Anti-ox40 antibodies and uses thereof

Abstract

This invention provides an anti-OX40 antibody or its antigen-binding fragment, a method for its preparation, and its use in treating OX40-related diseases or symptoms.

Description

[0001] This application is based on and claims priority to Chinese application CN 202010304381.8, filed on April 17, 2020, which is hereby incorporated herein by reference in its entirety for all purposes. Technical Field

[0002] This invention relates to antibodies, and more particularly to anti-OX40 antibodies and their antigen-binding fragments, methods for preparing the antibodies, and uses thereof for the treatment or prevention of OX40-related diseases or symptoms. Background Technology

[0003] OX40 (also known as CD134, TNFRSF4, and ACT35) is a member of the tumor necrosis factor superfamily, primarily expressed on the surface of activated CD4+ T cells, CD8+ T cells, and regulatory T cells, and also expressed on the surface of natural killer (NK) cells. Within activated T cells, OX40L-OX40-mediated co-stimulatory signaling can stimulate helper T cells to produce and secrete cytokines, stimulate effector T cells to release granzymes and perforin, and induce the proliferation of effector T cells and memory T cells. Simultaneously, OX40L-OX40 signaling can also inhibit the differentiation and activity of regulatory T cells and reduce their immunosuppressive function, thereby further enhancing the immune response. The crucial role of OX40 in T cell immune responses makes OX40 agonists important targets for tumor immunotherapy, while OX40 inhibitors have potential applications in inflammatory, allergic, and autoimmune diseases.

[0004] In recent years, with the widespread application of monoclonal antibody preparation technology, monoclonal antibodies that specifically bind to OX40 have emerged, including OX40 agonists and OX40 inhibitors. Under physiological conditions, OX40 activates corresponding intracellular signaling pathways by binding to its ligand OX40L and trimerizing. Therefore, OX40-agonist monoclonal antibodies usually require other means to cross-link to exert their agonist function. In vitro and in vivo, antibody cross-linking can be achieved through antibody coating and Fc receptors, respectively. Fc receptors are a class of receptor proteins that specifically bind to the Fc fragment of antibodies. Among them, Fcγ receptors can specifically bind to IgG and perform functions such as ADCC and ADCP. Fcγ receptors mainly include FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcγRIIIB, which are expressed on the surface of various blood cells, including B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, mast cells, and platelets. Under physiological conditions, Fcγ receptors can simultaneously bind to the Fc fragments of one or more IgG molecules, activating receptor-mediated functions while simultaneously achieving IgG molecule cross-linking. Agonistic antibodies against OX40 activate the function of OX40 molecules through the binding and cross-linking of Fcγ receptors. Conversely, inhibitory antibodies against OX40 inhibit the activation of T cells and related inflammatory responses induced by OX40 activation by blocking the binding of OX40L to OX40, thus preventing OX40 trimerization.

[0005] Tumor cells can evade the immune system's recognition and attack through various mechanisms, thus achieving immune escape and surviving and proliferating excessively within the body. A key mechanism mediating tumor immune escape is the co-stimulatory molecules, or immune checkpoints, highly expressed by immune cells or tumor cells in the tumor microenvironment. Based on their function, these checkpoints can be divided into inhibitory immune checkpoints, represented by PD-1, PD-L1, and CTLA-4, and agonistic immune checkpoints, represented by OX40 and 4-1BB. Inhibitory immune checkpoints can be targeted with antibodies and other drugs to block their inhibitory function, essentially releasing the brakes on immune cells, allowing them to kill tumor cells. Tumor immunotherapy, represented by PD-1, PD-L1, and CTLA-4, is becoming a very important treatment approach and has shown promising therapeutic effects in clinical applications. Activating agonistic immune checkpoints with agonists is like pressing the accelerator after releasing the brakes, further increasing the activity of immune cells, making them more effective at killing tumor cells, ultimately resulting in a broader and more effective therapeutic effect against tumors.

[0006] Studies have found that OX40 is expressed in various tumor-infiltrating T cells, and OX40-positive tumor patients have relatively longer survival, indicating its function in tumor immunity. In various preclinical animal models, activation of OX40 function has consistently shown results in stimulating T cell proliferation, enhancing effector T cell function, and inhibiting regulatory T cell function. In a clinical trial using the OX40 agonist (9B12) to treat patients with metastatic solid tumors, significant immune upregulation was observed in patients, and 12 out of 30 patients experienced a reduction in metastatic lesions. Furthermore, tumor patients treated with the OX40 antibody showed good tolerability. Currently, several OX40 agonist monoclonal antibodies, such as MOXR0916, PF-04518600, BMS986178, GSK3174998, MEDI0562, and MEDI6469, are undergoing clinical trials as monotherapy or in combination with other immunomodulators.

[0007] Autoimmune diseases are a major medical challenge facing humanity today, and OX40 inhibitors hold promise as a potential treatment for these diseases. Preclinical studies have shown that mice deficient in OX40 or OX40L exhibit significantly weakened Th2 cell function in an allergic asthma model. OX40L inhibitors can alleviate symptoms associated with T cell suppression in a mouse asthma model, a result that can be replicated in in vivo experiments in monkeys. Furthermore, blocking the OX40-OX40L signaling pathway has demonstrated immunosuppression and symptom relief in various other classic inflammatory and autoimmune disease models, including experimental allergic encephalomyelitis (EAE), rheumatoid arthritis (RA), and diseases such as colitis, transplant anti-host disease, and type 1 diabetes caused by CD4+. + Or CD8 + A model for T cell function. Currently, preliminary results have been obtained from clinical trials of OX40 antagonist monoclonal antibody drugs. GRB830, developed by Glenmark, is a humanized human IgG1 monoclonal antibody. This antibody inhibits T cell activation induced by OX40L by binding to the second cysteine-rich domain of OX40. GBR830 has shown some efficacy in an ongoing clinical trial for moderate to severe atopic dermatitis (Phage IIa, NCT02683928). Additionally, KHK4083, an OX40 antagonist monoclonal antibody developed by Kyowa Hakko, demonstrated good tolerability and efficacy in a phase I clinical trial for atopic dermatitis, and a phase II clinical trial for moderate to severe atopic dermatitis was initiated in October 2018 (NCT03703102).

[0008] To date, no anti-OX40 antibody with proven efficacy has been approved for the treatment of any human disease. Further development of this type of drug to meet the enormous clinical demand is of great significance. Summary of the Invention

[0009] This invention provides anti-OX40 antibodies or antigen-binding fragments thereof, and methods for their preparation and use, including methods for treating OX40-related diseases or symptoms.

[0010] On one hand, the present invention provides isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising one to three HCDR1, HCDR2 and HCDR3 selected from the heavy chain variable region (VH), wherein the amino acid sequence of said VH is as shown in SEQ ID NO: 1, 2, 3, 4 or 5.

[0011] On one hand, the present invention provides isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising one to three LCDR1, LCDR2 and LCDR3 selected from light chain variable regions (VL), wherein the amino acid sequence of said VL is as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0012] In some embodiments, the present invention provides an isolated anti-OX40 antibody or its antigen-binding fragment comprising three CDRs of a heavy chain variable region (VH), namely HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL), namely LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the VH is as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and the amino acid sequence of the VL is as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0013] In some embodiments, the present invention provides an isolated anti-OX40 antibody or its antigen-binding fragment comprising three CDRs of the heavy chain variable region (VH), namely HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region (VL), namely LCDR1, LCDR2, and LCDR3; wherein the VH and VL are selected from:

[0014] (1) VH contains the amino acid sequence shown in SEQ ID NO:1 and VL contains the amino acid sequence shown in SEQ ID NO:6;

[0015] (2) VH contains the amino acid sequence shown in SEQ ID NO:2 and VL contains the amino acid sequence shown in SEQ ID NO:7 or 9;

[0016] (3) VH contains the amino acid sequence shown in SEQ ID NO:3 and VL contains the amino acid sequence shown in SEQ ID NO:9; or

[0017] (4) VH contains the amino acid sequence shown in SEQ ID NO:4 and VL contains the amino acid sequence shown in SEQ ID NO:7 or 8.

[0018] On one hand, the present invention provides isolated anti-OX40 antibody or antigen-binding fragment thereof, which includes heavy chain complementarity-determining regions (HCDRs), one to three of HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO:11, HCDR2 contains the amino acid sequence shown in SEQ ID NO:12 and HCDR3 contains the amino acid sequence shown in SEQ ID NO:13.

[0019] On one hand, the present invention provides isolated anti-OX40 antibody or antigen-binding fragment thereof, which includes light chain complementarity-determining regions (LCDRs), one to three of LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains the amino acid sequence shown in SEQ ID NO:14, LCDR2 contains the amino acid sequence shown in SEQ ID NO:15 and LCDR3 contains the amino acid sequence shown in SEQ ID NO:16.

[0020] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises heavy chain complementarity-determining regions (HCDRs), HCDR1, HCDR2 and HCDR3, and light chain complementarity-determining regions (LCDRs), LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12, HCDR3 comprises the amino acid sequence shown in SEQ ID NO:13, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:14, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:15 and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:16.

[0021] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 5.

[0022] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, 7, 8, 9, or 10.

[0023] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and wherein the VL comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:6.

[0024] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 3, 4, or 5, and wherein the VL comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, 8, 9, or 10.

[0025] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:1 and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:6.

[0026] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:2, 3, 4 or 5 and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:7, 8, 9 or 10.

[0027] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:2 and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:7.

[0028] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:2 and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:9.

[0029] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:3 and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:9.

[0030] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:4 and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:7.

[0031] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:4 and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:8.

[0032] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention is a murine antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the present invention provides an anti-OX40 antibody or its antigen-binding fragment, which is a full-length antibody, a single-domain antibody (such as VHH), Fab, Fab', Fab'-SH, (Fab')2, a single-chain antibody such as scFv, Fv, dAb (domain antibody), or a bispecific (multispecific) antibody.

[0033] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention includes an Fc region. In some embodiments, the amino acid sequence of the Fc region is identical to or a variant of the Fc region sequence of human IgG1, IgG2, or IgG4.

[0034] On the other hand, the present invention provides an anti-OX40 antibody agonist comprising the CDR of the antibody provided by the present invention and including an Fc region having an FcγR binding region. In some embodiments, the Fc region of the anti-OX40 antibody agonist has an amino acid sequence identical to the Fc region of human IgG1 or IgG2.

[0035] On the other hand, the present invention provides an anti-OX40 antibody antagonist comprising the CDR of the antibody provided by the present invention. In some embodiments, the anti-OX40 antibody antagonist comprises an Fc region variant that reduces or eliminates the binding of the Fc region to FcγR. In some embodiments, the anti-OX40 antibody antagonist comprises an Fc region variant, wherein the variant is IgG1N297A.

[0036] In a preferred embodiment, the present invention provides an isolated anti-OX40 antibody comprising...

[0037] (1) Heavy chain complementarity-determining regions (HCDRs), HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO:11, HCDR2 contains the amino acid sequence shown in SEQ ID NO:12 and HCDR3 contains the amino acid sequence shown in SEQ ID NO:13.

[0038] (2) Light chain complementarity-determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains the amino acid sequence shown in SEQ ID NO:14, LCDR2 contains the amino acid sequence shown in SEQ ID NO:15, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:16; and

[0039] (3) An Fc variant, which is human IgG1N297A. Preferably, the binding of the Fc variant to FcγR is reduced or eliminated. In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:2 and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:7. In one embodiment, the antibody is a full-length antibody. In other embodiments, the antibody has a high affinity (K0) for binding to human OX40. D The concentration is below 10 nM. In some further embodiments, the antibody is an OX40 antibody antagonist that blocks OX40-mediated signal transduction activity.

[0040] On the other hand, the present invention provides an isolated nucleic acid that encodes any antibody or fragment thereof provided by the present invention. Preferably, the nucleic acid encodes the heavy chain or light chain of the antibody of the present invention, or the variable region of the heavy chain or the variable region of the light chain.

[0041] On the other hand, the present invention provides a recombinant vector or expression vector comprising one or more nucleic acids provided by the present invention, wherein the vector is adapted for recombination to generate any of the antibodies or antigen-binding fragments provided by the present invention. In some embodiments, the vector is an expression vector.

[0042] On the other hand, the present invention provides a host cell comprising one or more nucleic acids, recombinant vectors or expression vectors provided by the present invention.

[0043] On the other hand, the present invention provides an immunoconjugate or immunofusion compound comprising the anti-OX40 antibody or its antigen-binding fragment provided by the present invention.

[0044] On the other hand, the present invention provides a pharmaceutical composition comprising the anti-OX40 antibody or its antigen-binding fragment provided by the present invention, nucleic acid, carrier, or host cell, and optionally comprising at least one pharmaceutically acceptable excipient (e.g., pharmaceutical carrier or pharmaceutical excipient).

[0045] On the other hand, the present invention also provides the use of the anti-OX40 antibody of the present invention or its antigen-binding fragment, nucleic acid, vector, host cell, immune conjugate or immune fusion in the preparation of a medicament for treating OX40-related diseases or symptoms.

[0046] On the other hand, the present invention also provides the use of the anti-OX40 antibody agonist of the present invention in the preparation of a medicament for treating cancer.

[0047] On the other hand, the present invention also provides the use of the anti-OX40 antibody antagonist of the present invention in the preparation of a medicament for treating inflammatory and / or autoimmune diseases.

[0048] On the other hand, the present invention provides a method for treating or preventing OX40-related diseases or symptoms, comprising administering to the individual an effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, immune conjugate or immune fusion, or pharmaceutical composition comprising the present invention. In some embodiments, the OX40-related disease or symptom is an inflammatory and / or autoimmune disease, such as graft-versus-host disease. In some embodiments, the OX40-related disease or symptom is cancer, such as melanoma, preferably metastatic melanoma.

[0049] The anti-OX40 antibody or its antigen-binding fragment of the present invention can also be combined with other therapeutic agents or treatment methods for the treatment or prevention of OX40-related diseases or symptoms.

[0050] On the other hand, the present invention also provides a method for detecting OX40 in a sample using the anti-OX40 antibody of the present invention or its antigen-binding fragment, for the purpose of diagnosing / detecting OX40-related diseases or symptoms.

[0051] This invention also covers any combination of any embodiments described herein. Any embodiment described herein, or any combination thereof, is applicable to any and all anti-OX40 antibodies or fragments thereof, methods, and uses of the invention described herein. Attached Figure Description

[0052] Figure 1 The study showed that the Hu38E11-IgG2 antibody promoted the secretion of IFNγ by human T cells activated by the anti-CD3 antibody.

[0053] Figure 2 This demonstrates the ability of the ELISA detection antibody Hu38E11 (IgG1N297A) to block the binding of OX40 and OX40L.

[0054] Figure 3 The results show the effect of ELISA detection of Hu38E11 (IgG1N297A) in blocking the activation of OX40L on T cells.

[0055] Figure 4 The antagonistic and agonistic activities of the anti-OX40 antibody Hu38E11 (IgG1N297A) were demonstrated based on a luciferase reporter gene assay.

[0056] Figure 5 The effects of Hu38E11 (IgG1N297A) on hPBMC-induced graft-versus-host disease were demonstrated. Invention Details

[0058] This invention provides an anti-OX40 antibody or its antigen-binding fragment, characterized by having a unique CDR sequence and exhibiting high affinity and high specificity for binding to human OX40. The anti-OX40 antibody or its antigen-binding fragment provided by this invention can be used as a standalone therapy or in combination with other therapies for the treatment of OX40-related diseases or conditions such as cancer, inflammation, or autoimmune diseases.

[0059] definition

[0060] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0061] To facilitate a better understanding of this invention, certain technical terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For specific definitions and terms in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0062] The term “about” refers to a value or composition within an acceptable range of error as determined by one of ordinary skill in the art, depending in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more standard deviations according to practice in the art. Alternatively, “about” can mean a range of up to 5%, 10%, or 20% (i.e., ±5%, ±10%, or ±20%).

[0063] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0064] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0065] The term "OX40" in this article refers to a type I transmembrane glycoprotein of about 50 kDa, which is a member of the tumor necrosis factor receptor superfamily. OX40 is also known as ACT35, CD134, or TNFRSF4. In this document, the term refers to any native OX40 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise stated. The term encompasses “full-length” unprocessed OX40 as well as any form of OX40 or any fragment thereof produced by intracellular processing. The term also includes variants of naturally occurring OX40, such as splice variants or allelic variants. In some embodiments, OX40 refers to the full-length OX40 or a fragment thereof from humans (such as a mature fragment lacking its signal peptide). In some embodiments, human OX40 refers to mature OX40 (with amino acid residues 1-28 as the leader peptide) consistent with the Uniprot#P43489 amino acid sequence or a fragment thereof (such as its extracellular domain). In some embodiments, the term also encompasses fusion proteins containing OX40 or a fragment thereof (such as its extracellular domain), such as fusion proteins containing the extracellular domain and Fc region of human OX40.

[0066] The term "OX40 ligand" or "OX40L" in this article refers to the unique ligand of OX40, also known as gp34, CD252, or TNFSF4. The human OX40 ligand is identical in amino acid sequence to uniprot#P23510 or a variant thereof. OX40L naturally forms a homologous trimeric complex on the cell surface and is primarily expressed on activated antigen-presenting cells (APCs), including activated B cells, mature conventional dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), macrophages, and Langerhans cells. It can also be expressed on other cell types, such as NK cells, mast cells, a subset of activated T cells, and vascular endothelial cells and smooth muscle cells.

[0067] The term "affinity" in this document refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed using the dissociation constant (K0). D The term is used to describe this. Examples of analyses known in the art for determining binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., ForteBio).

[0068] The terms “OX40 antagonist,” “OX40 inhibitor,” “OX40 antagonistic antibody,” “antagonistic OX40 antibody,” or “OX40 antibody antagonist” are used interchangeably herein and include antibodies capable of inhibiting and / or neutralizing OX40-mediated biological signal transduction activities. In some embodiments, for example by blocking or substantially reducing the binding of OX40 to OX40 ligands, OX40 antagonistic antibodies inhibit or reduce OX40-triggered signal transduction pathways and / or inhibit or reduce OX40-mediated cellular responses such as lymphocyte proliferation, cytokine expression, or lymphocyte survival.

[0069] The terms “OX40 agonist,” “OX40 agonist antibody,” “OX40 agonist antibody,” or “OX40 antibody agonist” are used interchangeably herein and include antibodies capable of promoting and / or enhancing OX40-mediated biological signal transduction activity. In some embodiments, for example by cross-linking an antibody to bind to OX40 and activate OX40-mediated biological signals, OX40 agonist antibodies promote or enhance OX40-triggered signal transduction pathways and / or promote or enhance OX40-mediated cellular responses such as lymphocyte proliferation, cytokine expression, or lymphocyte survival.

[0070] The term "OX40-related disease or symptom" in this document refers to non-physiological states related to the expression, function, or activity of OX40 or to OX40-mediated signal transduction activity, including but not limited to cancer, inflammation, and autoimmune diseases. In some embodiments, the disease will benefit from blocking OX40-mediated signal transduction. In other embodiments, the disease will benefit from activating OX40-mediated signal transduction.

[0071] The terms "immune response" or "immune reaction" are used interchangeably herein and refer to the action produced by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, resulting in the selective damage, destruction, or clearance from the body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation. In some embodiments, the OX40 antibody antagonist of the present invention can inhibit or reduce the immune response, for example, by reducing immune rejection in graft-versus-host disease. In some embodiments, the OX40 antibody agonist of the present invention can enhance anti-tumor immune responses.

[0072] The term "signal transduction" in this article refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of OX40L (ligand) to OX40 (receptor), that results in the transmission of a signal from one part of the cell to another. Generally, transduction involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that induce signal transduction. The penultimate process typically involves nuclear events that lead to changes in gene expression.

[0073] The terms "enhanced T cell function" or "T cell activating activity" in this article include inducing, evoking, or stimulating the renewal of effector or memory T cells, and / or maintaining or amplifying, and / or inducing, evoking, or stimulating the biological functions of effector or memory T cells. Examples of enhanced T cell function include: elevated levels of CD8+ cells relative to pre-intervention levels. + Increased interferon-gamma (IFN-γ) secretion from effector T cells, elevated INF-γ secretion from CD4+ memory and / or effector T cells, elevated CD4+ effector and / or memory T cell proliferation, elevated CD8+ effector T cell proliferation, and elevated antigen responsiveness (e.g., clearance). In one embodiment, the enhancement level relative to pre-intervention is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 500%, or higher. The method of measuring this enhancement is known to those skilled in the art. In some embodiments, the agonistic activity of the antibody against T cells is assessed by detecting the inflammatory factor IFNγ released by activated T cells in the presence of the antibody. In some embodiments, the EC50 value of the antibody promoting IFNγ release from T cells is determined, with a lower value indicating higher T cell agonistic activity. In some embodiments, the antibody exhibits higher T cell agonistic activity compared to a control OX40 agonist antibody (e.g., OX40mAb24).

[0074] The terms "reduced T cell function" or "T cell antagonistic activity" in this article include reducing, blocking, or diminishing the turnover of effector or memory T cells, and / or reducing, blocking, or diminishing the biological function of effector or memory T cells. Examples of reduced T cell function include: reduced levels of CD8+ cells relative to pre-intervention levels. +The reduction in interferon-gamma (IFN-γ) secretion from effector T cells, decreased INF-γ secretion from CD4+ memory and / or effector T cells, decreased CD4+ effector and / or memory T cell proliferation, decreased CD8+ effector T cell proliferation, and decreased antigen responsiveness (e.g., clearance) are observed. In one embodiment, the reduction level relative to pre-intervention is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 500%, or higher. The method of measuring this reduction is known to those skilled in the art. In some embodiments, the antagonistic activity of the antibody against T cells is assessed by detecting the inflammatory factor IFNγ released by activated T cells in the presence of the antibody and the OX40 ligand OX40L. In some embodiments, the IC50 value of the antibody blocking OX40-OX40L-mediated T cell release of IFNγ is determined, with a lower value indicating higher T cell antagonistic activity. In some embodiments, the antibodies of the present invention exhibit higher T-cell antagonistic activity compared to control OX40 antagonist antibodies (e.g., GBR830).

[0075] The terms “activity” or “bioactivity”, or “biological property” or “biocharacteristic”, used herein are interchangeable and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize OX40 activity in vivo or in vitro, the ability to enhance or activate OX40 in vivo or in vitro, T cell agonistic activity, and the IC50 of blocking OX40 binding to OX40L. 50 Blocking OX40-OX40L-mediated T cell activation IC 50 The in vivo stability and immunogenic properties of antibodies. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high expression levels of antibodies in mammalian cells. The aforementioned properties or characteristics can be observed, measured, or evaluated using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasma resonance analysis, in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0076] The term "antibody" in this document refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, CrossMab antibodies, or camel-derived single-domain antibodies.

[0077] The terms "whole antibody," "full-length antibody," and "intact antibody" are used interchangeably herein to refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity-determining regions (CDRs), interspersed with more conserved regions (framework regions (FRs)). A "complementarity-determining region" or "CDR" is a region within the antibody's variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. Heavy and light chain CDRs are typically referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. Sequential numbering. CDRs located within the variable domain of the antibody heavy chain are sequentially named HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of the antibody light chain are sequentially named LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions.

[0078] In a given VH or VL amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known schemes or combinations thereof, including, for example: the Chothia scheme (Chothia et al., Canonical structures for the hypervariable regions of immunoglobulins, Journal of Molecular Biology, 196, 901-917 (1987)); the Kabat scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), and Contact (University College London); and the North scheme (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406, 228-256 (2011)). The boundaries of the CDRs of the anti-OX40 antibody in this invention can be determined according to any scheme or combination thereof in the art and through human evaluation.

[0079] Antibody light chains can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of their constant domains. Antibody heavy chains can be classified into five main types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. Several of these types can be further subdivided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2.

[0080] "Antibody in IgG form" refers to an antibody whose heavy chain constant region is derived from IgG2. For example, an antibody in IgG2 form means that its heavy chain constant region is derived from IgG2.

[0081] The term "antigen-binding fragment" in this document refers to a fragment or derivative of an antibody. Typically, an antigen-binding fragment comprises at least one fragment (e.g., one or more CDRs) of the antigen-binding region or variable region of the antibody and retains at least some of the binding properties of the antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of the antigen-binding activity of its source antibody. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding activity of its source antibody.

[0082] It is anticipated that antibodies or their antigen-binding fragments may include conserved or non-conserved amino acid substitutions that do not significantly alter their biological activity (referred to as “conserved variants” or “functionally conserved variants” of antibodies). In a preferred aspect, the conserved substitutions are derived from the conserved substituted residues shown in Table A below, preferably the preferred conserved amino acid substituted residues shown in Table A.

[0083] Table A

[0084]

[0085]

[0086] An epitope is an antigenic region to which an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein.

[0087] The term “isolated anti-OX-40 antibody or antigen-binding fragment” used herein refers to the purified state of an anti-OX-40 antibody or antigen-binding fragment. For example, “isolated” may mean that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. However, as those skilled in the art will appreciate, the term “isolated” does not mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic application of the antibody described herein. In some embodiments, the isolated antibody or antigen-binding fragment may have a purity greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0088] The term "monoclonal antibody" in this document refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies through any particular method.

[0089] The term "chimeric antibody" in this article refers to an antibody possessing a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies originate from different species. Typically, the variable domain is derived from antibodies from laboratory animals such as rodents, while the constant domain sequence is derived from human antibodies. This results in chimeric antibodies being less likely to induce adverse immune responses in human subjects compared to antibodies from the aforementioned laboratory animals.

[0090] The term "humanized antibody" as used herein refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, a humanized antibody comprises at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of a human immunoglobulin. Optionally, a humanized antibody may contain at least a portion of the constant region (Fc) of a human immunoglobulin. In some cases, as is known to those skilled in the art, amino acid mutations may be introduced into the humanized antibody (e.g., variable domains, framework regions, and / or constant regions (if present)) to, for example, improve certain properties of the antibody; such antibody forms still fall within the scope of the "humanized antibody" of this invention.

[0091] As those skilled in the art will appreciate, antibodies can have a glycan form suitable for the cells used to produce the antibody. For example, when produced in mice, in mouse cells, or in hybridomas derived from mouse cells, antibodies may contain mouse glycans. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, antibodies may contain rat glycans.

[0092] The term "Fc region" as used herein is used to define a C-terminal region in an immunoglobulin heavy chain that contains at least a portion of a constant region. This term includes native sequence Fc regions and Fc region variants. Native sequence Fc regions encompass the various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0093] The terms "Fc variant" or "variant Fc region" used herein are interchangeable and refer to a polypeptide containing a modified Fc region relative to the native sequence Fc region. The Fc variants of the present invention are defined according to the amino acid modifications that constitute them. Thus, for example, N297A is an Fc variant in which asparagine is substituted for alanine at position 297 relative to the parent polypeptide, wherein the numbering follows the EU index. For example, human IgG1 N297A refers to such an Fc variant having a sequence having a human IgG1 Fc region substituted with N297A. Modifications can be additions, deletions, or substitutions. Substitutions can include naturally occurring and non-naturally occurring amino acids. Variants may contain non-natural amino acids.

[0094] The term "Fc receptor" or "FcR" as used herein describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is an FcγR (γ receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, as well as allelic variants and alternative splicing forms of those receptors. FcγRII includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immune receptor with a tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immune receptor with a tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Annu. Rev. Immunol. 15:203-234 (1997). Reviews of FcRs can be found, for example, Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and And de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" in this paper encompasses other FcRs, including those to be identified in the future. The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.). (al., J. Immunol. 24:249 (1994)) and regulate the in vivo homeostasis of immunoglobulins. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)). The in vivo binding and serum half-life of human FcRn to high-affinity binding peptides can be determined, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered peptides with a variant Fc region. WO 2000 / 42072 (Presta) describes antibody variants that increase or decrease binding to FcR.See also, for example, Shields et al., J.Biol.Chem.9(2):6591-6604 (2001).

[0095] The term "pharmaceuticalally acceptable excipient" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), pharmaceutical excipients, pharmaceutical carriers, or stabilizers that are administered together with the active substance.

[0096] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0097] In this article, "immunoconjugate" is an antibody conjugated to one or more other substances (including but not limited to cytotoxic agents or labels). "Immunofusion" is an antibody fused to one or more other peptides or polypeptides via covalent linkage.

[0098] The term "therapeutic agent" as used in this article covers any substance that is effective in preventing or treating related diseases, such as cancer.

[0099] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0100] "Chemotherapy agents" include small chemical molecule drugs that are useful in treating cancer or immune system diseases.

[0101] The term "small molecule drug" refers to low-molecular-weight compounds capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0102] As used herein, the term "immunomodulator" refers to a natural or synthetic active agent or drug that modulates (e.g., suppresses or enhances) an immune response. An immune response can be a humoral or cellular response. In some embodiments, the immunomodulator comprises an immunosuppressant that suppresses the immune response, such as an immunosuppressant beneficial for suppressing the immune response in the treatment of inflammation and autoimmune diseases. In some embodiments, the immunomodulator comprises an active agent or drug that enhances the immune response, such as an active agent or drug beneficial for enhancing the anticancer immune response in cancer treatment.

[0103] The terms “cancer” and “cancer malignancy” refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. This definition includes benign and malignant tumors, as well as dormant tumors or micrometastases. Cancer includes, but is not limited to, solid tumors and hematologic malignancies. Examples of various cancers include, but are not limited to, carcinomas, lymphomas, germ cell tumors, sarcomas, and leukemias.

[0104] "Inflammatory and / or autoimmune diseases" broadly encompasses any inflammatory or immune-related condition (e.g., pathological inflammation and autoimmune diseases). An "autoimmune disease" is a disease or symptom that arises from and targets an individual's own tissues or organs, or a co-isolation or manifestation thereof, or a condition arising from it. Autoimmune diseases can refer to conditions caused or exacerbated by the production of B cells with antibodies that react to normal body tissues and antigens. Similarly, autoimmune diseases can be diseases involving the secretion of autoantibodies specific to epitopes derived from self-antigens (e.g., nuclear antigens).

[0105] The term "vector" in this document refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is the viral vector, in which an additional DNA segment can be ligated into a viral genome. Some vectors are capable of autonomous replication in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). After introduction into the host cell, other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors can guide the expression of operatively linked genes. This document refers to such vectors as "expression vectors," which are nucleic acids capable of replicating and expressing a target gene upon transformation, transfection, or transduction into host cells. Expression vectors contain one or more phenotypic selection markers and origins of replication to ensure the maintenance of the vector and to provide amplification within the host when needed.

[0106] The terms “subject” or “patient” or “individual” in this article include any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc.

[0107] The terms "therapeutic effective amount," "therapeutic effective dose," and "effective amount" in this document refer to the amount of the anti-OX40 antibody or its antigen-binding fragment of the present invention, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions. Therapeutic effective dose also refers to an amount of antibody or its antigen-binding fragment sufficient to cause symptom improvement, such as the amount that treats, cures, prevents, or improves the associated medical condition, or accelerates the treatment, cure, prevention, or improvement of such condition. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in diagnostic criteria or parameters.

[0108] In this document, "treatment" includes 1) therapeutic measures that cure, alleviate, or reduce the symptoms of a diagnosed pathological condition or disease and / or halt the progression of the diagnosed pathological condition or disease, and 2) preventive or preventative measures that prevent and / or alleviate the development of a pathological condition or disease. Therefore, the treater includes an individual who has already contracted the disease, an individual who is susceptible to the disease, and an individual who wishes to prevent the disease. In some embodiments, the present invention relates to the treatment of a disease or symptom; in other embodiments, the present invention relates to the prevention of a disease or symptom.

[0109] In some embodiments of the invention, “treatment” for a disease or symptom means improving the disease or symptom (i.e., slowing or halting or reducing the progression of the disease or at least one of its clinical symptoms). In other embodiments, “treatment” means alleviating or improving at least one bodily parameter, including those physical parameters that may not be identifiable by the patient. In still other embodiments, “treatment” means regulating the disease or symptom physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of bodily parameters), or in both ways. Unless explicitly described herein, methods for assessing the treatment and / or prevention of diseases are generally known in the art.

[0110] In further embodiments of the invention, “prevention” of a disease or symptom includes the suppression of the occurrence or development of a disease or symptom, or a symptom of a particular disease or symptom. In some embodiments, subjects with a family history of cancer are candidates for preventative programs. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the occurrence of signs or symptoms of cancer, particularly prior to their occurrence in subjects at risk of cancer.

[0111] In some embodiments, cancer is considered successfully treated by the method of the present invention if the patient exhibits one or more of the following: a reduction or complete disappearance of the number of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs, including, for example, cancer spread to soft tissues and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduction in morbidity and mortality; improvement in quality of life; reduction in tumorigenesis, tumorigenesis frequency, or tumorigenesis capacity; reduction in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenesis cells into a non-tumorigenesis state; or a combination of some of these effects.

[0112] "Inhibition of tumor growth" refers to any mechanism by which tumor cell growth can be suppressed. In some embodiments, tumor cell growth is suppressed by delaying tumor cell proliferation. In some embodiments, tumor cell growth is suppressed by stopping tumor cell proliferation. In some embodiments, tumor cell growth is suppressed by killing tumor cells. In some embodiments, tumor cell growth is suppressed by inducing tumor cell apoptosis. In some embodiments, tumor cell growth is suppressed by inducing tumor cell differentiation. In some embodiments, tumor cell growth is suppressed by depriving tumor cells of nutrients. In some embodiments, tumor cell growth is suppressed by preventing tumor cell migration. In some embodiments, tumor cell growth is suppressed by preventing tumor cell invasion.

[0113] In this document, “sequence identity” refers to the degree of sequence similarity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. “(Percentage) sequence identity” can be calculated as follows: Two optimally aligned sequences are compared within a comparison window, and the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is determined to obtain the number of matching positions. The number of matching positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to produce the percentage of sequence identity. Optimal alignments for determining the percentage of sequence identity can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. In this invention, for antibody sequences, the percentage of amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with a reference antibody sequence, preferably according to the Kabat numbering rules.

[0114] In this article, "GBR830" refers to an OX40 antagonistic antibody obtained by transient expression based on the VH6 / VL9 heavy and light chain sequences disclosed in patent WO 2013008171; "OX40mAb24" refers to an OX40 agonist antibody obtained by transient expression based on the OX40mAb24 antibody heavy and light chain sequences disclosed in patent WO 2016057667; and "11D4" refers to an OX40 agonist antibody obtained by transient expression based on the 11D4 antibody heavy and light chain sequences disclosed in patent WO2009079335.

[0115] Anti-OX40 antibodies and their production

[0116] The antibodies of the present invention can be produced using any suitable method for antibody production. Any suitable form of OX40 can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any OX40 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells that produce murine monoclonal anti-human OX40 antibodies can be produced by methods known in the art. These methods include, but are not limited to, the hybridoma technique originally developed by Kohler et al. (1975) (Nature 256:495-497). Preferably, according to a standard protocol, mouse spleen cells are isolated and fused with mouse myeloma cell lines using PEG or by electrofusion. Hybridoma cells that secrete antibodies with OX40 binding activity are then screened. The DNA sequence of the immunoglobulin variable region of the hybridoma cells of the present invention can be determined using a degenerate primer-based PCR method.

[0117] Antibodies derived from rodents (such as mice) can induce unwanted antibody immunogenicity when used as therapeutic agents in vivo. Repeated use can lead to an immune response against the therapeutic antibody, which at least results in loss of therapeutic efficacy and, in severe cases, a potentially fatal allergic reaction. One approach to reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies, in which a mouse variable region is fused with a human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, the preservation of the intact rodent variable region in chimeric antibodies can still induce harmful immunogenicity in patients.

[0118] Transplanting rodent variable region CDRs onto human frameworks (i.e., humanization) has been used to further minimize rodent sequences. The humanized antibody described in this invention allows insertion of mouse CDR regions into human germline framework regions using methods known in the art. See U.S. Patent No. 5,225,539 to Winter et al. and U.S. Patents 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0119] The precise amino acid sequence boundary of the variable region CDR of the antibody of the present invention can be determined using any of many well-known schemes (e.g., Kabat, Chothia, AbM, Contact, or North). It should be noted that the boundaries of the CDR of the variable region of the same antibody may differ based on different definition systems. That is, the CDR sequence of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined with a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundary differs from the specific CDR boundary defined in this invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0120] Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs. However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining portion of the CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, any variant of the CDR given herein is also contemplated in this invention. In some embodiments, in a variant of a CDR of the anti-OX40 antibody of the present invention or its antigen-binding fragment, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or IMGT may be replaced by conserved amino acid residues.

[0121] In some embodiments, the present invention provides an anti-OX40 antibody or an antigen-binding fragment thereof comprising one or three of the heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence identical to or having at least one and no more than three, two, or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared to the amino acid sequence shown in SEQ ID NO: 11; HCDR2 comprises an amino acid sequence identical to or having at least one and no more than three, two, or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared to the amino acid sequence shown in SEQ ID NO: 12; and HCDR3 comprises an amino acid sequence identical to or having at least one and no more than three, two, or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared to the amino acid sequence shown in SEQ ID NO: 13.

[0122] In some embodiments, the present invention provides an anti-OX40 antibody or an antigen-binding fragment thereof comprising one or three of light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence identical to or having at least one and no more than three, two, or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared to the amino acid sequence shown in SEQ ID NO:14; LCDR2 comprises an amino acid sequence identical to or having at least one and no more than three, two, or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared to the amino acid sequence shown in SEQ ID NO:15; and LCDR3 comprises an amino acid sequence identical to or having at least one and no more than three, two, or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared to the amino acid sequence shown in SEQ ID NO:16.

[0123] In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof provided by the present invention further encompasses an antibody or antigen-binding fragment thereof wherein, on the three CDRs of the heavy chain variable region, relative to the three CDRs specifically disclosed herein, there are at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) and / or on the three CDRs of the light chain variable region, relative to the three CDRs specifically disclosed herein, there are at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).

[0124] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention further encompasses an antibody or its antigen-binding fragment wherein, compared with the heavy chain variable region and / or light chain variable region of the antibody specifically disclosed herein, the heavy chain variable region and / or light chain variable region has one or more (preferably no more than 10, more preferably no more than 6, 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conserved amino acid substitutions) in the amino acid sequence, preferably, the amino acid changes do not occur in the CDR region.

[0125] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 5.

[0126] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment provided by the present invention comprises a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, 7, 8, 9, or 10.

[0127] In one embodiment of the invention, the amino acid alterations described herein include substitution, insertion, or deletion of amino acids. Preferably, the amino acid alterations described herein are amino acid substitutions, and more preferably, conservative substitutions.

[0128] In a preferred embodiment, the amino acid change described in this invention occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid change described in this invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid change occurs in the heavy chain constant region and / or the light chain constant region.

[0129] In some embodiments, the antibodies of the present invention containing amino acid modifications have properties comparable to or similar to the specific antibodies disclosed herein.

[0130] In some embodiments, the anti-OX40 antibody of the present invention includes post-translational modifications to the CDR, light chain variable region, heavy chain variable region, light chain, or heavy chain.

[0131] In some embodiments, the anti-OX40 antibody provided by the present invention is a full-length antibody, a single-domain antibody such as VHH, Fab, Fab', Fab'-SH, (Fab')2, a single-chain antibody such as scFv, Fv, dAb (domain antibody), or a bispecific (multispecific) antibody.

[0132] In some embodiments, the anti-OX40 antibody provided by the present invention is any IgG isotype antibody, such as IgG1, IgG2, IgG3 or IgG4.

[0133] In some embodiments, the present invention also provides antibodies with altered effector functions. The term "effector function" refers to those biological activities attributable to the Fc region of an antibody that vary with antibody class. There are five main antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; recruitment of immune cells; and antibody cross-linking mediated by binding of the Fc region to cell surface FcR receptors. As those skilled in the art will understand, an appropriate antibody Fc region sequence can be selected based on needs, such as whether it is necessary to recruit the immune system to kill target cells or whether it is necessary to induce antibody cross-linking through interaction with FcR. For example, when immune system recruitment and target cell killing are desired properties of a target antibody, the Fc region of the antibody can be selected or further modified to provide enhanced binding to activated FcγR receptors and / or complement to promote effector functions such as ADCC or CDC. As another example, when immune system recruitment is not desired, the Fc region of the antibody can be selected or further modified to reduce this effector function; for example, the Fc region of human IgG2 or IgG4 subtypes can be used, or the Fc region of IgG1 subtypes with mutations such as N297A can be used. Furthermore, by selecting or mutating the Fc region, binding of the antibody to one or more Fc receptors can be selectively provided, while binding to another or more Fc receptors can be reduced or eliminated, thereby achieving modulation of antibody effector function, such as enhancing antibody crosslinking while altering ADCC activity intensity. See, for example, Xinhua Wang et al., IgG Fc engineering to modulate antibody effector functions, Protein Cell 2018, 9(1): 63-73, DOI 10.1007 / s13238-017-0473-8; ShieldsRL, High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn and Design of IgG1Variants with Improved Binding to theFcγR,2001,J Biol Chem.2001Mar2;276(9):6591-604.Epub 2000Nov 28.

[0134] This invention provides antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, and certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / attenuation of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity or antibody crosslinking activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Tmmunol. 9:457-492 (1991). The binding sites on human IgG1 with FcγRI, FcγRII, FcγRIII and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276: 6591-6604, 2001).

[0135] In some embodiments, the Fc region of the antibody provided by the present invention may be modified with one or more amino acid modifications to produce Fc region variants. Fc region variants may comprise human Fc region sequences (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions. For example, in Bruhns and... In an article published in Immunol Rev. 2015 Nov; 268(1):25-51, on page 44, several modifications to human IgG1 to enhance or reduce its binding to FcγR and enhance or reduce the corresponding function were summarized.

[0136] In some embodiments, the antibody provided by the present invention comprises a human IgG1 Fc region variant having reduced or absent FcγR binding activity (such as antibody crosslinking activity). In some embodiments, the human IgG1 Fc region variant comprises one or more amino acid substitutions, specifically, the amino acid substitutions may be selected from amino acid substitutions at positions E233, L234, L235, N297, and P331 of the immunoglobulin heavy chain. In some embodiments, the human IgG1 Fc region variant comprises one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297G, N297D, and P331S. In some embodiments, the amino acid substitution of the human IgG1 Fc region variant is N297A.

[0137] On the one hand, the antibodies presented herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. Glycosylation can be modified to, for example, increase the antibody's affinity for an "antigen." This modification of the carbohydrate can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be performed, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This glycosylation-free modification can increase the antibody's affinity for the antigen. Such methods are described, for example, in U.S. Patent No. 5,426,300. When an antibody contains an Fc region, the sugars attached to it can be modified. In some applications, modification to remove unwanted glycosylation sites can be useful, for example, removing the fucose module to improve antibody-dependent cell-mediated cytotoxicity (ADCC) function. In other applications, galactosylation modification can be performed to modify complement-dependent cytotoxicity (CDC).

[0138] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0139] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0140] In some embodiments, the antibodies of the present invention have one or more of the following characteristics:

[0141] (i) K with high affinity, for example, less than 100 nM, for example, less than 50 nM, for example, less than 30 nM, preferably less than 10 nM or 5 nM. D Value, binds to human OX40, especially to the extracellular domain of human OX40, wherein K is preferred. D The values ​​were measured using the surface plasmon resonance method.

[0142] (ii) Binding to human OX40 expressed on the surface of cells (such as T cells) with high affinity, for example, with an EC50 value of less than 100 nM, for example, less than 50 nM, for example, less than 40 nM, preferably less than 20 nM, more preferably less than 10 or 5 nM, wherein the EC50 value is preferably measured by FACS assay.

[0143] (iii) Blocking the binding of human OX40 and its ligand OX40L, for example, as determined by ELISA, with an inhibition rate of at least 50%, for example, at least 60%, 70%, 80%, 85% or 90%, and preferably with an IC50 value of less than 10 nM, more preferably less than 1 nM;

[0144] (iv) Demonstrates the same or similar binding affinity and / or specificity as any of the antibodies listed in Table 2;

[0145] (v) Inhibit (e.g., competitively inhibit) the binding of any of the antibody molecules shown in Table 2 to OX40;

[0146] (vi) binds to the same or overlapping epitopes as any of the antibodies shown in Table 2;

[0147] (vii) has the same or similar biological activity as any of the antibodies shown in Table 2.

[0148] In some embodiments, the OX40 antibody of the present invention is an agonist antibody comprising an Fc region having an Fc region, such as a human IgG1, IgG2, or IgG4 Fc region or a variant thereof, binding to an FcR, such as FcγR, preferably a human IgG1 or IgG2 Fc region or a variant thereof. The variant preferably has an FcγR binding affinity equivalent to or stronger than that of the parental Fc region (e.g., the natural sequence Fc region). Preferably, the antibody cross-links with FcγR expressed on the cell surface through the binding of its Fc region. Preferably, the antibody comprises a human IgG1 or IgG2 Fc region sequence with a constant region sequence as shown in SEQ ID NO: 21 or 22, or comprises a human IgG1 or IgG2 Fc region variant having at least 95%, 96%, 97%, or 99% identity with the constant region sequence as shown in SEQ ID NO: 21 or 22, or having an Fc region modified with no more than 10, 5, or 1-3 amino acids as shown in the constant region sequence as shown in SEQ ID NO: 21 or 22.

[0149] In some embodiments, the OX40 agonist antibody of the present invention has one or more of the following characteristics:

[0150] (i) K with high affinity, for example less than 10 nM, more preferably less than 5 nM D Value, combined with human OX40, wherein K is preferably DThe values ​​were measured using the surface plasmon resonance method.

[0151] (ii) Binding to human OX40 expressed on the surface of cells (e.g., activated CD4+ T cells) with high affinity, for example, less than 10 nM, more preferably less than 5 nM EC50 value, wherein preferably the EC50 value is measured by FACS assay;

[0152] (iii) Activation of OX40-mediated signal transduction activity;

[0153] (iv) It has T cell agonistic activity, for example, the agonistic activity of the antibody on T cells can be assessed by detecting cytokines such as IFNγ released by activated T cells in the presence of the antibody. In some embodiments, the EC50 value of the antibody is less than 10 nM, preferably less than 5 nM.

[0154] (v) Inhibit tumor growth, such as inhibiting the growth of melanoma cells.

[0155] In some embodiments, the OX40 antibody of the present invention is an antagonistic antibody. In some embodiments, the antibody comprises an Fc region variant, wherein, for example, relative to the parental Fc region (e.g., the natural sequence Fc region), the Fc region variant has reduced or substantially eliminated binding affinity to FCγR. In some embodiments, the antibody of the present invention substantially does not bind to FcγR expressed on the cell surface, and no FcγR-mediated antibody crosslinking occurs. In some embodiments, the antibody of the present invention comprising an Fc region variant has reduced or eliminated FcγR-mediated effector function relative to a corresponding antibody comprising the parental Fc region (e.g., the natural sequence Fc region). Preferably, the Fc region of the antibody comprises a mutation selected from E233P, L234A, L235A, L235E, N297A, N297G, N297D, P331S, or combinations thereof. More preferably, the Fc region of the antibody is a human IgG1 Fc region comprising the N297A mutation. In some embodiments, the antibody comprises a human IgG1 Fc region sequence that is identical to the Fc region sequence of the constant region sequence shown in SEQ ID NO:21, or comprises a human IgG1 Fc region variant having at least 95%, 96%, 97%, 98%, or 99% identity with the Fc region sequence of the constant region sequence shown in SEQ ID NO:21, or having no more than 10, 5, or 1-3 amino acid modifications to the Fc region sequence of the constant region sequence shown in SEQ ID NO:21, and comprises a mutation that reduces the affinity of the Fc region for binding to FcγR, preferably the N297 mutation, more preferably the N297A.

[0156] In some embodiments, the OX40 antagonistic antibody of the present invention has one or more of the following characteristics:

[0157] (i) K with high affinity, for example less than 10 nM, more preferably less than 5 nM D Value, combined with human OX40, wherein K is preferably D The values ​​were measured using the surface plasmon resonance method.

[0158] (ii) Binding to human OX40 expressed on the surface of cells (e.g., activated CD4+ T cells) with high affinity, for example, less than 10 nM, more preferably less than 5 nM EC50 value, wherein preferably the EC50 value is measured by FACS assay;

[0159] (iii) Blocking the binding of OX40 and its ligand OX40L, for example, as determined by ELISA, the inhibition rate reaches at least 70%, preferably at least 80%, 85% or 90%, and preferably the IC50 value is less than 10 nM, more preferably less than 1 nM;

[0160] (iv) Blocking OX40-mediated signal transduction activity;

[0161] (v) It has T cell antagonistic activity, for example, the antibody’s blocking effect on OX40L-mediated T cell activation can be evaluated by detecting cytokines such as IFNγ released by T cells in the presence of the antibody and ligand OX40L, and in some embodiments, the antibody’s IC50 value is less than 5 nM, preferably less than 1 nM.

[0162] (vi) It exhibits anti-immune rejection activity, such as reducing immune rejection in graft-versus-host disease.

[0163] antibody expression

[0164] The present invention relates to host cells comprising one or more expression vectors and methods for generating any antibody or antigen-binding fragment thereof of the present invention, the methods comprising culturing the host cells, purifying and recovering the antibody or antigen-binding fragment.

[0165] In one aspect, the present invention provides nucleic acids encoding any of the above-described anti-OX40 antibodies or their antigen-binding fragments. For example, the present invention provides nucleic acids encoding segments comprising the heavy chain, light chain, variable region, or complementarity-determining region described herein. In some aspects, the nucleic acid encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 17 or 18. In some aspects, the nucleic acid encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 19 or 20.

[0166] In one aspect, one or more vectors containing the nucleic acid are provided. In some embodiments, the vector is an expression vector. The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operatively linked to a nucleic acid encoding an anti-OX40 antibody or an antigen-binding fragment thereof. In some embodiments, the expression vector also contains a sequence encoding the constant region of the antibody.

[0167] In one aspect, the present invention provides host cells, including prokaryotic or eukaryotic cells, for expressing the recombinant antibodies of the present invention. In some embodiments, *Escherichia coli* is a prokaryotic host that can be used to clone and express the nucleic acids of the present invention. Other suitable microbial hosts include bacilli, such as *Bacillus subtilis*, and other Enterobacteriaceae, such as *Salmonella*, *Serratia marcescens*, and various *Pseudomonas*. Expression vectors can also be prepared in these prokaryotic hosts, which typically contain expression control sequences (e.g., origin of replication) compatible with the host cells. In some embodiments, mammalian host cells are used to express and produce the anti-OX40 antibody peptides of the present invention. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines with exogenous expression vectors, including normal human cells or immortalized animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas.

[0168] In one aspect, the present invention provides a method for preparing anti-OX40 antibodies, wherein the method includes introducing an expression vector into mammalian host cells, culturing the host cells for a sufficient period of time to allow the antibody to be expressed in the host cells, or more preferably, secreting the antibody into the culture medium in which the host cells grow, to produce the antibody. The antibody can be recovered from the culture medium using standard protein purification methods. The antibody molecules prepared as described herein can be purified using known prior art techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these are obvious to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc.

[0169] Antibodies expressed in different cell lines or in transgenic animals are likely to have different glycosylations. However, all antibodies encoded by the nucleic acids provided herein or containing the amino acid sequences provided herein are part of this invention, regardless of their glycosylation.

[0170] Determination method

[0171] The anti-OX40 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. On one hand, the antigen-binding activity of the antibodies of the present invention can be tested, for example by known methods such as ELISA, Western blotting, etc. Binding to OX40 can be determined using methods known in the art, and exemplary methods are disclosed herein.

[0172] The present invention also provides an assay for identifying biologically active anti-OX40 antibodies. Biological activity may include, for example, binding to OX40 (e.g., binding to human OX40), enhancing OX40-mediated signal transduction (e.g., enhancing NF-κB-mediated transcription), enhancing T effector cell function (e.g., by enhancing effector T cell proliferation and / or enhancing effector T cell cytokine production (e.g., interferon-gamma)), etc. Antibodies exhibiting such biological activity in vivo and / or in vitro are also provided.

[0173] In some embodiments, the antibodies of the present invention are tested for such biological activity.

[0174] Cells used for any of the above in vitro assays include cell lines that naturally express OX40 or are engineered to express OX40, such as tumor cell lines. This class of cells also includes cell lines that express OX40 and, if not normally, cell lines transfected with DNA encoding OX40.

[0175] It is understood that the immunoconjugates or immunofusions of the present invention can be used to replace or supplement the anti-OX40 antibody for any of the above-described assays.

[0176] Understandably, any of the above assays can be performed using a combination of anti-OX40 antibodies and other active agents.

[0177] Immunoconjugates and Immunofusions

[0178] In some embodiments, the present invention provides an immunoconjugate comprising any anti-OX40 antibody or its antigen-binding fragment provided herein, and other substances. In one embodiment, the other substances are, for example, cytotoxic agents.

[0179] In some embodiments, the present invention provides an immunofusion comprising any provided anti-OX40 antibody or its antigen-binding fragment.

[0180] In some embodiments, the immune conjugate and the immune fusion are used to prevent or treat OX40-related diseases or symptoms.

[0181] Pharmaceutical Composition

[0182] The pharmaceutical compositions of the present invention may include the antibodies of the present invention and pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions of the present invention may be included in a kit, and in other embodiments, the pharmaceutical compositions of the present invention may be included in a reagent kit, such as a diagnostic kit.

[0183] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Pharmaceutical carriers suitable for this invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when administering pharmaceutical compositions intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0184] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For information on the use and applications of excipients, see "Handbook of Pharmaceutical Excipients," 5th Edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago. The compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, saccharin, etc.

[0185] This invention provides pharmaceutical compositions comprising one or more monoclonal antibodies or antigen-binding fragments thereof that bind to OX40, nucleic acids, vectors or host cells or immune conjugates or immune fusions. It should be understood that the anti-OX40 antibodies or antigen-binding fragments thereof, nucleic acids, vectors or host cells or immune conjugates or fusions provided by this invention can be integrated into pharmaceutical compositions with suitable pharmaceutical carriers, excipients and other reagents for co-administration, thereby providing improved transfer, delivery, tolerability, etc.

[0186] Pharmaceutical formulations comprising the anti-OX40 antibody described herein can be prepared by mixing the anti-OX40 antibody of the present invention, or an antigen-binding fragment thereof, having the desired purity, with one or more optional pharmaceutically acceptable excipients, preferably in the form of an aqueous solution or a lyophilized formulation. Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, the latter comprising a histidine-acetate buffer.

[0187] The pharmaceutical compositions or formulations of the present invention may also contain one or more other active ingredients required for the treatment of a specific disease, preferably those having complementary activities that do not adversely affect each other. For example, it is desirable to also contain other therapeutic agents. In some embodiments, the other therapeutic agents are chemotherapeutic agents, radiotherapy agents, cytokines, vaccines, other antibodies, immunomodulators, or other biological macromolecular drugs.

[0188] In some embodiments, the pharmaceutical compositions of the present invention further comprise a composition encoding a nucleic acid encoding an anti-OX40 antibody or an antigen-binding fragment thereof.

[0189] Method or use

[0190] This invention provides methods for preventing, diagnosing, or treating OX40-related diseases or symptoms. The method comprises administering to a patient in need an effective amount of an anti-OX40 antibody or its antigen-binding fragment, or an immunoconjugate or immunofusion containing the same, or a pharmaceutical composition, or a nucleic acid, vector, or host cell as described herein.

[0191] In one aspect, the present invention provides the use of an anti-OX40 antibody or an antigen-binding fragment thereof, or an immunoconjugate or immunofusion thereof, or a pharmaceutical composition comprising the same, in the production or preparation of a medicament for the prevention or treatment of OX40-related diseases or symptoms in a subject.

[0192] In one aspect, the anti-OX40 antibody and its antigen-binding fragment, and the pharmaceutical composition comprising thereof provided by the present invention can be used as a therapeutic agent for the prevention or treatment of OX40-related diseases or symptoms in subjects. For OX40-related diseases or symptoms identified in subjects using standard methods, the anti-OX40 antibody and its antigen-binding fragment, and the pharmaceutical composition or immunoconjugate or immunofusion product disclosed in this invention, or the nucleic acid, vector, or host cell described herein, can be administered.

[0193] In some embodiments, the methods and uses described herein further include administering an effective amount of at least one additional therapeutic agent or treatment to the individual. In some embodiments, the therapeutic agent may be, for example, a chemotherapeutic agent, a radiotherapy agent, a cytokine, a vaccine, other antibodies, immunomodulators, or other biological macromolecular drugs. In some embodiments, the treatment may include surgery; radiotherapy, local irradiation, or focused irradiation, etc.

[0194] The aforementioned combination therapy includes combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, wherein administration of the anti-OX40 antibody of the present invention or its antigen-binding fragment may occur before, simultaneously with, and / or after administration of other therapeutic agents and / or adjuvants and / or modes of treatment.

[0195] In some embodiments, the OX40-related diseases or symptoms described in this invention refer to diseases or symptoms associated with abnormalities in OX40 expression, activity, and / or signaling in a subject, including but not limited to cancer, inflammation, and autoimmune diseases. In some embodiments, in OX40-related diseases or symptoms, there is an increase in the level or content of nucleic acid encoding OX40, or an increase in OX40 expression, or an increase in the level or activity of OX40 protein, or an enhancement of OX40-mediated signaling. In other embodiments, in OX40-related diseases or symptoms, there is a decrease in the level or content of nucleic acid encoding OX40, or a decrease in OX40 expression, or a decrease in the level or activity of OX40 protein, or a decrease in OX40-mediated signaling.

[0196] In some implementations, treatment of the disease or symptom will benefit from inhibiting OX40 levels in nucleic acids or proteins, or from blocking the binding of OX40 to its ligands, or from inhibiting OX40-mediated signal transduction.

[0197] In other implementations, treatment of the disease or symptom will benefit from increased levels of OX40 nucleic acids or proteins, or from enhanced OX40-mediated signaling.

[0198] In some implementations, OX40-related diseases or symptoms are cancer. Specifically, cancers include, but are not limited to, solid tumors, breast cancer, urothelial carcinoma, melanoma, kidney cancer, ovarian cancer, head and neck cancer, stomach cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, prostate cancer, lymphocytic leukemia, and sarcoma. Preferably, the antibody used for the prevention, diagnosis, or treatment of OX40-related cancers is an OX40 agonist.

[0199] In some embodiments, OX40-related diseases or symptoms are inflammatory and / or autoimmune diseases. In some embodiments, OX40-related inflammatory and / or autoimmune diseases are selected from atopic dermatitis, rheumatoid arthritis, asthma (e.g., allergic asthma), COPD, autoimmune uveitis, multiple sclerosis, lupus (e.g., systemic lupus erythematosus), ulcerative colitis, scleroderma, and graft-versus-host disease (GVHD). Preferably, the antibody used to treat or prevent OX40-related inflammatory and / or autoimmune diseases is an OX40 antagonist.

[0200] In some embodiments, the subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein (e.g., cancer). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy.

[0201] The antibodies or antigen-binding fragments of the present invention can be administered by any suitable route, including oral, parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be carried out by any suitable route, such as by injection, for example, intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing regimens are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusion.

[0202] The antibody or antigen-binding fragment of the present invention will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to those skilled in the art. Optionally, the antibody is formulated with one or more agents currently used for the prevention or treatment of said disease. The effective amount of these other agents depends on the amount of antibody present in the formulation, the type of symptom or treatment, and other factors discussed above.

[0203] For the prevention or treatment of disease, the appropriate dosage of the antibody or antigen-binding fragment of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody may be appropriately administered to the patient in a single treatment or a series of treatments.

[0204] In some embodiments, any anti-OX40 antibody or its antigen-binding fragment provided herein can be used to detect the presence of OX40 in a biological sample. The term "detection" as used herein includes both quantitative and qualitative detection. In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is derived from a lesion associated with hyperplastic or cancerous lesions.

[0205] In one embodiment, the antibodies of the present invention or their antigen-binding fragments can be used to diagnose OX40-related diseases or symptoms, such as cancer, for example, to evaluate (e.g., monitor) the treatment or progression of the diseases described herein in an individual, their diagnosis, and / or staging. In some embodiments, labeled anti-OX40 antibodies or their antigen-binding fragments are provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions. In some embodiments, kits for diagnosing OX40-related diseases are provided herein, which contain the antibodies of the present invention or their antigen-binding fragments.

[0206] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-OX40 antibody or its antigen-binding fragment. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0207] This invention includes any combination of the specific embodiments described herein. It should be understood that although specific content and examples have been described to illustrate preferred embodiments of the invention, this is merely illustrative and for example purposes, and the invention also covers embodiments that, upon which modifications to the preferred embodiments of the invention will be obvious to those skilled in the art. For all purposes, all disclosures, patents, and patent applications cited herein, including in citations, are incorporated herein by reference in their entirety. Example

[0208] Example 1: Preparation and Screening of Hybridoma Antibodies

[0209] OX40 antibodies were obtained using hybridoma technology, and recombinant protein OX40-Fc (R&D, Cat 3388-OX), a human OX40 extracellular domain tagged with Fc, was used as an antigen to immunize mice. In short, OX40-Fc was mixed with complete or incomplete Freund's adjuvant (Sigma-Aldrich) and emulsified before immunizing C57BL / 6 and BALB / c mice. After one round of immunization (complete Freund's adjuvant) and two rounds of booster immunization (incomplete Freund's adjuvant), blood was collected after each booster immunization. The binding activity of the mouse serum to recombinant human OX40-His (R&D Systems, Cat 9969-OX) protein was detected by ELISA, and the binding titer of mouse serum to CHO cells (GenScript construction) overexpressing human OX40 was detected by flow cytometry (FACS). Mice with high serum titers were selected for fusion. Four days prior to fusion, mice were intraperitoneally injected with the recombinant protein OX40-Fc, containing the extracellular domain of human OX40, to enhance immunization. On the day of fusion, mice were euthanized, and their spleens were homogenized to obtain a single-cell suspension. Mouse spleen cells were fused with the mouse myeloma cell line SP2 / 0 (purchased from ATCC) using an electrofusion apparatus. The fused cells were resuspended in HAT (hypoxanthine, aminopterin, and thymidine deoxyribonucleoside, GIBCO, Cat 21060017) medium and seeded into 96-well plates. The cells were cultured at 37°C for 7 days. The activity of the hybridoma secretory supernatant was assessed using OX40-related functional platforms (such as specificity for binding to human OX40 and T-cell activation function). Positive hybridoma clones were subjected to single or multiple rounds of subcloning to obtain single clones. After screening, 38E11 was selected as the preferred hybridoma clone (its secreted antibody is designated 38E11).

[0210] Candidate hybridoma cells 38E11 were cultured for 7 to 10 days. The supernatant was collected, centrifuged, and filtered to remove cells and debris. The supernatant was passed through a protein A purification column (Genscript), followed by washing and equilibration with buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0), eluting with 0.1 M sodium citrate (pH 3.5), and immediately neutralized with 1 / 9 volume of 1 M Tris-HCl (pH 9), then dialyzed against PBS buffer. The resulting hybridoma antibody 38E11 was used for further characterization.

[0211] 1.1 ELISA detection of antibody binding activity to OX40 extracellular domain protein

[0212] Recombinant human OX40-His (R&D, Cat 9969-OX) was coated onto 96-well plates, blocked, and then serially diluted mouse serum or antibody was added and incubated. The plates were washed with PBS containing 0.5% Tween 20, and then incubated with HRP-labeled anti-mouse IgG secondary antibody. The plates were then developed with TMD and the OD450 was read using a microplate reader.

[0213] As shown in Table 1, the final hybridoma antibody 38E11 exhibits high binding activity to human OX40 protein, EC 50 It is 0.276 nM.

[0214] 1.2 FACS detection of antibody binding activity to OX40 on activated T cells

[0215] Primary PBMCs were obtained by collecting the intermediate layer from whole blood of healthy individuals using density gradient centrifugation with a Ficoll-Paque PLUS (GE Healthcare, Cat 17-1440-02) and washing three times with PBS. T cells were then isolated using the Pan T Cell Isolation Kit (Miltenyi biotec, Cat 130-096-535) according to the manufacturer's instructions via magnetic bead separation. The T cells were resuspended in RPMI 1640 medium (containing 10% FBS and penicillin / streptomycin antibiotics), and PHA-L and IL-2 (or Con-A and hIL-2) were added. Stimulation was performed for 2 days to induce OX40 expression in the T cells. The activated T cells were washed once with PBS containing 2% FBS, and serially diluted OX40 antibody was added. The cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS containing 2% FBS, and then PE-labeled anti-human IgG secondary antibody (Biolegend, Cat 409304) (or PE-labeled anti-mouse IgG secondary antibody (Biolegend, Cat 405307)) and APC-CY7-labeled anti-human CD4 antibody (Biolegend, Cat 300518) were added. The binding of OX40 antibody to the surface of CD4-positive T cells was detected using a BD CantoII flow cytometer. EC50 was calculated based on a curve fitted with the average fluorescence intensity values. 50 .

[0216] As shown in Table 1, hybridoma antibody 38E11 has binding activity to OX40 on human activated T cells, EC 50 It is 0.8 nM.

[0217] 1.3 Assay of antibody-mediated T-cell agonistic activity

[0218] The agonistic activity of antibodies against T cells was assessed by detecting the release of the cytokine IFNγ from T cells after activation. In short, the intermediate layer of whole blood from healthy individuals was collected by density gradient centrifugation using a Ficoll-Paque PLUS (GE Healthcare, Cat 17-1440-02) and washed three times with PBS to obtain primary PBMCs. Human primary T cells were then isolated using the Pan T Cell Isolation Kit (Miltenyi biotec, Cat 130-096-535) according to the manufacturer's instructions via magnetic bead separation. The T cells were resuspended in RPMI 1640 medium (containing 10% FBS and penicillin / streptomycin). Anti-CD3 antibody (eBioscience, Cat 16-0037-85) and serially diluted OX40 antibody were mixed and added at 100 μL per well to a 96-well plate, and the plates were incubated at 37°C for 2 h. Wash away the uncoated antibodies with PBS, add the isolated T cells to the wells, and collect the supernatant after culturing for 3 days. Detect the IFNγ concentration in the supernatant using ELISA (R&D, Ct SIF50) according to the standard detection method recommended in the instruction manual.

[0219] As shown in Table 1, hybridoma antibody 38E11 promotes the secretion of IFNγ by T cells, and its EC50 50 The value is 1.4nM.

[0220] Table 1 also shows the functional activities of the control anti-OX40 antibody 11D4 and OX40mAb24 obtained through transient expression.

[0221] Table 1. Functional activity identification of hybridoma antibody 38E11

[0222]

[0223] *Background: Control group without corresponding anti-OX40 antibody

[0224] Example 2: Humanization of Hybridoma Antibodies

[0225] 2.1 Determination of the variable region sequence of hybridoma antibody

[0226] Using hybridoma sequencing, cells of hybridoma clone 38E11 were expanded and cultured, and total RNA was extracted using TRIzol (purchased from Ambio). This RNA was then reverse transcribed into DNA using antibody-specific primers (Takara, PrimerScript 1). stThe Strand cDNA Synthesis Kit was used to amplify and clone the gene fragment encoding the V-region of mouse immunoglobulin using antibody-specific primers. The variable region sequence was obtained by sequencing analysis. The nucleotide sequence of the 38E11 heavy chain variable region is shown in SEQ.ID No.:17, and the nucleotide sequence of the light chain variable region is shown in SEQ.ID No.:18.

[0227] 2.2 Humanization Design of Hybridoma Antibodies

[0228] For antibody humanization, the PDB Antibody database was first used to search for human germline immunoglobulin genes with sequences highly homologous to the variable regions of murine antibodies. The 38E11 heavy chain variable region and light chain variable region showed high sequence homology with human germline IGHV1-46*01 and human germline IGKV4-1*01, respectively. The amino acid sequence and precise boundaries of the variable region CDR were then defined using the Kabat numbering system. In principle, human IGVH and IGVk, which have high homology to the murine antibody variable regions, were selected as templates for humanization, and humanization was achieved through CDR grafting.

[0229] To maintain the activity of humanized antibodies, computer simulation techniques are generally used to analyze the variable region and its surrounding framework amino acid sequences through molecular docking, examining their spatial and stereobinding mechanisms. By calculating electrostatic forces, van der Waals forces, hydrophilicity / hydrophobicity, and entropy, key amino acid individuals in each candidate antibody gene sequence that can interact with OX40 and maintain the spatial framework are analyzed. These individuals are then grafted back into the selected human antibody gene framework, and the amino acid sites in the framework region that must be retained are identified, leading to the synthesis of humanized antibodies. Seven sites were selected in the variable region of the 38E11 antibody heavy chain for reversion mutations: V20L, M48I, R67K, M70L, R72V, V79A, and T91S. Based on the number and arrangement of reversion mutations, four different humanized heavy chains were designed: VH1 (SEQ.ID No.:2), VH2 (SEQ.ID No.:3), VH3 (SEQ.ID No.:4), and VH4 (SEQ.ID No.:5). Three sites were selected in the variable region of the 38E11 antibody light chain for corresponding reversion mutations: M4L, V62I, and L82V, to design four different humanized light chains: VL1 (SEQ.ID No.:7), VL2 (SEQ.ID No.:8), VL3 (SEQ.ID No.:9), and VL4 (SEQ.ID No.:10). Thus, a total of 38E11 humanized antibody Hu38E11 and its variants Hu38E11-v1, Hu38E11-v2, Hu38E11-v3, and Hu38E11-v4 were designed and further characterized. The amino acid sequences of each antibody are shown in Tables 2 and 3.

[0230] 2.3 Expression of humanized antibodies

[0231] The variable region derived from hybridoma antibody 38E11 or its humanized sequence was amplified and cloned into a vector containing the constant region of human IgG to obtain the expression plasmid. The heavy chain constant region of the antibody can be selected from any subtype of human IgG (such as human IgG1, whose heavy chain constant region amino acid sequence is shown in SEQ.ID No.:21, and human IgG2, whose heavy chain constant region amino acid sequence is shown in SEQ.ID No.:22) or its variants. Unless specifically marked, the heavy chain constant region of Hu38E11 and its variants is identical to the heavy chain constant region sequence of human IgG1. The expression vector containing the heavy and light chains was co-transfected into 293 cells. After culturing at 37°C for 4-6 days, the supernatant was collected and purified by protein A affinity according to the aforementioned method to obtain the recombinant antibody for further antibody characterization.

[0232] Table 2. Amino acid sequences contained in anti-OX40 antibodies.

[0233] 38E11 and its variants VH amino acid sequence VL amino acid sequence 38E11 SEQ ID No.:1 SEQ ID No.:6 Hu38E11 SEQ ID No.:2 SEQ ID No.:7 Hu38E11-v1 SEQ ID No.:3 SEQ ID No.:9 Hu38E11-v2 SEQ ID No.:4 SEQ ID No.:7 Hu38E11-v3 SEQ ID No.:2 SEQ ID No.:9 Hu38E11-v4 SEQ ID No.:4 SEQ ID No.:8

[0234] Table 3. CDR amino acid sequence of anti-OX40 antibody (Kabat definition)

[0235] CDR amino acid sequence HCDR1 SEQ ID No.:11 HCDR2 SEQ ID No.:12 HCDR3 SEQ ID No.:13 LCDR1 SEQ ID No.:14 LCDR2 SEQ ID No.:15 LCDR3 SEQ ID No.:16

[0236] Example 3: FACS detection of the binding activity of humanized antibody to OX40 on activated T cells.

[0237] As described in Example 1.2 above, the detection method utilizes FACS to analyze the binding activity of candidate-derived antibodies to OX40 on activated human T cells.

[0238] Results: As shown in Table 4, the humanized antibody Hu38E11 and its variants exhibited good binding activity to OX40 on the surface of activated human T cells.

[0239] Table 4. Binding activity of antibody Hu38E11 and its variants to OX40 on activated T cells.

[0240] Antibody <![CDATA[EC 50 ,nM]]> OX40mAb24 6.23 Hu38E11 4.56 Hu38E11-v1 5.03 Hu38E11-v2 3.35 Hu38E11-v3 3.48 Hu38E11-v4 3.02

[0241] Example 4: Determination of the agonistic activity of humanized antibodies against T cells

[0242] As described in Example 1.3 above, the agonistic activity of the humanized antibody on T cells was assessed by detecting the inflammatory factor IFNγ released by activated T cells in the presence of the antibody.

[0243] As shown in Table 5, the humanized antibody Hu38E11 and its variants effectively promoted the release of IFNγ from activated T cells, indicating that they possess agonistic activity against T cells. Compared to the control antibody OX40mAb24, the humanized antibody Hu38E11 and its variants showed significantly higher ECGs in promoting the release of IFNγ from T cells. 50 The value is lower, and the agonist activity is more obvious.

[0244] Table 5. Agonistaltic activity of antibody Hu38E11 and its variants on T cells.

[0245]

[0246]

[0247] Example 5: Biacore detection of the binding activity of humanized antibody to human OX40.

[0248] Biacore determines binding kinetic parameters by measuring surface plasmon resonance (SPR). This technique detects the binding of antibodies to antigens (kJ). a ) and dissociation (k dThe microscopic rate constant of the antibody was used to calculate the affinity between the antibody and the antigen. Biacore instruments and reagents were purchased from GE Healthcare. Specifically, anti-human Fc antibody was immobilized on a CM5 sensor chip. Expression supernatant or purified antibody containing the antibody was diluted in mobile phase buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 7.4) and flowed through the CM5 chip coated with anti-human Fc antibody. Then, serially diluted human OX40-His fusion protein was flowed through the detection chip to measure antigen-antibody binding, followed by flow of mobile phase buffer through the chip to detect antigen-antibody dissociation. Binding and dissociation signal data of antigen and antibody at different concentrations were collected, and the affinity between the antigen and antibody was calculated using a 1:1 Langmuir model.

[0249] As shown in Table 6, Hu38E11 binds to human OX40 with high affinity, and the KD value is 2.36E-09(M).

[0250] Table 6. Kinetic constants for Biacore detection of humanized antibody binding to human OX40.

[0251]

[0252] Example 6: T cell agonistic activity of humanized antibody Hu38E11

[0253] As described in Example 1.3 above, the agonistic activity of Hu38E11 (IgG2) antibody subtype IgG2 on T cells was assessed by detecting the cytokine IFNγ released by T cells.

[0254] Result: As Figure 1 As shown, under antibody-coated conditions, Hu38E11 (IgG2) also exhibits T-cell agonistic activity, and its EC50... 50 It is 1.6 nM, compared to the control antibody 11D4 (EC). 50 =5.3nM) is even lower.

[0255] Example 7: Humanized antibody blocks the binding of OX40 and OX40L

[0256] This experiment uses ELISA to determine the blocking effect of antibody Hu38E11 on the binding of OX40 to its ligand OX40L. In short, OX40 (R&D systems, Cat 3388-OX) was diluted with PBS and added to a 96-well plate, incubated overnight at 4°C. The plate was washed three times with PBS containing 0.5% Tween-20 to remove uncoated protein, and then 200 μL of PBS containing 1% BSA was added to each well for blocking at room temperature for 1 h. After washing three times with PBS containing 0.5% Tween-20, serially diluted anti-OX40 antibody was added to each well at 100 μL, and incubated at room temperature for 1 h. After washing three times with PBS containing 0.5% Tween-20, OX40L (R&D systems, Cat 1054-OX) was added to a final concentration of 50 ng / mL, and incubated at room temperature for 1 h. After washing the plate three times, biotin-labeled anti-OX40L antibody (R&D systems, Cat BAF1054) was added, and the plate was incubated at room temperature for 1 hour. After washing again, HRP-labeled streptavidin (R&D systems, Cat DY998) was added, and the plate was incubated at room temperature for 1 hour. After washing, 200 μL of TMB chromogenic buffer was added to each well for development, and the reaction was stopped with 2NH₂SO₄. On a microplate reader, with a background absorbance of 570 nM, the detection wavelength was 450 nM.

[0257] like Figure 2 As shown, Hu38E11 (IgG1N297A) blocks the binding of OX40 to OX40L, achieving a maximum inhibition rate of 90%, which is higher than the maximum inhibition rate of 50% of the control antibodies GBR830 and OX40mAb24. The IC50 values ​​of Hu38E11 (IgG1N297A) and GBR830 are... 50 The values ​​are all around 0.3 nM, IC50 of 0.040 mAb24 50 The value is approximately 1.2 nM.

[0258] Example 8: Humanized Antibody Blocks OX40-OX40L-Mediated T Cell Activation

[0259] Primary PBMCs were obtained from healthy human blood using density gradient centrifugation with a Ficoll-Paque PLUS (GE Healthcare, Cat 17-1440-02) and washed three times with PBS. Human primary T cells were then isolated using the Pan T Cell Isolation Kit (Miltenyi Biotec, Cat 130-096-535) via magnetic bead separation according to the manufacturer's instructions. The T cells were resuspended in RPMI 1640 medium (containing 10% FBS and penicillin / streptomycin). Anti-CD3 antibody OKT3 (eBioscience, Cat 16-0037-85) was added at 100 μL per well to a 96-well plate, and the plates were coated at 37°C for 2 hours. Uncoated antibodies were washed away with PBS. The serially diluted OX40 antibody was mixed with OX40L (R&D systems, Cat 1054-OX) to a final concentration of 664 ng / ml and added to a coated 96-well plate. The isolated T cells were added to the wells and cultured for 3 days. The supernatant was collected and the IFNγ concentration in the supernatant was detected by ELISA (R&D, CatSIF50) according to the standard detection method recommended in the manufacturer's instructions.

[0260] Functional experiment of Hu38E11 (IgG1N297A) blocking OX40L-OX40 interaction: Anti-CD3 antibody was plated, and the natural ligand of OX40, OX40L, was added to stimulate T cells. Simultaneously, free anti-OX40 antibody was added to the culture system to detect the antibody's function in blocking the binding mediated by OX40L and OX40. Because the antibody was not plated, the lack of cross-linking prevented the anti-OX40 antibody molecules from stimulating T cells to secrete IFNγ. Furthermore, anti-OX40, by binding to OX40 on the cell surface, blocked the binding of OX40L and OX40, thus blocking OX40L-induced IFNγ secretion by T cells.

[0261] according to Figure 3 The results showed that antibody Hu38E11 (IgG1N297A) could inhibit OX40L-induced IFNγ secretion at higher concentrations. This indicates that the antibody blocks OX40L-mediated T cell activation. Compared with GBR830, Hu38E11 (IgG1N297A) has a stronger inhibitory effect on OX40L-mediated T cell activation, and its IC50 value is higher. 50 The values ​​are even smaller (Hu38E11 (IgG1N297A) is 0.3 nM, and GBR830 is 1.1 nM).

[0262] Example 9: Effect of the humanized antibody Fc region on the antibody's T-cell agonistic activity

[0263] The T cell activating activity of Hu38E11 cells with different Fc structures can be assessed by measuring the promotion of NF-κB-mediated transcriptional activation in a luciferase reporter gene assay. Recombinant Jurkat cells (Jurkat-OX40-NF-κB-Luc; purchased from Chempartner) overexpressing human OX40 and possessing a luciferase reporter gene (Luc) regulated by the NF-κB gene were constructed. Briefly, anti-CD3 antibody (eBioscience, Cat 16-0037-85) was added to 100 μL per well of a 96-well plate and incubated overnight at 4°C. Uncoated antibody was washed away with PBS. Jurkat-OX40-NF-κB-Luc was then mixed with Raji cells at a 1:1 ratio and added to the coated 96-well plates, followed by serially diluted Hu38E11 (IgG1), Hu38E11 (IgG1N297A), or hIgG1. After 5 hours of incubation, Steady-Glo (Promega) assay reagent was added to determine the relative expression level of luciferase.

[0264] In this experiment, Hu38E11 (IgG1) cross-linked with the FcγR receptor on the Raji surface, activated the downstream signaling of OX40, inducing the expression of reporter genes regulated by NF-κB (EC). 50 =0.70 nM). Conversely, due to the N297A mutation, Hu38E11 (IgG1N297A) cannot cross-link and cannot bind to the FcγR receptor, thus failing to activate downstream OX40 signaling. Furthermore, by blocking the binding of OX40 to OX40L expressed on the Raji surface, this antibody exhibits significant inhibition of NF-κB-regulated luciferase reporter gene expression (IC50). 50 =0.20nM)(e.g. Figure 4 (As shown).

[0265] Example 10: Humanized antibody Hu38E11 inhibits the growth of B16F10 subcutaneous xenografts.

[0266] C57BL / 6-Tnfrsf4 expressing human OX40 em1Clin(hTBFRSF4) A B16-F10 subcutaneous tumor model was established in transgenic mice to study the antitumor activity of the antibody of this invention.

[0267] Mouse melanoma cells B16-F10 were cultured in RPMI 1640 medium containing 10% fetal bovine serum. CRL-6475 TM Tumor cells were suspended in RPMI 1640 at a concentration of 1×10⁻⁶. 5 Cells / mouse dose were implanted subcutaneously in the right flank of female transgenic mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.).

[0268] On the day of tumor cell inoculation (Day 1), mice were randomly divided into three groups according to body weight: Group 1 (h-IgG2) (n=12), Group 2 (11D4(hIgG2)) (n=13), and Group 3 (Hu38E11(hIgG2)) (n=13). Antibodies from each group were diluted with DPBS and administered via a single intraperitoneal injection at a dose of 10 mg / kg. Tumor volume was measured periodically (tumor volume = 0.5 × major axis × minor axis). 2 Mouse body weight. The tumor inhibition rate in the antibody treatment group was calculated on day 15 or 16 after administration.

[0269] The tumor inhibition rate was calculated using the formula: [(Tumor volume in the control group - Tumor volume in the treatment group) / Tumor volume in the control group] × 100%. The relative body weight of mice was calculated using the formula: (Mice's body weight on the day of measurement / Mouse's body weight at the time of grouping) × 100%.

[0270] Results: At a dose of 10 mg / kg, the tumor growth inhibition rates of the control antibody 11D4 and the antibody Hu38E11 (IgG2) of this invention were 33.2% and 48.1%, respectively. Furthermore, the mice in each group showed rapid weight gain and normal behavior during the observation period, indicating good animal tolerance.

[0271] Example 11: Anti-immune rejection activity of humanized antibody Hu38E11 (IgG1N297A)

[0272] In immunodeficiency NOD-Prkdc em26Cd52 Il2rg em26Cd22 A graft-versus-host disease (GVHD) model was established by transplanting human peripheral blood mononuclear cells (hPBMCs) from Nju (NCG) mice, which was used to study the anti-immune rejection activity of the antibodies of this invention.

[0273] Primary human PBMCs were isolated from whole blood of healthy individuals by Ficoll-Paque density gradient centrifugation and then suspended in phosphate-buffered saline (PBS).

[0274] One day before PBMC transplantation (Day -1), mice were randomly divided into 7 groups according to body weight, as shown in Table 7. On the day of transplantation (Day 0), all mice received a single 1.5 Gy systemic injection. 137Csγ-ray irradiation (total body irradiation, TBI) was followed by dilution of Hu38E11 (IgG1N297A) and GBR830 antibody with PBS, administered weekly via tail vein injection at a dose of 1 mg / kg, in a volume of 5 mL / kg. Finally, a single tail vein injection of 0.2 mL / animal (2.5 × 10⁻⁶) was administered. 7 Cells / mL PBMCs. Mouse survival status was observed daily, and mouse weight was measured periodically. Euthanasia was defined as a 20% relative weight loss, and survival time was recorded.

[0275] The formula for calculating the relative body weight of mice is: (body weight of mice on the day of measurement / body weight of mice at the time of grouping) × 100%.

[0276] Table 7. Grouping and Dosing Regimens of Hu38E11 (IgG1N297A) in the Treatment of hPBMC-Induced Graft-versus-Host Model

[0277]

[0278]

[0279] Experimental results are as follows Figure 5 As shown in the figure. In this experiment, all mice in the model control group (hPBMC+hIgG1 group) died on day 48, with a median survival time of 32.5 days. All mice treated with the antibody Hu38E11 (IgG1N297A) 1 mg / kg survived on day 64, but the median survival time could not be calculated, showing a statistically significant difference compared to the model control group (hPBMC+hIgG1 group) (**: p<0.01). The survival rate of mice treated with the positive control antibody GBR830 1 mg / kg was 66.7% on day 64, but the median survival time could not be calculated, and there was no statistically significant difference compared to the model control group.

[0280] Sequence List Description

[0281]

[0282]

[0283] sequence list <110> Hutchison MediPharma (Shanghai) Co., Ltd. <120> Anti-OX40 antibodies and their uses <130> PF210196CNP <160> twenty two <170> PatentIn version 3.3 <210> 1 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Gln Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Val Asp Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Gln Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Tyr Gly Tyr Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 2 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Val Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Tyr Gly Tyr Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Val Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Tyr Gly Tyr Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 4 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Val Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Tyr Gly Tyr Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Val Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Tyr Gly Tyr Tyr Gly Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 111 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 6 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Ser 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 7 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Ser Ser 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 8 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 8 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Ser Ser 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 9 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Ser Ser 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 10 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 10 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Ser Ser 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Ser Tyr Trp Val Asp 1 5 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Asn Ile Tyr Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 13 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 13 Ser Tyr Gly Tyr Tyr Gly Thr Trp Phe Ala Tyr 1 5 10 <210> 14 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 14 Arg Ala Ser Glu Ser Val Asp Ser Ser Gly Asn Ser Phe Met His 1 5 10 15 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 15 Arg Ala Ser Asn Leu Glu Ser 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 16 Gln Gln Ser Asn Glu Asp Pro Trp Thr 1 5 <210> 17 <211> 360 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 17 Cys Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Gly Cys Ala Gly Cys 1 5 10 15 Ala Gly Cys Cys Thr Gly Gly Gly Gly Cys Thr Gly Ala Gly Cys Thr 20 25 30 Gly Gly Thr Gly Ala Gly Gly Cys Cys Thr Gly Gly Gly Thr Cys Thr 35 40 45 Thr Cys Ala Gly Thr Gly Cys Ala Gly Thr Thr Gly Thr Cys Cys Thr 50 55 60 Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Thr Gly Gly Cys Thr Ala 65 70 75 80 Cys Ala Cys Cys Thr Thr Cys Ala Cys Cys Ala Gly Cys Thr Ala Cys 85 90 95 Thr Gly Gly Gly Thr Gly Gly Ala Thr Thr Gly Gly Gly Thr Gly Ala 100 105 110 Ala Gly Cys Ala Gly Ala Gly Gly Cys Cys Thr Gly Gly Ala Cys Ala 115 120 125 Ala Gly Gly Cys Cys Thr Thr Cys Ala Ala Thr Gly Gly Ala Thr Thr 130 135 140 Gly Gly Thr Ala Ala Cys Ala Thr Thr Thr Ala Cys Cys Cys Thr Thr 145 150 155 160 Cys Thr Gly Ala Thr Ala Gly Thr Gly Ala Ala Ala Cys Thr Cys Ala 165 170 175 Cys Thr Ala Cys Ala Ala Thr Cys Ala Ala Ala Ala Gly Thr Thr Cys 180 185 190 Ala Ala Gly Gly Ala Cys Ala Ala Gly Gly Cys Cys Ala Cys Ala Thr 195 200 205 Thr Gly Ala Cys Thr Gly Thr Ala Gly Ala Cys Ala Ala Ala Thr Cys 210 215 220 Cys Thr Cys Cys Ala Gly Cys Ala Cys Ala Gly Cys Cys Thr Ala Cys 225 230 235 240 Ala Thr Gly Cys Ala Gly Cys Thr Cys Ala Gly Cys Ala Gly Cys Cys 245 250 255 Thr Gly Ala Cys Ala Thr Cys Thr Gly Ala Ala Gly Ala Cys Thr Cys 260 265 270 Thr Gly Cys Gly Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr 275 280 285 Gly Cys Ala Ala Gly Ala Thr Cys Thr Thr Ala Thr Gly Gly Thr Thr 290 295 300 Ala Cys Thr Ala Cys Gly Gly Gly Ala Cys Cys Thr Gly Gly Thr Thr 305 310 315 320 Thr Gly Cys Thr Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Ala 325 330 335 Gly Gly Gly Ala Cys Thr Cys Thr Gly Gly Thr Cys Ala Cys Thr Gly 340 345 350 Thr Cys Thr Cys Thr Gly Cys Ala 355 360 <210> 18 <211> 360 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 18 Cys Ala Gly Gly Thr Thr Cys Ala Gly Thr Thr Gly Gly Thr Thr Cys 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly Cys Gly Cys Cys Gly Ala Ala Gly Thr 20 25 30 Gly Ala Ala Gly Ala Ala Ala Cys Cys Thr Gly Gly Cys Gly Cys Cys 35 40 45 Thr Cys Thr Gly Thr Gly Ala Ala Gly Gly Thr Gly Thr Cys Cys Thr 50 55 60 Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Cys Gly Gly Cys Thr Ala 65 70 75 80 Cys Ala Cys Cys Thr Thr Thr Ala Cys Cys Ala Gly Cys Thr Ala Cys 85 90 95 Thr Gly Gly Gly Thr Cys Gly Ala Cys Thr Gly Gly Gly Thr Cys Cys 100 105 110 Gly Ala Cys Ala Gly Gly Cys Thr Cys Cys Thr Gly Gly Ala Cys Ala 115 120 125 Ala Gly Gly Ala Cys Thr Gly Gly Ala Ala Thr Gly Gly Ala Thr Gly 130 135 140 Gly Gly Cys Ala Ala Cys Ala Thr Cys Thr Ala Cys Cys Cys Cys Thr 145 150 155 160 Cys Cys Gly Ala Cys Thr Cys Cys Gly Ala Gly Ala Cys Ala Cys Ala 165 170 175 Cys Thr Ala Cys Ala Ala Cys Cys Ala Gly Ala Ala Ala Thr Thr Cys 180 185 190 Ala Ala Gly Gly Ala Cys Cys Gly Cys Gly Thr Gly Ala Cys Cys Ala 195 200 205 Thr Gly Ala Cys Cys Ala Gly Ala Gly Ala Cys Ala Cys Cys Thr Cys 210 215 220 Cys Ala Cys Cys Ala Gly Cys Ala Cys Cys Gly Thr Gly Thr Ala Cys 225 230 235 240 Ala Thr Gly Gly Ala Ala Cys Thr Gly Thr Cys Cys Ala Gly Cys Cys 245 250 255 Thr Gly Ala Gly Ala Thr Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys 260 265 270 Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala Cys Thr Gly Cys 275 280 285 Gly Cys Cys Ala Gly Ala Thr Cys Cys Thr Ala Cys Gly Gly Cys Thr 290 295 300 Ala Cys Thr Ala Cys Gly Gly Cys Ala Cys Thr Thr Gly Gly Thr Thr 305 310 315 320 Thr Gly Cys Cys Thr Ala Thr Thr Gly Gly Gly Gly Cys Cys Ala Gly 325 330 335 Gly Gly Cys Ala Cys Ala Cys Thr Gly Gly Thr Cys Ala Cys Cys Gly 340 345 350 Thr Thr Thr Cys Thr Thr Cys Cys 355 360 <210> 19 <211> 333 <212> PRT <213> Artificial Sequence <220> <223> Synthesis <400> 19 Gly Ala Cys Ala Thr Thr Gly Thr Gly Cys Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Ala Thr Cys Thr Cys Cys Ala Gly Cys Thr Thr Cys Thr Thr Thr 20 25 30 Gly Gly Cys Thr Gly Thr Gly Thr Cys Thr Cys Thr Ala Gly Gly Gly 35 40 45 Cys Ala Gly Ala Gly Gly Gly Cys Cys Ala Cys Cys Ala Thr Ala Thr 50 55 60 Cys Cys Thr Gly Cys Ala Gly Ala Gly Cys Cys Ala Gly Thr Gly Ala 65 70 75 80 Ala Ala Gly Thr Gly Thr Thr Gly Ala Thr Ala Gly Thr Thr Cys Thr 85 90 95 Gly Gly Cys Ala Ala Thr Ala Gly Thr Thr Thr Thr Ala Thr Gly Cys 100 105 110 Ala Cys Thr Gly Gly Thr Ala Cys Cys Ala Gly Cys Ala Gly Ala Ala 115 120 125 Ala Cys Cys Ala Gly Gly Ala Cys Ala Gly Cys Cys Ala Cys Cys Cys 130 135 140 Ala Ala Ala Cys Thr Cys Cys Thr Cys Ala Thr Cys Thr Ala Thr Cys 145 150 155 160 Gly Thr Gly Cys Ala Thr Cys Cys Ala Ala Cys Cys Thr Ala Gly Ala 165 170 175 Ala Thr Cys Thr Gly Gly Gly Ala Thr Cys Cys Cys Thr Gly Cys Cys 180 185 190 Ala Gly Gly Thr Thr Cys Ala Gly Thr Gly Gly Cys Ala Gly Thr Gly 195 200 205 Gly Gly Thr Cys Thr Ala Gly Gly Ala Cys Ala Gly Ala Cys Thr Thr 210 215 220 Cys Ala Cys Cys Cys Thr Cys Ala Cys Cys Ala Thr Thr Ala Ala Thr 225 230 235 240 Cys Cys Thr Gly Thr Gly Gly Ala Gly Gly Cys Thr Gly Ala Thr Gly 245 250 255 Ala Thr Gly Thr Thr Gly Cys Ala Ala Cys Cys Thr Ala Thr Thr Ala 260 265 270 Cys Thr Gly Thr Cys Ala Gly Cys Ala Ala Ala Gly Thr Ala Ala Thr 275 280 285 Gly Ala Gly Gly Ala Thr Cys Cys Gly Thr Gly Gly Ala Cys Gly Thr 290 295 300 Thr Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Ala Cys Cys Ala Ala 305 310 315 320 Ala Cys Thr Gly Gly Ala Ala Ala Thr Cys Ala Ala Ala 325 330 <210> 20 <211> 333 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 20 Gly Ala Thr Ala Thr Cys Gly Thr Gly Ala Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys Thr Gly Ala Cys Ala Gly Cys Cys Thr 20 25 30 Gly Gly Cys Thr Gly Thr Gly Thr Cys Thr Cys Thr Gly Gly Gly Cys 35 40 45 Gly Ala Gly Ala Gly Ala Gly Cys Cys Ala Cys Cys Ala Thr Cys Ala 50 55 60 Ala Cys Thr Gly Cys Ala Gly Ala Gly Cys Cys Thr Cys Thr Gly Ala 65 70 75 80 Gly Thr Cys Cys Gly Thr Gly Gly Ala Cys Thr Cys Cys Thr Cys Cys 85 90 95 Gly Gly Cys Ala Ala Cys Thr Cys Thr Thr Thr Cys Ala Thr Gly Cys 100 105 110 Ala Cys Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys Ala Gly Ala Ala 115 120 125 Gly Cys Cys Cys Gly Gly Cys Cys Ala Gly Cys Cys Thr Cys Cys Thr 130 135 140 Ala Ala Gly Cys Thr Gly Cys Thr Gly Ala Thr Cys Thr Ala Cys Ala 145 150 155 160 Gly Ala Gly Cys Cys Thr Cys Cys Ala Ala Cys Cys Thr Gly Gly Ala 165 170 175 Ala Thr Cys Thr Gly Gly Cys Gly Thr Gly Cys Cys Cys Gly Ala Cys 180 185 190 Ala Gly Ala Thr Thr Cys Thr Cys Cys Gly Gly Cys Thr Cys Thr Gly 195 200 205 Gly Cys Thr Cys Thr Gly Gly Cys Ala Cys Ala Gly Ala Cys Thr Thr 210 215 220 Thr Ala Cys Cys Cys Thr Gly Ala Cys Cys Ala Thr Cys Ala Gly Cys 225 230 235 240 Thr Cys Cys Cys Thr Gly Cys Ala Gly Gly Cys Cys Gly Ala Gly Gly 245 250 255 Ala Thr Gly Thr Gly Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala 260 265 270 Cys Thr Gly Cys Cys Ala Gly Cys Ala Gly Thr Cys Cys Ala Ala Cys 275 280 285 Gly Ala Gly Gly Ala Cys Cys Cys Cys Thr Gly Gly Ala Cys Ala Thr 290 295 300 Thr Thr Gly Gly Cys Gly Gly Cys Gly Gly Ala Ala Cys Ala Ala Ala 305 310 315 320 Gly Cys Thr Gly Gly Ala Ala Ala Thr Cys Ala Ala Gly 325 330 <210> 21 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 21 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 22 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 22 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325

Claims

1. An isolated anti-OX40 antibody, comprising... (1) Heavy chain complementarity-determining regions (HCDRs), HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:11, the amino acid sequence of HCDR2 is shown in SEQ ID NO:12 and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

13. (2) Light chain complementarity-determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:14, the amino acid sequence of LCDR2 is shown in SEQ ID NO:15, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:16; and (3) Fc region variant, which is human IgG1 N297A.

2. The antibody of claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of the VH comprises SEQ ID NO:2 and the amino acid sequence of the VL comprises SEQ ID NO:

7.

3. The antibody of claim 1, comprising a human IgG1 constant region, wherein the amino acid sequence of the human IgG1 constant region comprises SEQ ID NO:21 having the N297A mutation.

4. The antibody as described in claim 1, wherein it is a full-length antibody.

5. The antibody as described in claim 1, wherein its affinity (K) for binding to human OX40 is [not specified]. D (Below 10 nM) 6. An isolated antagonistic anti-OX40 antibody, which is a full-length antibody and comprises: - Heavy chain variable region (VH), the amino acid sequence of which is shown in SEQ ID NO:2; - Light chain variable region (VL), the amino acid sequence of which is shown in SEQ ID NO:7; and - Human IgG1 heavy chain constant region, wherein the amino acid sequence of the human IgG1 heavy chain constant region contains SEQ ID NO:21 with the N297A mutation.

7. An isolated nucleic acid encoding an antibody as described in any one of claims 1-6.

8. A recombinant vector or expression vector comprising one or more nucleic acids as described in claim 7, wherein the vector is suitable for recombinant production of the antibody as described in any one of claims 1-6.

9. A host cell comprising one or more recombinant vectors or expression vectors as described in claim 8.

10. An immunoconjugate comprising an antibody conjugated to a marker as described in any one of claims 1-6.

11. A pharmaceutical composition comprising an antibody as claimed in any one of claims 1-6, a nucleic acid as claimed in claim 7, a carrier as claimed in claim 8, a host cell as claimed in claim 9, or an immunoconjugate as claimed in claim 10, and optionally comprising at least one pharmaceutically acceptable excipient.

12. Use of an antibody as described in any one of claims 1-6, a nucleic acid as described in claim 7, a vector as described in claim 8, a host cell as described in claim 9, or an immunoconjugate as described in claim 10 in the preparation of a medicament for treating OX40-related diseases or symptoms, wherein the OX40-related diseases or symptoms are selected from atopic dermatitis, rheumatoid arthritis, asthma, COPD, autoimmune uveitis, multiple sclerosis, lupus, ulcerative colitis, scleroderma, and graft-versus-host disease (GVHD).

13. The use as described in claim 12, wherein the OX40-related disease or symptom is graft-versus-host disease.

14. An in vitro method for detecting OX40 in a sample for non-diagnostic purposes, comprising: (a) Contact the sample with the antibody of any one of claims 1-6, or the immunoconjugate of claim 10; and (b) Detect the formation of a complex between the antibody or immunoconjugate and the OX40 protein.

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