anti-CD40 antibody or various concentrations of control (4-fold serial dilutions starting at 80 nM) in FACS buffer on ice for 40 minutes. Cells were washed twice with FACS buffer, and 100 μL / well of R-phycoerythrin Affini Pure F(ab')2 fragment goat anti-mouse IgG(H+L) (1:1000 dilution in FACS buffer, Jackson Immuno Research, Cat#115-116-146) was added. After 40 minutes of incubation at 4°C in the dark, cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument and plotted against antibody concentration. Data were analyzed using Graphpad Prism software, and EC 50 The values ​​were reported.

[0142] The results are shown in Figures 1A to 1B and Figures 2A to 2B.

[0143] The results show that the murine antibodies of the present disclosure specifically bind to human CD40, with 1A3, 1D1, and C1H1 having lower EC2s than either BM1 or BM2, or both. 50These results suggest that they bind more efficiently to human CD40 protein. Furthermore, as can be seen from Figures 1A-1B and 2A-2B, the maximum binding of murine antibody 1A3 / C1H1 was comparable to that of BM1 or BM2.

[0144] Example 4 Blocking activity of murine anti-CD40 antibodies against CD40-CD40L or CD40-benchmark binding 4.1 Ligand-Blocking ELISA The ability of anti-CD40 antibodies to block CD40-CD40L binding was measured in a competitive ELISA assay. Briefly, 100 μl of human CD40-Fc protein (amino acid residues 21-193 of SEQ ID NO: 30 linked to the N-terminus of amino acid residues 99-330 of SEQ ID NO: 28) was coated onto a 96-well microplate at 2 μg / mL in coating buffer (carbonate / bicarbonate buffer) and incubated overnight at 4°C. The next day, the plate was washed once with wash buffer (PBS + 0.05% v / v Tween-20, PBST) and blocked with 5% w / v nonfat milk in PBST for 2 hours at 37°C. The plate was then washed four times with wash buffer.

[0145] Serially diluted anti-CD40 antibodies of the present disclosure or controls (starting at 200 nM in 5-fold serial dilutions) in PBST with 2.5% w / v nonfat milk were added to the CD40-Fc-bound plate at 100 μl / well and incubated at 37° C. for 40 minutes. The plate was washed four times again with wash buffer, and then 100 μl / well of 95 ng / mL biotin-labeled human CD40L-his protein (Sino biological Inc., Cat# 10239-H08E) was added and incubated at 37° C. for 40 minutes. The plate was washed again with wash buffer. Streptavidin-conjugated HRP (1:10,000 dilution in PBST buffer, Jackson Immunoresearch, Cat# 016-030-084) was then added to the plate at 100 μl / well and incubated at 37° C. for 40 minutes. The plate was washed again with wash buffer. Finally, TMB was added, the reaction was stopped with 1 M H2SO4, and the absorbance of each well was read on a microplate reader using dual wavelength mode with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values ​​were then plotted against the antibody concentration. The data were analyzed using Graphpad Prism software, and the IC 50 The values ​​were reported.

[0146] 4.2 Benchmark Blocking ELISA The ability of anti-CD40 antibodies of the present disclosure to block benchmark-human CD40 binding was measured in a competitive ELISA assay. Briefly, BM2 antibody was coated at 1 μg / mL in PBS at 100 μl / well onto a 96-well microplate and incubated overnight at 4°C. The next day, the plate was washed once with wash buffer and blocked with blocking buffer (5% w / v nonfat milk in PBST) for 2 hours at 37°C. While blocking, anti-CD40 antibodies of the present disclosure or controls were diluted in 5-fold serial dilutions starting at 66.7 nM with biotin-labeled human CD40-Fc protein (amino acid residues 21-193 of SEQ ID NO: 30 linked to the N-terminus of amino acid residues 99-330 of SEQ ID NO: 28, 0.23 nM in PBST with 2.5% v / v nonfat milk) and incubated at room temperature for 40 minutes. After washing the plate four times, 100 μl / well of the antibody / human CD40-Fc-biotin mixture was added to the BM2-coated plate. After incubation at 37°C for 40 minutes, the plate was washed four times again with wash buffer. Next, 100 μl / well of streptavidin-conjugated HRP was added to the plate and incubated at 37°C for 40 minutes to detect plate-bound biotin-labeled human CD40-Fc. The plate was washed four times again with wash buffer. Finally, TMB was added, the reaction was stopped with 1 M H2SO4, and the absorbance was read on a microplate reader using dual wavelength mode with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values ​​were then plotted against the antibody concentration. Data were analyzed using Graphpad Prism software, and IC 50 The values ​​were reported.

[0147] The results of the two assays are shown in Figures 3A-3B and 4A-4B.

[0148] The murine antibody C1H1 has comparable or even lower IC compared to BM1 and BM2. 503B, it can be seen that the mouse antibody C1H1 was able to block human CD40-human CD40L binding at values ​​of 0.01 and 0.02, respectively. Furthermore, as shown in FIG. 3B, the mouse antibody C1H1 showed higher maximal blocking than BM1 and BM2.

[0149] Figures 4A and 4B show that mouse antibody C1H1 was able to block human CD40-BM2 binding, suggesting that it bound to the same or equivalent epitope as that bound by BM2. Mouse antibodies 1A3 and 1D1, which did not show blocking, may bind to different epitopes.

[0150] Example 5 Cell-based reporter assay of mouse anti-CD40 antibodies The anti-CD40 antibodies of the present disclosure were further tested for their agonistic activity using a CD40-expressing reporter cell line, 293T-NF-κB-Luc-CD40, which stably expresses full-length human CD40 (uniprot number P25942-1, SEQ ID NO: 30). 293T-NF-κB-Luc-CD40 cells were prepared by transfecting 293T cells with the pGL4.32[luc2P NF-κB-RE Hygro] vector (Promega, Genbank® accession number: EU581860) followed by the pCMV-TP plasmid, in which the CD40 coding sequence was inserted between the EcoRI and XbaI sites, according to the Lipofectamine 3000 transfection reagent (Thermo Fisher) instructions. When a CD40 agonist was added to these cells, CD40 signaling was activated and luciferase expression was upregulated, which can be measured in a luminescent assay.

[0151] Briefly, 5 × 10 log-phase cells were cultured in 20 μL of DMEM medium (Gibco Inc., Cat. #10566-016) supplemented with 10% FBS (Gibco, Cat. #10099-141). 3293T-NF-κB-Luc-CD40 cells were plated in a 384-well cell culture plate (Corning, Cat#3707). Next, 20 μl / well of serially diluted anti-CD40 antibodies of the present disclosure or controls (including an in-house produced anti-CD22 antibody as a negative control) were added to the plate (starting at 200 nM in 3-fold serial dilutions in culture medium) and incubated at 37°C for 6 hours. Next, ONE-Glo™ Luciferase Assay System reagents (Promega, Cat#E6120, 30 μl / well) were added to the plate and incubated for 5 minutes at room temperature. Chemiluminescence was measured using a Tecan Infinit® 200Pro instrument. Data were analyzed using Graphpad Prism software, and EC 50 The values ​​were reported.

[0152] The results are shown in Figures 5A and 5B.

[0153] It can be seen that murine antibodies 1A3, 1D1 and C1H1 had comparable agonistic activity compared to BM1, but not as good as BM2.

[0154] Example 6 Generation and Characterization of Chimeric Antibodies The heavy and light chain variable domains of the anti-CD40 murine mAbs were sequenced and summarized in Table 1.

[0155] The heavy and light chain variable domains of anti-CD40 murine mAbs C1H1, 1D1, and 1A3 were cloned in frame into the human IgG1 heavy chain (SEQ ID NO: 27) and human kappa light chain constant region (SEQ ID NO: 29), respectively, with the C-terminus of the variable regions linked to the N-terminus of the respective constant regions.

[0156] Vectors containing nucleotides encoding the heavy chain variable region linked to the human IgG1 heavy chain constant region and vectors containing nucleotides encoding the light chain variable region linked to the human kappa light chain constant region were transiently transfected into 50 ml 293F suspension cell cultures with 1 mg / mL PEI at a ratio of 1.1:1 light chain to heavy chain constructs.

[0157] The cell supernatant was collected after 6 days in the shake flask and spun down to pellet the cells, and the chimeric antibody was purified from the cell supernatant as described above. The purified chimeric antibody was tested in capture ELISA, BIAcore affinity studies, and cell-based reporter assays according to the protocols in the examples above and below, with minor modifications.

[0158] For BIAcore, goat anti-human IgG (GE healthcare, Cat#BR100839, Human Antibody Capture Kit) was covalently coupled to a CM5 chip instead of goat anti-mouse IgG, and the CM5 chip was used for BM1 and BM2 instead of the protein G chip. The results are shown in Table 3.

[0159] For the capture ELISA, AffiniPure goat anti-human IgG F(ab')2 fragment specific (Jackson Immuno Research, Cat#109-005-097) was used at 100 μl / well instead of AffiniPure goat anti-mouse IgG F(ab')2 fragment specific.

[0160] The results are shown in Table 3, Figures 6A to 6C, and Figures 7A to 7C.

[0161] The data showed that the chimeric antibodies had binding capacities and agonistic activities comparable to their parental antibodies. In particular, the chimeric C1H1 antibody exhibited higher binding affinity and potency to human CD40 and higher binding affinity to cynomolgus monkey CD40 than BM1.

[0162] [Table 3]

[0163] Example 7 Humanization of anti-CD40 mouse monoclonal antibody C1H1 The murine anti-CD40 antibody C1H1 was selected for humanization and further study. Humanization of this antibody was performed using well-established CDR grafting methods as detailed below.

[0164] To select an acceptor framework for humanization of the mouse antibody C1H1, the light and heavy chain variable region sequences of this mouse C1H1 were blasted against the human immunoglobulin gene database. The human germline with the highest homology to mouse C1H1 was selected as the acceptor framework for humanization. The mouse antibody heavy and light chain variable region CDRs were inserted into the selected framework, and the residues in the framework were further mutated to obtain more candidate heavy and light chain variable regions. A total of four humanized C1H1 antibodies (i.e., huC1H1-V1 to huC1H1-V4) were obtained, and their heavy and light chain variable region sequences are shown in Table 1.

[0165] Vectors containing nucleotides encoding the heavy chain variable region linked to the human IgG2 heavy chain constant region (SEQ ID NO: 28) and vectors containing nucleotides encoding the humanized light chain variable region linked to the human kappa light chain constant region (SEQ ID NO: 29) were transiently transfected into 50 ml 293F suspension cell cultures with 1 mg / ml PEI at a ratio of 60% to 40% light chain to heavy chain constructs.

[0166] Example 8 Characterization of humanized antibodies Cell supernatants containing humanized antibodies huC1H1-V1 to huC1H1-V4 were collected after 6 days in shake flasks and tested for binding affinity to human CD40 using the Octet system (ForteBio, Octet RED96) by Octect according to the protocol described below. Briefly, AHC biosensors (anti-human IgG Fc capture from ForteBio) were presoaked with 10 mM glycine (pH 1.5) for 3 seconds, then dipped into the wells with running buffer (0.5% w / v BSA in PBST) for 3 seconds. The soaking and dipping steps were repeated three times. The sensors were then presoaked with humanized anti-CD40 antibodies, 5 μg / ml in HBS-EP for 120 seconds. + chimeric C1H1 antibody in HBS-EP at 5 μg / ml + The wells were dipped with cell supernatant containing benchmarks in the running buffer for 5 minutes, followed by dipping in a well containing running buffer for 5 minutes. A new baseline was tested for 180 seconds in another well containing running buffer. Next, the sensor was dipped in a well with serially diluted human CD40-his protein (Acro biosystems, Cat#CD0-H5228, 2-fold serial dilutions starting from 40 nM) in running buffer for 120 seconds, followed by dipping in the baseline well for 10 minutes. Finally, the sensor was pre-soaked with 10 mM glycine (pH 1.5) for 3 seconds, then dipped in the well with running buffer for 3 seconds; the dipping and dipping steps were repeated three times. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis 8.1. a , K. d and K. D The values ​​were determined and are summarized in Table 4 below.

[0167] The results showed that the tested huC1H1-V1 and huC1H1-V2 had comparable human CD40 binding affinity compared to the chimeric antibody C1H1, which was better than that of BM2.

[0168] [Table 4]

[0169] Humanized antibodies huC1H1-V1 and huC1H1-V2 were purified as described above and tested in Biacore, capture ELISA, cell-based binding FACS, competitive ELISA, cell-based reporter assays, and protein thermal shift assays according to the protocols in the examples above and below, with minor modifications.

[0170] In the capture ELISA, AffiniPure goat anti-human IgG, F(ab')2 fragment specific (Jackson Immuno Research, Cat#109-005-097) was used at 100 μl / well instead of AffiniPure goat anti-mouse IgG, F(ab')2 fragment specific.

[0171] In the BIAcore, goat anti-human IgG (GE healthcare, Cat#BR100839, Human Antibody Capture Kit) was covalently bound to a CM5 chip instead of goat anti-mouse IgG, and the CM5 chip was used for BM1 and BM2 instead of a protein G chip.

[0172] For cell-based binding FACS, R-Phycoerythrin AffiniPure Goat Anti-Human IgG Fcγ Fragment Specific (Jackson Immuno Research, Cat#109-115-098) was used at 100 μl / well instead of R-Phycoerythrin AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG(H+L).

[0173] Additionally, humanized antibody huC1H1-V2 was tested for thermal stability. Protein thermal shift assays were used to determine Tm (melting temperature) using the GloMelt™ Thermal Shift Protein Stability Kit (Biotium, Cat# 33022-T). Briefly, GloMelt™ dye was thawed and allowed to reach room temperature. The vial containing the dye was vortexed and centrifuged. Next, 10x dye was prepared by adding 5 μL of 200x dye to 95 μL of PBS. 2 μL of 10x dye and 10 μg of humanized antibody were added, and PBS was added to a total reaction volume of 20 μL. The tube containing the dye and antibody was briefly spun and placed in a real-time PCR thermocycler (Roche, LightCycler 480 II) set using a melting curve program with the parameters in Table 5.

[0174] [Table 5]

[0175] The assay results are shown in Table 6 and Figures 8 to 12.

[0176] It can be seen from Table 6 that the humanized antibodies huC1H1-V1 and huC1H1-V2 exhibited comparable binding affinities to human and cynomolgus CD40 compared to the chimeric antibody C1H1. In other words, the binding affinities of huC1H1-V1 and huC1H1-V2 to human and cynomolgus CD40 were higher than those of BM1 and BM2.

[0177] FIG. 10 shows that the humanized antibodies huC1H1-V1 and huC1H1-V2 of the present disclosure can block CD40-CD40L binding, with comparable or slightly lower blocking activity compared to BM1 and BM2.

[0178] Figure 11 shows that the humanized antibodies huC1H1-V1 and huC1H1-V2 of the present disclosure can block human CD40-BM2 binding, suggesting that the antibodies huC1H1-V1 and huC1H1-V2 of the present disclosure can bind to an epitope similar to that of BM2.

[0179] As shown in FIG. 12, the humanized antibodies huC1H1-V1 and huC1H1-V2 of the present disclosure had higher agonist activity compared to BM1 and BM2 in a cell-based reporter assay.

[0180] [Table 6]

[0181] Example 9 In vivo antitumor efficacy of huC1H1-V2 antibody in the human B-cell lymphoma Ramos xenograft model The in vivo antitumor activity of the huC1H1-V2 antibody was tested in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Briefly, 3 × 10 7 Human B-cell lymphoma Ramos cells (Chinese Academy of Sciences) were injected subcutaneously into the right axilla. Tumor volume was measured using electronic calipers and expressed as (length × width). 2 ) / 2. 3 When tumors reached an average volume of 1000 mg / kg, 40 tumor-bearing mice were selected and randomized into 4 groups (10 mice / group), and on the day of administration, the animal groupings were designated as day 0. Starting on day 0, the animals were intravenously injected via the tail vein with an isotype control antibody (anti-HEL-human IgG2 isotype control, also referred to as IgG2, Biointron Inc) or huC1H1-V2 antibody according to the administration regimen shown in Table 7.

[0182] [Table 7]

[0183] The data are shown in Table 8.

[0184] All treatments were well tolerated by tumor-bearing animals, with no significant weight loss or other symptoms. huC1H1-V2 antibody treatment at 5 mg / kg demonstrated the strongest antitumor activity, with tumor mass of 230.2 mm at day 14. 3 The mean tumor volume in Group 3 on day 14 was statistically smaller than that in the control group. However, huC1H1-V2 antibody treatment at 15 mg / kg did not demonstrate further enhanced efficacy; i.e., tumor volume in Group 4 was not significantly different from that in Group 3.

[0185] [Table 8]

[0186] Example 10 In vivo antitumor efficacy of huC1H1-V2 antibody in a murine colon cancer MC38 xenograft model The in vivo anti-tumor activity of the huC1H1-V2 antibody was further tested in CD40 humanized transgenic mice (also referred to as hCD40 mice). Briefly, 1×10 6 Mouse colon cancer MC38 cells (Shanghai Lanli Biological Technology Co., Ltd.) were subcutaneously injected into the right flank. Tumor volume was measured using electronic calipers (length × width). 2 ) / 2. 3 When tumors reached an average volume of 1000 mg / kg, 18 tumor-bearing mice were selected and randomized into 3 groups (6 mice / group), and on the day of dosing, animal groupings were designated as day 0. Starting on day 1, animals were intravenously injected via the tail vein with vehicle (phosphate-buffered saline, also known as PBS) or huC1H1-V2 antibody according to the dosing regimen shown in Table 9.

[0187] [Table 9]

[0188] The data are summarized in Table 10.

[0189] All treatments were well tolerated by tumor-bearing animals, with no significant weight loss or symptoms. huC1H1-V2 antibody treatment at 3 mg / kg demonstrated potent antitumor activity, with tumor mass of 1169 mm at day 14. 3 The tumor size in Group 2 on Day 14 was statistically smaller than that in the vehicle group. huC1H1-V2 antibody treatment at 10 mg / kg also demonstrated potent antitumor activity, with a mean tumor size of 1446 mm on Day 14. 3 The mean tumor size in Group 3 on Day 14 was statistically smaller than that in the vehicle group.

[0190] [Table 10]

[0191] While the present disclosure has been described above in conjunction with one or more embodiments, it is to be understood that the disclosure is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and equivalents as may be included within the spirit and scope of the appended claims. All references cited herein are further incorporated by reference in their entirety.

[0192] The sequences in this application are summarized below.

[0193] [Table 11]

[0194] [Table 12]

[0195] [Table 13]

[0196] [Table 14]

[0197] [Table 15]

[0198] Having thus described in detail the preferred embodiments of the present invention, it should be understood that the invention defined by the above paragraphs should not be limited to the specific details set forth in the above description, as many obvious variations thereof may be made without departing from the spirit or scope of the invention.

Claims

1. 1. An isolated monoclonal antibody, or antigen-binding portion thereof, that binds to CD40, comprising: (i) a heavy chain variable region comprising a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region; and (ii) a light chain variable region comprising a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, the VH CDR3 region, the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region comprise the amino acid sequences of SEQ ID NOs: 1, 4, 7, 10, 13, and 16, respectively.

2. 2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 19 or 20, and the 49th amino acid residue of SEQ ID NO: 20 is A or S.

3. 3. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 23 or 24, and the amino acid residues at positions 49 and 87 of SEQ ID NO: 24 are K and F, respectively, or Y and Y, respectively.

4. the heavy chain variable region and the light chain variable region comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to (1) SEQ ID NOs: 19 and 23, respectively; or (2) SEQ ID NOs: 20 and 24, respectively; The 49th amino acid residue of SEQ ID NO:20 is A, and the 49th and 87th amino acid residues of SEQ ID NO:24 are K and F, respectively; or The 49th amino acid residue of SEQ ID NO:20 is S, and the 49th and 87th amino acid residues of SEQ ID NO:24 are K and F, respectively; or the 49th amino acid residue of SEQ ID NO:20 is A, and the 49th and 87th amino acid residues of SEQ ID NO:24 are Y and Y, respectively; or The 49th amino acid residue of SEQ ID NO:20 is S, and the 49th and 87th amino acid residues of SEQ ID NO:24 are Y and Y, respectively. An isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3.

5. 5. The isolated monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 4, comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 27 or 28 linked to the heavy chain variable region, and a light chain constant region having the amino acid sequence of SEQ ID NO: 29 linked to the light chain variable region.

6. 6. The isolated monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 5, which is a murine, chimeric or humanized antibody, or an antigen-binding portion thereof.

7. A nucleotide encoding the isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 6.

8. An expression vector containing the nucleotide of claim 7.

9. A host cell containing the nucleotide of claim 7 or the expression vector of claim 8.

10. A pharmaceutical composition comprising an antibody or antigen-binding portion thereof according to any one of claims 1 to 6, a nucleotide according to claim 7, an expression vector according to claim 8, or a host cell according to claim 9, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10 for use in treating cancer in a subject in need thereof.

12. 12. The pharmaceutical composition for use according to claim 11, wherein the cancer is a solid or non-solid tumor.

13. 13. The pharmaceutical composition for use according to claim 11 or 12, wherein the cancer is selected from the group consisting of B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, renal cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal cancer.

14. The pharmaceutical composition of claim 10 for use in treating an infectious disease in a subject in need thereof.

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