Anti-OX40 antibody and application thereof

Abstract

The invention provides antibodies and antigen-binding fragments thereof that bind to human OX40, pharmaceutical compositions comprising said antibodies and the use of said antibodies or said compositions for the treatment of diseases, such as cancer.

Description

Anti-OX40 antibodies and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to an anti-OX40 antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising the anti-OX40 antibody and the antigen-binding fragment thereof, and the use of the anti-OX40 antibody and the antigen-binding fragment thereof as an anticancer drug. Background Art

[0002] Cancer is a serious, long-standing health challenge facing human society. Cancer therapy encompasses a broad range of treatment options, including surgery, radiation, and chemotherapy. While these diverse approaches offer medical practitioners a wider range of options for treating cancer, existing treatments also have numerous drawbacks, such as a lack of selectivity for targeting cancer cells relative to normal, healthy cells, and the development of resistance to treatments as cancer progresses. Furthermore, for solid tumors that have already metastasized, traditional therapies such as surgery, chemotherapy, and radiation often have minimal efficacy.

[0003] Increasing evidence from preclinical and clinical results indicates that targeting immune checkpoints is becoming one of the most promising approaches for treating cancer. By enhancing anti-tumor T cell function, it can be used to prevent cancer progression and even cure the disease. Tumor necrosis factor receptor superfamily member 4 (TNFRSF4, also known as OX40, CD134, and ACT35) is one of the immune checkpoint proteins.

[0004] OX40, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a glycoprotein with a molecular weight of approximately 50 kDa expressed on the cell surface. The extracellular ligand-binding domain of OX40 consists of four cysteine-rich peptides (CRDs). The natural ligand of OX40 is OX40L (CD252), with which it forms the OX40-OX40L complex.

[0005] OX40 is primarily expressed on activated T cells. OX40 is a secondary co-stimulatory molecule expressed 24-72 hours after activation. Its ligand, OX40L, is primarily expressed on activated antigen-presenting cells. In normal tissues, OX40 expression is low and primarily on lymphocytes of lymphoid organs. However, upregulation of OX40 expression on immune cells is frequently observed in animal models of pathological conditions or cancer, as well as in human patients. OX40-expressing T lymphocytes have been demonstrated in the draining lymph nodes of various human malignancies and cancer patients.

[0006] In a severe combined immunodeficiency mouse model, the interaction between the OX40 and OX40L binding domains can enhance tumor immunity, leading to the inhibition of tumor growth in various human malignant tumor cell lines, such as lymphoma, prostate cancer, colon cancer, and breast cancer.

[0007] Currently, multiple preclinical mouse models and clinical trials have demonstrated that OX40 agonists are a promising strategy for treating cancer and infectious diseases. Agonistic OX40 antibodies specifically stimulate and activate the immune system, enhancing the patient's own immune response to tumors, thereby killing tumor cells.

[0008] Summary of the Invention

[0009] The present invention is directed to agonistic anti-OX40 antibodies and antigen-binding fragments that activate OX40 and induce signal transduction in immune cells, thereby promoting anti-tumor immunity.

[0010] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to OX40, comprising a heavy chain variable region and / or a light chain variable region, wherein:

[0011] 1) The heavy chain variable region comprises

[0012] (i) the three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained in the peptide segment having the amino acid sequence of SEQ ID NO: 7, 9, 11, 12, 13, 14, 15, 23, 25, 27, 28, 29, 31 or 32; or

[0013] (ii) relative to the sequences of the three heavy chain complementary determining regions (HCDRs) described in (i), comprising at least one and no more than five amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in the three heavy chain complementary determining regions (HCDRs);

[0014] and / or

[0015] 2) The light chain variable region comprises,

[0016] (i) three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained in a peptide segment having an amino acid sequence of SEQ ID NO: 8, 10, 16, 24, 26, 30 or 33; or

[0017] (ii) relative to the sequences of the three light chain complementary determining regions (LCDRs) described in (i), a sequence comprising at least one and no more than five amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in the three light chain complementary determining regions (LCDRs).

[0018] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to OX40, comprising:

[0019] The three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained in the peptide segment with the amino acid sequence of SEQ ID NO: 7, 9, 11, 12, 13, 14, 15, 23, 25, 27, 28, 29, 31 or 32; and / or

[0020] The peptide segment with the amino acid sequence of SEQ ID NO: 8, 10, 16, 24, 26, 30 or 33 contains three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3.

[0021] Optionally, the antibody or antigen-binding fragment thereof, which specifically binds to OX40, comprises:

[0022] a) a heavy chain complementarity determining region HCDR3, said HCDR3 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 3 or 19, or said HCDR3 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 3 or 19; and

[0023] b) a light chain complementary determining region LCDR3, wherein the LCDR3 comprises the amino acids as shown in SEQ ID NO: 6 or 22, or consists of the amino acid sequence as shown in SEQ ID NO: 6 or 22, or the LCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 6 or 22.

[0024] Optionally, it further comprises:

[0025] c) a heavy chain complementarity determining region HCDR1, said HCDR1 comprising, or consisting of, the amino acid sequence as shown in SEQ ID NO: 1 or 17, or said HCDR1 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 1 or 17;

[0026] d) a heavy chain complementarity determining region HCDR2, said HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 2 or 18, or said HCDR2 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 2 or 18;

[0027] e) a light chain complementary determining region LCDR1, said LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 4 or 20, or consisting of the amino acid sequence as shown in SEQ ID NO: 4 or 20, or said LCDR1 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 4 or 20; and

[0028] f) a light chain complementary determining region LCDR2, said LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 5 or 21, or consisting of the amino acid sequence as shown in SEQ ID NO: 5 or 21, or said LCDR2 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 5 or 21.

[0029] Optionally, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3; and three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein:

[0030] HCDR1 comprises, or consists of, SEQ ID NO: 1 or SEQ ID NO: 17, or the HCDR1 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 1 or 17;

[0031] HCDR2 comprises SEQ ID NO: 2 or SEQ ID NO: 18, or consists thereof, or the HCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 2 or 18;

[0032] HCDR3 comprises, or consists of, SEQ ID NO: 3 or SEQ ID NO: 19, or the HCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 3 or 19;

[0033] LCDR1 comprises SEQ ID NO: 4 or SEQ ID NO: 20, or consists thereof, or the LCDR1 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 4 or 20;

[0034] LCDR2 comprises SEQ ID NO: 5 or SEQ ID NO: 21, or consists thereof, or the LCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 5 or 21;

[0035] LCDR3 comprises SEQ ID NO: 6 or SEQ ID NO: 22, or consists of them, or the LCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 6 or 22.

[0036] Optionally, the antibody or antigen-binding fragment thereof comprises:

[0037] (1) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 contained in the peptide segment represented by the amino acid sequence of SEQ ID NO: 7, 9, 11, 12, 13, 14, or 15, and

[0038] The three light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained in the peptide segment with the amino acid sequence shown in SEQ ID NO: 8, 10 or 16; or

[0039] (2) three heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 contained in the peptide segment represented by the amino acid sequence of SEQ ID NO: 23, 25, 27, 28, 29, 31, or 32, and

[0040] The amino acid sequence of the peptide segment shown in SEQ ID NO: 24, 26, 30 or 33 comprises three light chain complementary determining regions (LCDRs): LCDR1, LCDR2 and LCDR3.

[0041] Optionally, the antibody or antigen-binding fragment thereof comprises:

[0042] (1) HCDR1 comprising or consisting of SEQ ID NO: 1,

[0043] HCDR2 comprising or consisting of SEQ ID NO: 2,

[0044] A HCDR3 comprising or consisting of SEQ ID NO: 3,

[0045] LCDR1 comprising or consisting of SEQ ID NO: 4,

[0046] LCDR2 comprising or consisting of SEQ ID NO: 5, and

[0047] LCDR3 comprising or consisting of SEQ ID NO: 6; or

[0048] (2) HCDR1 comprising SEQ ID NO: 17 or consisting thereof,

[0049] A HCDR2 comprising or consisting of SEQ ID NO: 18,

[0050] HCDR3 comprising or consisting of SEQ ID NO: 19,

[0051] LCDR1 comprising or consisting of SEQ ID NO: 20,

[0052] LCDR2 comprising or consisting of SEQ ID NO: 21, and

[0053] LCDR3 comprising or consisting of SEQ ID NO: 22.

[0054] The present invention also discloses an antibody or an antigen-binding fragment thereof, which specifically binds to OX40, comprising:

[0055] A heavy chain variable region comprising or consisting of SEQ ID NO: 7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence of SEQ ID NO: 7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32. NO:7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; or the heavy chain variable region comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to SEQ ID NO:7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32, preferably, the amino acid mutations do not occur in the heavy chain complementarity determining region; and / or

[0056] A light chain variable region comprising SEQ ID NO: 8, 24, 10, 16, 26, 30 or 33, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence of SEQ ID NO: 8, 24, 10, 16, 26, 30 or 33; or consisting of SEQ ID NO: 8, 24, 10, 16, 26, 30 or 33, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence of SEQ ID NO: 8, 24, 10, 16, 26, 30 or 33. NO:8, 24, 10, 16, 26, 30 or 33, an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions), or consisting of the same, preferably, the amino acid mutations do not occur in the complementary determining region of the light chain.

[0057] Optionally, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence pairs of the heavy chain variable region and the light chain variable region are selected from any one of the groups consisting of the following amino acid sequence pairs: SEQ ID NO: 7 and 8, SEQ ID NO: 23 and 24, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 16, SEQ ID NO: 12 and 16, SEQ ID NO: 13 and 16, SEQ ID NO: 14 and 16, SEQ ID NO: 15 and 16, SEQ ID NO: 25 and 26, SEQ ID NO: 27 and 26, SEQ ID NO: 28 and 26, SEQ ID NO: 29 and 30, SEQ ID NO: 31 and 33 and SEQ ID NO: 32 and 33.

[0058] Optionally, the antibody or antigen-binding fragment thereof, wherein the CDR region is defined according to the Kabat definition scheme, the Clothia definition scheme, the Abm definition scheme and / or the Contact scheme, preferably the Kabat definition scheme or the Clothia definition scheme.

[0059] Optionally, the antibody or antigen-binding fragment thereof further comprises a constant region, and the constant region is derived from an IgG antibody, an IgM antibody, an IgA antibody, an IgD antibody or an IgE antibody. Preferably, the constant region is derived from an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0060] Optionally, the antibody or antigen-binding fragment thereof, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.

[0061] Optionally, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region comprising: SEQ ID NO: 42 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 42; or the heavy chain constant region consists of SEQ ID NO: 42, or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 42; and / or

[0062] A light chain constant region comprising or consisting of SEQ ID NO: 43, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 43.

[0063] Optionally, the antibody or antigen-binding fragment thereof is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (preferably scFv) or (Fab')2, single-domain antibody, diabody (dAb) or linear antibody.

[0064] Optionally, the antibody or antigen-binding fragment thereof, wherein the antibody is a bispecific or multispecific antibody molecule, preferably, the bispecific antibody molecule binds to OX40 and PD-1, PD-L1 or PD-L2.

[0065] The second aspect of the present invention discloses a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the first aspect and a pharmaceutically acceptable excipient.

[0066] The third aspect of the present invention discloses a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of the first aspects.

[0067] The fourth aspect of the present invention discloses an expression vector comprising the nucleic acid molecule as described in any one of the third aspects.

[0068] The fifth aspect of the present invention discloses a host cell comprising the expression vector as described in any one of the fourth aspects.

[0069] Optionally, the host cell is a prokaryotic cell or a eukaryotic cell.

[0070] Optionally, the host cell is Escherichia coli, yeast cell, insect cell, plant cell or mammalian cell.

[0071] Optionally, the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell or an NSO cell.

[0072] The sixth aspect of the present invention discloses a method for producing an antibody or an antigen-binding fragment thereof, which comprises culturing the host cell as described in any one of the sixth aspects, and recovering the antibody or antigen-binding fragment thereof expressed by the host cell from the culture.

[0073] The seventh aspect of the present invention discloses use of the antibody or antigen-binding fragment according to any one of the first aspects in the preparation of a medicament for treating cancer.

[0074] The present invention also discloses the antibody or antigen-binding fragment according to any one of the first aspects, which is used for treating cancer.

[0075] The present invention also discloses a method for treating cancer, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of the first aspects.

[0076] definition

[0077] For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0078] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be divided into kappa and lambda light chains. Heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Antibodies also have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region comprises three domains (CH1, CH2, and CH3). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into highly variable regions (called complementarity determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus, of which the three CDRs of VH are HCDR1, HCDR2 and HCDR3, and the three CDRs of VL are LCDR1, LCDR2 and LCDR3. The variable regions of each heavy chain / light chain pair (VH and VL) form the antigen binding site, respectively.

[0079] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, which can be used interchangeably in the context of this application, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be achieved by certain fragments of a full-length antibody. Under some conditions, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Antigen-binding fragments of an antibody can be obtained from a given antibody by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) and can be screened for specificity in the same manner as intact antibodies.

[0080] Different analyses can be used to determine or roughly estimate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition. The Kabat definition is a standard for numbering residues in antibodies and is commonly used to determine CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the position of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group that simulates antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from its primary sequence using known databases and ab initio methods, such as those described in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). Contact definitions are based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0081] The term "monoclonal antibody" or "monoantibody" or "mAb" refers to an antibody molecule or preparation thereof of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0082] The term "chimeric antibody" refers to antibodies in which the variable region sequences are from one species and the constant region sequences are from another species, for example, antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0083] The term "humanized antibody" refers to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0084] The term "amino acid mutation" refers to an amino acid substitution, insertion, deletion, or modification. Specifically, it refers to a mutation between a polypeptide and its variant at one or more amino acid sites within the polypeptide fragment. A variant can be obtained by substitution, insertion, deletion, or modification of an amino acid at one or more sites within the polypeptide.

[0085] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence, and the variant of the antibody obtained by conservatively substituted amino acids fully retains the biological activity of its source sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.

[0086] The term "anti-OX40 antibody" or "OX40 antibody" refers to an antibody as defined herein that is capable of binding to, for example, the human OX40 receptor.

[0087] The terms "OX40," "OX40 receptor," "OX40 protein," "tumor necrosis factor receptor superfamily member 4 (TNFRSF4) or CD134," used interchangeably herein, are members of the tumor necrosis factor (TNF) receptor superfamily. The term "OX40" may include human OX40 receptor, as well as variants, isoforms and species homologs thereof. Thus, the antibodies or antigen-binding portions thereof as defined and disclosed herein may also bind to OX40 from species other than human.

[0088] The term "human OX40" refers to the complete amino acid sequence of human OX40, for example, human OX40 having Genbank Accession No. CAE11757.1. The human OX40 sequence may differ from the human OX40 of Genbank Accession No. CAE11757.1, for example, by having conservative mutations in non-conserved regions, and the OX40 may have substantially the same biological function as the human OX40 of Genbank Accession No. CAE11757.1.

[0089] The term "affinity" refers to the strength of the sum of 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, "affinity" refers to the intrinsic binding affinity of a 1:1 interaction between members of a reactive binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ), dissociation constant (K d ) or binding constant (K a Affinity can be measured by general methods known in the art, including those described herein, preferably using Biacore TM System analysis.

[0090] The term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements carried are expressed in the host cell. Vectors are well known to those skilled in the art and include but are not limited to plasmids, phages, artificial chromosomes such as yeast artificial chromosomes, bacterial artificial chromosomes, viruses; phages such as lambda phage or M13 phage. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may contain an origin of replication.

[0091] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, such as cultured mammalian cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only specific test cells, but also the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may be different from the parent cell, but are also included within the scope of the term "host cell".

[0092] The terms "identity" or "homology" refer to the sequence similarity between two nucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the total number of positions compared × 100. For example, if 6 out of 10 positions in the two sequences match or are homologous, the two sequences are 60% homologous when the sequences are optimally aligned; if 95 out of 100 positions in the two sequences match or are homologous, the two sequences are 95% homologous.

[0093] The term "treatment" includes therapeutic treatment, prophylactic treatment, and use in reducing the risk of a subject developing a disease or other risk factors. Treatment includes, but is not limited to, complete cure of the disease, as well as alleviation of symptoms or mitigation of potential risks.

[0094] The term "cancer" refers to any neoplastic or malignant cell growth, proliferation, or metastasis-mediated medical condition, both solid tumors and non-solid tumors such as leukemia.

[0095] The term "therapeutically effective amount" refers to an amount of an antibody that, when administered to a subject for treatment of at least one clinical symptom of a disease or disorder, is sufficient to produce an effect on such treatment of the disease, disorder, or symptom. The therapeutically effective amount may vary depending on factors such as the severity of the disease or disorder, or the age and / or weight of the subject being treated.

[0096] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, which is well known in the art and includes but is not limited to pH adjusters, surfactants, adjuvants, and ionic strength enhancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 ELISA test results of affinity between humanized antibodies and human OX40. The horizontal axis is the sample concentration Log C (ng / mL), and the vertical axis is the corresponding OD 450 value;

[0098] Figure 2a-2c shows the activation of CD4 by anti-OX40 antibodies + For T cell results, the horizontal axis represents sample concentration Log C (ng / mL) and the vertical axis represents T cell activation efficiency (%).

[0099] Figure 3a-c shows the activation of CD8 by anti-OX40 antibodies + For T cell results, the horizontal axis represents sample concentration Log C (ng / mL) and the vertical axis represents T cell activation efficiency (%).

[0100] Figure 4 shows the changes in tumor volume in different mice after drug administration, with the horizontal axis representing time (days) and the vertical axis representing tumor volume (mm 3 );

[0101] FIG5 shows the effects of different anti-OX40 antibodies on tumor mass in mice after administration, with the horizontal axis representing time (days) and the vertical axis representing tumor mass (g). DETAILED DESCRIPTION

[0102] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0103] Abbreviations

[0104] DMSO refers to dimethyl sulfoxide.

[0105] TMB refers to 3,3',5,5'-Tetramethylbenzidine

[0106] solution.

[0107] PBMC refers to peripheral blood mononuclear cells (PMBC).

[0108] PBST refers to phosphate buffer solution (PBS) with Tween-20 added.

[0109] BSA refers to bovine serum albumin.

[0110] Example 1 Preparation of hybridoma monoclonal antibodies

[0111] Immunized animals

[0112] Anti-OX40 antibodies were produced by immunizing mice. The immunizing antigen was a fusion protein formed by linking the C-terminus of the extracellular domain of human OX40 to hFc. The experimental animals were 6-8 week old female BALB / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.).

[0113] Immunization scheme: The above antigens were mixed with adjuvants (adjuvant system, Sigma) in a 1:1 ratio, and inoculated after the antigen was emulsified. The dose for the first immunization of mice was 100 μg / mouse / time. Two booster immunizations were performed in sequence 3 weeks after the first immunization, of which the dose for both booster immunizations was 50 μg / mouse / time, and the interval between the two booster immunizations was 3 weeks. Blood was collected after the second booster immunization, and the antibody titer in the mouse serum was determined by protein level ELISA method. Another booster immunization was performed three days before spleen cell fusion. The booster immunization scheme for this time was an intraperitoneal injection of 50 μg / mouse of an antigen solution prepared with normal saline.

[0114] Spleen cell fusion: Hybridoma cells were generated by fusing splenic lymphocytes (ground from mouse spleens) with myeloma Sp2 / 0 cells (ATCC) using an optimized electrofusion procedure. The fused hybridoma cells were seeded into 96-well plates and incubated at 37°C, 5% CO2 for 7 days. The medium was changed one day in advance. As much of the old medium as possible was aspirated without removing the hybridoma cells, and 180 μL of HAT selection medium (Thermo) was added. After overnight incubation, 100 μL of the supernatant was collected for protein analysis by ELISA.

[0115] Hybridoma Cell Screening: Based on the hybridoma cell growth density, the supernatant of the hybridoma cell culture was tested using a protein ELISA assay. The supernatant from the cell lines that tested positive for the protein ELISA assay was then subjected to a cell binding assay. The cells from the positive wells were expanded, frozen, and subcloned. Each subclone was then subjected to an ELISA assay for OX40 protein levels and a cell binding assay. Hybridoma clones screened through these assays were then used to further produce antibodies using serum-free cell culture for testing.

[0116] The OX40 protein level ELISA test and cell binding test cell level ELISA test are as follows:

[0117] Protein level ELISA detection: add 100 μL of cell supernatant to the Elisa plate coated with OX40-His protein, set up a positive control (positive mouse serum, diluted at 1:1000), incubate in a 37°C biochemical incubator for 1 hour, wash three times with PBST as the washing solution, shake off the residual solution, and pat dry; add enzyme-labeled secondary antibody diluted with PBST + 2% BSA (1:10000), 100 μL per well, incubate in a 37°C biochemical incubator for 1 hour, wash four times with PBST as the washing solution, shake off the residual solution, and pat dry; add TMB single-component color development solution, 100 μL / well, protect from light and color for about 8 minutes; add stop solution 2M H2SO4 to stop the reaction, 50 μL / well; read the OD value on a microplate reader. 450 Select the positive wells and transfer them to 24-well plates for further culture.

[0118] Cell level ELISA detection: add the supernatant of cells expanded to 24-well plate to the coated

[0119] In the detection plate of L929-OX40EDC overexpression cell line (constructed in-house in the laboratory), 80 μL was added to each well, and a positive control (positive mouse serum, diluted at 1:1000) was set up. The cells were incubated in a 37°C biochemical incubator for 1 hour, washed 3 times with PBS + 2% FBS as a washing solution, and the residual solution was shaken off. The enzyme-labeled secondary antibody (diluted at 1:10000) with PBS + 2% FBS was added, 100 μL per well, incubated in a 37°C biochemical incubator for 1 hour, washed 4 times with PBS + 2% FBS as a washing solution, and the residual solution was shaken off. TMB single-component colorimetric solution was added, 100 μL / well, and color was developed in the dark for about 8 minutes. The stop solution 2M H2SO4 was added to terminate the reaction, 50 μL / well; the OD was read on a microplate reader. 450 Select positive wells for freezing.

[0120] Hybridoma antibody sequence determination

[0121] Monoclonal hybridoma cell lines with good activity were screened and RNA was extracted, reverse transcribed, and amplified for sequencing. The murine OX40 antibodies 12G9 and 23F8 were sequenced. The hybridoma cell line sequencing results are shown in Table 1:

[0122] Table 1 Sequencing results of hybridoma cell lines

[0123] Example 2 Humanization of murine antibodies

[0124] To reduce the immunogenicity of murine antibodies, the screened antibodies 12G9 and 23F8, which exhibited excellent in vitro and in vivo activity, were humanized. Humanization of murine monoclonal antibodies follows methods published in the literature. This involves replacing the constant regions of murine antibodies with those of human antibodies. Human germline antibody sequences are selected based on homology between the murine and human antibodies, and CDR grafting is performed. Based on the three-dimensional structure of the murine antibody, key amino acids in the FR regions of the humanized antibodies are backmutated to obtain highly humanized and highly active humanized antibodies.

[0125] The constant region sequences of the following humanized antibodies are shown as SEQ ID NO: 42 and SEQ ID NO: 43.

[0126] Heavy chain constant region:

[0127] Light chain constant region:

[0128] 2.1 Selection and backmutation of the human FR region of antibody 12G9

[0129] (1) Selection of human FR regions

[0130] The humanized VH templates for 12G9 were IGHV1-2*02 (sequence such as SEQ ID NO: 34, see Table 2) and IGHJ6*01 (sequence such as SEQ ID NO: 40, see Table 2), and the humanized VL templates were IGKV2-30*01 (sequence such as SEQ ID NO: 35, see Table 2) and IGKJ5*01 (sequence such as SEQ ID NO: 41, see Table 2). The CDRs of 12G9 were transplanted onto the humanized templates. The variable region sequences obtained after transplantation are as follows:

[0131] hu12G9 VH-CDR:

[0132] hu12G9 VL-CDR: Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The ununderlined part of the sequence is the FR sequence, and the underlined part is the CDR sequence.

[0133] Table 2 Humanized template sequences

[0134] (2) Reversion mutation of humanized antibodies

[0135] Individual site mutations were performed on the above humanized antibodies. The specific back mutation sites are shown in Table 3 below:

[0136] Table 3 Back mutations of humanized antibody h12G9 Note: A24T in the table indicates that the amino acid A at position 24 of the humanized antibody was backmutated to T.

[0137] The VH and VL of the humanized antibody hu12G9 were subjected to single mutation and multi-point mutation according to the mutation sites in Table 3, and the constructed heavy and light chains with different backmutations were combined. The resulting humanized antibodies are shown in Table 4. The expressed proteins were subjected to affinity and cell activity measurements as described in Examples 3, 4, 5, and 6.

[0138] Table 4 12G9 humanized antibody sequence

[0139] 2.2 Selection and backmutation of the human FR region of 23F8

[0140] (1) Selection of human FR regions

[0141] The humanized VH templates of 23F8 were IGHV1-2*02 (sequence such as SEQ ID NO: 36, see Table 2) and IGHJ6*01 (sequence such as SEQ ID NO: 40, see Table 2), and the humanized VL templates were IGKV1-33*01 (sequence such as SEQ ID NO: 37, see Table 2) and IGKJ5*01 (sequence such as SEQ ID NO: 41, see Table 2). The CDRs of 23F8 were transplanted onto the humanized templates. The variable region sequences obtained after transplantation are as follows:

[0142] hu23F8-1 VH-CDR:

[0143] hu23F8-1 VL-CDR: Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The ununderlined part of the sequence is the FR sequence, and the underlined part is the CDR sequence.

[0144] (2) Reversion mutation of humanized antibodies

[0145] The back mutation sites of the humanized antibody hu23F8-1 are shown in Table 5:

[0146] Table 5 Back mutations of 23F8 humanized antibody Note: In the table, M48I indicates that the amino acid M at position 48 of the humanized antibody was mutated back to I.

[0147] The VH and VL of the humanized antibody hu23F8-1 were subjected to single and multiple mutations according to the mutation sites listed in Table 5. Different backmutated heavy and light chains were constructed and combined. The resulting humanized antibodies are shown in Table 6. The expressed proteins were subjected to affinity and cell viability assays as described in Examples 3, 4, 5, and 6 below:

[0148] Table 6 23F8 humanized antibody sequence

[0149] 2.3 Selection of high expression template for 23F8 humanized antibody and back mutation

[0150] (1) Selection of high expression template for 23F8 humanized antibody:

[0151] To further increase the expression of humanized antibodies, a high-expression template was selected to humanize 23F8. The high-expression humanized VH templates of 23F8 were IGHV1-46*03 (sequence such as SEQ ID NO: 38, see Table 2) and IGHJ6*01 (sequence such as SEQ ID NO: 40, see Table 2), and the humanized VL templates were IGKV1-5*01 (sequence such as SEQ ID NO: 39, see Table 2) and IGKJ5*01 (sequence such as SEQ ID NO: 41, see Table 2). The CDRs of 23F8 were transplanted onto the high-expression humanized templates. The variable region sequences obtained after transplantation are as follows:

[0152] hu23F8-2 VH-CDR:

[0153] hu23F8-2 VL-CDR:

[0154] (2) Reversion mutation of humanized antibodies

[0155] The back mutation sites of humanized antibody h23F8-2 are shown in Table 7:

[0156] Table 7 Back mutations of humanized antibody h23F8-2 Note: In the table, M48I indicates that the amino acid M at position 48 of the humanized antibody was mutated back to I.

[0157] The VH and VL of the humanized antibody hu23F8-2 were subjected to single and multiple mutations according to the mutation sites in Table 7. The constructed different backmutated heavy and light chains were combined to obtain the humanized antibodies described in Table 8 below. The expressed proteins were then subjected to affinity, cell viability, and other assays as described in Examples 3, 4, 5, and 6 below.

[0158] Table 8 23F8 humanized antibody sequence

[0159] Example 3 Antibody Affinity Determination

[0160] The Biacore method is a well-known method for detecting the affinity and kinetics of protein-protein interactions. We used Biacore T200 to analyze the affinity and binding kinetics of the OX40 antibodies tested in the present invention.

[0161] The Biacore binding activity of each humanized antibody obtained in Example 2 was measured. The results are shown in Table 9:

[0162] Table 9 Antibody affinity results

[0163] The affinity of the humanized antibodies obtained in the present invention is higher than nM (10 -9 ) level, which can effectively bind to OX40.

[0164] Example 4 Protein Binding Assay of Antibodies

[0165] OX40-His protein (Sino Biological) was coated at 50 ng / well at 4°C overnight and blocked at 37°C for 2 h. Antibodies AH1-12, 5HT6, and 6HA5L obtained in Example 2 were diluted to 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.008, 0.004, 0.002, 0.001, 0.0005, 0.0002, 0.0001, and 0 μg / ml, respectively, and added to the ELISA plate. The plate was incubated at 37°C for 1 h. The secondary antibody mouse anti-human Fc HRP (GenScript) was diluted 1:10,000 and incubated at 37°C for 1 h. The color was developed with TMB substrate and terminated with 2 M H2SO4. The OD 450 The data were analyzed using GraphPad Prism. The results are shown in Table 10 and in Figure 1:

[0166] Table 10 Binding IC of antibodies 50 result

[0167] The results showed that the humanized antibody developed by the present invention has a high affinity for OX40.

[0168] Example 5 Antibody Cell Activation Experiment

[0169] The humanized antibody obtained in Example 2 was used to perform a T cell activation experiment according to the following steps:

[0170] The antibodies of Example 2 were diluted according to the following concentration gradient: 0.001 ug / mL, 0.01 ug / mL, 0.1 ug / mL, 0.5 ug / mL, 1 ug / mL, 5 ug / mL, 10 ug / mL, and 20 ug / mL, respectively. The CD3 antibody was diluted to 5 ug / mL. The CD3 antibody and the above-mentioned OX40 antibodies were mixed and coated on a plate according to the following conditions: 100 ul / well, and coated overnight at 4°C.

[0171] Resuscitate PBMC cells: Remove the cells from liquid nitrogen and place them in a 37°C water bath. Shake to dissolve, then transfer them to the prepared culture medium. Centrifuge to remove DMSO and discard the supernatant for later use.

[0172] Resuspend the cells in 1 ml serum-free medium (X-VIVO medium, Lonza) and transfer them to a sorting flow tube. Add 50 μl of cocktail reagent (T cell sorting kit, Stemcell) and mix well. Incubate at room temperature for 5 min. TM Dextran RapidSpheres TM After swirling the reagent (T cell sorting kit, Stemcell) for 30 seconds in advance, pipette 40ul into the sorting flow tube and pipette evenly, add culture medium to 2.5ml, and pipette 2-3 times; open the sorting flow tube and place it in the magnetic pole, incubate at room temperature for 3 minutes, and the poured supernatant is the sorted T cells.

[0173] Prepare cellTrace TM (Thermo) stock solution: add one Celltrace TM Dissolve the reagent in 20 μL DMSO and mix well (5 mM); add 1 μL Cell Trace per milliliter of cell suspension (X-VIVO medium) TM The cells were incubated in the dark at 37°C for 20 minutes. Five times the volume of culture medium was added and incubated for 5 minutes. The cells were centrifuged and resuspended in preheated full culture medium and incubated for 10 minutes. 5 The cells were plated into a pre-coated 96-well plate / well and incubated in a CO2 incubator for 5-7 days before flow cytometry analysis. The data were analyzed using GraphPad Prism.

[0174] The results of the cell activation experiments are shown in Figures 2a-2c and 3a-3c. 50 See Tables 11 and 12.

[0175] Table 11 Cell activation IC of 12G9 humanized antibody 50

[0176] Table 12 Cell activation IC of 23F8 humanized antibody 50

[0177] The results showed that the anti-OX40 antibody developed by the present invention can enhance T cell activation.

[0178] Example 6 Animal Experiment

[0179] Each mouse (OX40 humanized mice, purchased from Biocytogen) was injected with 1 million units of the mouse colon cancer cell line MC38 (National Laboratory Cell Resource Sharing Platform). Seven days after colon cancer cell inoculation, the mice were intraperitoneally injected with the humanized antibody obtained in Example 2 at a dose of 3 mg / kg every three days. After 21 days, the mice were sacrificed and the tumors were dissected. The results are shown in Figures 4 and 5.

[0180] The results showed that compared with the blank control group, the tumor volume and mass of the drug-treated group were significantly reduced, and the antibody of the present invention can significantly inhibit the growth of MC38 tumors.

[0181] The present invention has been described through the above-described embodiments. However, it should be understood that the above-described embodiments are for illustrative and illustrative purposes only and are not intended to limit the present invention to the described embodiments. Furthermore, it will be understood by those skilled in the art that the present invention is not limited to the above-described embodiments and that further variations and modifications may be made based on the teachings of the present invention, all of which fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof, which specifically binds to OX40, comprising a heavy chain variable region and / or a light chain variable region, wherein: 1) The heavy chain variable region comprises (i) three heavy chain complementary determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained in a peptide segment having an amino acid sequence of SEQ ID NO: 7, 9, 11, 12, 13, 14, 15, 23, 25, 27, 28, 29, 31 or 32; or (ii) relative to the sequences of the three heavy chain complementary determining regions (HCDRs) described in (i), a sequence comprising at least one and no more than 5 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in the three heavy chain complementary determining regions (HCDRs); and / or 2) The light chain variable region comprises, (i) three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained in a peptide segment having an amino acid sequence of SEQ ID NO: 8, 10, 16, 24, 26, 30 or 33; or (ii) relative to the sequences of the three light chain complementary determining regions (LCDRs) described in (i), a sequence comprising at least one and no more than 5 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in the three light chain complementary determining regions (LCDRs).

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: The three heavy chain complementary determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained in the peptide segment with the amino acid sequence of SEQ ID NO: 7, 9, 11, 12, 13, 14, 15, 23, 25, 27, 28, 29, 31 or 32; and / or The peptide segment with the amino acid sequence of SEQ ID NO: 8, 10, 16, 24, 26, 30 or 33 contains three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3.

3. An antibody or antigen-binding fragment thereof that specifically binds to OX40, comprising: a) a heavy chain complementarity determining region HCDR3, said HCDR3 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 3 or 19, or said HCDR3 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 3 or 19; and / or b) a light chain complementary determining region LCDR3, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 6 or 22, or consisting of an amino acid sequence as shown in SEQ ID NO: 6 or 22, or said LCDR3 comprising 1 or 2 amino acids compared to the amino acid sequence as shown in SEQ ID NO: 6 or 22. Or an amino acid sequence having three amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions).

4. The antibody or antigen-binding fragment thereof according to claim 3, further comprising: c) a heavy chain complementarity determining region HCDR1, wherein the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 1 or 17, or consists of the amino acid sequence as shown in SEQ ID NO: 1 or 17, or the HCDR1 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 1 or 17; d) a heavy chain complementarity determining region HCDR2, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO: 2 or 18, or consisting of an amino acid sequence as shown in SEQ ID NO: 2 or 18, or said HCDR2 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 2 or 18; e) a light chain complementary determining region LCDR1, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO: 4 or 20, or consisting of an amino acid sequence as shown in SEQ ID NO: 4 or 20, or said LCDR1 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 4 or 20; and / or f) a light chain complementary determining region LCDR2, said LCDR2 comprising an amino acid sequence as shown in SEQ ID NO: 5 or 21, or consisting of an amino acid sequence as shown in SEQ ID NO: 5 or 21, or said LCDR2 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 5 or 21.

5. An antibody or antigen-binding fragment thereof, comprising three heavy chain complementary determining regions (HCDRs) HCDR1, HCDR2 and HCDR3; and three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein: HCDR1 comprises SEQ ID NO: 1 or SEQ ID NO: 17, or consists thereof, or the HCDR1 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 1 or 17; HCDR2 comprises SEQ ID NO: 2 or SEQ ID NO: 18, or consists thereof, or the HCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 2 or 18; HCDR3 comprises SEQ ID NO: 3 or SEQ ID NO: 19, or consists thereof, or the HCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in SEQ ID NO: 3 or 19; LCDR1 comprises SEQ ID NO: 4 or SEQ ID NO: 20, or consists thereof, or the LCDR1 An amino acid sequence comprising 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 4 or 20; LCDR2 comprises SEQ ID NO: 5 or SEQ ID NO: 21, or consists of it, or the LCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 5 or 21; and LCDR3 comprises SEQ ID NO: 6 or SEQ ID NO: 22, or consists of them, or the LCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 6 or 22.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, comprising: (1) three heavy chain complementary determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained in the peptide segment with the amino acid sequence shown in SEQ ID NO:7, 9, 11, 12, 13, 14 or 15, and three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained in the peptide segment with the amino acid sequence shown in SEQ ID NO:8, 10 or 16; or (2) three heavy chain complementary determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 contained in the peptide segment with an amino acid sequence such as SEQ ID NO: 23, 25, 27, 28, 29, 31 or 32, and three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained in the peptide segment with an amino acid sequence such as SEQ ID NO: 24, 26, 30 or 33.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising: (1) HCDR1 comprising or consisting of SEQ ID NO: 1, HCDR2 comprising or consisting of SEQ ID NO:2, A HCDR3 comprising or consisting of SEQ ID NO: 3, LCDR1 comprising or consisting of SEQ ID NO:4, LCDR2 comprising or consisting of SEQ ID NO:5, and LCDR3 comprising or consisting of SEQ ID NO: 6; or (2) HCDR1 comprising SEQ ID NO: 17 or consisting thereof, A HCDR2 comprising or consisting of SEQ ID NO: 18, A HCDR3 comprising or consisting of SEQ ID NO: 19, LCDR1 comprising or consisting of SEQ ID NO:20, LCDR2 comprising or consisting of SEQ ID NO: 21, and LCDR3 comprising or consisting of SEQ ID NO:

22.

8. An antibody or antigen-binding fragment thereof that specifically binds to OX40, comprising: A heavy chain variable region comprising: SEQ ID NO: 7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32; or the heavy chain variable region consists of SEQ ID NO: 7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32, or consists of the sequence of SEQ ID NO:7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; or the heavy chain variable region comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to SEQ ID NO7, 23, 9, 11, 12, 13, 14, 15, 25, 27, 28, 29, 31 or 32, preferably, the amino acid mutations do not occur in the heavy chain complementary determining region; and / or a light chain variable region comprising: SEQ ID NO:8, 24, 10, 16, 26, 30 or 33, or a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having a sequence having NO:8, 24, 10, 16, 26, 30 or 33 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence; or the light chain variable region consists of SEQ ID NO:8, 24, 10, 16, 26, 30 or 33, or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence of SEQ ID NO:8, 24, 10, 16, 26, 30 or 33; or the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence of SEQ ID NO:8, 24, 10, 16, 26, 30 or 33 NO:8, 24, 10, 16, 26, 30 or 33, an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions), or consisting of the same, preferably, the amino acid mutations do not occur in the complementary determining region of the light chain.

9. The antibody or antigen-binding fragment thereof of claim 8, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence pairs of the heavy chain variable region and the light chain variable region are selected from any one of the groups consisting of the following amino acid sequence pairs: SEQ ID NOs: 7 and 8, SEQ ID NOs: 23 and 24, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 16, SEQ ID NOs: 12 and 16, SEQ ID NOs: 13 and 16, SEQ ID NOs: 14 and 16, SEQ ID NOs: 15 and 16, SEQ ID NOs: 25 and 26, SEQ ID NOs: 27 and 26, SEQ ID NOs: 28 and 26, SEQ ID NOs: 29 and 30, SEQ ID NOs: 31 and 33, and SEQ ID NOs: 32 and 33.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2 or 6, wherein the CDR regions are defined according to the Kabat definition scheme, the Clothia definition scheme, the Abm definition scheme, the IMGT definition scheme and / or the Contact definition scheme.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, further comprising a constant region, wherein the constant region is derived from an IgG antibody, an IgM antibody, an IgA antibody, an IgD antibody or an IgE antibody, preferably, the constant region is derived from an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, further comprising a heavy chain constant region comprising: SEQ ID NO: 42 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 42; or the heavy chain constant region consists of SEQ ID NO: 42, or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 42; and / or A light chain constant region, comprising: SEQ ID NO:43, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:43; or the light chain constant region consists of SEQ ID NO:43, or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:

43.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (preferably scFv) or (Fab')2, single domain antibody, diabody (dAb) or linear antibody.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody is a bispecific or multispecific antibody molecule, preferably, the bispecific antibody molecule binds to OX40 and PD-1, PD-L1 or PD-L2.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 and a pharmaceutically acceptable excipient.

17. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

18. An expression vector comprising the nucleic acid of claim 17.

19. A host cell comprising the expression vector according to claim 18.

20. The host cell of claim 19, which is a prokaryotic cell or a eukaryotic cell.

21. The host cell of claim 19, wherein the host cell is Escherichia coli, a yeast cell, an insect cell, a plant cell or a mammalian cell.

22. The host cell of claim 19, wherein the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell or a NSO cell.

23. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell according to any one of claims 19 to 22, and recovering the antibody or the antigen-binding fragment thereof expressed by the host cell from the culture.

24. Use of the antibody or antigen-binding fragment of any one of claims 1 to 15 in the preparation of a medicament for treating cancer, preferably, the cancer is colon cancer.

25. The antibody or antigen-binding fragment of any one of claims 1-15, for use in treating cancer, preferably colon cancer.

26. A method for treating cancer, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, preferably the cancer is colon cancer.