An anti-OX40 antibody, its preparation method and application

Abstract

The present invention provides an anti-OX40 antibody or an antigen-binding fragment thereof, as well as a preparation method and applications thereof. A new anti-OX40 antibody was unexpectedly screened in this application, and its activity is significantly better than existing drugs that have been clinically tested, and it can be used for the treatment of inflammation and tumors, especially autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to an anti-OX40 antibody or its antigen-binding fragment, as well as a preparation method and application thereof. Background Art

[0002] Tumor necrosis factor receptor OX40 (CD134) functions as a T cell co-stimulatory molecule by binding to its ligand OX40L (CD134L, CD252). The ligand of OX40, OX40L (CD252), is usually expressed on antigen-presenting cells such as dendritic cells, and OX40 (CD134) is generally expressed on activated CD4 + and CD8 + T cells and Treg cells. The expression of both molecules increases after antigen presentation, and their interaction promotes T cell survival, tends to reduce regulatory functions, increases the production of effector cytokines, and enhances the activity of cells.

[0003] The binding of OX40 and OX40L is associated with many diseases. There is evidence that agonistic anti-OX40 antibodies that activate the OX40 signaling pathway have been used in preclinical studies for the treatment of tumors and infectious diseases. However, antagonistic antibodies that block the OX40 signaling pathway are used to treat autoimmune diseases or inflammation. For example, atopic dermatitis is a type II immune disease. After the epidermal barrier of patients is ruptured, allergens and irritants penetrate, stimulating keratinocytes to produce pro-inflammatory cytokines. Thymic stromal lymphopoietin (TSLP) stimulates DC cells to express OX40L, and OX40L + DC cells induce the differentiation of OX40+ T cells, and the Th2 cells of OX40 + release IL-4 and IL-13 after activation, promoting the IgE class switch of B cells and generating antigen-specific IgE through the signal transducer and activator of transcription (STAT) pathway. Therefore, OX40 and OX40L play an important role in the pathogenic mechanism of atopic dermatitis, and blocking the binding of OX40 and OX40L can be used to treat autoimmune diseases or inflammation.

[0004] Existing technologies show that OX40 antibodies weaken the activation of T cells by blocking the binding of OX40L and OX40, and at the same time kill over-activated T cells through the ADCC effect. Currently, two anti-OX40 monoclonal antibody drugs for the treatment of moderate to severe atopic dermatitis have entered phase II clinical trials, namely GBR830 and KHK4083, both of which have shown good therapeutic effects. Anti-OX40 monoclonal antibodies show good application prospects in the treatment of moderate to severe atopic dermatitis. Summary of the Invention

[0005] Based on the progress of the prior art, a new anti-OX40 antibody was unexpectedly screened in this application. Its activity is significantly better than that of GBR830 and KHK4083. Especially after Fc engineering modification, the activity is further improved, and it can be used for better treatment of inflammation or tumors, especially autoimmune diseases. The specific scheme is as follows:

[0006] In the first aspect of the present invention, there is provided an anti-OX40 antibody or an antigen-binding fragment thereof, and the anti-OX40 antibody or an antigen-binding fragment thereof comprises VHCDR1-3 of the heavy-chain variable region and / or VLCDR1-3 of the light-chain variable region. Among them:

[0007] The amino acid sequence of VHCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, or, comprises an amino acid sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% with the amino acid sequence shown in SEQ ID NO: 1, or, comprises an amino acid sequence having at least one, two or more than three or at most five, four, three or two amino acid substitutions, deletions or mutations on SEQ ID NO: 1, and has the same or similar activity as SEQ ID NO: 1 (such as the binding activity to OX40).

[0008] The length of the said VHCDR1 is 3-30 amino acids, preferably 5-15 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0009] The amino acid sequence of VHCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, or, comprises an amino acid sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% with the amino acid sequence shown in SEQ ID NO: 2, or, comprises an amino acid sequence having at least one, two or more than three or at most five, four, three or two amino acid substitutions, deletions or mutations on SEQ ID NO: 2, and has the same or similar activity as SEQ ID NO: 2 (such as the binding activity to OX40).

[0010] The length of the said VHCDR2 is 3-30 amino acids, preferably 17-25 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0011] The amino acid sequence of VHCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, or, comprises an amino acid sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% with the amino acid sequence shown in SEQ ID NO: 3, or, comprises an amino acid sequence having at least one, two or more than three or at most five, four, three or two amino acid substitutions, deletions or mutations on SEQ ID NO: 3, and has the same or similar activity as SEQ ID NO: 3 (such as the binding activity to OX40).

[0012] The length of the VHCDR3 described above is 3 - 30 amino acids, preferably 5 - 15 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0013] The amino acid sequence of VLCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, or, comprises an amino acid sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% with the amino acid sequence shown in SEQ ID NO: 4, or, comprises an amino acid sequence having at least one, two or more than three or at most five, four, three or two amino acid substitutions, deletions or mutations on SEQ ID NO: 4, and has the same or similar activity as SEQ ID NO: 4 (such as the binding activity to OX40).

[0014] The length of the VLCDR1 described above is 3 - 30 amino acids, preferably 10 - 20 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0015] The amino acid sequence of VLCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, or, comprises an amino acid sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% with the amino acid sequence shown in SEQ ID NO: 5, or, comprises an amino acid sequence having at least one, two or more than three or at most five, four, three or two amino acid substitutions, deletions or mutations on SEQ ID NO: 5, and has the same or similar activity as SEQ ID NO: 5 (such as the binding activity to OX40).

[0016] The length of the VLCDR2 is 3 - 30 amino acids, preferably 5 - 15 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0017] The amino acid sequence of VLCDR3 contains the amino acid sequence as shown in SEQ ID NO: 6, or contains an amino acid sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% with the amino acid sequence shown in SEQ ID NO: 6, or contains an amino acid sequence having substitution, deletion or mutation of at least one, two or more than three or at most five, four, three or two amino acids on SEQ ID NO: 6, and has the same or similar activity as SEQ ID NO: 6 (such as the binding activity to OX40).

[0018] The length of the VLCDR3 is 3 - 30 amino acids, preferably 8 - 20 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0019] In a specific embodiment of the present invention, the amino acid sequence of VHCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of VHCDR2 is as shown in SEQ ID NO: 2, the amino acid sequence of VHCDR3 is as shown in SEQ ID NO: 3, the amino acid sequence of VLCDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of VLCDR2 is as shown in SEQ ID NO: 5, and the amino acid sequence of VLCDR3 is as shown in SEQ ID NO: 6.

[0020] Preferably, the anti - OX40 antibody or its antigen - binding fragment further contains the FR region, and more preferably includes VHFR1, VHFR2, VHFR3, VHFR4 of the heavy - chain variable region, and / or VLFR1, VLFR2, VLFR3, VLFR4 of the light - chain variable region.

[0021] In a specific embodiment of the present invention, the heavy - chain variable region includes VHFR1, VHCDR1, VHFR2, VHCDR2, VHFR3, VHCDR3, VHFR4 in sequence from the N - terminal to the C - terminal.

[0022] In a specific embodiment of the present invention, the light chain variable region sequentially includes VLFR1, VLCDR1, VLFR2, VLCDR2, VLFR3, VLCDR3, and VLFR4 from the N-terminus to the C-terminus.

[0023] Preferably, the FR region is derived from or is the FR region of a human or non-human animal (such as a non-human mammal), preferably derived from or is the FR region of a human, rodent, or primate.

[0024] Preferably, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and VLFR4 are each independently derived from or are the corresponding FR regions of a human or non-human animal (such as a non-human mammal), preferably derived from or are the corresponding FR regions of a human, rodent, or primate.

[0025] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 7, or, includes an amino acid sequence having a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the amino acid sequence shown in SEQ ID NO: 7, or, includes an amino acid sequence having at least one, two, or three or more up to ten, nine, eight, seven, six, five, four, three, or two amino acid substitutions, deletions, or mutations on SEQ ID NO: 7, and having the same or similar activity as SEQ ID NO: 7 (such as the binding activity to OX40).

[0026] In a specific embodiment of the present invention, the nucleotide sequence encoding the heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO: 8, or, includes a nucleotide sequence having a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the nucleotide sequence shown in SEQ ID NO: 8, or, includes a nucleotide sequence having at least one, two, or three or more up to ten, nine, eight, seven, six, five, four, three, or two nucleotide substitutions, deletions, or mutations on SEQ ID NO: 8, and having the same or similar activity as SEQ ID NO: 8 (such as the activity of expressing SEQ ID NO: 7).

[0027] In a specific embodiment of the present invention, the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, or, comprises an amino acid sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% with the amino acid sequence shown in SEQ ID NO: 9, or, comprises an amino acid sequence having at least one, two or more than three or at most ten, nine, eight, seven, six, five, four, three or two amino acid substitutions, deletions or mutations on SEQ ID NO: 9, and having the same or similar activity as SEQ ID NO: 9 (such as the binding activity to OX40).

[0028] In a specific embodiment of the present invention, the nucleotide sequence encoding the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 10, or, comprises a nucleotide sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% with the nucleotide sequence shown in SEQ ID NO: 10, or, comprises a nucleotide sequence having at least one, two or more than three or at most ten, nine, eight, seven, six, five, four, three or two nucleotide substitutions, deletions or mutations on SEQ ID NO: 10, and having the same or similar activity as SEQ ID NO: 10 (such as the activity of expressing SEQ ID NO: 9).

[0029] The anti - OX40 antibody or its antigen - binding fragment comprises a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region and / or the light chain constant region is derived from or is the heavy chain constant region and / or the light chain constant region of a human or non - human animal (such as a non - human mammal), preferably derived from or is the heavy chain constant region and / or the light chain constant region of a human, a rodent or a primate.

[0030] Preferably, the heavy chain constant region includes but is not limited to the constant region of any one of IgG, IgD, IgA, IgM or IgE heavy chains. Further preferably, the IgG heavy chain includes IgG1 heavy chain, IgG2 heavy chain, IgG2b heavy chain, IgG3 heavy chain or IgG4 heavy chain. Further preferably, the IgA heavy chain includes IgA1 or IgA2.

[0031] Preferably, the light chain constant region is selected from the constant region of any one of λ - type light chain or κ - type light chain.

[0032] In a specific embodiment of the present invention, the heavy chain constant region is the constant region of an IgG heavy chain (IgG1 or IgG4), and the light chain constant region is the constant region of a κ light chain.

[0033] Preferably, the amino acid sequence of the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 11 or its D153E, M311L, and / or N317S mutants, or, comprises an amino acid sequence having a homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% with the amino acid sequence of SEQ ID NO: 11 or its D153E, M311L, and / or N317S mutants, or, comprises an amino acid sequence having substitution, deletion, or mutation of at least one, two, or more than three, or at most ten, nine, eight, seven, six, five, four, three, or two amino acids on SEQ ID NO: 11 and having the same or similar activity as SEQ ID NO: 11.

[0034] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain constant region is any one of the amino acid sequences of SEQ ID NO: 11-14.

[0035] Preferably, the anti-OX40 antibody or its antigen-binding fragment includes Fab, Fd, Fab’, Fab’-SH, Fv, scFv, F(ab’)2, or a single-domain antibody.

[0036] Preferably, the anti-OX40 antibody or its antigen-binding fragment can be a murine antibody, a chimeric antibody, or a humanized antibody.

[0037] Preferably, the antibody anti-OX40 antibody or its antigen-binding fragment can be monospecific, bispecific, trispecific, or more multi-specific.

[0038] Preferably, the anti-OX40 antibody or its antigen-binding fragment can be a monoclonal antibody or a polyclonal antibody.

[0039] The anti-OX40 antibody or its antigen-binding fragment specifically binds to the OX40 protein or its fragment of a human or non-human animal. The OX40 fragment can be an extracellular region.

[0040] In the second aspect of the present invention, there is provided a VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain, or light chain.

[0041] In the third aspect of the present invention, there is provided an antigen receptor, and the antigen receptor comprises the above-mentioned anti-OX40 antibody or its antigen-binding fragment, or, the above-mentioned VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain, or light chain.

[0042] The antigen receptor described above is selected from TCR, STAR or CAR.

[0043] The STAR described above includes the α-chain and β-chain of TCR, and the variable regions of the α-chain and β-chain are replaced with the light chain variable region and heavy chain variable region described in the first aspect, or are replaced with the heavy chain variable region and light chain variable region described in the first aspect.

[0044] In a specific embodiment of the present invention, the STAR includes the connection of the constant region of the TCR α-chain with the light chain variable region, and the connection of the constant region of the TCR β-chain with the heavy chain variable region.

[0045] In a specific embodiment of the present invention, the STAR includes the connection of the constant region of the TCR α-chain with the heavy chain variable region, and the connection of the constant region of the TCR β-chain with the light chain variable region.

[0046] Preferably, the constant region of the α-chain is connected to the constant region of the β-chain by a disulfide bond.

[0047] Preferably, the constant region of the α-chain is derived from the constant region of the TCR α-chain of a human or non-human animal.

[0048] Preferably, the constant region of the β-chain is derived from the constant region of the TCR β-chain of a human or non-human animal.

[0049] The extracellular antigen-binding domain of the CAR includes the anti-OX40 antibody or its antigen-binding fragment described above, the heavy chain variable region described above, the light chain variable region described above, the heavy chain described above, the light chain described above, including the antibody or its antigen-binding fragment described above.

[0050] Preferably, the CAR has one or more than two identical or partially identical or different extracellular antigen-binding domains.

[0051] Preferably, the CAR further includes a transmembrane domain, an intracellular domain and / or a hinge region.

[0052] More preferably, the hinge region connects the extracellular antigen-binding domain and the transmembrane domain.

[0053] Preferably, the transmembrane domain is selected from one or a combination of two or more of CD3ζ polypeptide, CD4 polypeptide, CD8 polypeptide, CD28 polypeptide, CD28-41BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide or BTLA polypeptide.

[0054] Preferably, the intracellular domain includes but is not limited to a signal transduction domain. More preferably, the intracellular domain further includes a co-stimulatory domain.

[0055] Preferably, the intracellular domain includes, but is not limited to, one or a combination of two or more of CD28, ICOS, 4-1BB, OX-40, CD27, or CD3ζ.

[0056] Preferably, the hinge region includes, but is not limited to, the extracellular hinge regions of CD8, CD8α, CD28, or IgG.

[0057] In a fourth aspect of the present invention, there is provided a nucleic acid encoding the anti-OX40 antibody or antigen-binding fragment thereof as described above, or the antigen receptor as described above, or the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain, or light chain as described above.

[0058] Preferably, the nucleotide sequence encoding the heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO: 8, or a nucleotide sequence having a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the nucleotide sequence shown in SEQ ID NO: 8, or a nucleotide sequence having at least one, two, or three or more up to ten, nine, eight, seven, six, five, four, three, or two nucleotide substitutions, deletions, or mutations on SEQ ID NO: 8 and having the same or similar activity as SEQ ID NO: 8 (such as the activity of expressing SEQ ID NO: 7).

[0059] Preferably, the nucleotide sequence encoding the light chain variable region includes the nucleotide sequence shown in SEQ ID NO: 10, or a nucleotide sequence having a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the nucleotide sequence shown in SEQ ID NO: 10, or a nucleotide sequence having at least one, two, or three or more up to ten, nine, eight, seven, six, five, four, three, or two nucleotide substitutions, deletions, or mutations on SEQ ID NO: 10 and having the same or similar activity as SEQ ID NO: 10 (such as the activity of expressing SEQ ID NO: 9).

[0060] In a fifth aspect of the present invention, there is provided a method for preparing the anti-OX40 antibody or antigen-binding fragment thereof as described above, or the antigen receptor as described above, or the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain, or light chain as described above, the method comprising transforming the above nucleic acid into a host cell and then inducing its expression.

[0061] In the sixth aspect of the present invention, a vector is provided, and the vector contains the above-mentioned nucleic acid.

[0062] The vector can be expressed in vivo, in vitro or ex vivo. Preferably, the vector is a prokaryotic expression vector, a viral expression vector or a eukaryotic expression vector. Further preferably, the prokaryotic expression vector includes but is not limited to any one of the vector series of the Escherichia coli expression system, the Bacillus subtilis expression system series vector or the streptomycin expression system series vector.

[0063] Further preferably, the eukaryotic expression vector includes but is not limited to the vector series of the yeast expression system, the fungal expression system series vector, the insect cell expression system series vector or the mammalian cell expression system series vector.

[0064] In the seventh aspect of the present invention, a host cell is provided, and the host cell contains the above-mentioned nucleic acid or the above-mentioned vector.

[0065] The host cell can be a eukaryotic cell or a prokaryotic cell.

[0066] Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells or CHO cells, etc.

[0067] Prokaryotic cells such as Escherichia coli, etc.

[0068] In the eighth aspect of the present invention, a method for preparing a host cell is provided, and the preparation method includes introducing the above-mentioned nucleic acid or vector into a host cell and then inducing its expression.

[0069] In the ninth aspect of the present invention, an immune cell is provided, and the immune cell expresses the above-mentioned anti-OX40 antibody or its antigen-binding fragment, the above-mentioned antigen receptor, the above-mentioned VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain or light chain.

[0070] Preferably, the immune cell includes one or more of lymphocytes, dendritic cells, peripheral blood mononuclear cells, macrophages, granulocytes or mast cells.

[0071] Further preferably, the lymphocytes are selected from T cells, TIL cells, NK cells, NKT cells or B cells.

[0072] In a specific embodiment of the present invention, the immune cell includes STAR-T cells or CAR-T cells.

[0073] In the tenth aspect of the present invention, a method for preparing the above-mentioned immune cells is provided, and the preparation method includes expressing the above-mentioned nucleic acid by transfecting it into immune cells.

[0074] In the eleventh aspect of the present invention, an antibody-drug conjugate (ADC) is provided, which comprises the above-mentioned anti-OX40 antibody or its antigen-binding fragment, the above-mentioned antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned immune cell, the above-mentioned VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain or light chain conjugated (such as covalently bound) with a therapeutic agent or a diagnostic reagent.

[0075] In the twelfth aspect of the present invention, an application of the above-mentioned anti-OX40 antibody or its antigen-binding fragment, the above-mentioned antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned immune cell, the above-mentioned VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain or light chain in blocking or reducing the binding of OX40 protein to the receptor, or an application in preparing a drug for treating and / or preventing diseases is provided.

[0076] Preferably, the disease is a disease in which targeting OX40 is beneficial for treatment.

[0077] Preferably, the disease is a disease in which blocking or reducing the binding of OX40 protein to the receptor is beneficial for treatment.

[0078] In a specific embodiment of the present invention, the disease is inflammation or tumor, preferably an autoimmune disease.

[0079] Preferably, the inflammation can be dermatitis.

[0080] In the thirteenth aspect of the present invention, a drug is provided, and the drug includes the above-mentioned anti-OX40 antibody or its antigen-binding fragment, the above-mentioned antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned immune cell, the above-mentioned VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain or light chain.

[0081] The drug further includes pharmaceutically acceptable excipients.

[0082] Preferably, the pharmaceutically acceptable excipients include one or more of excipients, diluents, binders, lubricants, wetting agents, emulsifiers, preservatives, antioxidants, buffers, bacteriostatic agents, disintegrants, surfactants, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and fragrances.

[0083] Preferably, the drug can be used alone or in combination with other therapeutic agents.

[0084] The "drug" described above can be used to treat humans or non-human animals, such as non-human mammals. The drug may contain pharmaceutically acceptable excipients or salts commonly used in the prior art. The drug can be administered by any suitable route, such as enteral administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, infusion, intracerebral, intrathecal, transdermal, rectal, etc.). The drug can be in any suitable dosage form, such as an enteral dosage form or a parenteral dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder aerosols, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, dripping pills, gels, etc. The various dosage forms of the drug can be prepared according to the conventional production methods in the pharmaceutical field. The drug may contain 0.01-99.5% by weight (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of an anti-OX40 antibody or its antigen-binding fragment, antigen receptor, nucleic acid, vector, host cell, ADC or immune cell, etc. The drug can be prepared as a reagent with a protein concentration of 1-300 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg / mL). The single-dose of the drug can be 0.1-1000 mg, such as 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, 10, 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg.

[0085] The drug treatment targets OX40 for treating diseases beneficial to treatment.

[0086] The drug treatment that blocks or reduces the binding of OX40 protein to the receptor is beneficial for treating diseases.

[0087] The drug treats inflammation or tumors, preferably autoimmune diseases. Preferably, the inflammation can be dermatitis.

[0088] In a fourteenth aspect of the present invention, a diagnostic kit is provided. The diagnostic kit includes the anti-OX40 antibody or its antigen-binding fragment, the antigen receptor, the nucleic acid, the vector, the host cell, the immune cell, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain or light chain as described above.

[0089] The diagnostic kit can diagnose diseases in which targeting OX40 is beneficial for treatment.

[0090] The diagnostic kit can diagnose diseases in which blocking or reducing the binding of OX40 protein to the receptor is beneficial for treatment.

[0091] The diagnostic kit can diagnose inflammation or tumors, preferably autoimmune diseases. Preferably, the inflammation can be dermatitis.

[0092] The diagnostic kit can diagnose diseases that express or overexpress OX40.

[0093] In a fifteenth aspect of the present invention, a method for detecting OX40 is provided. The method includes contacting a sample to be tested with the anti-OX40 antibody or its antigen-binding fragment, the antigen receptor, the nucleic acid, the vector, the host cell, the immune cell, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain or light chain as described above, and then detecting the content of the complex.

[0094] The detection of OX40 is to detect the presence or content of OX40. Among them, the presence indicates whether there is any, and the content can be the expression level or protein concentration, etc.

[0095] In a sixteenth aspect of the present invention, a method for diagnosing a disease is provided. The method includes sampling, contacting the sample with the anti-OX40 antibody or its antigen-binding fragment, the antigen receptor, the nucleic acid, the vector, the host cell, the immune cell, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain or light chain as described above, and then detecting the content of the complex.

[0096] Preferably, the above-mentioned diagnostic kit is used to detect OX40.

[0097] Preferably, the disease is a disease in which targeting OX40 is beneficial for treatment.

[0098] Preferably, the disease is a disease in which blocking or reducing the binding of OX40 protein to the receptor is beneficial for treatment.

[0099] In a specific embodiment of the present invention, the disease is inflammation or tumor, preferably an autoimmune disease. Preferably, the inflammation can be dermatitis.

[0100] In the seventeenth aspect of the present invention, there is provided a method for blocking or reducing the binding of OX40 protein to the receptor, or preventing and / or treating a disease, the method comprising administering an effective amount of the above-mentioned anti-OX40 antibody or its antigen-binding fragment, the above-mentioned antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, ADC, the above-mentioned immune cell, the above-mentioned drug, the above-mentioned VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, heavy chain variable region, light chain variable region, heavy chain or light chain to an individual in need.

[0101] Preferably, the disease is a disease in which targeting OX40 is beneficial for treatment.

[0102] Preferably, the disease is a disease in which blocking or reducing the binding of OX40 protein to the receptor is beneficial for treatment.

[0103] In a specific embodiment of the present invention, the disease is inflammation or tumor, preferably an autoimmune disease. Preferably, the inflammation can be dermatitis.

[0104] The "antigen-binding fragment" as described in the present invention is a part of an antibody that retains the specific binding activity of the antibody, that is, any part of the antibody capable of specifically binding to an epitope on the target molecule of the antibody. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd, Fab'-SH, and variants of these fragments. For example, the heavy chain and / or light chain of an antibody, the variable region of the heavy chain and / or the variable region of the light chain of an antibody, or one or more than two CDRs (Complementarity Determining Region) from the heavy chain or light chain of an antibody. Among them, Fab is a monovalent fragment composed of the VL, VH, CL, and CH1 domains. F(ab')2 is a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region. Fd is an Fd fragment composed of the VH and CH1 domains. Fv is an Fv fragment composed of the VL and VH domains of a single arm of an antibody. Fab' is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain. Fab'-SH is a Fab' in which the cysteine residue in the constant domain has at least one free thiol group. Among them, VH represents the variable region of the heavy chain, VL represents the variable region of the light chain, and CL represents the light chain.

[0105] The "scFv" as described in the present invention, that is, a single chain antibody (single chain antibody fragment), is composed of the variable region of the heavy chain and the variable region of the light chain of an antibody, and the variable region of the heavy chain and the variable region of the light chain can be linked by a short peptide (linker), and the length of the linker is generally about 15 - 20 amino acids.

[0106] The "single-domain antibody" as described in the present invention can be an antibody that contains only one of the variable region of the heavy chain or the variable region of the light chain. For example, a nanobody contains only the variable region domain of the heavy chain (VHH).

[0107] The "murine antibody" as described in the present invention refers to the framework region of the variable region of the heavy chain or the framework region of the variable region of the light chain or the constant region part (such as the CH region) of the antibody, or the entire antibody is encoded by murine antibody genes.

[0108] The "chimeric antibody" as described in the present invention refers to that the framework region of the variable region of the heavy chain or the framework region of the variable region of the light chain or the constant region part (such as the CH region) of the antibody is independently derived from different species, for example, independently derived from humans or non-human animals respectively. Preferably, the framework region of the variable region of the heavy chain or the framework region of the variable region of the light chain is derived from a mouse, and the constant region part (such as the CH region) is derived from a human.

[0109] The "humanized antibody" as described in the present invention refers to that the framework region of the variable region of the heavy chain or the framework region of the variable region of the light chain or the constant region part (such as the CH region) of the antibody, or the entire antibody is encoded by human antibody genes.

[0110] As used herein, "CDR" refers to the complementarity determining regions within the variable sequences of an antibody. For each variable region, there are three CDRs in each variable region of the heavy and light chains, which are designated CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat et al. (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to antibody variable regions, but also provides the residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Each complementarity determining region may contain amino acid residues from the "complementarity determining region" as defined by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)) found that certain subparts within the Kabat CDRs adopt nearly identical peptide backbone conformations, despite significant diversity at the amino acid sequence level. These subparts are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" represent the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. There are other CDR boundary definitions that do not strictly follow one of the above systems, but still overlap with the Kabat CDRs. The methods used herein can utilize CDRs defined according to any of these systems, although the preferred embodiments use Kabat or Chothia defined CDRs.

[0111] As used herein, "antibody variable region" refers to the portion of the amino acid sequence of the light and heavy chains of an antibody molecule that includes the complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0112] The "inflammation" described in the present invention can be inflammation occurring in any tissue, and the tissues include but are not limited to adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (such as renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, white blood cell. Further preferably, the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergy (such as allergic rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivity, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, transplant rejection, vasculitis or interstitial cystitis.

[0113] The "tumor" as described in the present invention can be any abnormal cell proliferation (or any disease that presents as abnormal cell proliferation itself), neoplasm, or an increased tendency or risk of abnormal cell proliferation, neoplasm, or tumor. It can be benign or malignant, and can also be primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (such as renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, white blood cell. Further preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (such as glioblastoma), colorectal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyosarcoma, squamous cell carcinoma of the tongue, nasopharyngeal cancer, ovarian cancer, placental choriocarcinoma, lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (such as neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic carcinoma, blood cancer, head and neck cancer, or oropharyngeal cancer.

[0114] "Pharmaceutically acceptable" as described in the present invention refers to an active substance that neither significantly stimulates the organism nor inhibits the biological activities and properties of the product administered (such as the anti-OX40 antibody or its antigen-binding fragment, nucleic acid, vector, host cell, immune cell, antigen receptor, ADC, or drug, etc.) described in the present invention.

[0115] The "… method" as described in the present invention can be for the purpose of diagnosing and treating diseases, or for the purpose of diagnosing and treating non-diseases.

[0116] "Comprising" or "including" as described in the present invention is an open-ended writing method. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid can consist of the described sequence, or can have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still has the same or similar activity or function as the original sequence.

[0117] As used herein, "homology" means that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, so that the sequences used have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with the sequences obtained from the prior art, and retain the same or similar activities or functions.

[0118] As used herein, "treatment" means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0119] As used herein, "effective amount" means the amount or dose of the anti-OX40 antibody or its antigen-binding fragment antigen receptor, nucleic acid, vector, host cell, immune cell, ADC, or drug of the present invention that provides the desired treatment or prevention after being administered to an individual or an organ in a single or multiple doses.

[0120] As used herein, "prevention" means a manner implemented to prevent or delay the occurrence of a disease or condition or symptom in an organism.

[0121] As used herein, "diagnosis" means to ascertain whether a patient has had, has, or will have a disease or condition, or to ascertain the progression or possible future progression of a disease.

[0122] As used herein, "individual" can be a human or a non-human animal (e.g., a non-human mammal), or a cell, tissue, or organ of a human or a non-human mammal.

[0123] The "non-human mammal" described in the present invention can be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cows, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys (such as cynomolgus monkeys, rhesus monkeys), etc.

[0124] The "and / or" described in the present invention includes all combinations of the items connected by this term, and each combination should be regarded as having been separately listed herein. For example, "A and / or B" includes "A", "A and B", and "B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C". BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0126] Figure 1 : ELISA binding activity of Ab18 antibody and hOX40-mFc.

[0127] Figure 2 : ELISA binding activity of Ab18 antibody and mOX40-mFc.

[0128] Figure 3 : ELISA binding activity of Ab18 antibody and RatX40-mFc.

[0129] Figure 4 : Detection experiment of ELISA binding activity of Ab18 antibody and CynOX40-mFc.

[0130] Figure 5 : Detection experiment of FCS binding of Ab18 antibody and hOX40-HEK293 cells.

[0131] Figure 6 : Detection experiment of ELISA binding of Ab18 antibody and OX40 proteins of different species.

[0132] Figure 7 : Detection experiment of the blocking effect of Ab18 antibody on the binding of OX40L and Jurkat-OX40.

[0133] Figure 8 : Detection experiment of FCS binding of Ab18 antibody and its mutants and hFcγRⅡB-CHO-K1 cells.

[0134] Figure 9 : Detection experiment of the agonistic activity of Ab18 antibody and its mutants on Jurkat-OX40 cells.

[0135] Figure 10 : FCS binding detection experiment of Ab18 antibody and its mutants with hFcRn-CHO-K1 cells under the condition of pH 6.0.

[0136] Figure 11 : FCS binding detection experiment of Ab18 antibody and its mutants with hFcRn-CHO-K1 cells under the condition of pH 7.4.

[0137] Figure 12 : ADCC activity detection experiment of Ab18 antibody and its mutants. Specific implementation mode

[0138] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present invention.

[0139] Cells, plasmids, animals used in the examples:

[0140] HGH-hOX40: The HGH sequence is derived from NCBI (NP_000506.2) (Met1–Phe217), the hOX40 sequence is derived from NCBI (AAB33944.1) (His27–Asp167), and the linker between HGH and hOX40 is SGHHHHHHHHDYDIPSSENLYFQGS (SEQ ID NO: 15). The whole gene is synthesized by GenScript (pSGHV0) expression vector, and the protein is prepared by this application.

[0141] Jurkat / NFAT-luc+FcγRIIIa cells: Purchased from Promega Corporation, USA.

[0142] Control antibodies: The sequences of the control antibodies GBR830 and KHK4083 in this application are derived from the WHO official website INN Proposed List 122 telazorlimab sequence and INN Proposed List 125 Rocatinlimab sequence. The heavy and light chain nucleic acid sequences are synthesized by GenScript and cloned into the expression vector pcDNA3.1 / Hygro(+), and the antibodies are prepared by this application.

[0143] hOX40-mFc: The hOX40 sequence is derived from NCBI (AAB33944.1) (His27–Asp167), and mFc is murine IgG1 Fc, whose sequence is derived from NCBI (AAK53870.1) (Val98–Lys324). It was synthesized by GenScript and constructed into the PTT5 expression vector (preserved by the First Research Division of Shanghai Institute of Biological Products), and the protein was prepared in this application.

[0144] mOX40-mFc: The mOX40 sequence is derived from NCBI (NP_035789) (Val20-Pro211), and mFc is murine IgG1 Fc, whose sequence is derived from NCBI (AAK53870.1) (Val98–Lys324). It was synthesized by GenScript (pcDNA3.1 / Hygro(+)) expression vector, and the protein was prepared in this application.

[0145] RatOX40-mFc: The rat (Rat) OX40 sequence is derived from NCBI (P15725) (Val20-Pro210), and mFc is murine IgG1 Fc, whose sequence is derived from NCBI (AAK53870.1) (Val98–Lys324). It was synthesized by GenScript (pcDNA3.1 / Hygro(+)) expression vector, and the protein was prepared in this application.

[0146] CynOX40-mFc: The cynomolgus monkey (Cyn) OX40 sequence is derived from NCBI (XP_001090870.1) (Lys28-Ala214), and mFc is murine IgG1 Fc, whose sequence is derived from NCBI (AAK53870.1) (Val98–Lys324). It was synthesized by GenScript (pcDNA3.1 / Hygro(+)) expression vector, and the protein was prepared in this application.

[0147] hFc-hOX40L: The hOX40L sequence is derived from NCBI (P23510.1) (Gln51–Leu183), and hFc is human IgG1 Fc, whose sequence is derived from NCBI (4CDH_A) (Asp29–Lys255). It was synthesized by GenScript and constructed into the PSGHV0 (preserved by the First Research Division of Shanghai Institute of Biological Products) expression vector, and the protein was prepared in this application.

[0148] hFcRn-CHO-K1 cells: Purchased from GeneCopoeia Biotechnology (Shanghai) Co., Ltd. and preserved by the First Research Division of Shanghai Institute of Biological Products.

[0149] hFcγRⅡB-CHO-K1 cells: Purchased from GeneMan Biotech (Shanghai) Co., Ltd. and stored in the First Research Laboratory of Shanghai Institute of Biological Products.

[0150] Jurkat-OX40-NFκB-luc cells: Purchased from the National Institutes for Food and Drug Control and stored in the First Research Laboratory of Shanghai Institute of Biological Products.

[0151] hOX40-HEK293 stable transfected cell line: Stored in the First Research Laboratory of Shanghai Institute of Biological Products.

[0152] Example 1: Antibody Screening

[0153] 1. Animal Immunization and Hybridoma Preparation

[0154] The BALB / c mice used in the experiment were purchased from Shanghai SIPPR-BK Lab Animal Co., Ltd. All immunized mice were 4 - 6 weeks old, female, standardized disease-free, and healthy purebred mice. The injection dose of antigen HGH-hOX40 (300 μg / ml) was 50 μg per mouse. After mixing and emulsifying with Freund's adjuvant, an injection solution was prepared. Immunize subcutaneously once every two weeks for a total of 6 times. After the injection cycle, the serum antibody titer was detected to evaluate the immunization effect of HGH-hOX40 on mice. Select mice with an antibody titer > 10 5 for booster immunization. The injection dose was 10 μg per mouse, administered intravenously once a day for 3 days, and electrofusion was performed on the fourth day.

[0155] The hybridoma cells were screened by ELISA method. Dilute the HGH-hOX40 antigen protein to 1 μg / mL and coat 100 μL per well onto a 96-well ELISA plate overnight at 4°C. After washing three times with PBST, pat dry the residual liquid. Block with 200 μL per well of 5% skim milk powder overnight at 4°C. After washing three times with PBST, pat dry the residual liquid. On the 14th day of hybridoma cell culture, take 100 μL of the culture supernatant and add it to the antigen-coated microplate, incubate at 37°C for 1 h. After washing three times with PBST, add the goat anti-mouse IgG secondary antibody (purchased from Seracare Company, USA) diluted 1:5000, incubate at 37°C for 45 min. After washing three times with PBST, pat dry the residual liquid. Add 100 μL of TMB chromogenic solution to each well, and let it develop color in the dark at room temperature for 30 min. Add 50 μL of 2M sulfuric acid termination solution to each well to terminate the color development. Read the OD450 with SpectraMax. The positive hybridoma cells detected were used for subsequent re-screening and subcloning.

[0156] 2. Antibody

[0157] Total mRNA of hybridoma was extracted using the RNA extraction kit TakaRa MiniBEST Universal RNA Extraction Kit (Takara, 9767). The variable region sequences of murine antibodies were obtained by PCR using the SMARTer RACE 5' / 3' Kit (Clontech, 634858). According to the murine monoclonal antibody subtype, 3' specific primers were designed according to the method in "Recombinant Antibodies" (Shen Beifen, 2005), and the variable region PCR of murine monoclonal antibodies was performed according to the instructions of the SMARTer RACE 5' / 3' Kit. Homologous recombination primers were designed based on the variable region sequencing results, and the variable region fragments amplified by PCR were recovered by gel extraction.

[0158] The antibody sequences obtained by screening are shown in Table 1.

[0159] Table 1

[0160]

[0161]

[0162] Example 2: Antibody Preparation

[0163] The variable region sequences of the antibodies screened in Example 1 were respectively subjected to homologous recombination with the expression vector PTT5 (preserved by the First Research Laboratory of Shanghai Institute of Biological Products) containing the human IgG1 / κ heavy and light chain constant regions after digestion, to obtain the heavy and light chain expression vectors of the antibody. A commercial plasmid large-scale extraction kit was used for plasmid extraction. One day before transfection, the density of Expi293F cells in the logarithmic growth phase was adjusted to 1.0 - 2.0×10 6 cells / mL. After culturing for 24 h, transfection was carried out according to the final concentration of the total plasmid of 1 μg / mL, and the ratio of the heavy chain plasmid: light chain plasmid: PEI was 1:1:3. After 7 d, the cells were centrifuged to collect the culture supernatant, and the antibody was purified using a Protein A affinity column.

[0164] Among them, the sequence of the constant region of the IgG1 heavy chain is shown in SEQ ID NO: 11. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0165] Example 3: Antibody Activity and Function

[0166] In this example, the OX40 antibody (abbreviated as Ab18 antibody) prepared in Example 2 was verified for its activity and function, especially in comparison with GBR830 and KHK4083.

[0167] I. Binding Activity to OX40 Proteins from Different Sources

[0168] 1. Binding Activity of OX40 Antibody to Human OX40 Protein

[0169] Dilute the hOX40-mFc antigen protein to 1 μg / mL and coat it onto a 96-well ELISA plate at 100 μL / well overnight at 4°C. After washing three times with PBST, pat dry the residual liquid. Block it with 200 μL / well of 5% skim milk overnight at 4°C. After washing three times with PBST, pat dry the residual liquid. Dilute the antibody to be tested from 30 μg / mL in 5-fold serial dilutions and incubate at 100 μL / well at 37°C for 1 h. After washing three times with PBST, add the goat anti-human IgG-Fc-HRP secondary antibody (purchased from Southern Biotech, USA) diluted 1:2000 and incubate at 37°C for 45 min. After washing three times with PBST, pat dry the residual liquid. Develop color with TMB at 100 μL / well (A solution: B solution = 39:1). Stop the color development with 50 μL / well of 2M sulfuric acid termination solution. Read the OD450 with SpectraMax.

[0170] The results are as Figure 1 shown. The EC of the Ab18 antibody binding to human OX40 protein 50It was 0.08746 μg / mL, and its binding activity was superior to GBR830 and KHK4083.

[0171] 2. Binding activity of OX40 antibody to mouse OX40 protein

[0172] Dilute the mOX40-mFc antigen protein to 1 μg / mL and coat it onto a 96-well ELISA plate at 100 μL / well overnight at 4°C. After washing three times with PBST, drain the residual liquid. Block it with 200 μL / well of 5% skim milk powder overnight at 4°C. After washing three times with PBST, drain the residual liquid. Dilute the antibody to be tested from 30 μg / mL in 5-fold serial dilutions, and incubate at 100 μL / well at 37°C for 1 h. After washing three times with PBST, add the secondary antibody of goat anti-human IgG-Fc-HRP diluted 1:2000 (purchased from Southern Biotech, USA), and incubate at 37°C for 45 min. After washing three times with PBST, drain the residual liquid. Add 100 μL / well of TMB for color development. Stop with 50 μL / well of 2M sulfuric acid termination solution. Read the OD450 with SpectraMax.

[0173] The results were as Figure 2 shown. The EC50 of the Ab18 antibody binding to mouse OX40 protein was 20.97 μg / mL, while no binding activity of GBR830 and KHK4083 to mOX40 was observed.

[0174] 3. Binding activity of OX40 antibody to rat OX40 protein

[0175] Dilute the RatOX40-mFc protein to 1 μg / mL and coat it onto a 96-well ELISA plate at 100 μL / well overnight at 4°C. After washing three times with PBST, drain the residual liquid. Block it with 200 μL of 5% skim milk powder overnight at 4°C. After washing three times with PBST, drain the residual liquid. Dilute the antibody to be tested from 30 μg / mL in 5-fold serial dilutions, and incubate at 100 μL / well at 37°C for 1 h. After washing three times with PBST, add the secondary antibody of goat anti-human IgG-Fc-HRP diluted 1:2000 (purchased from Southern Biotech, USA), and incubate at 37°C for 45 min. After washing three times with PBST, drain the residual liquid. Add 100 μL / well of TMB for color development. Stop with 50 μL / well of 2M sulfuric acid termination solution. Read the OD450 with SpectraMax.

[0176] The results were as Figure 3 shown. It was observed that the antibody Ab18 had weak binding activity to rat OX40 protein at high concentrations, while no binding activity of GBR830 and KHK4083 to RatOX40 was observed.

[0177] 4. Binding Activity of OX40 Antibody to Cynomolgus Monkey OX40 Protein

[0178] Dilute CynOX40-mFc protein to 1 μg / mL and coat it onto a 96-well ELISA plate at 100 μL / well overnight at 4°C. After washing three times with PBST, pat dry the residual liquid, and block it with 200 μL of 5% skim milk overnight at 4°C. After washing three times with PBST, pat dry the residual liquid. Dilute the antibody to be tested from 30 μg / mL in 5-fold serial dilutions and incubate 100 μL / well at 37°C for 1 h. After washing three times with PBST, add goat anti-human IgG-Fc-HRP secondary antibody (purchased from Southern Biotech, USA) diluted 1:2000 and incubate at 37°C for 45 min. After washing three times with PBST, pat dry the residual liquid, add 100 μL / well of TMB chromogenic solution. Stop with 50 μL / well of 2M sulfuric acid termination solution. Read the OD450 with SpectraMax.

[0179] The results are as Figure 4 shown. The EC 50 of Ab18 antibody binding to cynomolgus monkey OX40 protein is 0.03789 μg / mL, which is similar to its binding activity to KHK4083. The binding activity of GBR830 to cynomolgus monkey OX40 protein is weak.

[0180] 5. Binding Activity of OX40 Antibody to hOX40-HEK293 Cells

[0181] After digesting hOX40-HEK293 cells, wash them twice with 4°C Stain Buffer (FBS) (purchased from BD), and prepare a cell suspension of 1E6 cells / mL. Add 500 μL / well to a deep well plate. Centrifuge at 2500 rpm for 5 min and then pour off the liquid. Dilute the OX40 antibody with Stain Buffer, starting from 60 μg / mL in 3-fold serial dilutions, and incubate 100 μL / well on ice for 1 h. After washing twice with PBA (PBS + 2% BSA), add PE-labeled goat anti-human IgG-Fc secondary antibody (diluted 1:600 with Stain Buffer) to the deep well plate and incubate on ice in the dark for 45 min. After washing twice with PBA, add 200 μL of PBA to resuspend the cells, and detect with a flow cytometer, recording the mean fluorescence intensity (MFI).

[0182] The results are as Figure 5 shown. The EC 50It is 0.3669 μg / mL, and its binding activity is similar to that of GBR830 and KHK4083, and even better than that of GBR830 and KHK4083.

[0183] 6. Binding activity of OX40 antigen proteins of different species to Ab18 antibody

[0184] Coat the Ab18 antibody at 1 μg / mL on a 96-well ELISA plate overnight at 4 °C. After washing three times with PBST, drain the residual liquid completely, and then block it with 200 μL of 5% skim milk overnight at 4 °C. After washing three times with PBST and draining the residual liquid completely, dilute the OX40 antigens of different species from 150 μg / mL in a 3-fold gradient, and incubate 100 μL per well at 37 °C for 1 h. After washing three times with PBST, add the goat anti-mouse IgG-Fc-HRP secondary antibody (purchased from KPL, USA) diluted 1:8000, and incubate at 37 °C for 45 min. After washing three times with PBST and draining the residual liquid completely, add 100 μL of TMB chromogenic solution per well. Terminate with 50 μL of 2M sulfuric acid termination solution per well. Read the OD450 with SpectraMax.

[0185] The results are as Figure 6 shown. The Ab18 antibody has strong binding activities to human and cynomolgus monkey OX40 proteins, and weak binding activities to mouse and rat OX40.

[0186] II. Detection of the blocking activity of Ab18 antibody against OX40 / OX40L

[0187] Seed Jurkat-OX40-NFκB-Luc cells at 2E6 cells / mL, 50 μL per well (previously added with 2 μg / mL of CD3 and CD28 each, and 120 μg / mL of hFc-OX40L). Dilute the Ab18 antibody from 80 μg / mL in a 2-fold gradient with the culture medium, and add 50 μL per well to the cell plate. Incubate the cell plate at 5% CO2 and 37 °C for 16 h. Take out the cell plate 15 min before adding the Bio Luciferase assay reagent and equilibrate it to room temperature. Read with SpectraMaxM5 5 min after adding 100 μL of the Bio Luciferase assay reagent per well, and record the RLUs value.

[0188] The results are as Figure 7 shown. The blocking IC50 of the Ab18 antibody against OX40L is 0.6573 μg / mL, which is similar to that of KHK4083, while GBR830 shows a weak blocking effect.

[0189] Example 4: Engineering design of Ab18 Fc

[0190] Studies have shown that OX40 monoclonal antibodies have the ability to activate OX40 mediated by the FcγRⅡB receptor. By reducing the binding ability between the OX40 antibody and the FcγRⅡB receptor, the agonistic effect of the OX40 antibody on T cells can be weakened; at the same time, in order to extend the half-life of the antibody in vivo, reduce the frequency of drug administration, and improve the compliance of patients with medication, this example introduces mutations that enhance the affinity for FcRn and reduce the binding ability to the FcγRⅡB receptor to extend its half-life in vivo.

[0191] The preparation method of the OX40 antibody in this example is the same as that in Example 2, except that mutations are designed in the heavy chain constant region, and the designed mutation sites are shown in Table 2.

[0192] Table 2

[0193] Antibody Mutation sites that reduce binding to FcγRⅡB Mutation sites that increase affinity for FcRn Ab18v1 D153E Ab18v2 M311L and N317S Ab18v3 D153E M311L and N317S

[0194] The specific sequences of each mutation site are shown in SEQ ID NO: 12-14.

[0195] Heavy chain constant region: D153E (SEQ ID NO: 12)

[0196] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0197] VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0198] SRTPEVTCVVVDVSHEEPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0199] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ

[0200] PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain constant region: M311L and N317S (SEQ ID NO: 13)

[0201] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0202] VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0203] SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0204] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK*

[0205] Heavy chain constant region: D153E, M311L and N317S (SEQ ID NO: 14)

[0206] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0207] VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0208] SRTPEVTCVVVDVSHEEPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0209] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK*

[0210] Example 5: Activity and function of the antibody after Ab18 Fc engineering

[0211] In this example, the activities and functions of the Ab18 antibody (prepared in Example 2) and the antibody after Fc mutation design (prepared in Example 4) were compared to determine the advantages of the mutation design.

[0212] 1. Binding activity (FCS) to hFcγRⅡB-CHO-K1

[0213] After digesting hFcγRⅡB-CHO-K1 cells, wash them twice with 4℃ Stain Buffer (FBS), and then prepare a cell suspension of 1×10 6 cells / mL. Add 500 μL / well to a deep well plate, with 5×10 5 cells / well. After centrifuging at 2500 rpm / min, pour off the liquid. Dilute the OX40 antibody with Stain Buffer, perform 3-fold serial dilution starting from an initial concentration of 60 μg / mL, and then add 100 μL / well to the deep well plate. Incubate on ice for 1 h. Wash twice with PBA, then add goat anti-human IgG-Fc-PE secondary antibody (1:600) (diluted with Stain Buffer) to the deep well plate and incubate in the dark on ice for 45 min. Wash twice with PBA, add 200 μL of PBA to resuspend the cells, and detect with a flow cytometer and record the readings.

[0214] The results are as Figure 8 shown. After the D153E mutation of the Ab18 antibody, its binding activity to hFcγRⅡB-CHO-K1 decreased significantly, and the mutant strains with triple mutations of M311L, N317S, and D153E also maintained a low binding activity to hFcγRⅡB.

[0215] 2. Agonistic activity detection

[0216] After digesting hFcγRⅡB-CHO-K1 cells, inoculate them into a 96-well cell plate at 5×10 5 cells / mL, 100 μL / well. After culturing at 37℃ in 5% CO2 for 24 h, aspirate 95 μL of the culture medium, and then add Jurkat-OX40-NFκB-Luc cells at 2×10 6 cells / mL, 50 μL / well. Dilute the antibody starting from an initial concentration of 5 μg / mL with 5-fold serial dilution, and then add 50 μL / well to the cell plate. Incubate the cell plate at 5% CO2, 37℃ for 16 h. Take out the cell plate 15 min before adding the Bio Luciferase assay reagent and equilibrate it to room temperature. After adding 100 μL / well of the Bio Luciferase assay reagent for 5 min, read with a SpectraMax M5 and record the RLUs value.

[0217] The results are as Figure 9As shown, the EC50 of the Ab18 antibody in the agonist activity detection experiment was 0.01488 μg / mL. The agonist activity of the mutated Ab18 (M311L, N317S, D153E) mutant against Jurkat-OX40 was significantly reduced compared to the Ab18 antibody.

[0218] 3. Binding to hFcRn-CHO-K1 at pH = 6.0 (FCS)

[0219] After digesting hFcRn-CHO-K1 cells, wash them twice with 4°C Stain Buffer (FBS), then prepare a cell suspension of 1E6 cells / mL, add 500 μL / well to a deep well plate, 5E5 cells / well. After centrifugation at 2500 rpm / min, pour off the liquid. Dilute the OX40 antibody with PBA (pH = 6.0), perform 4-fold serial dilution starting from 80 μg / mL, and incubate 100 μL / well on ice for 1 h. After washing twice with PBA (pH = 6.0), add the goat anti-human IgG-Fc-PE secondary antibody (1:600) (diluted with PBA (pH = 6.0)) to the deep well plate and incubate in the dark on ice for 45 min. After washing twice with PBA (pH = 6.0), add 200 μL of PBA to resuspend the cells, detect with a flow cytometer, and record the readings.

[0220] The results are as Figure 10 shown, the binding activity of the Ab18 antibody with hFcRn at pH 6.0 was significantly enhanced after M311L and N317S point mutations.

[0221] 4. Binding to hFcRn-CHO-K1 at pH = 7.4 (FCS)

[0222] After digesting hFcRn-CHO-K1 cells, wash them twice with 4°C Stain Buffer (FBS), then prepare a cell suspension of 1E6 cells / mL, add 500 μL / well to a deep well plate, 5E5 cells / well. After centrifugation at 2500 rpm / min, pour off the liquid. Dilute the OX40 antibody with PBA (pH = 7.4), perform 4-fold serial dilution starting from 80 μg / mL, and incubate 100 μL / well on ice for 1 h. After washing twice with PBA (pH = 7.4), add the goat anti-human IgG-Fc-PE secondary antibody (1:600) (diluted with PBA (pH = 7.4)) to the deep well plate and incubate in the dark on ice for 45 min. After washing twice with PBA (pH = 7.4), add 200 μL of PBA to resuspend the cells, detect with a flow cytometer, and record the readings.

[0223] The results are as Figure 11As shown, after the Ab18 antibody undergoes M311L and N317S point mutations, its binding activity with hFcRn at pH 7.4 remains unchanged.

[0224] 5. ADCC Activity Detection

[0225] Plate hOX40-HEK293 cells (constructed in this application) 24 hours in advance at a density of 5×10 5 cells / mL, 100 μL per well. After aspirating 95 μL of the culture medium, add 6×10 6 Jurkat / NFAT-luc+FcγRIIIa cells at a density of cells / mL, 50 μL per well. Starting from an initial concentration of 6 μg / mL, serially dilute the antibody by a factor of 3 and add 50 μL per well to the cell plate. Incubate the cell plate at 5% CO2 and 37°C for 6 hours. Remove the cell plate 15 minutes before adding the Bio Luciferase assay reagent and equilibrate it to room temperature. Five minutes after adding 100 μL per well of the BioLuciferase assay reagent, read the RLUs value using a SpectraMax M5.

[0226] The results are as Figure 12 shown. The EC50 value of the Ab18 antibody in the ADCC activity experiment is 0.02568 μg / mL. After mutation, its ADCC activity is not found to decrease, and the ADCC activities of both the Ab18 antibody and its mutants are stronger than those of GBR830 and KHK4083.

[0227] Taken together, the above results indicate that this antibody treats atopic dermatitis or autoimmune diseases by blocking the binding of immune cell OX40 and OX40L and clearing activated OX40-positive immune cells through ADCC.

[0228] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0229] In addition, it should be noted that in the above specific embodiments, the various specific technical features described can be combined in any appropriate manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. An anti-OX40 antibody or an antigen-binding fragment thereof, characterized in that, The anti - OX40 antibody or its antigen - binding fragment comprises VHCDR1 - 3 of the heavy - chain variable region and VLCDR1 - 3 of the light - chain variable region, wherein: The heavy - chain variable region comprises: VHCDR1 with the amino - acid sequence shown in SEQ ID NO: 1; VHCDR2 with the amino - acid sequence shown in SEQ ID NO: 2; VHCDR3 with the amino - acid sequence shown in SEQ ID NO: 3; The light - chain variable region comprises: VLCDR1 with the amino - acid sequence shown in SEQ ID NO: 4; VLCDR2 with the amino - acid sequence shown in SEQ ID NO: 5; VLCDR3 with the amino - acid sequence shown in SEQ ID NO:

6.

2. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, wherein The anti - OX40 antibody or its antigen - binding fragment further comprises FR regions.

3. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, wherein The anti - OX40 antibody or its antigen - binding fragment comprises VHFR1, VHFR2, VHFR3, VHFR4 of the heavy - chain variable region, and VLFR1, VLFR2, VLFR3, VLFR4 of the light - chain variable region.

4. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy - chain variable region sequentially comprises VHFR1, VHCDR1, VHFR2, VHCDR2, VHFR3, VHCDR3, VHFR4 from the N - terminus to the C - terminus; and The light - chain variable region sequentially comprises VLFR1, VLCDR1, VLFR2, VLCDR2, VLFR3, VLCDR3, VLFR4 from the N - terminus to the C - terminus.

5. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 2, wherein The FR region is a FR region of a human or non - human animal.

6. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 5, wherein, The FR region is a FR region of a human, rodent or primate.

7. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino - acid sequence of the heavy - chain variable region comprises the amino - acid sequence shown in SEQ ID NO: 7, or comprises an amino - acid sequence having a homology of more than 95% with the amino - acid sequence shown in SEQ ID NO: 7 and having the activity of binding to OX40.

8. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 7, wherein, The nucleotide sequence encoding the heavy - chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 8, or comprises a nucleotide sequence having a homology of more than 90% with the nucleotide sequence shown in SEQ ID NO:

8.

9. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino - acid sequence of the light - chain variable region comprises the amino - acid sequence shown in SEQ ID NO: 9, or comprises an amino - acid sequence having a homology of more than 95% with the amino - acid sequence shown in SEQ ID NO: 9 and having the activity of binding to OX40.

10. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 9, wherein The nucleotide sequence encoding the light - chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 10, or comprises a nucleotide sequence having a homology of more than 90% with the nucleotide sequence shown in SEQ ID NO:

10.

11. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The anti - OX40 antibody or its antigen - binding fragment comprises a heavy - chain constant region and a light - chain constant region.

12. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 11, wherein The heavy - chain constant region and / or the light - chain constant region is / are the heavy - chain constant region and / or the light - chain constant region of a human or non - human animal.

13. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 11, characterized in that, The heavy - chain constant region and / or the light - chain constant region is / are the heavy - chain constant region and / or the light - chain constant region of a human, rodent or primate.

14. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 11, wherein The heavy - chain constant region is selected from the constant regions of any one of IgG, IgD, IgA, IgM or IgE heavy chains.

15. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 11, wherein The light chain constant region described above is selected from the constant regions of any one of the λ-type light chain or κ-type light chain.

16. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 11, wherein, The heavy chain constant region described above is an IgG heavy chain constant region, and the light chain constant region described above is a κ light chain constant region.

17. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 16, wherein The IgG heavy chain described above includes IgG1 heavy chain, IgG2 heavy chain, IgG2b heavy chain, IgG3 heavy chain or IgG4 heavy chain.

18. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 11, wherein The amino acid sequence of the heavy chain constant region described above includes the amino acid sequence such as SEQ ID NO: 11 or its D153E, M311L and / or N317S mutants, or, includes an amino acid sequence having more than 90% homology with the amino acid sequence of SEQ ID NO: 11 or its D153E, M311L and / or N317S mutants.

19. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 11, wherein, The amino acid sequence of the heavy chain constant region described above is as shown in any one of SEQ ID NO: 11-14.

20. The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-19, wherein The anti-OX40 antibody or its antigen-binding fragment described above includes Fab, Fab’, Fab’-SH, Fv, scFv or F(ab’)2.

21. The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-OX40 antibody or its antigen-binding fragment described above specifically binds to the OX40 protein of human or non-human animals.

22. A nucleic acid, characterized in that, The nucleic acid described above encodes the anti-OX40 antibody or its antigen-binding fragment according to any one of claims 1-21.

23. The nucleic acid according to claim 22, wherein The nucleotide sequence encoding the heavy chain variable region includes the nucleotide sequence as shown in SEQ ID NO: 8, or, includes a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO:

8.

24. The nucleic acid according to claim 22, characterized in that, The nucleotide sequence encoding the light chain variable region includes the nucleotide sequence as shown in SEQ ID NO: 10, or, includes a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO:

10.

25. A method for preparing the anti-OX40 antibody or its antigen-binding fragment according to any one of claims 1-21, characterized in that, The preparation method described above includes transforming the nucleic acid according to any one of claims 22-24 into a host cell, and then inducing its expression.

26. A carrier, characterized in that, The vector described above contains the nucleic acid according to any one of claims 22-24.

27. A host cell, characterized in that The host cell described above contains the nucleic acid according to any one of claims 22-24 or the vector according to claim 26.

28. An immune cell, characterized in that, The immune cell described above expresses the anti-OX40 antibody or its antigen-binding fragment according to any one of claims 1-21.

29. The immune cell according to claim 28, wherein The immune cell described above includes one or more of lymphocytes, dendritic cells, peripheral blood mononuclear cells, macrophages, granulocytes or mast cells.

30. The immune cell according to claim 29, wherein The lymphocyte described above is a TIL cell.

31. The immune cell according to claim 29, characterized in that, The lymphocyte described above is selected from T cells, NK cells, NKT cells or B cells.

32. Use of the anti-OX40 antibody or its antigen-binding fragment according to any one of claims 1-21, the nucleic acid according to any one of claims 22-24, the vector according to claim 26, the host cell according to claim 27 or the immune cell according to any one of claims 28-31 in the preparation of a medicament for treating and / or preventing atopic dermatitis or autoimmune diseases.

33. The application according to claim 32, characterized in that, The autoimmune disease described above is asthma, systemic lupus erythematosus, rheumatoid arthritis or scleroderma.

34. A drug, characterized in that, The medicament described above comprises the anti-OX40 antibody or its antigen-binding fragment as described in any one of claims 1-21, the nucleic acid as described in any one of claims 22-24, the vector as described in claim 26, the host cell as described in claim 27, or the immune cell as described in any one of claims 28-31.

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