CN114685658A

CN114685658A - OX40 targeted antibody and preparation method and application thereof

Info

Publication number: CN114685658A
Application number: CN202011645723.9A
Authority: CN
Inventors: 夏冰; 周宇虹; 魏紫萍
Original assignee: Bailisikang Biomedical Hangzhou Co ltd
Current assignee: Bailisikang Biomedical Hangzhou Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-07-01

Abstract

The invention discloses a targeted antibody of OX40 and a preparation method and application thereof. The antibody comprises a VH comprising the CDRs of: VH CDR1, VH CDR2 and VH CDR3 as shown in the amino acid sequences of SEQ ID NOs 1, 2 and 3; the VL comprises the following CDRs: VL CDR1, VL CDR2 and VL CDR3 as set forth in the amino acid sequences of SEQ ID NOS 4, 5 and 6. Compared with the prior art antibody, the antibody of the invention also has excellent capability of activating OX40 signal pathway under the condition of equivalent affinity and specificity, thereby achieving the balance between drug effect and side effect when used in clinic at later stage.

Description

OX40 targeted antibody and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an OX40 targeted antibody and a preparation method and application thereof.

Background

OX40(CD134, TNFRSF4), originally defined as a T cell activation marker, was later found to be a member of the NGFR/TNFR superfamily with co-activation functions, mainly expressed on activated effector T cells (Teffs) and regulatory T cells (Tregs), and also on NKT cells, NK cells and neutrophils (D.J. Paterson, et al, "antibodies of activated Rat T lymphocytes encapsulating a Molecular of 50,000M (r) protected only on CD4positive T blasts," Molecular immunology.1987; 24(12): 1281-1290). OX40 transmitted a costimulatory signal in combination with the ligand OX40L (CD252, TNFSF 4). OX40L may be present on Antigen Presenting Cells (APCs), such as: b cells, dendritic cells, macrophages; expression can also be induced in other cell types such as Langerhans cells, endothelial cells, smooth muscle cells, mast cells, and NK cells (Flynn S, et al, CD 4T cell cytokine differentiation: the B cell activation molecule, OX40 ligand, templates CD 4T cells to expression 4and expression of the chemokine receptor, Blr-1.J Exp Med.1998; 188: 297-; (Ohshima Y, et al, Expression and function of OX40 ligand on human dendritic cells. J Immunol. 1997; 159: 3838-. It can be seen that the binding of OX40 and OX40L is involved in various physiological responses between T cells and lymphocytes and non-lymphocytes. Increases survival and expansion of effector and memory T cells, increases secretion of cytokines (e.g., IL-2, IL-4, IL-5, IFN- γ) when OX40 binds to its ligand OX 40L; reducing the immunosuppressive activity of Tregs and further amplifying the T cell activation effect (Gramaglia I, et al., Ox-40ligand: a patent synergy molecule for preserving primary CD 4T cell responses. J Immunol.1998; 161: 6510-; (Ohshima Y, et al, OX40 simulation industries inter leukin-4(IL-4) expression at printing and mobility the differentiation of negative human CD4(+) T cells inter high IL-4-producing effectors. blood 1998; 92:3338 and 3345.); (Ruby CE, et al, IL-12is required for anti-OX40-media CD 4T cell subset. J Immunol.2008; 180: 2140-. In the tumor microenvironment, immune activation can lead to OX40 expression. Can enhance the activation and proliferation of effector T cells and suppress Tregs, resulting in a complex anti-tumor immune response (reviewed in Jensen SM, et al, Semin Oncol.2010 Oct; 37(5): 524-32).

A portion of the OX40 antibody with activation function may mimic its ligand OX40L, activating the OX40 signaling pathway, thereby enhancing the activation and proliferation of effector T cells. The field of tumor immunization currently uses the ratio of the effects of PD1 inhibitors as "brake release" for the immune system, and the ratio of anti-OX 40-activating antibodies as "throttle down" for the immune system (Linch SN, et al, Front Oncol.2015; 16; 5: 34).

OX40 is a type I membrane protein, and almost all prior art anti-OX40 antibodies have been used to immunize animals with extracellular domain fusion proteins of OX40 as immunizing antigens. Whereas the OX40 on the cell membrane binds its ligand in its trimer form and activates the signaling pathway. It is clearly not an optimal solution to immunize animals with monomeric forms of ectodomain fusion proteins as immunizing antigens. Firstly, the monomer OX40 exposes a large number of immunogens which are masked in the trimeric state and which produce antibodies which generally have the effect of blocking the formation of trimers, so-called antagonistic antibodies; second, monomer OX40 lacks those specific conformational immunogens which are only available in trimeric form, and the antibodies generated by these immunogens generally have the effect of stabilizing trimer formation, so-called activating antibodies. Currently, a variety of anti-OX40 antibody drugs are being used in clinical trials against tumors and immune system diseases. For the control was von lerolizumab, roche, with the target in the clinical study stage, the antibody was now in the clinical phase II trial, but multiple clinical trials were declared to be terminated in 2019, and no reason was published. It may be difficult to strike a balance between drug efficacy and side effects due to the weaker ability of von lerolizumab to activate the OX40 signaling pathway.

Therefore, there remains a need to develop new anti-OX40 antibodies that have a modest ability to activate the OX40 signaling pathway compared to anti-OX40 antibodies known in the prior art, with comparable affinity and specificity, so that a balance between drug efficacy and side effects, etc., is more likely to be achieved later in the clinic.

Disclosure of Invention

The invention aims to overcome the defect that a targeted antibody of OX40 with higher affinity and higher endocytosis activity is absent in the prior art, and provides a targeted antibody of OX40(CD134/TNFRSF4) or an antigen binding fragment thereof, and a preparation method and application thereof.

In the present invention, the targeted antibody to OX40 or an antigen-binding fragment thereof is obtained by immunizing an animal with an immunogen comprising an expression cell of a transient OX40 protein, rather than by de-immunizing an animal with an extracellular domain fusion protein of OX40 as an immunizing antigen. Thus, stronger affinity and specificity are obtained, and simultaneously, the OX40 targeted antibody or the antigen-binding fragment thereof has excellent capability of activating an OX40 signal pathway.

In order to solve the above technical problems, the present invention provides, in a first aspect, an OX 40-targeting antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and/or a light chain variable region (VL),

the VH comprises the following Complementarity Determining Regions (CDRs): VH CDR1 as shown in the amino acid sequence of SEQ ID NO. 1; and/or a VH CDR2 as shown in the amino acid sequence of SEQ ID NO 2; and/or, a VH CDR3 as set forth in the amino acid sequence of SEQ ID NO. 3;

the VL comprises the following CDRs: VL CDR1 as shown in the amino acid sequence of SEQ ID NO. 4; and/or, a VL CDR2 as set forth in the amino acid sequence of SEQ ID NO. 5; and/or, a VL CDR3 as set forth in the amino acid sequence of SEQ ID NO 6;

alternatively, said VH has 3, 2 or 1 amino acid mutations in the amino acid sequences of said VH CDR1, VH CDR2, VH CDR3, respectively, and/or said VL has 3, 2 or 1 amino acid mutations in the amino acid sequences of said VL CDR1, VL CDR2, VL CDR3, respectively.

"amino acid mutation" in the analogous "mutation with 3, 2 or 1 amino acids" means that there is a mutation of amino acids in the sequence of the variant as compared with the original amino acid sequence, including insertion, deletion or substitution of amino acids based on the original amino acid sequence. An exemplary explanation is that the mutations to the CDRs may comprise 3, 2 or 1 amino acid mutations, and that the CDRs may optionally be mutated by selecting the same or different number of amino acid residues between them, e.g. 1 amino acid mutation to CDR1 and no amino acid mutation to CDR2 and CDR 3.

In the present invention, the mutations may include mutations that are currently known to those skilled in the art, such as mutations that are made to the antibody during the production or use of the antibody, for example, mutations made at sites that may be present, particularly post-transcriptional modifications (PTMs) of the CDR regions, including related mutations such as aggregation, deamidation sensitivity (ASPARAGAMIDIDATION) sites (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N- { P } S/T) sensitive sites, and oxidation sensitive sites of the antibody.

In the present application, the amino acid sequences of the above-listed CDRs are shown according to the IMGT definition (sequences shown in the claims of the present invention according to the IMGT definition are also shown, and can be analyzed in https:// www.imgt.org/3D structure-DB/cgi/DomainGapAlign. However, it is well known to those skilled in the art that the CDRs of an antibody can be defined in the art by a variety of methods, such as the Kabat definition rule based on sequence variability (see Kabat et al, immunological protein sequences, fifth edition, national institutes of health, Besserda, Md. (1991)) and the Chothia definition rule based on the position of the structural loop region (see Jmol Biol 273:927-48, 1997). It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are understood to encompass complementarity determining regions as defined by any of the above-described known schemes described by the present invention. Although the scope of the claims of the present invention is based on the sequence shown in the definition rules of IMGT, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of the present invention (see https:// qininjanhan. com/biology/antibody/antibody-number-description /).

Table 1-1 methods for CDR definition of antibodies of the present application

Wherein Laa-Lbb can refer to the amino acid sequence from aa to bb, starting from the N-terminus of the antibody light chain; Haa-Hbb may refer to the amino acid sequence from aa to bb, starting from the N-terminus of the heavy chain of the antibody. For example, L27-L32 may refer to the amino acid sequence from position 27 to 32, beginning at the N-terminus of the antibody light chain, according to the IMGT coding rules; H26-H33 may refer to the amino acid sequence from position 26 to 33 according to the IMGT encoding rules, starting from the N-terminus of the antibody heavy chain. It is well known to those skilled in the art that there are positions where insertion sites will occur when encoding CDRs using these encoding rules.

Preferably, the targeting antibody or antigen binding fragment thereof of OX40 is a murine antibody or antigen binding fragment thereof.

More preferably, the VH of the murine antibody further comprises a heavy chain variable region framework region (VH FWR), and/or the VL of the murine antibody further comprises a light chain variable region framework region (VL FWR). Said VH FWR is selected from 1) germline IGHV1S81 x 02, IGHV1-53 x 01, IGHV1-69 x 02, IGHV1-64 x 01, FR1, FR2 and FR3 regions in IGHV1-74 x 04, and 2) FR4 region in germline IGHJ2 x 01, or a back-mutation thereof; and/or, said VL FWR is selected from 1) FR1, FR2 and FR3 regions in germline IGKV10-96 × 01, IGKV10-96 × 02, IGKV10-96 × 03, IGKV10-96 × 04, IGKV10-96 × 05, and 2) FR4 region in germline IGKJ5 × 01, or a back-mutation thereof.

Even more preferably, the VH FWRs include VH FWR1 as shown in SEQ ID nos. 11, 29 or mutations thereof, VH FWR2 as shown in SEQ ID nos. 12, 30 or mutations thereof, VH FWR3 as shown in SEQ ID nos. 13, 31 or mutations thereof, and VH FWR4 as shown in SEQ ID nos. 14, 32 or mutations thereof; and/or the VL FWR comprises VL FWR1 shown as SEQ ID NO. 15, 33 or a mutation thereof, VL FWR2 shown as SEQ ID NO. 16, 34 or a mutation thereof, VL FWR3 shown as SEQ ID NO. 17, 35 or a mutation thereof, and VL FWR4 shown as SEQ ID NO. 18, 36 or a mutation thereof.

Even more preferably, the amino acid sequence of the VH of the murine antibody is the amino acid sequence shown as SEQ ID NO. 7 or a mutation thereof; and/or the amino acid sequence of the VL is the amino acid sequence shown as SEQ ID NO. 8 or mutation thereof; the mutation is a deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of the VH and/or the VL, and the mutated amino acid sequence has at least 85% sequence identity to the amino acid sequence of SEQ ID NO. 7 and/or SEQ ID NO. 8 and maintains or improves the binding of the antibody to OX 40; the at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.

Even more preferably, the nucleotide sequence of VH of the murine antibody is shown as SEQ ID NO 9; and/or the nucleotide sequence of the VL is an amino acid sequence shown as SEQ ID NO. 10.

Preferably, the targeting antibody or antigen binding fragment thereof of OX40 further comprises a murine antibody constant region or a human antibody constant region; the murine antibody constant regions comprise the heavy chain constant region of murine IgG1, IgG2a, IgG2b3, or IgG3, or a mutation thereof, and the light chain constant region of the kappa or lambda type, or a mutation thereof, and the human antibody constant regions comprise the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a mutation thereof, and the light chain constant region of the kappa or lambda type, or a mutation thereof.

Preferably, the targeting antibody or antigen binding fragment thereof of OX40 is a humanized antibody or antigen binding fragment thereof;

more preferably, the framework regions of the variable region of the humanized antibody comprise the framework regions of a human antibody heavy chain variable region and the framework regions of a human antibody light chain variable region; the framework regions of the human antibody light chain variable region are selected from 1) germline IGHV1-46 x 01, IGHV1-46 x 02, IGHV1-46 x 03, IGHV1-46 x 04, FR1, FR2 and FR3 regions in IGHV1-2 x 06, and 2) germline IGHJ4 x 01, IGHJ4 x 01, FR4 regions in IGHJ4 x 01, or back mutations thereof; and/or, the framework regions of the heavy chain variable regions of said human antibodies are selected from 1) germline IGKV1-33 x 01, IGKV1D-33 x 01, IGKV1-39 x 01, IGKV1D-39 x 01, FR1, FR2 and FR3 regions in IGKV1-27 x 01, and 2) germline IGKJ2 x 01, FR4 regions in IGKJ2 x 02, or back mutations thereof; the number of the amino acid sites of the back mutation is 0-10;

preferably, the light chain of the antibody comprises a human antibody kappa or lambda type light chain constant region or a mutation thereof; and/or the heavy chain of the antibody comprises the heavy chain constant region of human IgG1, IgG2, IgG3, and IgG4, or a mutation thereof.

Preferably, the OX 40-targeting antibody or antigen-binding fragment thereof comprises a full-length antibody, Fab ', F (ab')₂Fv, scFv, VHH, HCAb, bispecific antibody, multispecific antibody, or monoclonal or polyclonal antibody made from the above antibodies.

In the present invention, a "Fab fragment" consists of one light chain and one heavy chain of CH1 and the variable domains. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain. A "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form F (ab ')₂A molecule. "F (ab')₂A fragment "comprises two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus F (ab')₂The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The term "Fv" means an antibody fragment consisting of the VL and VH domains directed to a single arm of an antibodySegment, but lacking a constant region.

In the present invention, the scFv (single chain antibody) can be a single chain antibody conventional in the art, and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids. Wherein the VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain [ see, e.g., Bird et al, Science 242:423-]. Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art joints are made of repeating G₄S amino acid sequence or a variant thereof. For example, a polypeptide having an amino acid sequence (G)₄S)₄Or (G)₄S)₃Linkers, but variants thereof may also be used.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; an antibody having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently, and the like.

The antibodies of the invention include monoclonal antibodies. The monoclonal antibody or mAb or Ab of the present invention refers to an antibody obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. The monoclonal antibody can be developed by various means and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the monoclonal antibody is prepared from wild-type or transgenic mice by the hybridoma technology in the mainstream.

In this application, the term "heavy chain antibody" refers to an antibody, also known as HCAb, comprising only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

"Single domain antibody", also known as "Nanobody", refers to the VHH structure cloned from a heavy chain antibody, which is the smallest unit known to bind the antigen of interest.

In order to solve the above technical problems, the second aspect of the present invention provides a bispecific antibody comprising a first protein functional region which is a targeting antibody of OX40 or an antigen-binding fragment thereof according to the first aspect of the present invention; the second protein functional region is an antibody that targets a non-OX 40 antigen.

Preferably, the non-OX 40 antigen is an immune checkpoint antigen, preferably comprising PD-1, PD-L1, PD-L2, 4-1BB, CD40, CD73, Tim3, LAG3 or CD47, or a tumor therapy target, preferably comprising OX 40.

In order to solve the above technical problem, the third aspect of the present invention provides an isolated nucleic acid encoding a targeted antibody to OX40 or an antigen-binding fragment thereof according to the first aspect of the present invention.

The preparation method of the nucleic acid is a preparation method which is conventional in the field, and preferably comprises the following steps: obtaining the nucleic acid molecule for coding the antibody by a gene cloning technology, or obtaining the nucleic acid molecule for coding the antibody by an artificial complete sequence synthesis method.

Those skilled in the art know that the base sequence encoding the amino acid sequence of the above antibody may be appropriately substituted with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homologue. The polynucleotide homologue of the present invention may be prepared by substituting, deleting or adding one or more bases of a gene encoding the antibody sequence within a range in which the activity of the antibody is maintained.

In order to solve the above technical problems, the fourth aspect of the present invention provides an expression vector comprising the isolated nucleic acid according to the third aspect of the present invention.

Preferably, the expression vector is a plasmid, cosmid, phage, or viral vector, preferably a retroviral, lentiviral, adenoviral, or adeno-associated viral vector.

In order to solve the above technical problems, a fifth aspect of the present invention provides a transformant comprising the recombinant expression vector according to the fourth aspect of the present invention in a host cell.

Preferably, the host cell is an e.coli TG1, BL21 cell, or CHO-K1 cell.

In order to solve the above-mentioned technical problems, the sixth aspect of the present invention provides a method for producing a targeted antibody to OX40 or an antigen-binding fragment thereof, which comprises culturing the transformant according to the fifth aspect of the present invention, and obtaining the targeted antibody to OX40 or an antigen-binding fragment thereof from the culture.

In order to solve the above technical problems, the seventh aspect of the present invention provides a chimeric antigen receptor comprising a targeted antibody to OX40 or an antigen-binding fragment thereof according to the first aspect of the present invention.

In order to solve the above technical problem, an eighth aspect of the present invention provides a genetically modified cell comprising a targeted antibody to OX40 or an antigen-binding fragment thereof according to the first aspect of the present invention, or a chimeric antigen receptor according to the seventh aspect of the present invention.

Preferably, the genetically modified cell is a eukaryotic cell, more preferably an isolated human cell; further more preferred are immune cells such as T cells, or NK cells such as NK92 cell line.

In order to solve the above technical problem, a ninth aspect of the present invention provides an antibody drug conjugate comprising a cytotoxic agent, and a targeting antibody to OX40 or an antigen binding fragment thereof according to the first aspect of the present invention.

In order to solve the above technical problems, the tenth aspect of the present invention provides a pharmaceutical combination (e.g. may be in the form of a kit of parts such as an antibody combination, an antibody equivalent, or may be in the form of a pharmaceutical composition) comprising a targeting antibody or antigen binding fragment thereof to OX40 according to the first aspect of the invention, a bispecific antibody according to the second aspect of the invention, a chimeric antigen receptor according to the seventh aspect of the invention, a genetically modified cell according to the eighth aspect of the invention, and/or an antibody drug conjugate according to the ninth aspect of the invention.

When present in kit form, it may be a kit comprising kit a and kit B, said kit a comprising a targeting antibody or antigen binding fragment thereof of OX40 according to the first aspect of the invention, a bispecific antibody according to the second aspect of the invention, a chimeric antigen receptor according to the seventh aspect of the invention, a genetically modified cell according to the eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, a drug combination according to the tenth aspect of the invention, and/or a kit according to the eleventh aspect of the invention. The kit B is another anti-tumor antibody or a pharmaceutical composition comprising the other anti-tumor antibody (e.g., antibody 1G10-9-13-11 as referred to herein). The medicine box A and the medicine box B can be used simultaneously, the medicine box A can be used firstly and then the medicine box B can be used, the medicine box B can be used firstly and then the medicine box A can be used, and the medicine box A can be determined according to actual requirements in specific application.

When the composition is in the form of a pharmaceutical composition, a pharmaceutically acceptable solvent and/or a carrier and/or an auxiliary material can be further included. The pharmaceutically acceptable carrier can be a carrier which is conventional in the art, and the carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical adjuvant is conventional in the field, and preferably comprises pharmaceutically acceptable excipient, filler or diluent and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the targeted antibody of OX40 or an antigen-binding fragment thereof, the bispecific antibody, the chimeric antigen receptor, the genetically modified cell, the antibody drug conjugate, and 0.01-99.99% of a pharmaceutically acceptable carrier, wherein the percentages are mass percentages of the pharmaceutical composition.

The pharmaceutical composition preferably further comprises other anti-tumor antibodies as active ingredients. Preferably, the pharmaceutical composition is an anti-tumor drug. More preferably a medicament for treating OX40 positive tumor, such as lung cancer, liver cancer, gastric cancer, breast cancer, head and neck cancer and/or colon cancer.

The route of administration of the pharmaceutical composition of the present invention is preferably parenteral, injection or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is various dosage forms which are conventional in the field, preferably in the form of solid, semisolid or liquid, namely aqueous solution, non-aqueous solution or suspension, and more preferably tablet, capsule, granule, injection or infusion and the like. More preferably via intravascular, subcutaneous, intraperitoneal or intramuscular administration. Preferably, the pharmaceutical composition may also be administered as an aerosol or a coarse spray, i.e. nasally; alternatively, intrathecal, intramedullary or intraventricular administration. More preferably, the pharmaceutical composition may also be administered transdermally, topically, enterally, intravaginally, sublingually or rectally.

The dosage level of a pharmaceutical composition of the invention administered may be adjusted depending on the amount of the composition to achieve a desired diagnostic or therapeutic result. The administration regimen may also be a single injection or multiple injections, or adjusted. The selected dose level and regimen will be reasonably adjusted depending on various factors including the activity and stability (i.e., half-life) of the pharmaceutical composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and/or treated, and the health and prior medical history of the subject to be treated.

A therapeutically effective dose for the pharmaceutical composition of the invention may be estimated initially in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and/or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. And can subsequently be used to determine useful doses and routes for administration in humans. In general, the determination and adjustment of the effective amount or dosage to be administered and the assessment of when and how to make such adjustments are known to those skilled in the art.

For combination therapy, the targeted antibody or antigen-binding fragment thereof of OX40, the bispecific antibody described above, the chimeric antigen receptor, the genetically modified cell, the antibody drug conjugate, the drug combination, the kit, and/or the additional therapeutic or diagnostic agent can each be used as a single agent, within any time frame suitable for performing the intended therapy or diagnosis. Thus, these single agents may be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours) or sequentially. For example, these single agents may be administered within a year, or within 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 week, or within 5, 4, 3, 2, or 1 day.

For additional guidance on formulation, dosage, administration regimen, and measurable therapeutic outcomes, see Berkow et al (2000) The Merck Manual of Medical Information (Merck handbook of Medical Information) and Merck & co.inc, Whitehouse Station, New Jersey; ebadi (1998) CRC Desk Reference of Clinical Pharmacology (handbook of Clinical Pharmacology) and the like.

In order to solve the above technical problems, an eleventh aspect of the present invention provides a kit comprising a targeting antibody or an antigen binding fragment thereof of OX40 according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a chimeric antigen receptor according to the seventh aspect of the present invention, a genetically modified cell according to the eighth aspect of the present invention, an antibody drug conjugate according to the ninth aspect of the present invention, and/or a drug combination according to the tenth aspect of the present invention.

Preferably, the kit further comprises (i) a device for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.

In order to solve the above technical problems, the twelfth aspect of the present invention provides a use of the targeted antibody or antigen-binding fragment thereof of OX40 according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the chimeric antigen receptor according to the seventh aspect of the present invention, the genetically modified cell according to the eighth aspect of the present invention, the antibody drug conjugate according to the ninth aspect of the present invention, the drug combination according to the tenth aspect of the present invention, and/or the kit according to the eleventh aspect of the present invention for the preparation of a medicament for the diagnosis, prevention and/or treatment of tumors.

Preferably, the tumor is an OX40 positive tumor; such as lung cancer, liver cancer, stomach cancer, breast cancer, head and neck cancer, and/or colon cancer.

To solve the above technical problem, the present invention also provides a targeting antibody or an antigen binding fragment thereof of OX40 according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a chimeric antigen receptor according to the seventh aspect of the present invention, a genetically modified cell according to the eighth aspect of the present invention, an antibody drug conjugate according to the ninth aspect of the present invention, a drug combination according to the tenth aspect of the present invention, and/or a kit according to the eleventh aspect of the present invention for use in the diagnosis, prevention and/or treatment of a tumor. The tumor is preferably as described above.

In one aspect, the present invention also provides a method for detecting the expression of OX40 protein in a sample to be tested, comprising the steps of: the target antibody or antigen-binding fragment thereof of OX40 according to the first aspect of the invention is contacted with the sample to be tested in vitro, and binding of the target antibody or antigen-binding fragment thereof of OX40 according to the first aspect of the invention to the sample to be tested is detected.

In the present invention, unless otherwise specified, scientific and technical terms used in the present invention have meanings commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in the present invention are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present invention, OX40, OX-40 and OX-40 represent the same meanings.

The three letter and one letter codes for amino acids used in the present invention are known to those skilled in the art or described in j.biol.chem,243, p3558 (1968).

As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements but do not exclude other elements, but, as the context dictates, also include the case of "consisting of … …".

In the present invention, the term "variable" generally refers to the fact that certain portions of the sequence of the variable domains of an antibody are strongly varied, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light and heavy chain variable regions, called Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portions of the variable domains are called the Framework (FWR). The variable domains of native heavy and light chains each comprise four FWR regions, largely in a β -sheet configuration, connected by three CDRs, forming a loop junction, and in some cases forming part of a β -sheet structure. The CDRs in each chain are held in close proximity by the FWR regions and together with the CDRs from the other chain form the antigen binding site of the antibody, and the constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, e.g. involved in antibody-dependent cytotoxicity of the antibody.

The term "epitope" refers to the portion of an antigen (e.g., OX40) that specifically interacts with an antibody molecule. The term "competes" in the present invention refers to the ability of an antibody molecule to interfere with the binding of an anti-OX40 antibody molecule to a target (e.g., OX 40). Interference with binding may be direct or indirect (e.g., via allosteric modulation of an antibody molecule or target). Competitive binding assays (e.g., FACS assays, ELISA, or BIACORE assays) can be used to determine the extent to which an antibody molecule is able to interfere with the binding of another antibody molecule to its target.

The term "antibody" as used herein includes immunoglobulins, which are tetrapeptide chain structures formed by two identical heavy chains and two identical light chains joined by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus in their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. In the five classes of igs, the second class of igs can have either kappa chains or lambda chains.

As used herein, the term "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is referred to as "isolated". The term "isolated" does not exclude the presence of other impurities which do not interfere with the activity of the substance, either mixed with artificial or synthetic substances.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and includes, but is not limited to, prokaryotic cells such as E.coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

The term "murine antibody" is in the present invention a monoclonal antibody to OX40 prepared according to the knowledge and skill in the art. Preparation is accomplished by injecting a subject with an OX40 antigen, and then isolating hybridomas that express antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine OX40 antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or IgG4 or variant thereof.

The term "humanized antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized antibodies").

As used herein, the term "specific" with respect to an antibody means an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to the antigen from one or more species. However, such interspecies cross-reactivity does not change the classification of antibodies by specificity itself. In another example, an antibody that specifically binds to an antigen may also bind to a different allelic form of the antigen. However, this cross-reactivity does not change the classification of antibodies by specificity itself. In some cases, the term "specificity" may be used to refer to the interaction of an antibody, protein or peptide with a second chemical, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical; for example, antibodies generally recognize and bind to a particular protein structure, not a protein. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of the epitope A-containing molecule (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

"identity", "mutation" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity between two sequences is a function of the number of matching or homologous positions common to both sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percent identity. "optimization" refers to a mutation that maintains or improves the binding of the antibody to the antigen, and in the present invention, refers to a mutation that maintains, maintains or improves the binding to CLDN 18.2.

The terms "polypeptide", "peptide" and "protein" (if single-chain) are used interchangeably herein. The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably.

The term "mutation" includes substitutions, additions and/or deletions of amino acids or nucleotides, "amino acid substitutions" and "conservative amino acid substitutions" are respectively in which an amino acid residue is substituted with another amino acid residue and with an amino acid residue having a similar side chain.

As used herein, "lentivirus" refers to a genus of the family Retroviridae (Retroviridae family). Lentiviruses are unique among retroviruses, which are capable of infecting non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, and are therefore one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are examples of lentiviruses. Vectors from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

The term "vector" as used herein is a composition comprising an isolated nucleic acid and useful for delivering the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

As used herein, the expressions "cell", "cell line" and "cell line" are used interchangeably and all such designations include progeny. The term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as E.coli, fungal cells such as yeast cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

The term "transfection" refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection may be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics (biolistics).

The term "immune cell" refers to a cell that can elicit an immune response, and "immune cell" and grammatical variations thereof can refer to an immune cell of any origin. "immune cells" include, for example, white blood cells (leukocytes), lymphocytes (T cells, B cells, Natural Killer (NK) cells, and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells) derived from Hematopoietic Stem Cells (HSCs) produced in the bone marrow.

As used in the present invention, the term EC₅₀Means the half-maximal effect concentration (concentration for 50% of the maximum effect), i.e. the concentration that causes 50% of the maximal effect.

The pharmaceutical composition of the present invention can be formulated into various dosage forms as required, and can be administered at a dose that is determined by a physician in consideration of the kind, age, weight and general condition of a patient, administration manner, and the like, which are beneficial to the patient. Administration may be by injection or other therapeutic means, for example.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: compared with the prior art antibody, the antibody of the invention also has excellent capability of activating OX40 signal pathway under the condition of equivalent affinity and specificity, thereby achieving the balance between drug effect and side effect when used in clinic at later stage. In a preferred embodiment of the invention, antibody activation of the invention potently activates the OX40 signaling pathway at a half maximal effective concentration (EC50) of 633 ng/mL.

Drawings

FIG. 1 is the binding activity of anti-OX40 antibody to OX40 and 4-1BB fusion proteins, respectively, in example 4.

FIG. 2is the binding activity of anti-OX40 antibodies to cell surface OX40 protein in example 4.

FIG. 3 shows the transcriptional activity of NF-. kappa.B of different positive cell lines in example 4.

FIG. 4 is a concentration-stimulatory activity response curve for the antibody of example 4.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 antigen preparation, mouse immunization and hybridoma preparation

a. Antigen (transient OX40 expressing cells) preparation

293F cells were used as transfected cells, and a commercial OX40 plasmid (HG 0481-UT, Chinesen, Yi) was transiently transfected into 293F cells, which were cultured for 48 hours and then taken as an immunogen. The method comprises the following specific steps: 293F cells were prepared and the cell density reached 1.0-1.2X 10 on the day of transfection⁶Per ml; the cells were counted and 1.0X 10 added to 6-well plates ⁶2 ml/well for 4 wells per cell/ml; a DNA transfection system was prepared. Adding 640 mu L of Opti-MEM into 8ug of DNA plasmid, and mixing gently; adding 16 mu l of PEI, mixing the mixture gently and uniformly, and standing the mixture for 20 minutes at room temperature; the DNA-293PEI mixture was added to 293F cells and placed in an Infors shaker at 115rpm, 37 ℃ and 5% CO₂Culturing for 2 days in a culture environment; and (4) collecting a sample. All samples were collected, centrifuged at 1500rpm for 3 minutes; the cells were taken and washed twice at 1500rpm for 3 minutes after resuspension in PBS.Finally resuspend to 5X 10⁶And (4) obtaining the antigen (namely, the expression cell of the transient OX40 or the OX40-293F cell) for immunization.

b. Immunization

Animals (mice and rabbits) were immunized with the above-prepared cells expressing transient OX40 as an immunogen.

After cell collection, the cells were used for cellular immunization of six week old mice (university of Hangzhou Master animal center). Animal immunization was performed by the animal center of Hangzhou university. The immune part is subcutaneous multipoint immunity, and each immunization is 5 multiplied by 10⁶Individual cells/mouse; a total of 4 immunizations were performed (following priming, a second immunization 21 days later, followed by an additional immunization every 14 days). Serum titers were measured by ELISA (similar to the ELISA procedure described below) after 4-immunization. Mice with acceptable serum titers will be boosted and spleens will be removed for storage or fusion.

c. Fusion

Firstly taking thymocytes of a 4-week mouse (animal center of Hangzhou university), and suspending the thymocytes in an IMDM culture medium to serve as feeder cells; SP2/0, which had been well grown, was resuspended in IMDM medium as a fused cell. Splenocytes were then prepared as follows: the spleens of the mice after the boosting were taken, placed in a sterile 10cm dish, excess adherent tissues were cut off, the spleens were placed in a cell strainer, and the spleens were ground with the core of a sterile 2mL syringe. Isolated spleen cells were collected and washed twice with IMDM medium as fusion cells for use. In preparation for cell fusion, SP2/0 cells and splenocytes were mixed in a volume ratio of 1:1 of the cell pellet, and IMDM medium was added to make the volume of the tube 30mL, and centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded. The cell pellet was carefully knocked off and the cells in the centrifuge tube were placed in a beaker containing warm water at 37 ℃. Adding 1mL of PEG dropwise into the cells at a constant speed within 1min, and standing in a water bath at 37 ℃ for 30 s; 10mL of IMDM medium was added to the fused cells, followed by addition of medium in time to make a total volume of 30 mL. Centrifuging at 1200rpm for 3 minutes, and discarding the supernatant; the cell pellet was gently knocked up and slowly transferred to hybridoma selection medium, and feeder cells were added to the selection medium, gently mixing the fused cells and feeder cells. Push button 200mu.L/well of plates, a total of 20 plates; standing at 37 deg.C for 5% CO₂Culturing for 7 days under the culture condition of (2), and then taking hybridoma supernatant for detection and screening.

Example 2 antibody screening and sequencing

1. Antibody screening

And (3) ELISA screening: the antigen OX40 fusion protein (OX40 fusion protein available from Hangzhou Huaan biotechnology Co., Ltd.) was first diluted to 1. mu.g/ml with a coating solution and added to a 96-well plate at 50. mu.l per well. After covering the cover of the ELISA plate, putting the ELISA plate into a 4-degree freezer overnight; after sealing, adding the hybridoma supernatant obtained in the example 1 into an enzyme label plate by a pipette according to 50 mul per hole, after shaking uniformly, putting into a constant temperature incubator, and standing for 1 hour; washing with TBST for 3 times, adding diluted secondary antibody (goat anti-mouse IgG-HRP, Pierce,31430) into the ELISA plate at a volume of 50 μ l per well by pipette, placing in a constant temperature incubator, and standing for 30 min; washing with TBST for 3 times, and developing with HRP developing solution. After the color development is finished, H is used₂SO₄(TMB) the reaction was stopped, the plate read and the data analyzed. And screening positive clones according to the OD value of the positive hole.

ELISA-positive clones were screened for double-positive clones by Flow (FACS) screening using OX40-293F cells obtained in example 1: transient OX40-293F cells were collected, added to a 96-well V-plate at 50000 wells, blocked with 1% BSA/PBS, and left at 4 ℃ for 30 minutes; adding 50 mu l of ELISA positive hybridoma supernatant into each hole, placing in a 4-degree freezer for 1 hour; after washing, diluted anti-mouse-FITC (goat anti-mouse IgG-FITC, Jackson, 115-; the incubated cells were washed, resuspended, detected on a flow sorter and MFI recorded. Positive clones were selected and subcloned.

Subcloning: and carrying out subcloning on the screened double-positive clone, and selecting a single clone for later research and development through detection. This example uses two methods for subcloning. Limiting dilution method and counting plates. All by routine operation in the field.

Subclone screening: the subclone screening method is the same as the screening method of the hybridoma supernatant, and the screening method utilizes an ELISA method and a FACS method for detection, and finally selects a double-positive monoclonal as a final clone for antibody recombination to obtain a sequence.

2. Sequencing

After the positive hybridoma cells are lysed, the sequences are obtained by RT (reverse transcription) and PCR of heavy and light chains and verified by sequencing.

The conditions for RT are shown in Table 1 below.

TABLE 1

The reaction system and the reaction procedure for heavy chain PCR are shown in tables 2 and 3 below.

TABLE 2 PCR reaction System

Reagent	10 μ l reaction System	Final concentration
			1×MAX Mixture	9.6μl	1×
Forward Primer 1，10μM	0.1μl	0.1μM
			Reverse Primer(M12H-R/M2A-R)，10μM	0.1μl	0.1μM
cDNA	0.2μl

TABLE 3 PCR reaction procedure

The reaction system and the reaction procedure of the light chain PCR are shown in tables 4and 5 below.

TABLE 4 PCR reaction System

TABLE 5 PCR reaction procedure

The obtained PCR product is sent to be sequenced, and the sequencing company is Hangzhou Youkang biotechnology limited.

3. Antibody sequence information

The numbering systems used for antibodies are Kabat, Chothia and IMGT, and in this example the numbering system used for IMGT. The amino acid sequences of the CDRs of the antibodies of the invention are shown in table 6 below.

TABLE 6 amino acid sequences of CDRs of antibodies of the invention

Table 7 shows a preferred combination of sequences of the framework regions.

TABLE 7 framework region sequence combinations

The variable regions of the light chain and the heavy chain of the antibody obtained by the present invention and the control antibody may be as shown in table 8 below.

TABLE 8 amino acid sequences of light and heavy chain variable regions

The nucleotide sequence of VH of the above antibody 5A3-6-15 may preferably (SEQ ID NO:9) be:

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATATTATGCACTGGGTGAAGCAGAAGCCTGAGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTTCTAAGTACAATGAGAACTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTTTTACTGTGCAAGGGGGGCCTACGGTTCTAGTTACAACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

the nucleotide sequence of VL of the above antibody 5A3-6-15 may preferably (SEQ ID NO:10) be:

GATATCCAGATGACACAGACTACTTCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAAGTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTATTATTGTCAGCAGTATAGTAAGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGAAATAAAAC

the nucleotide sequence of the VH of the above antibody 1G10-9-13-11 may preferably (SEQ ID NO:27) be:

GAGGTTCAGCTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATACACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAACGGTCGTACTAACTCCAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAGAGGCGGTGCCGTACTACTTTGTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA；

the nucleotide sequence of VL of the above antibody 1G10-9-13-11 may preferably (SEQ ID NO:28) be:

GATATTGTGATGACGCAGGCTGCATCCTCCCTGTCTGCCTCTCTGGGAGACAGGGTCACCATCAGTTGTAGGGCAAGTCAGGACATTAACAATTATTTAAACTGGTATCAGCAGAAACCTGATGGAACTGTTAAACTCCTGATCTACTTCACATCAGGATTACGCTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTAAAC

selection of antibody light and heavy chain constant regions: the light chain constant region and the heavy chain constant region of the antibody OX40 of the invention can be murine, rabbit, human, etc., e.g., the heavy chain constant region and the light chain constant region are murine.

EXAMPLE 3 antibody production, purification

The antibody of the invention is purified by ascites in mice. The operation steps are all routine in the field, and mainly comprise culturing and expanding hybridoma cells, and then directly inoculating the hybridoma cells into the abdominal cavity of a pre-sensitized mouse. Ascites fluid was collected after 10 days and purified by proG, and the purified antibody was quantified and stained with SDS-pAGE.

Example 4 Effect identification

1. OX40 binding affinity and specificity identification

1.1 construction of OX40 and specific control (4-1BB) fusion proteins

The OX40 gene fragment is purchased from Qiao Shenzhou, and the OX40 extracellular region fragment is obtained by a PCR method by taking the purchased gene fragment as a template and is constructed on a pTT5 vector for protein expression. Wherein the restriction sites of OX40-rFc fusion protein (prepared by conventional method in the art) are EcoRI and XhoI, and the restriction sites of OX40-His fusion protein (prepared by conventional method in the art) are EcoRI and NheI. After the construction is successful, the plasmid is transfected into 293F cells for expression and purification, and OX40-rFc fusion protein and OX40-His fusion protein are obtained.

4-1BB gene (4-1BB is another molecule of the same gene family as OX40, but the sequence is completely different, and therefore, the fragment serves as an experimental control of binding specificity) fragment was purchased from Qiao Shen, and 4-1BB extracellular region fragment was obtained by PCR method using the purchased gene fragment as a template and constructed on pTT5 vector for protein expression. Wherein the restriction enzyme cutting sites of the 4-1BB-rFc fusion protein are EcoRI and XhoI, and the restriction enzyme cutting sites of the 4-1BB-His fusion protein are EcoRI and NheI. After the construction is successful, the plasmid is transfected into 293F cells for expression and purification, and the 4-1BB-rFc fusion protein and the 4-1BB-His fusion protein are obtained.

1.2 binding affinity and specificity identification

The binding affinity and specificity were identified by both ELISA and FACS methods, respectively.

The binding affinities of the positive antibody (i.e., Vonllerlizumab) and OX40-rFc fusion protein and OX40-His fusion protein were first identified by ELISA, and the binding specificity of the antibody was identified using 4-1BB-rFc fusion protein and 4-1BB-His fusion protein as controls.

The antigen OX40 fusion protein or 4-1BB fusion protein was diluted to 1. mu.g/ml with a coating solution, and 50. mu.l per well was added to a 96-well microplate. After covering the enzyme label plate with a cover, putting the enzyme label plate into a freezer at 4 ℃ for overnight; after sealing, adding the purified antibody (1 microgram/mL) into an enzyme label plate, uniformly vibrating, putting into a constant-temperature incubator, and standing for 1 hour; washing with TBST for 3 times, adding diluted secondary antibody into the ELISA plate by a pipette according to 50 μ l per hole, placing in a constant temperature incubator, and standing for 30 minutes; washing with TBST for 3 times, and developing with HRP developing solution. After the color development is finished, H is used₂The reaction was stopped with SO4(TMB), plates were read and data analyzed.

The results of the experiment are shown in FIG. 1. As can be seen in the figure, both 1G0-9-13-11 and 5A3-6-15 antibodies specifically recognized the OX40 fusion protein, but not the 4-1BB fusion protein. Wherein the OX40 positive control antibody is an anti-OX40 antibody synthetically expressed according to the sequence of Vonllerolizumab in a clinical trial by Roche; the 4-1BB positive control antibody was an anti-4-1 BB antibody (purchased from Guanke Biotechnology Ltd.) which was synthetically expressed based on the sequence of Utomillumab in clinical trials of pfeiffer. As can be seen, the anti-OX40 antibodies of the invention specifically recognized OX40 fusion protein, but not 4-1BB fusion protein, with binding capacity comparable to the positive control antibody Vonlrolizumab.

2. Binding affinity and specificity identification for binding to OX40 expressing cells

2.1 construction of OX 40-expressing cells and specific control (4-1BB) cells

OX40/4-1BB expression vector was purchased from Yi Qiao Shen (OX-40: HG 10481-UT). The expression vector can be directly transfected (the transfection procedure is conventional in the art, and is specifically described in the following: CHO-K1 cells are prepared, and the cell density reaches 1.0-1.2X 10 on the day of transfection⁶Per mL; the cells were counted and 1.0X 10 added to 6-well plates⁶cells/mL, 2 mL/well, 4 wells total; a DNA transfection system was prepared. Adding 640 mu L of Opti-MEM into 8 mu g of DNA plasmid, and mixing the mixture gently; adding 16 mu LPEI, mixing evenly and mildly, and standing for 20 minutes at room temperature; the DNA-PEI mixture was added to CHO-K1 cells and placed in an Infors shaker at 115rpm, 37 ℃ and 5% CO₂Culturing for 2 days in a culture environment; and (4) collecting a sample. All samples were collected, centrifuged at 1500rpm for 3 minutes; the cells were taken, resuspended in PBS, washed twice at 1500rpm for 3 minutes, and prepared for FACS experiments). After identification by CHO-K1 cell transfection, the vector was confirmed to be able to correctly express OX40 and 4-1BB molecules on the surface of CHO-K1 cells, i.e., OX40-CHO-K1 cells and 4-1BB-CHO-K1 cells were successfully obtained.

2.2 binding affinity and specificity identification

The anti-OX40 antibodies of the invention were further characterized by Flow (FACS) using the resulting OX40-CHO-K1 cells as described above: transient OX40-CHO-K1 cells were collected, added to a 96-well V-plate at 50000 wells, blocked with 1% BSA/PBS, and left at 4 ℃ for 30 minutes; diluting ELISA positive antibody by 3 times from 10 μ g/mL, adding diluted antibody 50 μ l into each well, placing in 4 degree refrigerator for 1 hr; after washing, diluted anti-mouse-FITC was added to the cells in 50. mu.l per well in a freezer at 4 ℃ for 30 minutes; the incubated cells were washed, resuspended, examined on a flow sorter, MFI recorded and data analyzed.

As shown in FIG. 2, it can be seen that both the 1G10-9-13-11 and 5A3-6-15 antibodies recognize OX40-CHO-K1 cells. Since the positive control antibody was humanized (i.e., von lerolizumab roche) and the anti-OX40 antibody of the invention was mouse, the secondary antibody used for detection was a completely different secondary antibody (goat anti-human IgG-HRP, Abcam, ab 98595; goat anti-human IgG-R-PE, Southern Biotech,204009), and thus the final MFI reading was also greatly different.

3. Specific activation of the OX40 Signal pathway

3.1 OX40 Signal pathway reporting cell line construction

Reporter gene vector pGL4.32[ luc 2P/NF-kB-RE/Hygro ] was purchased from Promega, HEK293 cells (Shanghai Guanguan Zhi bioengineering # C028) were transfected by the reporter gene vector and were screened by hygromycin (conventional screening procedure is available), positive cell lines with stronger drug resistance were selected, TNF α was used to stimulate the positive cells to activate NF-kB signaling pathway, and single luciferase reporter gene detection kit (Beijing Prohah Ming Hehao Bio # LF102-01) was used to treat the samples, RLU was measured by a multifunctional microplate reader to detect the transcriptional activity of luciferase, and finally three positive cell lines with highest transcriptional activity, namely 1C2-2, 1C6-3, and 1B1-2 (Table 9), were selected.

OX40 expression vector is purchased from Qiao Shen, and OX40 expression plasmid is used for transfecting the three positive cell strains, and hygromycin and G418 are used for screening to select the positive cell strain with stronger drug resistance. Respectively detecting the expression level of OX40 on the cell line surface by FACS (the specific experimental steps are 2.2 except for different detection samples); the ability of anti-OX40 antibodies to activate NF- κ B signaling pathway after binding to different positive cell surface OX40 was assayed using a multifunctional microplate reader assay. FACS showed no significant difference in the expression level of OX40 for each positive cell line, with the 19B3 cell line having the strongest NF- κ B transcriptional activity (FIG. 3). Thus, positive cell line 19B3 was selected as the OX40 signaling pathway reporter cell line.

TABLE 9 transcriptional Activity of NF- κ B in different Positive cell lines

3.2 identification of specific activation of the OX40 Signal pathway

The positive cell line 19B3 cells obtained above were inoculated into a 96-well plate and incubated overnight in a CO2 incubator. OX40 antibodies 1G10-9-13-11 and 5A3-6-15 were subjected to gradient dilution, added to the corresponding wells, incubated in a CO2 incubator for 5 hours, then treated with a single luciferase reporter assay kit, Relative Luminescence Units (RLU) were read by a multifunctional microplate reader, and the concentration-stimulatory activity effect curves of the antibodies were fitted by analytical software according to a four-parameter logistic equation.

As shown in FIG. 4, both 1G10-9-13-11 and 5A3-6-15 antibodies were effective in activating OX40 signaling pathways at half effective concentrations (EC50) of 833.9ng/mL and 633.0ng/mL, respectively, and with increasing antibody concentrations, 5A3-6-15 antibody was much more potent than 1G10-9-13-11 antibody at the same concentrations; in contrast, positive control antibodies showed only relatively weak activation capacity under the same conditions so that EC50 could not fit effectively.

SEQUENCE LISTING

<110> Bailixikang biomedical (Hangzhou) Co., Ltd

<120> OX40 targeted antibody, preparation method and application thereof

<130> P20017287C

<160> 36

<170> PatentIn version 3.5

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<400> 6

Gln Gln Tyr Ser Lys Leu Pro Tyr Thr

1 5

<210> 7

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VH amino acids

<400> 7

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ile Met His Trp Val Lys Gln Lys Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys

85 90 95

Ala Arg Gly Ala Tyr Gly Ser Ser Tyr Asn Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 8

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VL amino acid

<400> 8

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Lys Asn

100 105

<210> 9

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VH nucleotides

<400> 9

cagatccagt tggtgcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact agctatatta tgcactgggt gaagcagaag 120

cctgagcagg gccttgagtg gattggatat attaatcctt acaatgatgg ttctaagtac 180

aatgagaact tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct tttactgtgc aaggggggcc 300

tacggttcta gttacaactt tgactactgg ggccaaggca ccactctcac agtctcctca 360

<210> 10

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VL nucleotides

<400> 10

gatatccaga tgacacagac tacttcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gtgcaagtca gggcattagc aattatttaa actggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctattac acatcaagtt tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tgggacagat tattctctca ccatcagcaa cctggaacct 240

gaagatattg ccacttatta ttgtcagcag tatagtaagc ttccgtacac gttcggaggg 300

gggaccaagc tgaaataaaa c 321

<210> 11

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VH FWR1

<400> 11

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser

20 25

<210> 12

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VH FWR2

<400> 12

Ile Met His Trp Val Lys Gln Lys Pro Glu Gln Gly Leu Glu Trp Ile

1 5 10 15

Gly Tyr Ile

<210> 13

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VH FWR3

<400> 13

Ser Lys Tyr Asn Glu Asn Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp

1 5 10 15

Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu

20 25 30

Asp Ser Ala Val Phe Tyr Cys Ala Arg

35 40

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VH FWR4

<400> 14

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

<210> 15

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VL FWR1

<400> 15

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys

20

<210> 16

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VL FWR2

<400> 16

Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr

1 5 10 15

<210> 17

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VL FWR3

<400> 17

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser

1 5 10 15

Leu Thr Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys

20 25 30

<210> 18

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 5A3-6-15 VL FWR4

<400> 18

Phe Gly Gly Gly Thr Lys Leu Lys Asn

1 5

<210> 19

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH CDR1

<400> 19

Gly Tyr Thr Phe Thr Asn Tyr Trp

1 5

<210> 20

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH CDR2

<400> 20

Ile Asn Pro Ser Asn Gly Arg Thr

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH CDR3

<400> 21

Glu Ala Val Pro Tyr Tyr Phe Val Tyr

1 5

<210> 22

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL CDR1

<400> 22

Gln Asp Ile Asn Asn Tyr

1 5

<210> 23

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL CDR2

<400> 23

Phe Thr Ser

1

<210> 24

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL CDR3

<400> 24

Gln Gln Gly Asn Thr Leu Pro Leu Thr

1 5

<210> 25

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH amino acids

<400> 25

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Ser Asn Glu Lys Phe

50 55 60

Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ala Val Pro Tyr Tyr Phe Val Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 26

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL amino acid

<400> 26

Asp Ile Val Met Thr Gln Ala Ala Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Phe Thr Ser Gly Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Asn

100 105

<210> 27

<211> 351

<212> DNA

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH nucleotides

<400> 27

gaggttcagc tgcagcagtc tggggctgaa ctggtgaagc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc aactactgga tacactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggagag attaatccta gcaacggtcg tactaactcc 180

aatgagaagt tcaagaacaa ggccacactg actgtagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc agaggcggtg 300

ccgtactact ttgtctactg gggccaaggc accactctca cagtctcctc a 351

<210> 28

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL nucleotides

<400> 28

gatattgtga tgacgcaggc tgcatcctcc ctgtctgcct ctctgggaga cagggtcacc 60

atcagttgta gggcaagtca ggacattaac aattatttaa actggtatca gcagaaacct 120

gatggaactg ttaaactcct gatctacttc acatcaggat tacgctcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttcctctcac gttcggtgct 300

gggaccaagc tggagctaaa c 321

<210> 29

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH FWR1

<400> 29

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser

20 25

<210> 30

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH FWR2

<400> 30

Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

1 5 10 15

Glu

<210> 31

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH FWR3

<400> 31

Asn Ser Asn Glu Lys Phe Lys Asn Lys Ala Thr Leu Thr Val Asp Lys

1 5 10 15

Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp

20 25 30

Ser Ala Val Tyr Tyr Cys Ala

35

<210> 32

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VH FWR4

<400> 32

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

<210> 33

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL FWR1

<400> 33

Asp Ile Val Met Thr Gln Ala Ala Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

20 25

<210> 34

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL FWR2

<400> 34

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

1 5 10 15

Tyr

<210> 35

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL FWR3

<400> 35

Gly Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala

20 25 30

Thr Tyr Phe Cys

35

<210> 36

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G10-9-13-11 VL FWR4

<400> 36

Phe Gly Ala Gly Thr Lys Leu Glu Leu Asn

1 5 10

Claims

1. A targeting antibody of OX40 or an antigen binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL),

the VH comprises the following Complementarity Determining Regions (CDRs): VH CDR1 shown as the amino acid sequence of SEQ ID NO. 1, VH CDR2 shown as the amino acid sequence of SEQ ID NO. 2, and VH CDR3 shown as the amino acid sequence of SEQ ID NO. 3;

the VL comprises the following CDRs: VL CDR1 as shown by the amino acid sequence of SEQ ID NO. 4, VL CDR2 as shown by the amino acid sequence of SEQ ID NO. 5, and VL CDR3 as shown by the amino acid sequence of SEQ ID NO. 6.

2. The OX40 targeting antibody or antigen binding fragment thereof of claim 1, wherein the antibody is a murine antibody;

preferably:

the VH of the murine antibody further comprises a heavy chain variable region framework region (VH FWR), and/or the VL of the murine antibody further comprises a light chain variable region framework region (VL FWR); the VH FWR is selected from 1) the FR1, FR2 and FR3 regions in germline IGHV1S81 x 02, IGHV1-53 x 01, IGHV1-69 x 02, IGHV1-64 x 01, IGHV1-74 x 04, and 2) the FR4 region in germline IGHJ2 x 01; and/or said VL FWR is selected from 1) FR1, FR2 and FR3 regions in germline IGKV10-96 x 01, IGKV10-96 x 02, IGKV10-96 x 03, IGKV10-96 x 04, IGKV10-96 x 05, and 2) FR4 region in germline IGKJ5 x 01;

more preferably:

the amino acid sequence of VH of the murine antibody is shown as SEQ ID NO. 7; and/or the amino acid sequence of the VL is the amino acid sequence shown as SEQ ID NO. 8;

3. The OX40 targeting antibody or antigen binding fragment thereof of claim 1, wherein the antibody is a humanized antibody;

preferably:

the framework regions of the variable regions of the humanized antibody comprise the framework regions of the human antibody heavy chain variable region and the framework regions of the human antibody light chain variable region; the framework regions of the human antibody light chain variable region are selected from 1) regions FR1, FR2 and FR3 in germline igh 1-46 x 01, IGHV1-46 x 02, IGHV1-46 x 03, IGHV1-46 x 04, IGHV1-2 x 06, and 2) regions FR4 in germline IGHJ4 x 01, IGHJ4 x 01, IGHJ4 x 01; and/or, the framework regions of the heavy chain variable regions of said human antibodies are selected from the group consisting of 1) the FR1, FR2 and FR3 regions in germline IGKV1-33 x 01, IGKV1D-33 x 01, IGKV1-39 x 01, IGKV1D-39 x 01, IGKV1-27 x 01, and 2) the FR4 regions in germline IGKJ2 x 01, IGKJ2 x 02.

4. The OX40 targeting antibody or antigen binding fragment thereof of any one of claims 1-3, comprising a full lengthAntibody, Fab ', F (ab')₂Fv, scFv, VHH, HCAb, bispecific antibody, multispecific antibody, or monoclonal or polyclonal antibody made from the above antibodies.

5. The OX40 targeting antibody or antigen binding fragment thereof of claim 4, wherein the antibody further comprises a murine antibody constant region or a human antibody constant region; the murine antibody constant region comprises a heavy chain constant region of murine IgG1, IgG2a, IgG2b3, or IgG3, and/or a light chain constant region of the kappa or lambda type; the human antibody constant regions comprise the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, and/or the light chain constant region of the kappa or lambda type;

preferably, the heavy chain constant region is hIgG1 and the light chain constant region is the kappa chain of a human antibody.

6. A bispecific antibody comprising a first protein functional region and a second protein functional region, wherein the first protein functional region is the targeted antibody to OX40 of any one of claims 1-5, or an antigen-binding fragment thereof; the second protein functional region is an antibody that targets a non-OX 40 antigen;

7. An isolated nucleic acid encoding the targeted antibody to OX40 of any one of claims 1-5, or an antigen-binding fragment thereof.

8. An expression vector comprising the isolated nucleic acid of claim 7; preferably, the expression vector is a plasmid, cosmid, phage, or viral vector, preferably a retroviral, lentiviral, adenoviral, or adeno-associated viral vector.

9. A transformant comprising the recombinant expression vector of claim 8 in a host cell; preferably, the host cell is an e.coli TG1, BL21 cell, or CHO-K1 cell.

10. A method of making a targeted antibody to OX40 or an antigen-binding fragment thereof, comprising culturing the transformant of claim 9, and obtaining the targeted antibody to OX40 or an antigen-binding fragment thereof from the culture.

11. A chimeric antigen receptor comprising the targeting antibody of OX40 or an antigen binding fragment thereof of any one of claims 1-5.

12. A genetically modified cell comprising the chimeric antigen receptor of claim 11; preferably, the genetically modified cell is a eukaryotic cell, more preferably an isolated human cell; further preferred are immune cells such as T cells, or NK cells such as the NK92 cell line.

13. An antibody drug conjugate comprising a cytotoxic agent, and the OX40 targeting antibody or antigen binding fragment thereof of any one of claims 1-5.

14. A pharmaceutical combination comprising a targeting antibody or antigen binding fragment thereof of OX40 according to any one of claims 1-5, a bispecific antibody according to claim 6, a chimeric antigen receptor according to claim 11, a genetically modified cell according to claim 12, and/or an antibody drug conjugate according to claim 13.

15. A kit comprising a targeting antibody or antigen binding fragment thereof of OX40 according to any one of claims 1-5, a bispecific antibody according to claim 6, a chimeric antigen receptor according to claim 11, a genetically modified cell according to claim 12, an antibody drug conjugate according to claim 13, and/or a pharmaceutical combination according to claim 14;

16. Use of a targeting antibody or antigen binding fragment thereof to OX40 according to any one of claims 1-5, a bispecific antibody according to claim 6, a chimeric antigen receptor according to claim 11, a genetically modified cell according to claim 12, an antibody drug conjugate according to claim 13, a pharmaceutical combination according to claim 14 and/or a kit according to claim 15 for the manufacture of a product for the diagnosis, prevention and/or treatment of a tumor; preferably, the tumor is an OX40 positive tumor, such as lung, liver, stomach, breast, head and neck and/or colon cancer.