CN111303285B

CN111303285B - Antibody targeting OX40, and preparation method and application thereof

Info

Publication number: CN111303285B
Application number: CN201911387302.8A
Authority: CN
Inventors: 周宇虹; 魏紫萍; 戴继鸿; 夏冰
Original assignee: Bailisikang Biomedical Hangzhou Co ltd
Current assignee: Bailisikang Biomedical Hangzhou Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2023-06-02
Anticipated expiration: 2039-12-27
Also published as: CN111303285A

Abstract

The invention discloses an antibody targeting OX40, a preparation method and application thereof. The antibody comprises a VH and/or a VL, the VH comprising CDRs of: VH CDR1 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 shown in the amino acid sequence of SEQ ID No. 3; the VL comprises CDRs of: VL CDR1 as shown in the amino acid sequence of SEQ ID NO. 4; and/or VL CDR2 as shown in the amino acid sequence of SEQ ID NO. 5; and/or VL CDR3 as shown in the amino acid sequence of SEQ ID NO. 6. Compared with the prior art antibodies, the antibody provided by the invention has moderate capacity of activating the OX40 signal path under the condition of equivalent affinity and specificity, so that the balance between drug effect and side effect can be achieved in later clinical use.

Description

Antibody targeting OX40, and preparation method and application thereof

Technical Field

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

Background

OX40 (CD 134, TNFRSF 4), initially defined as a T cell activation marker, was later found to be a member of the NGFR/TNFR superfamily with co-activation function, expressed primarily on activated effector T cells (Teffs) and regulatory T cells (Tregs), as well as on NKT cells, NK cells and neutrophils (D.J. Paterson, et al, "Antigens of activated Rat T lymphocytes including a molecule of 50,000M (r) detected only on CD4positive T blasts," Molecular immunology.1987;24 (12): 1281-1290). OX40 binds to ligand OX40L (CD 252, TNFSF 4) delivering a co-stimulatory signal. OX40L may be found in Antigen Presenting Cells (APC), such as: b cells, dendritic cells, macrophages; in addition, inducible expression is also possible 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, instructs CD 4T cells to express interleukin 4and upregulates expression of the chemokine receptor,Blr-1.J Exp Med.1998;188:297-304.); (Ohshima Y, et al Expression and function of OX ligand on human dendritic cells J Immunol 1997; 159:3838-3848). It can be seen that binding of OX40 and OX40L is involved in a variety of physiological responses between T cells and lymphocytes and non-lymphocytes. When OX40 binds to its ligand OX40L, it increases the survival and expansion of effector T cells and memory T cells, increases the secretion of cytokines (e.g., IL-2, IL-4, IL-5, IFN-gamma); decreasing the immunosuppressive activity of Tregs, further amplifying T cell activation effects (Gramaglia I, et al, ox-40ligand:a potent costimulatory molecule for sustaining primary CD4T cell responses.J Immunol.1998;161:6510-6517); (Ohshima Y, et al, OX40 costimulation enhances interleukin-4 (IL-4) expression at priming and promotes the differentiation of naive human CD (+) T cells into high IL-4-producing effects. Blood 1998; 92:3338-3345.); (Ruby CE, et al, IL-12is required for anti-OX40-mediated CD 4T cell survivin.J Immunol.2008; 180:2140-2148). In tumor microenvironments, immune activation can lead to OX40 expression. Can enhance activation and proliferation of effector T cells and inhibit Tregs, resulting in a complex anti-tumor immune response (reviewed in Jensen SM, et al, semin Oncol.2010Oct;37 (5): 524-32).

Some OX40 antibodies with activating function may mimic their ligand OX40L, activating OX40 signaling pathways, thereby enhancing activation and proliferation of effector T cells. In the current tumor immunity field, the action ratio of PD1 inhibitors is regarded as "brake release" to the immune system, while anti-OX 40 agonist antibodies are regarded as "accelerator depression" to the immune system (Linch SN et al, front Oncol.2015;16; 5:34).

OX40 is a type of membrane protein and almost all prior art anti-OX 40 antibodies use extracellular domain fusion proteins of OX40 as immunizing antigens to immunize animals. However, OX40 on the cell membrane binds to its ligand in trimeric form and activates the signaling pathway. The use of monomeric extracellular domain fusion proteins as immunizing antigens for the immunization of animals is clearly not the optimal solution. First, monomeric OX40 exposes a large number of immunogens that are masked in the trimeric state, and antibodies produced by these immunogens typically have the effect of blocking trimer formation, so-called antagonistic antibodies; second, monomeric OX40 lacks those specific conformational immunogens that are present only in trimeric form, and antibodies produced by these immunogens typically have the effect of stabilizing trimer formation, so-called activating antibodies. A variety of anti-OX 40 antibody drugs are currently being used in clinical trials for tumors and immune system diseases. For control, von lerolizumab, roche, whose target was in the clinical study phase, the antibody was now in clinical phase II trials, but many clinical trials in 2019 announced termination and did not disclose the cause. It may be difficult to balance drug effects with side effects due to the weaker capacity of von lerolizumab to activate the OX40 signaling pathway.

Thus, there remains a need to develop new anti-OX 40 antibodies that have comparable affinity and specificity compared to anti-OX 40 antibodies known in the art, yet have modest ability to activate OX40 signaling pathways to better balance drug effects with side effects for later clinical use, etc.

Disclosure of Invention

The invention aims to overcome the defect that an OX 40-targeted antibody with higher affinity and higher endocytic activity is lacking in the prior art, and provides an OX40 (CD 134/TNFRSF 4) -targeted antibody, a preparation method and application thereof.

In the present invention, the antibody targeting OX40 is obtained by immunizing an animal using an expression cell transiently transformed with OX40 protein as an immunogen, rather than deimmunizing using an extracellular domain fusion protein of OX40 as an immunizing antigen. Thus, while achieving greater affinity and specificity, antibodies targeting OX40 have modest ability to activate OX40 signaling pathway.

To solve the above technical problem, the first aspect of the present invention provides an antibody targeting OX40, comprising a heavy chain variable region (VH) and/or a light chain variable region (VL),

the VH comprises the following Complementarity Determining Regions (CDRs): VHCDR1 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 shown in the amino acid sequence of SEQ ID No. 3;

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

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

"amino acid mutation" in "having a 3, 2, or 1 amino acid mutation" is intended to mean that the sequence of the variant has an amino acid mutation, including an insertion, deletion, or substitution of an amino acid based on the original amino acid sequence, as compared to the original amino acid sequence. An exemplary interpretation is that mutations to a CDR may comprise 3, 2 or 1 amino acid mutations, and optionally the same or different numbers of amino acid residues may be selected between these CDRs for mutation, 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 which are currently known to those skilled in the art, such as some mutations performed on the antibody during the production or use of the antibody, such as mutations performed on the site of post-transcriptional modification (post-translational modifications, PTMs) of the CDR regions which may be present, including aggregation, deamidation-sensitive (asparagine deamidation) sites (NG, NS, NH, etc.) of the antibody, aspartic acid isomerism (DG, DP) sensitive sites, N-glycosylation (N- { P } S/T) sensitive sites, oxidation sensitive sites, and the like.

In this application, the amino acid sequences of the CDRs listed above are all shown according to the IMGT definition rules (the sequences shown in the claim of the present invention also according to the IMGT definition rules can be analyzed in http:// www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. Cgi# opennewwindow). However, it is well known to those skilled in the art that CDRs of antibodies can be defined in the art by a variety of methods, such as Kabat definition rules based on sequence variability (see, kabat et al, protein sequences for immunology, fifth edition, national institutes of health, besseda, malyland (1991)) and Chothia definition rules based on structural loop region position (see, jmol Biol 273:927-48,1997). It will be appreciated 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 to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention. Although the scope of the claimed invention is based on the sequences shown by IMGT definition rules, amino acid sequences corresponding to definition rules according to other CDRs should also fall within the scope of the invention (see also https:// qinqianshan. Com/biology/anti-number-description /).

TABLE 1-1 method for defining CDRs of antibodies of the present application

	IMGT definition rules
		VL CDR1	L27--L32
VL CDR2	L50--L52
		VL CDR3	L89--L97
VH CDR1	H26--H33
		VH CDR2	H51—H58
VH CDR3	H98--H106

Wherein Laa-Lbb may 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 position 32 starting from 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 position 33 starting from the N-terminus of the heavy chain of the antibody, according to the IMGT coding rules. It is well known to those skilled in the art that some positions may have insertion sites when coding CDRs using these coding rules.

Preferably, the OX 40-targeting antibody is a murine antibody.

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). The VH FWR is selected from 1) the FR1, FR2 and FR3 regions in the 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 the germline IGHJ2 x 01, or a back mutation thereof; and/or, the VL FWR is selected from 1) the FR1, FR2 and FR3 regions in the germline IGKV10-96 x 01, IGKV10-96 x 02, IGKV10-96 x 03, IGKV10-96 x 04, IGKV10-96 x 05, and 2) the intermediate FR4 region in the germline IGKJ5 x 01, or a back mutation thereof.

Even more preferably, the VH FWR comprises VH FWR1 shown as SEQ ID NO. 11 or a mutation thereof, VH FWR2 shown as SEQ ID NO. 12 or a mutation thereof, VH FWR3 shown as SEQ ID NO. 13 or a mutation thereof, and VH FWR4 shown as SEQ ID NO. 14 or a mutation thereof; and/or, the VL FWR comprises VL FWR1 shown as SEQ ID NO. 15 or mutation thereof, VL FWR2 shown as SEQ ID NO. 16 or mutation thereof, VL FWR3 shown as SEQ ID NO. 17 or mutation thereof, and VL FWR4 shown as SEQ ID NO. 18 or 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 shown as SEQ ID NO. 8 or a mutation thereof; said mutation is a deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of said VH and/or said VL, and said mutated amino acid sequence has at least 85% sequence identity to the amino acid sequence of said SEQ ID No. 7 and/or said SEQ ID No. 8, and retains or improves binding of said 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 the VH of the murine antibody is the amino acid sequence 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 OX 40-targeting antibody further comprises a murine antibody constant region or a human antibody constant region; the murine antibody constant region comprises a murine IgG1, igG2a, igG2b3, or IgG3 heavy chain constant region or mutation thereof and a kappa or lambda type light chain constant region or mutation thereof, and the human antibody constant region comprises a human IgG1, igG2, igG3, or IgG4 heavy chain constant region or mutation thereof and a kappa or lambda type light chain constant region or mutation thereof.

Preferably, the OX 40-targeting antibody is a humanized antibody;

more preferably, the framework regions of the variable region of the humanized antibody comprise a framework region of a heavy chain variable region of a human antibody and a framework region of a light chain variable region of a human antibody; the framework region of the human antibody light chain variable region is selected from the group consisting of 1) the FR1, FR2 and FR3 regions of the germline IGHV1-46 x 01, IGHV1-46 x 02, IGHV1-46 x 03, IGHV1-46 x 04, IGHV1-2 x 06, and 2) the FR4 region in the germline IGHJ4 x 01, or a back mutation thereof; and/or the framework regions of the human antibody heavy chain variable region are selected from 1) the FR1, FR2 and FR3 regions in the 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 region in the germline IGKJ2 x 01, IGKJ2 x 02, or a back mutation 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 mutation thereof; and/or the heavy chain of the antibody comprises the heavy chain constant regions of human IgG1, igG2, igG3 and IgG4 or mutations thereof.

Preferably, the antibody targeting OX40 comprises an immunoglobulin, fab ', F (ab') ₂ Fv, scFv, bispecific antibodies, multispecific antibodies, single domain antibodies or single domain antibodies, or monoclonal or polyclonal antibodies made from such antibodies.

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

In the present invention, the scFv (single chain antibody fragment, single chain antibody) may be a conventional single chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids. Wherein the VL and VH domains are paired to form monovalent molecules by making them capable of producing a linker that is a single polypeptide chain [ see, e.g., bird et al, science 242:423-426 (1988) and Huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art joints are made up of repeating G ₄ S amino acid sequence or variants thereof. For example, a polypeptide having an amino acid sequence (G ₄ S) ₄ Or (G) ₄ S) ₃ A linker, but variants thereof may also be used.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. Such 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 units have polyepitopic specificity; antibodies 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; antibodies 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.

Antibodies of the invention include monoclonal antibodies. The monoclonal antibodies or mabs or abs described herein refer to antibodies derived from a single clonal cell line, which is not limited to eukaryotic, prokaryotic or phage clonal cell lines. The monoclonal antibodies can be developed by a variety of routes and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the main stream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.

In the present invention, the single domain antibody may be a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

In the present invention, the single domain antibody may be a conventional single domain antibody in the art, which includes only a heavy chain variable region.

In order to solve the technical problem described above, a second aspect of the present invention provides a bispecific antibody comprising a first protein functional region and a second protein functional region, the first protein functional region being an antibody targeting OX40 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-L2, 4-1BB, CD40, CD73, tim3, LAG3 or CD47, or a tumor therapeutic target, preferably comprising OX40.

To solve the above technical problem, a third aspect of the present invention provides an isolated nucleic acid encoding an antibody targeting OX40 according to the first aspect of the present invention.

In order to solve the above technical problem, 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 vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

In order to solve the above technical problem, the 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 technical problem, a sixth aspect of the present invention provides a method for producing an antibody targeting OX40, comprising culturing the transformant according to the fifth aspect of the present invention, and obtaining the antibody targeting OX40 from the culture.

In order to solve the above technical problem, a seventh aspect of the present invention provides a genetically modified cell comprising an antibody targeting OX40 according to the first aspect of the present invention.

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

To solve the above technical problem, the eighth aspect of the present invention provides a pharmaceutical composition comprising an antibody targeting OX40 according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention and/or a genetically modified cell according to the seventh aspect of the present invention, and a pharmaceutically acceptable carrier.

The pharmaceutical composition preferably further comprises other anti-tumor antibodies as active ingredients.

The pharmaceutically acceptable carrier may be any carrier conventional in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the OX40 targeting antibody and/or the bispecific antibody and/or the genetically modified cell described above, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, said percentages being mass percentages of the pharmaceutical composition.

Preferably, the pharmaceutical composition is an anti-tumor drug. More preferably an OX40 positive tumor, for example a lung cancer, liver cancer, stomach cancer, breast cancer, head and neck cancer and/or colon cancer etc.

The route of administration of the pharmaceutical composition of the present invention is preferably parenteral, injectable or oral. The administration by injection preferably comprises intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, i.e. in the form of an aqueous solution, non-aqueous solution or suspension, more preferably in the form of tablets, capsules, granules, injections or infusions, etc. 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 the pharmaceutical composition of the present invention may be adjusted depending on the amount of the composition that achieves the desired diagnostic or therapeutic result. The administration regimen may also be single or multiple injections, or may be modified. The dosage level and regimen selected is 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, the 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.

The 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 the appropriate concentration ranges and routes of administration. And can then be used to determine useful dosages and routes of administration in humans. In general, the determination and adjustment of the administration of an effective amount or dose, and the assessment of when and how such adjustment is made, are known to those skilled in the art.

For combination therapy, the above-described OX 40-targeting antibodies, the above-described bispecific antibodies, genetically modified cells, pharmaceutical compositions, and/or additional therapeutic or diagnostic agents may each be used as a single agent, within any time frame suitable for performing the intended treatment or diagnosis. Thus, these single agents may be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours) or sequentially and continuously. For example, these single agents may be administered within one 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.

Additional guidance regarding formulation, dose, dosing regimen, and measurable therapeutic results, see Berkow et al (2000) The Merck Manual of Medical Information (Merck medical information handbook) and Merck & co.inc., whitehouse Station, new Jersey; ebadi (1998) CRC Desk Reference of Clinical Pharmacology (handbook of clinical pharmacology) et al.

In order to solve the above technical problem, the ninth aspect of the present invention provides the use of an antibody targeting OX40 according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a genetically modified cell according to the seventh aspect of the present invention, a pharmaceutical composition according to the eighth aspect of the present invention for the preparation of a medicament for the diagnosis, prevention and/or treatment of a tumor.

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, etc.

To solve the above technical problem, the present invention also provides the use of an OX 40-targeting antibody according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a genetically modified cell according to the seventh aspect of the present invention, a pharmaceutical composition according to the eighth aspect of the present invention for the diagnosis, prevention and/or treatment of a tumor.

To solve the above technical problem, the present invention further provides a kit comprising a kit a and a kit B, wherein the kit a is an OX40 targeting antibody according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a genetically modified cell according to the seventh aspect of the present invention, and the kit B is another anti-tumor antibody or a pharmaceutical composition comprising the other anti-tumor antibody. The medicine box A and the medicine box B can be used simultaneously, the medicine box A can be used first and then the medicine box B can be used, the medicine box B can be used first and then the medicine box A can be used according to the actual requirements in specific application.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological 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, definitions and explanations of related terms are provided below.

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

The three-letter and one-letter codes for amino acids used in the present invention are known to the person skilled in the art or are 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 that the terms "consisting of … …" are also included, as the context dictates otherwise.

In the present invention, the term "variable" generally refers to the fact that certain portions of the sequence of the variable domain of an antibody vary strongly, which results in the binding and specificity of various specific antibodies for their specific antigens. However, variability is not evenly distributed throughout the variable regions of antibodies. It focuses on three segments in the light and heavy chain variable regions, known as Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs). The more highly conserved parts of the variable domain are called the Framework (FWR). The variable domains of the natural heavy and light chains each comprise four FWR regions, mostly in a β -sheet configuration, connected by three CDRs, forming a loop connection, and in some cases forming part of a β -sheet structure. The CDRs in each chain are in close proximity by the FWR region and together with 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, such as participation in antibody-dependent cytotoxicity.

The term "epitope" refers to the portion of an antigen (e.g., OX 40) that specifically interacts with an antibody molecule. The term "compete" in the present invention refers to the ability of an antibody molecule to interfere with the binding of an anti-OX 40 antibody molecule to a target (e.g., OX 40). The interference with binding may be direct or indirect (e.g., through allosteric modulation of the antibody molecule or target). A competitive binding assay (e.g., FACS assay, ELISA, or BIACORE assay) can be used to determine whether an antibody molecule is capable of interfering with the extent to which another antibody molecule binds to its target.

The term "antibody" as used herein includes immunoglobulins which are tetrapeptide chain structures formed by joining two identical heavy chains and two identical light chains by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, i.e., igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be further divided into different subclasses, e.g., igG can be divided into IgG1, igG2, igG3 and IgG4, depending on the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each class Ig of the five classes of Igs may have either a kappa chain or a lambda chain.

As used herein, the term "isolated" refers to being obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide is said to be "isolated" in a high purity from its natural state. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.

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, a prokaryotic cell such as E.coli, a fungal cell such as a yeast cell, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, HEK 293 cell or human cell.

The term "murine antibody" is herein a monoclonal antibody to OX40 made according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with the OX40 antigen and then isolating hybridomas expressing 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 murine kappa, lambda chains or variants thereof, or further comprise a heavy chain constant region of murine IgG1, igG2, igG3 or IgG4 or variants 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., a "humanized antibody").

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

"identity", "mutation" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example 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 matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the maximum percent identity. "optimizing" refers to a mutation that maintains or improves binding of the antibody to an antigen, and in the present invention, refers to a mutation that maintains, maintains or improves binding to CLDN 18.2.

The terms "polypeptide", "peptide" and "protein" (if single-chain) are used interchangeably in the present invention. 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, and "amino acid substitutions" and "conservative amino acid substitutions" are those in which an amino acid residue is replaced with another amino acid residue and with an amino acid residue having a similar side chain, respectively.

As used herein, "lentivirus" refers to the genus retrovirus (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 therefore they are one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived 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 into 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 autonomously replicating plasmids or viruses. 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, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.

The expressions "cell", "cell line" as used herein are used interchangeably and all such designations include offspring. The term "host cell" refers to a cell that can be used to introduce a vector, and includes, but is not limited to, a prokaryotic cell such as E.coli, a fungal cell such as a yeast cell, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, HEK 293 cell, or human cell.

The term "transfection" refers to the introduction of an 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 any source of immune cells. "immune cells" include, for example, white blood cells (leukocytes) derived from Hematopoietic Stem Cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural Killer (NK) cells, and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). The term "immune cells" may also be human or non-human.

As used in the present invention, the term EC ₅₀ Refers to the half maximal effect concentration (concentration for 50%of maximal 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 needed, and the dosage beneficial to the patient can be determined by the physician according to the type, age, weight and general disease condition of the patient, the administration mode, etc. The administration may be, for example, injection or other therapeutic means.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

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

The invention has the positive progress effects that: compared with the prior art antibodies, the antibody provided by the invention has moderate capacity of activating the OX40 signal path under the condition of equivalent affinity and specificity, so that the balance between drug effect and side effect can be achieved in later clinical use. In a preferred embodiment of the invention, the antibody activation of the invention can powerfully activate the OX40 signaling pathway at a half-effective concentration (EC 50) of 803.4ng/mL.

Drawings

FIG. 1 shows the results of the affinity and specificity assays for fusion proteins in example 4.

FIG. 2 is a graph showing the results of the affinity and specificity assays for OX 40-expressing cells in example 4.

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

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

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 antigen preparation, mouse immunization and hybridoma preparation

a. Antigen (transient OX40 expressing cells) preparation

The commercial OX40 plasmid (HG 0481-UT, yinqiao) was transiently transfected into 293F cells using 293F cells as transfected cells, and after 48 hours of culture, the cells were taken as immunogens. The method comprises the following specific steps: 293F cells were prepared and the cell density reached 1.0-1.2X10 on the day of transfection ⁶ Individual/ml; cells were counted and 1.0X10 were added to 6-well plates ⁶ Individual cells/ml, 2 ml/well, 4 wells total; a DNA transfection system was prepared. Taking 640 mu L of Opti-MEM, adding 8ug of DNA plasmid, and gently mixing; adding 16 μl PEI, mixing, and standing at room temperature for 20 min; adding the DNA-293PEI mixture into 293F cells, placing into Infoster shaker, 115rpm,37℃and 5% CO ₂ Culturing in a culture environment for 2 days; and (5) collecting a sample. All samples were collected and centrifuged at 1500rpm for 3 minutes; cells were taken and washed twice at 1500rpm for 3 minutes after PBS was resuspended. Finally re-suspended to 5×10 ⁶ Each ml, the antigen (i.e., transient OX40 expressing cells or OX40-293F cells) was obtained for immunization.

b. Immunization

Animals (mice and rabbits) were deimmunized with the expression cells of the transiently transformed OX40 prepared as described above as immunogens.

After cell collection, the cells were used to immunize six week old mice (Hangzhou university animal center). Animal immunization was performed by the university of Hangzhou animal center. The immunization part is subcutaneous multipoint immunization, and each immunization is 5×10 ⁶ Individual cells/mice; a total of 4 immunizations were performed (after the first immunization, after 21 days, two-immunization was performed, and then one-boost was performed every 14 days). Serum titers were detected by ELISA (similar to the ELISA procedure described below) after the 4-release. Mice with acceptable serum titers will be boosted and the spleen taken for storage or fusion.

c. Fusion of

Firstly, taking thymus cells of a 4-week mouse (the center of Hangzhou university animals), and re-suspending the thymus cells in an IMDM culture medium to serve as feeder cells; the SP2/0 with good growth state is resuspended in IMDM culture medium, As a fused cell. Spleen cells were then prepared as follows: spleens of the mice after the above-mentioned booster immunization were taken, placed in a sterile 10cm dish, excess adhesion tissues were cut off, 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 confluent cells for later use. In preparation for cell fusion, SP2/0 cells and spleen cells were mixed uniformly in a volume ratio of 1:1 of cell pellet, IMDM medium was added so that the liquid in the tube was 30mL, centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded. The cell pellet was carefully knocked out and the cells in the centrifuge tube were placed in a beaker containing warm water at 37 ℃. 1mL of PEG is added into the cells dropwise at a constant speed within 1min, and the mixture is kept stand for 30s in a water bath at 37 ℃; 10mL of IMDM medium was added to the fused cells, followed by timely addition of medium to make the total volume 30mL. Centrifuging at 1200rpm for 3 min, and discarding the supernatant; the cell pellet is tapped up, slowly transferred to hybridoma selection medium, and feeder cells are added to the selection medium, and the fusion cells and feeder cells are gently mixed. Plating at 200. Mu.L/well for a total of 20 plates; placed at 37 ℃ and 5% CO ₂ After culturing for 7 days under the culture condition, hybridoma supernatants are taken for detection and screening.

Example 2 antibody screening and sequencing

1. Antibody screening

ELISA screening: the antigen OX40 fusion protein (OX 40 fusion protein was purchased from Hangzhou Hua An Biotechnology Co., ltd.) was first diluted to 1. Mu.g/ml with a coating solution and added to a 96-well ELISA plate at 50. Mu.l per well. After the cover is covered by the ELISA plate, the ELISA plate is placed into a 4-DEG refrigerator for overnight; after sealing, 50 μl of the hybridoma supernatant obtained in example 1 was added into an ELISA plate by a pipette per well, and after shaking, the mixture was placed in a constant temperature incubator and allowed to stand for 1 hour; after washing 3 times with TBST, adding 50 μl of diluted secondary antibody (goat anti-mouse IgG-HRP, pierce, 31430) into an ELISA plate with a pipettor, placing into a constant temperature incubator, and standing for 30 min; after washing 3 times with TBST, the color was developed with HRP-developed solution. After the end of the color development, H is used for ₂ SO ₄ (TMB) terminate the reaction, read the plate and analyze the data. Positive clones were screened according to the OD value of the positive wells.

ELISA-positive clones were screened by flow-through (FACS) using the OX40-293F cells obtained in example 1 to screen for double-positive clones: transient OX40-293F cells were collected, added to 96-well V-plates at 50000 wells per well, blocked with 1% BSA/PBS, and left at 4℃for 30 min; 50 μl of ELISA positive hybridoma supernatant was added to the cell well per well, and the mixture was placed in a 4-degree freezer for 1 hour; after washing, diluted anti-mouse-FITC (goat anti-mouse IgG-FITC, jackson, 115-095-003) was added to the cells, 50. Mu.l per well, and placed in a 4 degree freezer for 30 minutes; the cells after incubation were resuspended by washing and the MFI was detected and recorded on a flow sorter. Positive clones were picked and subcloned.

Subcloning: and subcloning the screened double-positive clones, and selecting a monoclonal for later development through detection. This example uses two methods for subcloning. Limiting dilution method and counting plating. All by conventional procedures in the art.

Subcloning screening: the subclone screening method is the same as the hybridoma supernatant screening method, and is to detect by using an ELISA method and a FACS method, and finally select double-positive monoclonal as the final clone to carry out antibody recombination to obtain a sequence.

2. Sequencing

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

The conditions for RT are shown in Table 1 below.

TABLE 1

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

TABLE 2 PCR reaction System

Reagent(s)	10 μl 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 reaction procedure for light chain PCR are shown in tables 4 and 5 below.

TABLE 4 PCR reaction System

TABLE 5 PCR reaction procedure

The obtained PCR product was sent to sequencing, and the sequencing company was Hangzhou Kangshen Biotechnology Co.

3. Antibody sequence information

Antibodies are commonly used in the numbering system Kabat, chothia and IMGT, the IMGT numbering system being used in this example. 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 framework sequences.

TABLE 7 frame region sequence combinations

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

TABLE 8 amino acid sequences of the light chain variable region and heavy chain variable region

Selection of antibody light and heavy chain constant regions: the light chain constant region and heavy chain constant region of antibody OX40 of the invention can be murine, rabbit, human, and the like.

The nucleotide sequence of the above VH may preferably be (SEQ ID NO: 9) as follows:

GAGGTTCAGCTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATACACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAACGGTCGTACTAACTCCAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAGAGGCGGTGCCGTACTACTTTGTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA；

the nucleotide sequence of the above VL may preferably be (SEQ ID NO: 10) as follows:

GATATTGTGATGACGCAGGCTGCATCCTCCCTGTCTGCCTCTCTGGGAGACAGGGTCACCATCAGTTGTAGGGCAAGTCAGGACATTAACAATTATTTAAACTGGTATCAGCAGAAACCTGATGGAACTGTTAAACTCCTGATCTACTTCACATCAGGATTACGCTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTAAAC

EXAMPLE 3 antibody production and purification

The antibody of the present invention is an antibody purified by ascites in mice. The operation steps are all routine in the field, and are mainly to inoculate the hybridoma cells into the abdominal cavity of the pre-sensitized mice after the hybridoma cells are cultured and expanded. Ascites was collected after 10 days and purified by proG, and the purified antibodies were quantified and stained by SDS-pAGE.

Example 4 Effect evaluation

1. OX40 binding affinity and specificity identification

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

OX40 gene fragment was purchased from Yinqiao Shenzhou, and the purchased gene fragment was used as a template to obtain OX40 extracellular region fragment by PCR method, and constructed on pTT5 vector for protein expression. Wherein the cleavage sites of the OX40-rFc fusion protein (prepared by methods conventional in the art) are EcoRI and XhoI, and the cleavage sites of the OX40-His fusion protein (prepared by methods conventional in the art) are EcoRI and NheI. After construction, the plasmid is transfected into 293F cells for expression and purified to obtain OX40-rFc fusion protein and OX40-His fusion protein.

The 4-1BB gene (4-1 BB is another molecule of the same gene family as OX40, but the sequence is completely different, thus being used as an experimental control for binding specificity) fragment was purchased from Yinqiao Shenzhou, and the 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 cleavage sites of the 4-1BB-rFc fusion protein are EcoRI and XhoI, and the cleavage sites of the 4-1BB-His fusion protein are EcoRI and NheI. After construction, the plasmid is transfected into 293F cells for expression and purified to obtain 4-1BB-rFc fusion protein and 4-1BB-His fusion protein.

1.2 binding affinity and specificity identification

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

First, ELISA was used to identify the binding affinities of positive antibodies (i.e., vonleizumab) and OX40-rFc fusion proteins and OX40-His fusion proteins, and 4-1BB-rFc fusion proteins and 4-1BB-His fusion proteins were used as controls to identify the binding specificities of the antibodies.

The antigen OX40 fusion protein or 4-1BB fusion protein was diluted to 1. Mu.g/ml with the coating solution and added to a 96-well ELISA plate at 50. Mu.l per well. After the cover is covered by the ELISA plate, the ELISA plate is placed into a refrigerator at 4 ℃ for overnight; adding purified antibody (1 mug/mL) into an ELISA plate after sealing, uniformly vibrating, and placing into a constant-temperature incubator for standing for 1 hour; after washing 3 times by TBST, adding 50 mu l of diluted secondary antibody into an ELISA plate by using a pipette according to each hole, placing the plate into a constant-temperature incubator, and standing for 30 minutes; after washing 3 times with TBST, the color was developed with HRP-developed solution. After the end of the color development, H is used for ₂ The reaction was quenched with SO4 (TMB), the plates were read and the data analyzed.

The experimental results are shown in FIG. 1. As can be seen from the figure, the anti-OX 40 antibody of the invention specifically recognizes OX40 fusion protein, but not 4-1BB fusion protein, and the binding capacity is comparable to that of the positive control antibody Vonllerlizumab. Wherein the OX40 positive control antibody is an anti-OX 40 antibody expressed according to the sequence synthesis of von lerolizumab (available from coronal biotechnology limited) in a clinical trial by roche; the 4-1BB positive control antibody was the anti-4-1 BB antibody Utomimumab from the clinical trial of the best, available from Corona, E4436-T1802, AB 180043).

2. Binding affinity and specificity identification of binding to OX 40-expressing cells

Construction of 2.1OX40-expressing cells and specific control (4-1 BB) cells

OX40/4-1BB expression vectors were purchased from Yinqiao Shenzhou (OX-40: HG10481-UT). The expression vector can be directly transfected (the transfection procedure is conventional in the art, and is specifically described as follows: 293F cells are prepared, and the cell density reaches 1.0-1.2X10 on the day of transfection) ⁶ individual/mL; cells were counted and 1.0X10 were added to 6-well plates ⁶ Individual cells/mL, 2 mL/well, 4 wells total; a DNA transfection system was prepared. Taking 640 mu L of Opti-MEM, adding 8 mu g of DNA plasmid, and gently mixing; then adding 16 mu LPEI, mixing gently, standing for 20 minutes at room temperature; adding the DNA-293PEI mixture into 293F cells, placing into Infoster shaker, 115rpm,37℃and 5% CO ₂ Culturing in a culture environment for 2 days; and (5) collecting a sample. All samples were collected and centrifuged at 1500rpm for 3 minutes; cells were taken, resuspended in PBS, and washed twice for 3 min at 1500rpm, in preparation for FACS experiments). After the identification by transfection of 293F cells, it was confirmed that the vector was able to correctly express OX40 and 4-1BB molecules on the surface of 293F cells, i.e., OX40-293F cells were successfully obtained.

2.2 binding affinity and specificity identification

The anti-OX 40 antibodies of the invention were further characterized by flow cytometry (FACS) using the OX40-293F cells obtained as described above: transient OX-40-293F cells were collected, added to a 96-well V-plate at 50000 wells per well, blocked with 1% BSA/PBS, and left at 4℃for 30 min; ELISA positive antibodies were diluted 3-fold from 10. Mu.g/mL, 50. Mu.l of diluted antibodies were added to the cell wells per well, and the mixture was placed in a 4-degree freezer for 1 hour; after washing, diluted anti-mouse-FITC was added to the cells, 50. Mu.l per well, and placed in a 4 degree freezer for 30 minutes; the cells after incubation were resuspended by washing, detected in a flow sorter, MFI recorded and the data analyzed.

The results of the experiment are shown in FIG. 2, from which it can be seen that the anti-OX 40 antibodies of the invention recognize OX40-293F cells. Since the positive control antibody was a humanized antibody (i.e., von willebrand), whereas the anti-OX 40 antibody of the present invention was a mouse antibody, the secondary antibodies used in the detection were completely different secondary antibodies (goat anti-human IgG-HRP, abcam, ab98595; goat anti-human IgG-R-PE, southern Biotech, 204009), and thus the final MFI reads were also greatly different.

3. Specific activation of OX40 signaling pathway

Construction of 3.1OX40 Signal pathway reporter cell line

The reporter vector pGL4.32[ luc 2P/NF-. Kappa.B-RE/Hygro ] was purchased from Promega, HEK293 cells were transfected with the reporter vector (Shanghai crown conductance bioengineering #C028), screened with hygromycin (conventional screening procedure), positive cell lines with stronger drug resistance were selected, NF-. Kappa.B signaling pathway was stimulated with TNF-. Alpha.stimulated positive cells, samples were treated with a single luciferase reporter gene detection kit (Beijing Yuan Ping Hao organism #LF102-01), the transcriptional activity of luciferases was detected by measuring RLU with a multifunctional enzyme-labeled instrument, and finally three positive cell lines 1C2-2, 1C6-3, 1B1-2 with the highest transcriptional activity were selected (Table 9).

OX40 expression vector was purchased from Yinqiao Shenzhou, the three positive cell lines were transfected with OX40 expression plasmid, and hygromycin and G418 were used for selection to select positive cell lines with stronger drug resistance. FACS is used to detect the expression level of OX40 on the cell surface (the specific experimental steps are the same as those of 2.2 except the detection samples); the ability of anti-OX 40 antibodies to activate NF- κB signaling pathway after binding to different positive cell surface OX40 was assayed using a multifunctional microplate reader. FACS showed no significant difference in expression levels of each positive cell line OX40, with the 19B3 cell line having the strongest NF- κB transcriptional activity (FIG. 3). Thus, positive cell line 19B3 was selected as an OX40 signaling pathway reporter cell line.

TABLE 9 transcriptional Activity of NF- κB in different positive cell lines

Numbering device	RLU
		Negative Control	9250
1C2-2	10513360
		1C6-3	6028090
1B1-2	5345550

3.2 specific activation of OX40 Signal pathway identification

Inoculating the obtained positive cell strain 19B3 cells into a 96-well plate, and placing CO ₂ Incubate overnight in incubator. The OX40 antibody of the invention is added into corresponding holes after being diluted in a gradient way, and CO is placed ₂ Incubators were incubated for 5 hours, then treated with a single luciferase reporter assay kit, relative Luminescence Units (RLU) were read by a multifunctional microplate reader, and concentration-stimulatory activity response curves of antibodies were fitted by analysis software according to a four parameter logistic equation.

As shown in FIG. 4, the OX40 antibody of the invention can strongly activate the OX40 signaling pathway with half-maximal effective concentration (EC 50) of 803.4ng/mL; in contrast, under the same conditions, the positive control antibody von lerolizumab only showed relatively weak activation capacity such that the EC50 could not be effectively fitted.

SEQUENCE LISTING

<110> Bailisi Kang Shengwu medicine (Hangzhou) Co., ltd

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

<130> P19014931C

<160> 18

<170> PatentIn version 3.5

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

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

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

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Ser Val Lys Leu Ser Cys Lys Ala Ser

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<400> 14

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

<210> 15

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> VL FWR1

<400> 15

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> 16

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> VL FWR2

<400> 16

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

1 5 10 15

Tyr

<210> 17

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> VL FWR3

<400> 17

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> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VL FWR4

<400> 18

Phe Gly Ala Gly Thr Lys Leu Glu Leu Asn

1 5 10

Claims

1. An antibody targeting OX40, 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 in the amino acid sequence of SEQ ID No. 1; VH CDR2 as shown in the amino acid sequence of SEQ ID No. 2; VH CDR3 shown in the amino acid sequence of SEQ ID No. 3;

The VL comprises CDRs of: VL CDR1 as shown in the amino acid sequence of SEQ ID NO. 4; VL CDR2 as shown in the amino acid sequence of SEQ ID NO. 5; VL CDR3 as shown in the amino acid sequence of SEQ ID NO. 6.

2. The OX 40-targeting antibody of claim 1, wherein the OX 40-targeting antibody is a murine antibody.

3. The OX 40-targeting antibody of claim 2, wherein 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 the 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 the germline IGHJ2 x 01, or a back mutation thereof; and/or, the VL FWR is selected from 1) the FR1, FR2 and FR3 regions in the germline IGKV10-96 x 01, IGKV10-96 x 02, IGKV10-96 x 03, IGKV10-96 x 04, IGKV10-96 x 05, and 2) the intermediate FR4 region in the germline IGKJ5 x 01, or a back mutation thereof.

4. An OX 40-targeting antibody according to claim 3, wherein the amino acid sequence of VH of the murine antibody is as shown in SEQ ID No. 7 or a mutation thereof; and/or the amino acid sequence of the VL is shown as SEQ ID NO. 8 or a 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 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 retains or improves binding of the antibody to OX 40.

5. The OX 40-targeting antibody of claim 4, wherein the sequence identity is at least 90% sequence identity.

6. The OX 40-targeting antibody of claim 5, wherein the sequence identity is at least 95% sequence identity.

7. The OX 40-targeting antibody of claim 6, wherein the sequence identity is at least 99% sequence identity.

8. The OX 40-targeting antibody of claim 4, wherein the VH of the murine antibody has the nucleotide sequence set forth in SEQ ID No. 9; and/or the nucleotide sequence of the VL is the nucleotide sequence shown as SEQ ID NO. 10.

9. The OX 40-targeting antibody of claim 2, further comprising a murine antibody constant region or a human antibody constant region; the murine antibody constant region comprises a murine IgG1, igG2a, igG2b3, or IgG3 heavy chain constant region or mutation thereof and a kappa or lambda type light chain constant region or mutation thereof, and the human antibody constant region comprises a human IgG1, igG2, igG3, or IgG4 heavy chain constant region or mutation thereof and a kappa or lambda type light chain constant region or mutation thereof.

10. The OX 40-targeting antibody of claim 1, wherein the OX 40-targeting antibody is a humanized antibody.

11. The OX 40-targeting antibody of claim 10, wherein the framework regions of the variable region of the humanized antibody comprise a framework region of a human antibody heavy chain variable region and a framework region of a human antibody light chain variable region; the framework region of the human antibody light chain variable region is selected from the group consisting of 1) the FR1, FR2 and FR3 regions of the germline IGHV1-46 x 01, IGHV1-46 x 02, IGHV1-46 x 03, IGHV1-46 x 04, IGHV1-2 x 06, and 2) the FR4 region in the germline IGHJ4 x 01, or a back mutation thereof; and/or the framework regions of the human antibody heavy chain variable region are selected from 1) the FR1, FR2 and FR3 regions in the 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 region in the germline IGKJ2 x 01, IGKJ2 x 02, or a back mutation thereof; the number of amino acid sites of the back mutation is 0-10.

12. The OX 40-targeted antibody of claim 10, wherein the light chain of the antibody comprises a human antibody kappa or lambda type light chain constant region or mutation thereof; and/or the heavy chain of the antibody comprises the heavy chain constant regions of human IgG1, igG2, igG3 and IgG4 or mutations thereof.

13. The OX 40-targeted antibody of any one of claims 1-12, wherein the OX 40-targeted antibody comprises or is a monoclonal or polyclonal antibody made from an immunoglobulin, fab ', F (ab') 2, fv, scFv, bispecific, multispecific, single domain or single region antibody.

14. A bispecific antibody comprising a first protein functional region and a second protein functional region, wherein the first protein functional region is an OX 40-targeting antibody of any one of claims 1-13; the second protein functional region is an antibody that targets a non-OX 40 antigen.

15. The bispecific antibody of claim 14, wherein the non-OX 40 antigen is an immune checkpoint antigen or a tumor therapeutic target.

16. The bispecific antibody of claim 15, wherein the immune checkpoint antigen comprises PD-1, PD-L2, 4-1bb, CD40, CD73, tim3, LAG3, or CD47, and the tumor therapeutic target comprises OX40.

17. An isolated nucleic acid encoding the OX 40-targeted antibody of any one of claims 1-13.

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

19. The expression vector of claim 18, wherein the expression vector is a plasmid, cosmid, phage, or viral vector.

20. The expression vector of claim 19, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

21. A transformant comprising the expression vector of any one of claims 18 to 20 in a host cell.

22. The transformant of claim 21, wherein the host cell is an e.coli TG1, BL21 cell, or CHO-K1 cell.

23. A method of making an antibody that targets OX40 comprising culturing a transformant as described in claim 21 or 22, and obtaining the antibody that targets OX40 from the culture.

24. Genetically modified cell, characterized in that it comprises an antibody targeting OX40 according to any one of claims 1-13.

25. The cell of claim 24, wherein the cell is a eukaryotic cell.

26. The cell of claim 25, wherein the cell is an isolated human cell.

27. The cell of claim 26, wherein the cell is an immune cell.

28. The cell of claim 27, wherein the cell is a T cell, or an NK cell.

29. The cell of claim 28, wherein the cell is an NK92 cell line.

30. A pharmaceutical composition comprising the OX 40-targeting antibody of any one of claims 1-13, the bispecific antibody of any one of claims 14-16, and/or the genetically modified cell of any one of claims 24-29, and a pharmaceutically acceptable carrier.

31. Use of an antibody targeting OX40 according to any one of claims 1-13, a bispecific antibody according to any one of claims 14-16, a genetically modified cell according to any one of claims 24-29 and/or a pharmaceutical composition according to claim 30 for the preparation of a medicament for the diagnosis, prevention and/or treatment of a tumor.

32. The use of claim 31, wherein the tumor is an OX40 positive tumor.

33. The use according to claim 32, wherein the tumour is lung cancer, liver cancer, gastric cancer, breast cancer, head and neck cancer and/or colon cancer.