Application of anti-OX40 antibody in preparation of medicine

Abstract

The invention relates to application of an anti-OX40 antibody or an antigen binding fragment thereof in preparation of a medicine, the medicine is used for treating autoimmune diseases, and the anti-OX40 antibody or the antigen binding fragment thereof comprises heavy chain variable regions CDR1, CDR2 and CDR3 and light chain variable regions CDR1, CDR2 and CDR3.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to the use of anti-OX40 antibodies or antigen-binding fragments thereof in the preparation of drugs. Background Art

[0002] The immune system is a disease defense system composed of a series of biological structures and processes in the body. Disorders in the immune system can lead to a variety of diseases. When the activity of the immune system is reduced, immunodeficiency will occur, leading to frequent and fatal infections. Abnormal immune systems will attack normal tissues as invaders, thus causing autoimmune diseases.

[0003] The exact cause and pathogenesis of autoimmune diseases are still unknown, and there is currently no complete cure. This has greatly affected patients' labor and quality of life, and even threatened their lives. It consumes a large amount of health resources and has become an important public health issue affecting human health.

[0004] Therefore, effective methods for treating autoimmune diseases are urgently needed. Summary of the invention

[0005] The present application provides an anti-OX40 antibody or an antigen-binding fragment thereof with good specificity, high affinity and stability, and a nucleic acid encoding the antibody, etc., for use in treating autoimmune diseases.

[0006] T cells in patients with autoimmune diseases are often in an over-activated state, so they will attack their own normal tissues. The anti-OX40 antibody or its antigen-binding fragment described in this application can bind to human OX40 with high affinity. OX40 is a type of membrane protein that is highly expressed after T cell activation. The antibody described in this application has an enhanced ADCC effect and can kill over-activated T cells through NK cells to achieve the effect of treating autoimmune diseases.

[0007] The anti-OX40 antibody or antigen-binding fragment thereof described in the present application has one or more of the following characteristics in the treatment of autoimmune diseases: high utilization efficiency, low side effects, wide range of action, and long-lasting therapeutic effect, and has a very broad application prospect.

[0008] In one aspect, the present application provides a use of an anti-OX40 antibody or an antigen-binding fragment thereof in the preparation of a medicament, wherein the anti-OX40 antibody or the antigen-binding fragment thereof comprises a CDR region selected from any one of the following groups:

[0009] 1) The sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 5-7, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 14-16, respectively;

[0010] 2) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 23-25, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 32-34, respectively;

[0011] 3) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 41-43, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 50-52, respectively;

[0012] 4) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 63, 42 and 43, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 50-52, respectively;

[0013] 5) the sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 41, 67, and 43, respectively, and the sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 50-52, respectively;

[0014] 6) the sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 41, 42, and 70, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 50-52, respectively;

[0015] 7) the sequences of heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 63, 67, and 70, respectively, and the sequences of light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 75-77, respectively;

[0016] 8) the sequences of heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 41, 42, and 70, respectively, and the sequences of light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 75-77, respectively;

[0017] The medicament is used to treat autoimmune diseases.

[0018] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises the following group of heavy chain variable regions, whose sequences are shown in SEQ ID NOs: 4, 22, 40, 62, 66, 72 and 79.

[0019] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises the following group of light chain variable regions, whose sequences are shown in SEQ ID NOs: 13, 31, 49, 74 and 81.

[0020] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof is a whole antibody, a bispecific antibody, a scFv, a Fab, a Fab', a F(ab')2 or a Fv.

[0021] In certain embodiments, when the anti-OX40 antibody or antigen-binding fragment thereof is scFv, a connecting peptide may be contained between the heavy chain and light chain variable regions.

[0022] In certain embodiments, the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof is selected from the group consisting of IgG, IgM, IgE, IgD and IgA.

[0023] In certain embodiments, the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof is selected from IgG1 or IgG4.

[0024] In certain embodiments, the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof is selected from IgG1, and the heavy chain constant region of the IgG1 comprises mutations of S239D, I332E and / or A330L.

[0025] In certain embodiments, the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 82-84.

[0026] In certain embodiments, the light chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof is κ or λ.

[0027] In certain embodiments, the light chain constant region of the anti-OX40 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:85.

[0028] In certain embodiments, the drug comprises a nucleic acid molecule encoding an anti-OX40 antibody or antigen-binding fragment thereof as described in any one of the present application.

[0029] In certain embodiments, the drug comprises a vector comprising the nucleic acid molecule.

[0030] In certain embodiments, the medicament comprises a cell comprising the nucleic acid molecule or the vector.

[0031] In certain embodiments, the drug comprises a conjugate comprising the anti-OX40 antibody or antigen-binding fragment thereof.

[0032] In certain embodiments, the drug comprises a pharmaceutical composition, which comprises the anti-OX40 antibody or antigen-binding fragment thereof described in any one of the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application and / or the conjugate described in the present application, and a pharmaceutically acceptable carrier.

[0033] In certain embodiments, the autoimmune disease comprises an OX40-mediated autoimmune disease.

[0034] In certain embodiments, the autoimmune disease comprises a T cell-mediated autoimmune disease.

[0035] In certain embodiments, the autoimmune disease comprises graft-versus-host disease.

[0036] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

[0038] Figure 1 This is the result of the binding of purified anti-hOX40scFv to hOX40.

[0039] Among them, the x-axis represents the fluorescence intensity of EGFP, and the Y-axis represents the fluorescence intensity of anti-hlg-PE;

[0040] Figure 2 It is a specific test for anti-OX40 antibodies.

[0041] Among them, the x-axis represents the fluorescence intensity of EGFP, and the Y-axis represents the fluorescence intensity of anti-hlg-PE;

[0042] Figure 3 This is the staining result of yeast monoclonal with improved affinity;

[0043] Figure 4 This is a graph showing the binding ability of three antibodies to hOX40 detected by ELISA;

[0044] Figure 5 It is a graph showing the binding ability of different heavy chain mutants after affinity maturation to hOX40 detected by ELISA;

[0045] Figure 6 This is a graph showing the ability of the H96 heavy chain mutant and different light chain mutants to bind to hOX40 after affinity maturation using ELISA;

[0046] Figure 7 This is a graph showing the test results of the in vitro agonist activity of the anti-hOX40 antibody;

[0047] Figure 8 This is a comparison chart of the in vitro agonist activity of the three IgG subtypes of H96-L80;

[0048] Fig. 9 This is a test chart of the ability of anti-OX40 antibody to bind to human and rhesus monkey CD4+T and CD8+T cells;

[0049] Fig.10 This is a graph showing the results of the O21 scFv antibody agonist activity test;

[0050] Fig.11 These are the results of the agonist activity test of two forms, O21 scFv and IgG;

[0051] Fig.12 The effects of the two subtypes of H96-L80, IgG1 and IgG4, on T cell subtypes in vitro;

[0052] Fig.13 This is a graph showing the results of the inhibitory effect of O21 mAb on tumor (PC-3) growth in vivo;

[0053] Fig.14 This is a graph showing the results of the inhibitory effect of O21 mAb on tumor (A375) growth in vivo;

[0054] Fig.15 This is the result of the 45℃ accelerated stability test to evaluate the stability of anti-OX40 antibody.

[0055] Among them, A is the concentration test, B is the monomer content test, and C is the test of in vitro activity by the NF-κb system;

[0056] Fig.16 These are the results of pharmacokinetic testing of the H96-L80 antibody.

[0057] Fig.17This is the test result that the anti-hOX40 antibody has an enhanced ADCC effect.

[0058] Fig.18 This is the grouping of the antibodies described in this application in the in vivo mouse testing experiment.

[0059] Figure 19 is a graph showing the therapeutic effect of the antibody described in this application on PBMC-induced M-NSG-hIL15 mouse GVHD. After treatment with the antibody described in this application, Fig.19A It showed that the proportion of hCD4 in mice was significantly decreased compared with the Abacept group; Fig.19B The survival time of mice was significantly prolonged compared with that of the PBS group; Fig.19C It is an evaluation of the degree of GVHD in mice. DETAILED DESCRIPTION

[0060] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0061] Definition of terms

[0062] In this application, the term "OX40" generally refers to a tumor necrosis factor family protein, also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4), ACT35, IMD16, TXGP1L and CD 134. OX40 refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. OX40 is a type I transmembrane glycoprotein that is primarily expressed on activated CD4+ T cells and CD8+ T cells. For example, the accession number of human OX40 in Uniprot may be P43489.

[0063] In this application, the term "OX40L" generally refers to the ligand of OX40, also known as ACT-4 receptor, CD134L, CD252 and Gp34. OX40L is a type II transmembrane glycoprotein expressed in activated antigen presenting cells, such as dendritic cells, B cells, etc. For example, the accession number of human OX40L in Uniprot may be P23510. OX40 / OX40L signaling plays a very important role in the activation, proliferation and inhibition of apoptosis of T cells.

[0064] In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to the target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In certain embodiments, specific binding may include but does not require exclusive binding.

[0065] In this application, the term "CDR", also known as "complementarity determining region", generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structurally defined loop. Typically, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camel antibodies consisting only of heavy chains can function normally and stably in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct.Biol.3:733-736 (1996). Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, Kabat / Chothia, etc. These coding systems are known in the art, and specific references include, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnumFor example, the amino acid sequence of the antigen binding protein can be numbered according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; http: / / imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310; Lefranc et al., 2005, Dev Comp Immunol 29:185-203). For example, the CDR of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The boundaries of the CDR of the variable region of the same antibody obtained based on different coding systems may be different. That is, the CDR sequences of the variable region of the same antibody defined under different coding systems are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers such antibodies, whose variable region sequences contain the specific CDR sequences, but due to the application of different schemes (such as different coding systems), the CDR boundaries claimed are different from the specific CDR boundaries defined in the present invention. The CDR of the antibody of the present invention can be manually evaluated and determined according to any scheme or combination thereof in the art. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

[0066] In this application, the term "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy chain and / or light chain. The variable domains of the heavy chain and light chain may be referred to as "V H ” and “V L” (or “VH” and “VL”, respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.

[0067] In this application, the term "variable" generally refers to certain segments of the variable domain between antibodies that may have large differences in sequence. The variable domain mediates antigen binding and determines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domain. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light chain and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, most of which adopt a β-folded configuration, connected by three CDRs, which form a ring connection and form a part of the β-folded structure in some cases. The CDRs in each chain are kept together in close proximity by the FR region, and the CDRs from the other chain together promote the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0068] In the present application, the term "antibody" generally refers to an immunoglobulin or its fragment or derivative thereof, covering any polypeptide including an antigen binding site, whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybridized, mutated and transplanted antibodies. Unless otherwise modified by the term "complete", as in "complete antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function (e.g., specifically binds OX40). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units with an additional polypeptide called J chain and contain 10 antigen binding sites, while IgA antibodies include 2-5 basic 4-chain units that can be combined with J chains to form multivalent combinations. For IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be divided into one of two distinct types based on the amino acid sequence of its constant domain, called κ and λ. According to the amino acid sequence of the heavy chain (CH) constant domain, immunoglobulins can be divided into different classes or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains named α, δ, ε, γ and μ, respectively.

[0069] In the present application, the term "antigen binding fragment" generally refers to one or more fragments that have the ability to specifically bind to an antigen (e.g., OX40). In the present application, the antigen binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0070] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, a whole antibody can be digested with papain. After papain digestion of an antibody, two identical antigen-binding fragments are produced, namely, a "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as above). The Fab fragment can be composed of a complete L chain with the variable region of a heavy chain and the H chain (V H )'s first constant region (C H 1) Composition.

[0071] In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0072] In the present application, the term "F(ab')2" generally refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F(ab')2 fragment contains two Fab fragments held together by a disulfide bond and a portion of the hinge region. The F(ab')2 fragment has a divalent antigen binding activity and is capable of cross-linking antigens.

[0073] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the heavy chain variable region and the light chain variable region, and lacking the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, an Fv fragment includes all or part of the amino terminal variable regions of about 110 amino acids of the heavy chain and the light chain.

[0074] In the present application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment including a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker), and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used in the present application, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0075] In the present application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, with reference to, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and with reference to, for example, WO 06 / 030220, WO 06 / 003388, and other published patent applications of Domantis Ltd. The term "dAb" generally includes sdAb. The term "sdAb" generally refers to a single-domain antibody. A single-domain antibody generally refers to an antibody fragment consisting only of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL).

[0076] In the present application, the term "VHH" generally refers to the variable antigen binding domains of heavy chain antibodies from Camelidae (camel, dromedary, llama, alpaca, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH may also be referred to as Nanobody (Nb).

[0077] In the present application, the term "monoclonal antibody" generally refers to an antibody molecule preparation of a single molecule composition. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (generally having different antibodies for different determinants), each monoclonal antibody is for a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins. The modifier "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody population, and is not to be construed as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibodies used in the present application can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.

[0078] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to induce an adverse immune response in a human individual than the parent (e.g., alpaca-derived) antibody.

[0079] In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., an alpaca antibody) are replaced by corresponding amino acids derived from human immunoglobulins. In the CDR region, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of a human immunoglobulin constant region. "Humanized antibodies" retain antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., alpaca) antibody may minimally contain a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as alpaca, mouse, rat, rabbit or non-human primate) having desired properties, affinity and / or ability. In some cases, FR region residues of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

[0080] In this application, the term "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. If it is produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain mouse sugar chains. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be generated in humans and in transgenic animals with human immunoglobulin germline sequences by phage display or other molecular biological methods. Exemplary techniques that can be used to make antibodies are described in U.S. Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as the use of libraries, are known in the art.

[0081] In the present application, "antibody-dependent cell-mediated cytotoxicity (ADCC)" generally refers to an immune mechanism that causes antibody-coated target cell lysis by immune effector cells. Target cells are cells to which antibodies or fragments thereof that contain an Fc region specifically bind, and the binding is generally via the protein portion of the N-terminus of the Fc region. As used herein, the term "improved / enhanced ADCC" is defined as an increase in the number of target cells lysed within a given time with a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and / or by the ADCC mechanism, a reduction in the antibody concentration in the medium surrounding the target cells required for the lysis of a given number of target cells within a given time.

[0082] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.

[0083] In the present application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries are expressed in the host cell. For example, the vector may include: plasmid; phagemid; cosmid; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. The types of animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. A vector may also include components that assist it in entering cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0084] In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original parent cell (in the form of total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described in the present application. Cells may be bacterial cells (e.g., Escherichia coli), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells.

[0085] In the present application, the term "conjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linked via a linker molecule) other agents (e.g., chemotherapeutic agents, radioactive elements, cytostatic agents and cytotoxic agents) to the antibody or its antigen-binding fragment, which can deliver the other agents to target cells (e.g., tumor cells) through the antibody or its antigen-binding fragment specifically binding to antigens on target cells.

[0086] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may include an antibody or antigen binding fragment described herein, a nucleic acid molecule described herein, a carrier described herein, and / or a cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also include a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable ingredients of the composition are preferably nontoxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen, and lyophilized compositions.

[0087] In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and / or nonionic surfactants.

[0088] In the present application, the term "autoimmune disease" generally refers to a disease caused by an immune response. Autoimmune diseases can include diseases caused by immune responses produced against any part, organ or substance in the body itself. For example, autoimmune diseases can include diseases caused by immune responses produced against the body's own tissues, organs, cells and / or cell components. For example, autoimmune diseases can include diseases caused by immune responses produced against any part, organ, substance in the body and / or grafts transplanted or imported into the body. For example, autoimmune diseases can include diseases caused by immune responses produced by any part, organ, substance in the body and / or grafts transplanted or imported into the body. For example, autoimmune diseases can include diseases caused by immune responses produced by any part, organ, substance in the body and / or grafts transplanted or imported into the body. For example, the immune response in an autoimmune disease can include inappropriate production and / or excessive production of antibodies. For example, the immune response in an autoimmune disease can include inappropriate immune cell activation levels. Autoimmune diseases are systemic diseases that can occur in almost all parts of the body (including the nervous system, gastrointestinal system, endocrine system, skin, skeletal system and vascular tissue). For example, autoimmune diseases can include immune inflammatory responses. For example, autoimmune diseases can include graft-versus-host disease (GVHD). For example, autoimmune diseases can include, but are not limited to, graft-versus-host disease (GVHD), transplant rejection, rheumatoid arthritis, autoimmune uveitis, multiple sclerosis, lupus (e.g., systemic lupus erythematosus), scleroderma, hidradenitis, diabetes, Crohn's disease, inflammatory bowel disease, celiac disease, psoriasis, proliferative lupus nephritis, granulomatous myopathy, and polymyositis, among others.

[0089] In the present application, the term "graft-versus-host disease (GVHD)" generally refers to the symptoms and / or diseases caused by immune cells from the donor (graft) recognizing the patient's normal cells and tissues (host) as foreign and attacking them. For example, GVHD can be classified according to the time of its occurrence. For example, GVHD can be classified according to pathological differences. For example, GVHD can be manifested clinically as 3 syndromes: acute GVHD (acute GVHD, aGVHD), chronic GVHD (chronic GVHD, cGVHD) and GVHD overlap syndrome.

[0090] In this application, the terms "patient", "subject", "individual" and the like are used interchangeably herein and include humans and other animals. For example, including mammals. For example, including humans.

[0091] In the present application, the term "prevention and / or treatment" includes not only the prevention and / or treatment of a disease, but also generally includes preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the treatment of patients.

[0092] In this application, the term "alleviate" generally refers to reducing, reducing or delaying a certain symptom, disease, condition or phenotype. The symptom, disease, condition or phenotype may include subjective perception of the subject such as pain, dizziness or other physiological disorders, or medically detectable indications, such as lesions detected by medical examination means, for example, abnormal B cell and / or plasma cell activity, autoantibody expression level, platelet count, bleeding.

[0093] In this application, the terms "patient", "subject", "body" can be used interchangeably, including human and other mammalian subjects receiving preventive or therapeutic treatment. In this application, the term "patient suffering from..." generally refers to a patient showing clinical signs of a disease.

[0094] In this application, the terms "administer" and "administer" are used interchangeably and generally refer to the act of applying a substance to a subject by a method described herein or otherwise known in the art. Any method known to those skilled in the art for contacting cells, organs or tissues with the substance can be used. For example, the dosage form for administration includes injections, tablets, capsules, oral liquids, granules, ointments, suppositories or aerosols. For example, the mode of administration includes aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. For example, administration includes intravenous injection, intraperitoneal injection, subcutaneous injection or intramuscular injection.

[0095] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs having the same or similar functions as the proteins or polypeptides.

[0096] In the present application, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of a drug that promotes disease regression (which is demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of the disease asymptomatic period, or the prevention of damage or disability caused by suffering from the disease) when used alone or in combination with another therapeutic agent. A "preventive effective amount" or "preventive effective dose" of a drug generally refers to an amount of a drug that inhibits the development or recurrence of a disease when administered alone or in combination with another therapeutic agent to a subject at risk of disease development or disease recurrence. The ability of a therapeutic agent or preventive agent to promote disease regression or inhibit disease development or recurrence can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting efficacy to humans, or by measuring the activity of a medicament in an in vitro assay.

[0097] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to OX40). For example, the functional variant may comprise a protein or polypeptide that has been subjected to amino acid changes by at least 1, for example, 1-30, 1-20 or 1-10, for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0098] In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to OX40).

[0099] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences. " sequence homology percentage ratio " can be calculated in the following manner: two sequences to be compared are compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number of positions in two sequences to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce sequence homology percentage ratio.Comparison carried out in order to determine the sequence homology percentage ratio can be realized in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and DJ. Lipman, "Improved tools for biological sequence comparisons," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and DJ. Lipman and W. R. Earson, "Rapid and sensitive protein similarity searches," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers and D. Lipman, "A basic local alignment search tool," Journal of Molecular Biology, 215: 403-410, 1990.

[0100] In this application, the term "comprising" generally means including, encompassing, containing or encompassing. In some cases, it also means "for", "consisting of...".

[0101] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. DETAILED DESCRIPTION OF THE INVENTION

[0103] Antibodies or antigen-binding fragments

[0104] In one aspect, the present application provides a use of an anti-OX40 or an antigen-binding portion thereof in the preparation of a medicament for treating an OX40-mediated disease. For example, the disease may include an autoimmune disease.

[0105] The present invention uses yeast display technology to obtain an anti-OX40 antibody with good specificity, high affinity and stability through screening and further affinity maturation. The antibody can specifically bind to human OX40, and can enhance the activation of T cells by binding to activated T cells, and has a significant inhibitory effect on tumor growth. In addition, the anti-OX40 antibody of the present invention is a fully human antibody, which has lower immunogenicity than traditional mouse-derived, chimeric antibodies and humanized antibodies, reduces the patient's rejection reaction, and has better drug-making ability.

[0106] The anti-OX40 antibody or antigen-binding portion thereof described in the present application comprises a CDR region selected from the following group:

[0107] 1) The sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 5-7, respectively, and the sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 14-16, respectively, or the heavy chain variable region and the light chain variable region satisfy at least one of the following two sequences compared with the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity of greater than 70%, 80%, 85%, 90%, or 97%;

[0108] 2) the sequences of its heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 23-25, respectively, and the sequences of its light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 32-34, respectively, or the heavy chain variable region and the light chain variable region satisfy at least one of the following two sequences compared with the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity of greater than 70%, 80%, 85%, 90%, or 97%;

[0109] 3) The sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 41-43, respectively, and the sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 50-52, respectively, or the heavy chain variable region and the light chain variable region satisfy at least one of the following two sequences compared with the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity of greater than 70%, 80%, 85%, 90%, or 97%;

[0110] 4) The sequences of its heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 63, 42, and 43, respectively, and the sequences of its light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 50-52, respectively, or the heavy chain variable region and the light chain variable region satisfy at least one of the following two sequences compared with the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity of greater than 70%, 80%, 85%, 90%, or 97%;

[0111] 5) The sequences of its heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 41, 67, and 43, respectively, and the sequences of its light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 50-52, respectively, or the heavy chain variable region and the light chain variable region satisfy at least one of the following two sequences compared with the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity of greater than 70%, 80%, 85%, 90%, or 97%;

[0112] 6) The sequences of its heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 41, 42, and 70, respectively, and the sequences of its light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 50-52, respectively, or the heavy chain variable region and the light chain variable region satisfy at least one of the following two sequences compared with the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity of greater than 70%, 80%, 85%, 90%, or 97%;

[0113] 7) The sequences of its heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 63, 67, and 70, respectively, and the sequences of its light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 75-77, respectively, or the heavy chain variable region and light chain variable region satisfy at least one of the following two sequences compared with the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity of greater than 70%, 80%, 85%, 90%, or 97%;

[0114] 8) The sequences of its heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 41, 42, and 70, respectively, and the sequences of its light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 75-77, respectively, or the heavy chain variable region and light chain variable region satisfy at least one of the following two sequences compared with the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity of greater than 70%, 80%, 85%, 90%, or 97%.

[0115] For example, the anti-OX40 antibody or antigen-binding portion thereof described in the present application further comprises a heavy chain variable region framework region selected from the following group:

[0116] 1) The sequences of the heavy chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 8-11, respectively;

[0117] 2) the sequences of the heavy chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 26-29, respectively;

[0118] 3) The sequences of the heavy chain variable regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 44-47, respectively.

[0119] For example, the anti-OX40 antibody or antigen-binding portion thereof described in the present application further comprises a light chain variable region framework region selected from the following group:

[0120] 1) The sequences of the light chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 17-20, respectively;

[0121] 2) the sequences of the light chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 35-38, respectively;

[0122] 3) The sequences of the light chain variable regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 53-56, respectively.

[0123] For example, the anti-OX40 antibody or antigen-binding portion thereof described in the present application comprises a heavy chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 4, 22 and 40, or a sequence whose heavy chain variable region satisfies at least one of the following two compared with the aforementioned sequence: a) binding to the same antigenic epitope, b) having an identity of greater than 70%, 80%, 85%, 90% or 97%.

[0124] Further, it includes a heavy chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 4, 22 and 40, or a sequence whose heavy chain variable region CDR region satisfies at least one of the following two compared with the aforementioned sequence: a) binding to the same antigenic epitope; b) the number of mutated amino acids does not exceed 3.

[0125] Further, it comprises a heavy chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 4, 22, 40, 62, 66, 69, 72 and 79.

[0126] For example, the anti-OX40 antibody or antigen-binding portion thereof described in the present application comprises a light chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 13, 31 and 49, or a sequence whose light chain variable region CDR region satisfies at least one of the following two compared with the aforementioned sequence: a) binds to the same antigen epitope, b) has an identity of greater than 70%, 80%, 85%, 90% or 97%.

[0127] Further, it includes a light chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 13, 31 and 49, or a sequence whose light chain variable region satisfies at least one of the following two compared with the aforementioned sequence: a) binding to the same antigenic epitope; b) the number of mutated amino acids does not exceed 3.

[0128] Further, it comprises a light chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 13, 31, 49, 74 and 81.

[0129] The anti-OX40 antibody or antigen-binding portion thereof described in the present application may be a whole antibody, a bispecific antibody, scFv, Fab, Fab', F(ab')2 or Fv. For example, when it is scFv, it may contain a connecting peptide between the heavy chain and light chain variable regions, and the sequence of the connecting peptide is shown in SEQ ID NO 1.

[0130] The anti-OX40 antibody or antigen-binding portion thereof described in the present application, its heavy chain constant region can be selected from IgG, IgM, IgE, IgD and IgA. For example, its heavy chain constant region is selected from IgG1, IgG2, IgG3 and IgG4. For example, its heavy chain constant region is IgG1 or IgG4. For example, its heavy chain constant region is selected from IgG1, and the heavy chain constant region of IgG1 comprises mutations of S239D, I332E and / or A330L. For example, the heavy chain constant region of the antibody is selected from IgG1, and the heavy chain constant region of IgG1 comprises mutations of S239D and I332E. For example, the heavy chain constant region of the antibody is selected from IgG1, and the heavy chain constant region of IgG1 comprises mutations of S239D, I332E and A330L. For example, the heavy chain constant region of the antibody can be selected from the amino acid sequence shown in any one of SEQ ID NOs: 82-84.

[0131] The anti-OX40 antibody or antigen-binding portion thereof described in the present application may have a light chain constant region of κ or λ. For example, the light chain constant region thereof comprises the amino acid sequence shown in SEQ ID NO: 85.

[0132] The CDR of an antibody is also called the complementary determining region, which is a part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. The antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, Kabat / Chothia, etc. These coding systems are known in the art, and specific references can be made to, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In the present application, the CDR covers the CDR sequence obtained by any CDR division method; it also covers its variants, which include the amino acid sequence of the CDR being substituted, deleted and / or added with one or more amino acids. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the CDR.

[0133] In addition, it should be noted that the antibody or antigen-binding fragment described in the present application may include a heavy chain and / or light chain sequence with one or more conservative sequence modifications. The so-called "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibody or antigen-binding fragment described in the present application by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are amino acid residues that are replaced with amino acid residues with similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR region of the antibody or antigen-binding fragment described herein can be replaced with other amino acid residues of the same side chain group. Those skilled in the art know that some conservative sequence modifications will not eliminate antigen binding. For details, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0134] The anti-OX40 antibodies or antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art. For example, the antigen binding activity of the antigen binding protein or fusion protein of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0135] In the present application, the antibody or antigen-binding fragment can specifically bind to OX40. In certain embodiments, the binding of the antibody or antigen-binding fragment to OX40 can be detected by an ELISA method. For example, the antigen-binding protein described in the present application can be less than or equal to about 0.010 μg / mL, less than or equal to about 0.009 μg / mL, less than or equal to about 0.008 μg / mL, less than or equal to about 0.007 μg / mL, less than or equal to about 0.006 μg / mL, less than or equal to about 0.005 μg / mL, less than or equal to about 0.004 μg / mL, less than or equal to about 0.003 μg / mL, less than or equal to about 0.002 μg / mL and less than or equal to about 0.001 μg / mL EC 50 Value combined with OX40.

[0136] In the present application, the antibody or antigen has an enhanced ADCC effect. For example, the enhanced ADCC effect may include mutating the Fc of the antibody. For example, the enhanced ADCC effect may include mutating the heavy chain constant region of the antibody. For example, the heavy chain constant region of the antibody includes mutations of S239D, I332E and / or A330L, which has an enhanced ADCC effect.

[0137] Nucleic acid molecules, vectors, cells, conjugates and pharmaceutical compositions

[0138] In another aspect, the present application provides an isolated nucleic acid molecule for use in preparing a medicament for treating an OX40-mediated disease, for example, an autoimmune disease.

[0139] The nucleic acid molecule described in the present application may encode the anti-OX40 antibody or antigen-binding fragment thereof described in the present application.

[0140] The nucleic acid molecule described in the present application may comprise a nucleic acid sequence capable of encoding the heavy chain variable region of the antibody described in the present application, wherein the heavy chain variable region comprises an amino acid sequence selected from the following group:

[0141] (1) SEQ ID NO: 5-7;

[0142] (2) SEQ ID NO: 23-25;

[0143] (3) SEQ ID NO: 41-43;

[0144] (4) A sequence that satisfies at least one of the following two conditions compared to the sequences in (1) to (3): a) binds to the same antigenic epitope; b) has an identity greater than 70%, 80%, 85%, 90% or 97%.

[0145] Furthermore, the heavy chain variable region comprises an amino acid sequence selected from the following group:

[0146] SEQ ID NO: 4, 22 and 40, or a sequence that satisfies at least one of the following two conditions compared to the aforementioned sequences: a) binds to the same antigenic epitope, b) has an identity of greater than 70%, 80%, 85%, 90% or 97%.

[0147] Further, it includes a heavy chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 4, 22 and 40, or a sequence whose heavy chain variable region satisfies at least one of the following two compared with the aforementioned sequence: a) binding to the same antigenic epitope; b) the number of mutated amino acids does not exceed 3.

[0148] Further, it comprises a heavy chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 4, 22, 40, 62, 66, 69, 72 and 79.

[0149] The nucleic acid molecule described in the present application further comprises a nucleic acid sequence capable of encoding a heavy chain constant region of an antibody; the heavy chain constant region is selected from IgG, IgM, IgE, IgD and IgA. For example, its heavy chain constant region is selected from IgG1, IgG2, IgG3 and IgG4. For example, its heavy chain constant region is IgG1 or IgG4. For example, its heavy chain constant region is selected from IgG1, and the heavy chain constant region of the IgG1 comprises mutations of S239D, I332E and / or A330L.

[0150] The nucleic acid molecule described in the present application may comprise a nucleic acid sequence capable of encoding an antibody light chain variable region, wherein the light chain variable region comprises an amino acid sequence selected from the following group:

[0151] (1) SEQ ID NO: 14-16;

[0152] (2) SEQ ID NO: 32-34;

[0153] (3) SEQ ID NO: 50-52;

[0154] (4) A sequence that satisfies at least one of the following two conditions compared to the sequences in (1) to (3): a) binds to the same antigenic epitope; b) has an identity greater than 70%, 80%, 85%, 90% or 97%.

[0155] Furthermore, the heavy chain variable region comprises an amino acid sequence selected from the following group:

[0156] SEQ ID NO: 13, 31 and 49, or a sequence that satisfies at least one of the following two conditions compared to the aforementioned sequences: a) binds to the same antigenic epitope; b) has an identity of greater than 70%, 80%, 85%, 90% or 97%.

[0157] Further, it includes a light chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 13, 31 and 49, or a sequence whose light chain variable region satisfies at least one of the following two compared with the aforementioned sequence: a) binding to the same antigenic epitope; b) the number of mutated amino acids does not exceed 3.

[0158] Further, it comprises a light chain variable region selected from the following group, whose sequence is shown in SEQ ID NO: 13, 31, 49, 74 and 81.

[0159] The nucleic acid molecule in the present application also comprises a nucleic acid sequence capable of encoding an antibody light chain constant region. For example, the light chain constant region is of κ type or λ type.

[0160] For example, the nucleic acid molecules described herein can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification; (ii) produced by cloning recombination; (iii) purified, such as by enzyme cleavage and gel electrophoresis fractionation; or (iv) synthesized, such as by chemical synthesis.

[0161] In another aspect, the present application provides a vector, which may contain the nucleic acid molecule described in the present application, and its use in preparing a drug for treating an OX40-mediated disease, for example, an autoimmune disease.

[0162] In addition, other genes may also be included in the vector, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector may also include expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art, and for example, may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The vector may be transformed, transduced, or transfected with a host cell so that the genetic material elements it carries are expressed in the host cell. The vector may include, for example, plasmids, cosmids, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may also include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not only these substances.

[0163] On the other hand, the application provides a kind of cell, it can comprise the nucleic acid molecule described in the application or the vector described in the application.In some embodiments, each or each host cell can comprise one or a nucleic acid molecule or vector described in the application.In some embodiments, each or each host cell can comprise multiple (for example, 2 or more) or multiple (for example, 2 or more) nucleic acid molecules or vectors described in the application.For example, the vector described in the application can be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi or yeast cells, etc.In some embodiments, the cell can be a bacterial cell (for example, Escherichia coli), a yeast cell or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NS0 cells or myeloma cells.The vector described in the application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0164] On the other hand, the present application also provides a conjugate, which may include the antibody or antigen-binding fragment described in the present application. For example, the conjugate may include the anti-OX40 antibody or antigen-binding portion thereof described in the present invention, and other biologically active substances, wherein the anti-OX40 antibody or antigen-binding portion thereof is coupled to the other biologically active substance directly or through a linker fragment.

[0165] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the antibody or antigen-binding fragment described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application and / or the conjugate described in the present application, and optionally a pharmaceutically acceptable carrier.

[0166] In certain embodiments, the pharmaceutical composition may also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0167] Methods and uses

[0168] On the other hand, the present application provides the use of the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease and / or condition.

[0169] On the other hand, the present application also provides a method for preventing and / or treating a disease and / or condition, which may include administering the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition described in the present application to a subject in need.

[0170] In the present application, the use can be carried out by different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0171] On the other hand, the present application provides use of the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition in preventing and / or treating diseases and / or disorders.

[0172] On the other hand, the present application provides use of the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition in the preparation of a drug for regulating an immune response.

[0173] On the other hand, the present application provides use of the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition in regulating an immune response.

[0174] On the other hand, the present application also provides a method for regulating an immune response, which may include administering the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition described in the present application to a subject in need.

[0175] The anti-OX40 antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition described in the present application can regulate an immune response. For example, the regulating immune response can include upregulating the level of the immune response. For example, the regulating immune response can include downregulating the level of the immune response. For example, the immune response includes an inflammatory response. For example, the immune response includes increased expression of MHC antigens. For example, the immune response includes the production and / or secretion of cytokines. For example, the immune response includes the activation of immune cells. For example, the immune response includes the proliferation and / or differentiation of immune cells. For example, the immune response includes the migration of immune cells. For example, the immune response includes the production and / or secretion of chemokines. For example, the immune response includes the production and / or secretion of antibodies.

[0176] On the other hand, the present application also provides a method for reducing transplant rejection in transplant patients while maintaining other immune responses, the method comprising administering an effective amount of the anti-OX40 antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition described in the present application to a patient in need thereof.

[0177] Subjects and Indications

[0178] On the other hand, the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate and / or the pharmaceutical composition described in the present application can be used to prevent and / or treat diseases and / or disorders.

[0179] In the present application, the disease and / or condition may include OX40-mediated diseases and / or conditions. In the present application, the disease and / or condition may include diseases and / or conditions associated with OX40 expression. In the present application, the disease and / or condition may include diseases and / or conditions associated with OX40 expression on T cells.

[0180] In the present application, the disease and / or condition may include an autoimmune disease. In the present application, the disease and / or condition may include an autoimmune disease mediated by OX40. In the present application, the disease and / or condition may include an autoimmune disease associated with OX40 expression. In the present application, the disease and / or condition may include an autoimmune disease associated with OX40 expression on T cells.

[0181] In the present application, the autoimmune disease may include, but is not limited to, graft-versus-host disease (GVHD), rheumatoid arthritis, autoimmune uveitis, multiple sclerosis, lupus (e.g., systemic lupus erythematosus), scleroderma, hidradenitis, and ulcerative colitis.

[0182] Without intending to be bound by any theory, the following examples are merely intended to illustrate the preparation method and use of the anti-OX40 antibody or antigen-binding fragment thereof of the present application, and are not intended to limit the scope of the present invention.

[0183] Example

[0184] Example 1 Expression of recombinant human OX40 and preparation of related EGFP cells

[0185] S1.1. Based on the amino acid sequence of human OX40 (P43489) on the protein database Uniprot, the amino acid sequence of the extracellular domain of human OX40 (hOX40) (i.e., residues 1 to 216 in P43489) was obtained;

[0186] S1.2. According to the amino acid sequence of the constant region of human immunoglobulin gamma1 (IgG1) (P01857) on the protein database Uniprot, the domain amino acid sequence of human IgG1-Fc (hFc) (i.e., residues 104 to 330 in P01857) was obtained;

[0187] S1.3. According to the constant region amino acid sequence of mouse immunoglobulin gamma1 (IgG1) on the protein database Uniprot (P01868), the domain amino acid sequence of mouse IgG1-Fc (muFc) (i.e., residues 98 to 324 in P01868) was obtained;

[0188] S1.4. The DNA fragments in steps S1.1 to S1.3 were obtained by artificial synthesis. The synthesized gene sequences were subcloned into the commercial vector pcDNA4 / myc-HisA (Invitrogen, V863-20) by HindIII and EcoRI of Fermentas Company, and the accuracy of the constructed plasmids was verified by sequencing to obtain recombinant plasmid DNA, namely: pcDNA4-hOX40-hFc, pcDNA4-hOX40-muFc;

[0189] S1.5, according to the information on the protein database Uniprot, the amino acid sequence of enhanced green fluorescent protein EGFP (C5MKY7), the amino acid sequence of human OX40 (P43489), the amino acid sequence of mouse OX40 (P47741), the amino acid sequence of human CD137 (Q07011), and the amino acid sequence of human CD27 (P26842) were obtained;

[0190] S1.6. The DNA fragment in step S1.5 was obtained by artificial synthesis. The synthesized gene sequences were double-digested with HindIII and EcoRI of Fermentas and subcloned into the commercial vector pcDNA4 / myc-HisA (Invitrogen, V863-20). The accuracy of the constructed plasmid was verified by sequencing to obtain recombinant plasmid DNA, namely: pcDNA4-hOX40-EGFP, pcDNA4-hCD137-EGFP, pcDNA4-mOX40-EGFP and pcDNA4-hCD27-EGFP.

[0191] S1.7, transfect the EGFP recombinant plasmid in step S1.6 into HEK293 (ATCC, CRL-1573 TM ) cells, and the expression of hOX40, hCD137, mOX40, and hCD27 was confirmed by fluorescence activated chromatin separation (FACS) 48 h after transfection.

[0192] S1.8, transiently transfect pcDNA4-hOX40-Fc and pcDNA4-hOX40-muFc into HEK293 cells for protein production. Dilute the recombinant expression plasmid with Freestyle293 medium and add the PEI (Polyethylenimine) solution required for transformation. Add each group of plasmid / PEI mixture to the cell suspension and culture at 37°C, 10% CO2, 90rpm; after 5-6 days of culture, collect the transient expression culture supernatant, and use Protein A affinity chromatography to preliminarily purify hOX40-Fc and hOX40-muFc protein samples for the following examples. The obtained protein samples were preliminarily detected by SDS-PAGE, and the target bands could be clearly seen.

[0193] Example 2: Screening of anti-hOX40 antibodies from yeast display libraries, cloning, expression and identification

[0194] 2.1 Methods

[0195] Yeast display technology was used to screen fully human antibodies against human OX40. The VH and VL genes in the IgM and IgG cDNAs from PBMCs of 150 healthy individuals were cloned into a specific vector to construct a scFV yeast display library (the connecting sequence between VH and VL is a GGGGSGGGGSGGGGS (SEQ ID NO: 1) connecting peptide) with a library capacity of 5×10 8 . The yeast library with 10 times the storage capacity was revived, and the yeast surface was induced to express antibodies. It was enriched twice by magnetic bead sorting with 100nM biotinylated hOX40 antigen, and then enriched twice by flow sorting with biotinylated hOX40. The obtained yeast was plated and single clones were picked. After amplification and induction of expression, the monoclonal yeast was stained and analyzed with anti-myc antibody and biotinylated hOX40 or control antigen hCD137. The yeast with antigen positive / control yeast negative was positive yeast.

[0196] The yeast clones confirmed by FACS were subjected to yeast colony PCR and sequencing, and the PCR primers were: sequence-F: CGTAGAATCGAGACCGAGGAGA (SEQ ID NO: 2); sequence-R: CTGGTGGTGGTGGTTCTGCTAGC (SEQ ID NO: 3); the sequencing primer was sequence-R. After the sequencing results were obtained, the sequences were compared and analyzed using BioEdit software.

[0197] The single-chain antibody scFv gene obtained above was fused with the human IgG1-Fc gene mentioned above and cloned into the commercial vector pcDNA4 / myc-HisA by Fermentas after double digestion with HindIII and EcoRI. Cloning and plasmid mini-extraction were performed according to the standard operation of molecular cloning. The extracted plasmid was transiently expressed in HEK293 cells and purified by protein A column.

[0198] Take hOX40-EGFP cells, resuspend them in 0.5% PBS-BSA Buffer, add 2μg of the purified anti-hOX40scFv antibody, and set up relevant controls. The negative control is 2μg of hIgG1 protein. The secondary antibody is eBioscience's anti-hIg-PE. After staining, flow cytometry is used for detection. This method is used to identify antibodies that can bind to the hOX40 antigen on the cell surface.

[0199] After screening and identification, three antibodies with better characteristics were obtained, namely O3scFv, O19scFv, and O21scFv. Figure 1 As shown, the three anti-hOX40 antibodies can bind to cell surface hOX40, while the negative control cannot bind to cell surface hOX40. The antibody heavy chain and light chain variable regions contain a connecting peptide sequence GGGGSGGGGSGGGGS (SEQ ID NO: 1).

[0200] 2.2 Sequence

[0201] 2.2.1O3 scFv heavy chain variable region amino acid sequence:

[0202] QVQLQQWGAGLLKPSETLSLTCGFNGEYFT DYNAMIC WVRQPPGEALEWLA LIYWDDDERYSPSLKN RLIITKDISKNQVVLTMTHMEPADTGTYYCAR WGGSLMNAFDV WGPGTMVTVSS (SEQ ID NO: 4)

[0203] The underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 5-7; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 8-11;

[0204] The corresponding DNA sequence is:

[0205] CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGGTTTCAATGGAGAATACTTCACTGATTACTTCTGGACCTGGGTCCGGCAGCCCCCCGGAGAGGCCCTGGAGTGGCTTGCACTCATTTATTGGGATGATGATGAGCGCTACAGC CCATCTCTGAAGAACAGACTCATCATCACCAGGACATTTCCAAAAACCAGGTGGTCCTTACAATGACCCACATGGAGCCTGCGGACACAGGCACCTATTACTGTGCGAGATGGGGTGGTTCTTTAATGAACGCTTTTGATGTCTGGGGCCCAGGGACAATGGTCACCGTCTCTTCA(SEQ ID NO:12)

[0206] Its light chain variable region amino acid sequence:

[0207] QSALIQPASVSGSPGQSITISC TGTSSDVGGYNYVS WYQRHPGKAPRLMIY DVTKRPS GVSNRFSGSSKSGNTASLTISGLQAEDEADYYC SSYTSSSSIAV FGGGTQLTVL (SEQ ID NO: 13)

[0208] The underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 14-16, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 17-20, respectively;

[0209] The corresponding DNA sequence is:

[0210] CAGTCTGCCCTGATTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGTAGTGACGTTGGTGGTTATAATTATGTCTCCTGGTACCAACGACACCCAGGCAAAGCCCCCAGACTCATGATTTATGATGTCACTAAGCG GCCCTCAGGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCATTGCTGTGTTCGGAGGAGGCACCCAGCTGACCGTCCTC(SEQ ID NO:21)

[0211] 2.2.2O19 scFv heavy chain variable region amino acid sequence:

[0212] QVQLVESEGGLVQPGGSLRLSCAASRFTFS NYWMS WVRQAPGKGLEWVA NIKQDGSEKYYMDSVKG RFTISRDNAKNSLFLQMNTLRAEDTAMYYCTR VSFGVPTYDDFWRSYATPAWYFDF WGRGTLVTVSS (SEQ ID NO: 22)

[0213] The underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 23-25, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 26-29, respectively;

[0214] The corresponding DNA sequence is:

[0215] CAGGGTGCAGCTGGTGGAGTCTGAGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGCGCAGCCTCTAGATTCACGTTTAGTAACTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAAGGGCTGGAGTGGGTGGCCAATATAAAGCAAGATGGAAGTGAGAAATATTATATGGACTCTGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTTTCTGCAGATGAACACCCTAAGAGCCGAGGACACGGCTATGTATTACTGTACGAGGGTTAGTTTCGGAGTGCCGACGTATGACGATTTTTGGAGGAGTTACGCGACGCCCGCTTGGTACTTCGATTTTTGGGGCCGTGGTACCCTGGTCACTGTCTCCTCA(SEQ ID NO: 30) Its light chain variable region amino acid sequence:

[0216] QSALIQPASVSGSPGQSITISC TGISSDDGYYKYVS WYQQYPGKAPKLMIY DVSKRPS GISFRFSGSKSGNTASLTISGLQAEDEADYYC SSYTSNMTPYV FGTGTKVTVL (SEQ ID NO: 31)

[0217] The underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 32-34, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 35-38, respectively;

[0218] The corresponding DNA sequence is:

[0219] CAGTCTGCTCTGATTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAATTAGTAGTGACGATGGTTATTATAAGTATGTCTCCTGGTACCAACAATATCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGG CCCTCAGGGATTTCTTTTCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGTAACATGACCCCCTATGTCTTCGGCACTGGGACCAAGGTCACCGTCCTA(SEQ ID NO:39)

[0220] 2.2.3O21scFv heavy chain variable region amino acid sequence:

[0221] QVQLQQSGPGLVKPSQTLSLTCAISGDSVS SNSVSWD WIRQSPSRGLEWLG RTYYRSKWYNEYAVSVE S RITINPDTSKNQFSLQLNSVTPEDTAIYFCVR NNYFFDL WGRGTLVTVSS (SEQ ID NO: 40)

[0222] The underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 41-43, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 44-47, respectively;

[0223] The corresponding DNA sequence is:

[0224] CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGTCTCTTGGGACTGGATCAGGCAGTCCCTCGAGGGGCCTTGAGTGGCTGGGAAGGACATACTATAGGTCCAAGTGGTAT AATGAGTATGCAGTATCTGTGGAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAACTGAACTCTGTGACTCCCGAGGACACGGCTATATATTTCTGTGTAAGAAATAACTACTTCTTCGATCTCTGGGGCCGTGGTACCCTGGTCACCGTCTCCTCA(SEQ ID NO:48)

[0225] Its light chain variable region amino acid sequence:

[0226] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASDRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QLRSNWPPGYT FGQGTKVEIK (SEQ ID NO: 49), wherein the underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 50-52, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 53-56, respectively;

[0227] The corresponding DNA sequence is:

[0228] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCGACAGGGCC ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTTGAGCCTGAAGATTTTGCAGTTTTATTACTGTCAGCTGCGTAGCAACTGGCCTCCGGGGTACACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 57)

[0229] Example 3: Formatting scFv-type antibodies of three antibody strains into IgG-type antibodies

[0230] According to the constant region amino acid sequence of human immunoglobulin gamma4 (IgG4) on the protein database Uniprot (P01861), the constant region amino acid sequence of human IgG4 was obtained. The heavy chain variable region VH sequences of O3, O19, and O21 obtained by screening were spliced ​​with the human IgG4 constant region gene sequence, and the spliced ​​genes were synthesized and subcloned into the vector pcDNA4 / myc-HisA after double restriction digestion with HindIII and EcoRI of Fermentas Company to obtain pcDNA4-O21HC, pcDNA4-O3HC, and pcDNA4-O19HC.

[0231] According to the amino acid sequence of the constant region of human immunoglobulin Kappa on the protein database Uniprot (P01834), the amino acid sequence of the human Kappa light chain constant region was obtained. The light chain variable region VL sequence of O21 obtained by screening was spliced ​​with the gene sequence of the human Kappa light chain constant region; according to the amino acid sequence of the constant region of human immunoglobulin lambda on the protein database Uniprot (A0M8Q6), the amino acid sequence of the human lambda light chain constant region was obtained, and the light chain variable region VL sequences of O3 and O19 obtained by screening were spliced ​​with the gene sequence of the human lambda light chain constant region, and the spliced ​​genes were synthesized, and subcloned into the vector pcDNA4 / myc-HisA after double restriction digestion with HindIII and EcoRI of Fermentas Company to obtain pcDNA4-O21LC; pcDNA4-O3LC; pcDNA4-O19LC.

[0232] The heavy chain and light chain plasmids obtained above were extracted using the plasmid extraction kit (PL14) provided by AidLab. The recombinant light chain and heavy chain plasmids were co-transfected into HEK293 cells for antibody expression. The recombinant expression plasmid was diluted with Freestyle293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each group of plasmid / PEI mixtures was added to the cell suspension, placed at 37°C, 10% CO2, 120rpm, and cultured for 5 to 6 days. The transient expression culture supernatant was collected and purified by ProteinA affinity chromatography to obtain anti-hOX40 antibodies: O3mAb, O19mAb and O21mAb.

[0233] Example 4: Characterization of anti-hOX40 Abs

[0234] Identification of whether the antibody specifically recognizes hOX40:

[0235] Binding of purified anti-hOX40 antibodies to hOX40, hCD137, and hCD27 proteins

[0236] Take the HEK293 cells expressing hOX40-EGFP, hCD137-EGFP and hCD27-EGFP constructed in Example 1, resuspend them in 0.5% PBS-BSA Buffer, add anti-hOX40 mAb protein, and incubate on ice for 20 minutes. After washing, add eBioscience secondary antibody anti-hIg-PE and incubate on ice for 20 minutes. After washing, resuspend the cells in 500μl 0.5% PBS-BSA Buffer and detect by flow cytometry. The results are shown in Figure 2. Figure 2 As shown, the three antibodies (O3mAb, O19mAb and O21mAb) can bind to hOX40-EGFP cells, but cannot bind to several other EGFP cells (hCD137-RGFP-293F, hCD27-RGFP-293F), showing good specificity.

[0237] Example 5: In vitro affinity enhancement of anti-OX40 antibodies

[0238] Since the three antibodies (O3mAb, O19mAb and O21mAb) can all specifically bind to hOX40-EGFP cells, and the experiment found that O21 scFv has a higher affinity for hOX40, and transient transfection has a stable and high expression level, the O21 scFv antibody was selected for further in vitro affinity improvement experiments; however, it should be noted that the purpose of the in vitro affinity improvement experiment is to study the effect of mutation rate on antibody affinity, and the test results should not be limited to the determination of O21 scFv, but should be understood as the effect of mutation rate on antibody affinity for antibodies including O3scFv, O19 scFv and O21 scFv.

[0239] 5.1 Construction of yeast library with improved affinity of anti-OX4021#ScFv

[0240] The pcDNA4-OX40-21-Fc plasmid constructed in Example 1 was used as a template, and pcDNA4-F: TCTGGTGGTGGTGGTTCTGCTAGC (SEQ ID NO: 58) and cMyc-BBXhoI: GCCAGATCTCGAGCTATTACAAGTCTTCTTCAGAAATAAGCTTTTGTTCTAGAAT TCCG (SEQ ID NO: 59) were used as primers for standard PCR reaction. The obtained PCR product was digested with NheI and BglII of Fermentas Company to construct a recombinant plasmid. Next, with reference to the method of Ginger et al. (2006) NatProtoc1(2):755-68, the PCR product of random mutation of scFv was obtained by error prone PCR method. The primers used were ep-F: TAATACGACTCACTATAGGG (SEQ ID NO: 60) and ep-R: GGCAGCCCCATAAACACACAGTAT (SEQ ID NO: 61). The obtained PCR product was purified by Fermentas GeneJET DNA purification Kit and then concentrated by ethanol precipitation to a concentration greater than 1 μg / μl. The remaining operation methods were based on the method of Ginger et al. (2006) Nat Protoc 1(2):755-68, and the affinity matured yeast library was obtained by yeast electroporation and in vivo recombination.

[0241] 5.2 Screening of yeast anti-OX40 21#scFv with improved affinity

[0242] The affinity-matured yeast library obtained above was subjected to two rounds of flow sorting with 10 nM and 1 nM hOX40-Fc proteins, and the yeast products obtained by sorting were plated and single clones were picked for identification. Using the low-concentration antigen staining method, the wild-type yeast obtained previously was used as a control, and the yeast single clones with improved affinity were determined by flow staining. The yeast clones confirmed by FACS were subjected to yeast colony PCR and sequencing, and the methods were the same as above. The sequences obtained by sequencing were analyzed for mutation sites using BioEdit software.

[0243] The mutation sites of O21 scFv that have no effect on antibody affinity or even improve affinity are sorted out, and the results are shown in Table 1. The results in this table should be understood as follows: one or more mutations as shown in the table on O21 scFv have no effect on affinity or even improve it; the corresponding results of yeast monoclonal sequence analysis are shown in Table 2, and this table should be understood as follows: the mutation sites listed in Table 1, when having several mutation combinations as shown in Table 2, can improve the affinity of the antibody; the yeast staining results shown in Table 2 are shown in Table 2. Figure 3 As shown, it can be seen that compared with O21 scFv, the affinity of the antibodies has been improved to varying degrees.

[0244] Table 1 O21 scFv mutation sites that have no effect on antibody affinity

[0245] Heavy chain Light chain H7 position: S mutates to L L30: S mutates to G H13: K mutates to M L90: L mutates to Q H28: S mutates to N H33: S mutates to G H43: R mutates to G H65: S mutates to N H67: I mutated to M H96: N mutates to D

[0246] Table 2 Sequence analysis results of yeast monoclonal clones with improved affinity

[0247]

[0248]

[0249] As can be seen from Table 2, for the heavy chain CDR region and light chain CDR region of O21 scFv, experiments have shown that 3 and 2 amino acids can be mutated respectively (the anti-OX40 21#H96-L80 mAb in Example 6 has a light chain mutation of up to 3 amino acids), and the affinity of the antibody can still be maintained or even improved. It is now known that the heavy chain variable region and light chain variable region of O21 scFv include 119 amino acids (including 32 amino acids in the CDR region and 87 amino acids in the FR region) and 109 amino acids (including 29 amino acids in the CDR region and 80 amino acids in the FR region), respectively. Therefore, it can be considered that if the homology with the heavy chain CDR region or the light chain CDR region is more than 90%, it still has the ability to maintain or even improve the affinity of the antibody. In practice, there is no decisive connection between the affinity of an antibody and the percentage of sequence homology. Instead, it is more affected by the key amino acid residues that determine the antibody epitope (or antigenic determinant site). Under the premise that these key amino acid residues have not mutated or the mutation does not have a decisive effect, the sequence homology can be as low as 70%.

[0250] Example 6: Formatting of scFv-type antibodies into IgG-type antibodies

[0251] The affinity-matured scFv antibody was formatted into an IgG antibody according to the method in Example 3 to obtain a series of anti-OX40 21#mAb variants, and the specific sequence information is as follows:

[0252] 6.1anti-OX40 21#H28H33 mAb

[0253] Its heavy chain variable region amino acid sequence:

[0254]

[0255] The underlined parts are CDR1, CDR2, and CDR3, and the parts in the boxes are mutated parts, and their sequence numbers are SEQ ID NOs: 63, 42, and 43, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 64, 45, 46, and 47, respectively;

[0256] The corresponding DNA sequence is:

[0257]

[0258]

[0259] Its light chain variable region amino acid sequence:

[0260] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLAWYQQKPGQAPRLLIY DASDRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QLRSNWPPGYT FGQGTKVEIK (SEQ ID NO: 49)

[0261] The underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 50-52, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 53-56, respectively;

[0262] The corresponding DNA sequence is:

[0263] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCGACAGGGCC ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTTGAGCCTGAAGATTTTGCAGTTTTATTACTGTCAGCTGCGTAGCAACTGGCCTCCGGGGTACACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 57)

[0264] 6.2. anti-OX40 21#H65 mAb

[0265] Its heavy chain variable region amino acid sequence:

[0266]

[0267] The underlined parts are CDR1, CDR2, and CDR3, and the parts in the boxes are mutated parts, and their sequence numbers are SEQ ID NOs: 41, 67, and 43, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 44-47, respectively;

[0268] The corresponding DNA sequence is:

[0269]

[0270]

[0271] Its light chain variable region amino acid sequence:

[0272] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASDRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QLRSNWPPGYT FGQGTKVEIK (SEQ ID NO: 49)

[0273] The underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 50-52, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 53-56, respectively;

[0274] The corresponding DNA sequence is:

[0275] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCGACAGGGCC ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTTGAGCCTGAAGATTTTGCAGTTTTATTACTGTCAGCTGCGTAGCAACTGGCCTCCGGGGTACACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 57)

[0276] 6.3. anti-OX40 21#H96 mAb

[0277] Its heavy chain variable region amino acid sequence:

[0278]

[0279] The underlined parts are CDR1, CDR2, and CDR3, and the parts in the boxes are mutated parts, and their sequence numbers are SEQ ID NOs: 41, 42, and 70, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 44-47, respectively;

[0280] The corresponding DNA sequence is:

[0281]

[0282]

[0283] Its light chain variable region amino acid sequence:

[0284] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASDRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QLRSNWPPGYT FGQGTKVEIK (SEQ ID NO: 49)

[0285] The underlined parts are CDR1, CDR2, and CDR3, and their sequence numbers are SEQ ID NOs: 50-52, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 53-56, respectively;

[0286] The corresponding DNA sequence is:

[0287] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCGACAGGGCC ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTTGAGCCTGAAGATTTTGCAGTTTTATTACTGTCAGCTGCGTAGCAACTGGCCTCCGGGGTACACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA(SEQ ID NO: 57)

[0288] 6.4, anti-OX40 21#VHnew-L80

[0289] Its heavy chain variable region amino acid sequence:

[0290]

[0291] The underlined parts are CDR1, CDR2, and CDR3, and the parts in the boxes are mutated parts, and their sequence numbers are SEQ ID NOs: 63, 67, and 70, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 44-47, respectively;

[0292] The corresponding DNA sequence is:

[0293]

[0294]

[0295] Its light chain variable region amino acid sequence:

[0296]

[0297] The underlined parts are CDR1, CDR2, and CDR3, and the parts in the boxes are mutated parts, and their sequence numbers are SEQ ID NOs: 75-77; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 53-56;

[0298] The corresponding DNA sequence is:

[0299]

[0300] 6.5, anti-OX40 21#H96-L80 mAb

[0301] Its heavy chain variable region amino acid sequence:

[0302]

[0303] The underlined parts are CDR1, CDR2, and CDR3, and the parts in the boxes are mutated parts, and their sequence numbers are SEQ ID NOs: 41, 42, and 70, respectively; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 44-47, respectively;

[0304] The corresponding DNA sequence is:

[0305]

[0306]

[0307] Its light chain variable region amino acid sequence:

[0308]

[0309] The underlined parts are CDR1, CDR2, and CDR3, and the parts in the boxes are mutated parts, and their sequence numbers are SEQ ID NOs: 75-77; the ununderlined parts are FR1, FR2, FR3, and FR4, and their sequence numbers are SEQ ID NOs: 53-56;

[0310] The corresponding DNA sequence is:

[0311]

[0312] Example 7: Characterization of anti-hOX40 Abs

[0313] 7.1 Detection of the binding ability of purified anti-hOX40 antibody to hOX40 (ELISA method)

[0314] Dilute hOX40-muFc to 2μg / ml with coating buffer (50mM Na2CO3, NaHCO3 pH9.6), 100μL / well, overnight at 4℃. After washing, block with 3% BSA-PBS at 37℃ for 1h. Dilute the anti-hOX40 antibody by 2-fold gradient starting from 2000ng / ml, for a total of 11 concentrations, and use the diluent (1% BSA-PBS) as a control, and incubate at 37℃ for 2h. Add goat anti-human IgG-HRP (Goat anti-human IgG-HRP conjugated) and incubate at 37℃ for 1h. Add soluble single-component TMB substrate colorimetric solution and color at room temperature in the dark for 5-10min. Terminate the color reaction with 2N H2SO4 50μL / well. The OD 450nm-650nm value was read on the MD SpectraMax Plus384 microplate reader, and the data was processed and analyzed using the SoftMax Pro v5.4 software. The results are shown in the figure. Figure 4-6 As shown. Figure 4 It can be seen that after the conversion from ScFv to IgG antibody, the affinity of O19 mAb is still relatively low; the affinity of O21 mAb has little change and is relatively good; the affinity of O3 mAb has been significantly improved. Figure 5 and Figure 6 It can be seen that the variants of the above anti-OX40 21#mAb can all bind to OX40, and the affinity of anti-OX4021#VHnew-L80 mAb and anti-OX40 21#H96-L80 mAb after in vitro affinity maturation is approximately twice that of anti-OX40 21#mAb.

[0315] 7.2 Analysis of kinetic affinity constants of a-hOX40 mAbs to hOX40 by surface plasmon resonance (SPR)

[0316] The binding kinetics of anti-hOX40 antibodies against recombinant human OX40 were measured by surface plasmon resonance (SRP) method using BIAcore X100 instrument. CM5 chip coupled with anti-human Fc antibody (no cross recognition of mouse Fc), the antibody to be tested was diluted to 5nM in running buffer and captured by the antibody on the chip as ligand. OX40-muFc was diluted to 1000-1.37nM in running buffer. The injection time was 180s, the dissociation time was 1800s, and the regeneration time was 60s. The running buffer was HBS-EP+, and the regeneration buffer was 10mM Mglycine-HCl (pH2.0). The association rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version3.2). The equilibrium dissociation constant (kD) was calculated as the ratio koff / kon. As shown in Table 3, the affinity of anti-OX40 21#H96-L80 mAb increased by about two times after affinity maturation.

[0317] Table 3 Kinetics of binding between anti-hOX40 antibody and hOX40

[0318] name Kon(1 / Ms) Koff(1 / s) KD(M) O3 mAb 2.84E+05 3.91E-04 1.378E-09 O21mAb 4.181E+4 6.077E-04 1.454E-08 VHnew-L80 3.329E+04 3.415E-04 1.026E-08 H96-L80 2.999E+04 2.368E-04 7.898E-09

[0319] 7.3 Detection of the agonist activity of anti-hOX40 antibodies in vitro using the NF-κb system

[0320] Construction of OX40-CD40 plasmid: Based on the amino acid sequence of human OX40 on the protein database Uniprot (P43489), the amino acid sequences of the extracellular domain and transmembrane region of human OX40 (i.e., residues 1 to 235 in P43489) were obtained; based on the amino acid sequence of human CD40 on the protein database Uniprot (P25942), the amino acid sequence of the intracellular segment of human CD40 (i.e., residues 216 to 277 in P25942) was obtained; the two were spliced ​​together by gene synthesis, and then subcloned into the commercial vector pcDNA4 / myc-HisA (Invitrogen, V863-20) of Fermentas Company after double restriction digestion with HindIII and EcoRI, and the accuracy of the constructed plasmid was verified by sequencing to obtain the recombinant plasmid DNA, namely: pcDNA4-OX40-CD40 (i.e., OX40-CD40 mentioned below).

[0321] 293T-NF-κB stable cell line: 293T cells were seeded in 24-well plates, 1x10 5 / well, 200μl DMEM complete medium per well, and 20μl NF-κB-luciferase-lentivirus (Qiagen, cat: CLS-013L) were added at the same time, MOI = 2. After 24h of infection, the supernatant was discarded and 1ml DMEM complete medium was added to continue culturing. After 24h, DMEM complete medium containing 0.3μg / ml promycin was used to continue culturing. The cells were expanded and frozen. 293T-NF-κB cells selected with promycin were inoculated in 24-well plates, and 1x10 5 / well, 10ng / ml TNF-α stimulation for 6h, lysed cells to detect luciferase, the results showed that the luciferase value of cells stimulated by TNF-α was significantly higher than that of unstimulated cells, proving that NF-κB-luciferase had been stably transferred into 293T cells.

[0322] Resuspend 5x10 in medium without dual antibody 5 293T-NF-κB cells were inoculated in 6-well plates. After 24 hours, the supernatant was discarded and washed with PBS. 1.8 ml of culture medium without anti-antibody and serum was added. 0.2 μg OX40-CD40 plasmid was transfected at a ratio of plasmid:liposome = 1:3. After 4 hours of transfection, the supernatant was discarded and replaced with fresh complete culture medium and culture was continued. The next day after transfection, the cells were cultured at 5x10 4 / well density was inoculated in a 96-well plate, and a series of antibody concentration gradients were added (initial concentration 10μg / ml, 10-fold dilution 7 gradients), and the same concentration of cross-link (Jackson ImmunoResearchLaboratories:109-006-008) was added and cultured for 6 hours, and the cells were lysed to detect luciferase. Figure 7 As shown in the figure, A, C, E, and G represent the in vitro agonist activity test results of O3 mAb, O21 mAb, anti-OX40 21#VHnew-L80 mAb and anti-OX40 21#H96-L80mAb at different concentrations, respectively. B, D, F, and H in the figure are the in vitro agonist activity test results after taking the log, respectively. The results show that O3 mAb and O21 mAb have good agonist activity in vitro and show good dose dependence. After the affinity is improved, the anti-OX40 21#VHnew-L80 mAb and anti-OX40 21#H96-L80mAb have an approximately two-fold increase in agonist activity.

[0323] Example 8 Effects of different subtypes on antibody agonist activity

[0324] Since the various variants of anti-OX40 21#mAb all have a specific high affinity for hOX40, and the binding kinetic results with hOX40 are all good, and one of them, anti-OX40 21#H96-L80 mAb, has an approximately two-fold improvement in Agonist activity, the present application uses anti-OX40 21#H96-L80 mAb (hereinafter referred to as H96-L80) as an example for further description, and the remaining variants are not described in detail. However, it should be noted that although only H96-L80 is described in the subsequent embodiments, it should not be understood that the various variants of anti-OX40 21#mAb do not have the corresponding characteristics, or that the other two antibodies (O3mAb, O19mAb) do not have the corresponding characteristics. The further experimental data for H96-L80 in the subsequent embodiments are only used to prove that the various anti-OX40 antibodies obtained according to the method of the present invention have higher activity, affinity, functionality or stability.

[0325] According to the constant region amino acid sequences of human immunoglobulin gamma1 (IgG1) and human immunoglobulin gamma2 (IgG2) on the protein database Uniprot (P01857 and P01859, respectively), the constant region amino acid sequences of human IgG1 and IgG2 were obtained. The rest of the operations were carried out according to the above method to obtain anti-OX40 21#H96-L80 IgG1 mAb and anti-OX40 21#H96-L80IgG2 mAb. The experiment was carried out according to the method of Example 7, and the results are as follows Figure 8 As shown, Figure 8 A and Figure 8 B respectively represents IgG1 and IgG2 subtypes. The results of in vitro agonist activity test at different concentrations are as follows: Figure 8 C is the comparison result of IgG1 and IgG2 subtypes after logarithm analysis. Figure 8 When D is at a concentration of 0.1ug / ml, it can be seen that the agonist activity of the three subtypes of antibodies of H96-L80 is comparable.

[0326] Example 9: Detection of the ability of the purified H96-L80 antibody to bind to activated CD4+ T and CD8+ T cells in humans and rhesus monkeys

[0327] Peripheral blood mononuclear cells (PBMCs) were separated from the concentrated leukocytes in the peripheral blood of healthy donors by density gradient centrifugation using human lymphocyte separation medium (Tianjin Haoyang) and inoculated into RPMI complete medium. PBMCs were activated for 48 hours with PHA at a final concentration of 5ug / ml. The cells were resuspended in 0.5% PBS-BSA Buffer, H96-L80 protein was added, and incubated on ice for 20 minutes. After washing, biolegend secondary antibody FITC anti-hIgG (Cat#409310) or Biolegend antibody PE anti-hOX40 (Cat#350003), and biolegend antibody APC anti-human CD4 (Cat#317416) were added and incubated on ice for 20 minutes. After washing, the cells were resuspended in 500μl 0.5% PBS-BSA Buffer and detected by flow cytometry. The results are shown in Figure 2. Fig. 9 As shown in A, H96-L80 can bind to activated CD4+ T cells well.

[0328] To detect the binding ability of H96-L80 to Treg and Teff in CD4+ cells, the above activated human PBMCs were used for staining. The cells were resuspended in 0.5% PBS-BSA Buffer, H96-L80 protein was added, and incubated on ice for 20 minutes. After washing, biolegend secondary antibody FITC anti-hIgG and biolegend antibody APC anti-human CD4 were added, incubated on ice for 20 minutes, washed, and then transmembrane fixative (BD, 51-2090KZ) was used for 1 hour, and then washed with transmembrane solution (eBioscience, 00-8333-56) and then resuspended with transmembrane solution, PE anti-human Foxp3 (Cat#320208) was added, and stained overnight at 4°C. After washing, the cells were resuspended in 500μl 0.5% PBS-BSA Buffer and detected by flow cytometry. The results are shown in Fig. 9 As shown in B, H96-L80 can bind well to activated CD4+Foxp3+Treg and CD4+Foxp3-Teff cells.

[0329] To detect the binding ability of H96-L80 to rhesus monkey OX40, activated rhesus monkey peripheral blood mononuclear cells PBMC were obtained using the above steps. The cells were resuspended in 0.5% PBS-BSA Buffer, H96-L80 protein was added, and incubated on ice for 20 minutes. After washing, biolegend secondary antibody FITC anti-hIgG, as well as biolegend antibodies APC anti-humanCD4 and PE anti-human CD8a (Cat#301008) were added and incubated on ice for 20 minutes. After washing, the cells were resuspended in 500μl 0.5% PBS-BSA Buffer and detected by flow cytometry. The results are shown in Figure 2. Fig. 9 As shown in C, H96-L80 can bind well to activated rhesus monkey CD4+T and CD8+T cells, suggesting that H96-L80 can bind to rhesus monkey OX40.

[0330] Example 10: Anti-OX40 antibodies promote T cell activation and proliferation

[0331] 10.1 T cell activation and proliferation were used to study the in vitro activity of anti-OX40 scFv antibodies and evaluate their agonist function.

[0332] Peripheral blood mononuclear cells (PBMCs) were separated from the concentrated leukocytes of the peripheral blood of healthy donors by density gradient centrifugation using human lymphocyte separation medium (Tianjin Haoyang) and inoculated into RPMI complete medium. 96-well plates were pre-coated with 50 μl 1 μg / ml anti-CD3 overnight at 4°C. The experimental group was coated with 50 μl 2 μg / ml O21 scFv at 37°C for 2 hours, and at the same time, a soluble form of O21 scFv with a final concentration of 2 μg / ml + a final concentration of 4 μg / ml cross-link (Jackson ImmunoResearch Laboratories: 109-006-008) was added. The negative control was RPMI complete medium. The amount of PBMCs was 2×10 5 / well, and the supernatant was collected after five days of culture. Fig.10 As shown, the IFN-γ level in the supernatant was detected using an IFN-γ ELISA kit (ebioscience) Fig.10 A), and T cell proliferation was detected using BrdU staining kit (Roche: 11647229001) Fig.10 B), it can be seen that O21 scFv has good activity in activating PBMC and promoting T cell proliferation under both coating and cross-link administration methods.

[0333] 10.2 Evaluation of the agonist activity of O21 scFv and IgG using in vitro PBMC and CD4+ T cell activation

[0334] Peripheral blood mononuclear cells (PBMCs) were separated from the concentrated leukocytes of the peripheral blood of healthy donors by density gradient centrifugation using human lymphocyte separation medium (Tianjin Haoyang) and inoculated into RPMI complete medium. CD4+T cells were separated from PBMCs using a CD4+T cell isolation kit (Miltenyi Biotec, cat#no.130-096-533). 96-well plates were pre-coated with 50μl 1μg / ml anti-CD3 at 4°C overnight. The experimental group was treated with 50μl 2μg / ml O21 scFv or O21 mAb

[0335] b, coated at 37℃ for 2h, negative control was hIgG-Fc at the same dose. The amount of PBMC and CD4+T cells was 2×10 5 / well, and the supernatant was taken after five days of culture, and the level of IFN-γ in the supernatant was detected using an IFN-γ ELISA detection kit (ebioscience).

[0336] like Fig.11 As shown, for PBMC and CD4+ T cells, it can be seen that the whole antibody mAb type of O21 antibody has better agonist activity than the scFv type in vitro.

[0337] Example 11: Effects of IgG1 and IgG4 subtype anti-OX40 antibody H96-L80 on T cell subsets

[0338] According to the operation of Example 10, human peripheral blood mononuclear cells (PBMC) were obtained, and the PBMC were stimulated with dissolved anti-CD28 (0.5μg / ml) and plate-bound anti-CD3 (3μg / ml) and anti-human OX40mAb H96-L80 IgG1 or IgG4 (10μg / ml). After 48 hours, the cells were harvested. Staining with biolegend antibodies APC anti-human CD4 and PE anti-human CD8a (Cat#301008), or staining with biolegend antibodies APC anti-human CD4 and PE anti-human Foxp3 (Cat#320208), and detected by flow cytometry. The results are shown in the figure below. Both subtypes of O21 mAb H96-L80 IgG1 and IgG4 can reduce the proportion of CD4+Foxp3+Treg in CD4+T cells ( Fig.12 A), but had no effect on the ratio of CD4+T to CD8+T cells ( Fig.12 B).

[0339] Example 12: Inhibitory effect of anti-OX40 antibody on tumor growth in mice

[0340] 12.1 Evaluation of the in vivo efficacy of anti-OX40 antibodies using a NOD-SCID mouse tumor model implanted with PC-3 tumor cells and human PBMCs

[0341] On day 0, PC-3 (ATCC CRL-1435 TM ) and human peripheral blood mononuclear cells (PBMCs) were subcutaneously (SC) injected into mice, and 10 mg / kg of O21 mAb or PBS was intraperitoneally injected on days 0 and 7, with PBS as a negative control. There were 5 mice in each group. Tumor formation was observed twice a week, and the long and short diameters of the tumors were measured with a vernier caliper, the tumor volume was calculated, and the tumor growth curve was plotted. The results are shown in Fig.13 As shown, it can be seen that the antibody O21 mAb can significantly inhibit tumor growth.

[0342] 12.2 Evaluation of the in vivo efficacy of anti-OX40 antibodies using a NOD-SCID mouse tumor model implanted with A375 tumor cells and human PBMCs

[0343] On day 0, 7 × 10 6 A375 (ATCC CRL-1619 TM ) and 1×10 6 Human peripheral blood mononuclear cells (PBMCs) were subcutaneously (SC) injected into mice, and 1 mg / kg of O21 mAb or PBS was intraperitoneally injected on days 0 and 7, with PBS as a negative control, with 5 mice in each group. Tumor formation was observed twice a week, and the long and short diameters of the tumors were measured with a vernier caliper, the tumor volume was calculated, and the tumor growth curve was plotted. Fig.14 As shown, it can be seen that the antibody O21 mAb can significantly inhibit tumor growth.

[0344] Example 13: Stability test of anti-OX40 Abs

[0345] The stability of the anti-hOX40 antibody O21 mAb was tested using a 45°C accelerated stability experiment. The specific experimental method is: the anti-O21 mAb antibody was concentrated to about 10 mg / ml, placed in a 45°C water bath, and samples were collected on days 0, 10, 20, and 30 for concentration, SEC-HPLC, and NF-κb analysis experiments. SEC-HPLC was performed using a Shimadzu LC20AT HPLC liquid chromatograph, the sample was concentrated to 1 mg / ml, the flow rate was 0.5 ml / min, the total sample load was 50 ug, and isocratic elution was performed for 30 minutes after loading; NF-κb was operated according to Example 7, and the results are as follows Fig.15 As shown, it can be seen that the anti-hOX40 antibody O21mAb has good stability in vitro.

[0346] Example 14 Pharmacokinetic evaluation of anti-hOX40 antibody O21 mAb

[0347] The pharmacokinetics of H96-L80 IgG1 antibody were evaluated at doses of 10 mg / kg and 1 mg / kg. Balb / C, female, 8-week-old mice were used, and 16 mice were taken. Each dose was divided into two groups, A / B, with four mice in each group. All mice were intravenously injected with 200 μg (10 mg / kg) or 20 ug (1 mg / kg) of H96-L80 IgG1 antibody. After administration, 100 μl of blood was collected at 14 time points, and one group of mice was taken at each time point, and the two groups were rotated. The serum was separated. The concentration of the test protein in the serum was detected by ELISA method: hOX40-muFc coated plate, serum samples of appropriate dilutions were added, and then Goat anti-Human IgG HRP (Sigma CatNO: A0170) was added, and TMB was used for color development. H96-L80 IgG1 antibody was used as the standard protein to make a standard curve. Pharmacokinetic parameters were calculated using WinNolin software. The average CT curve is shown in the figure. Fig.16 As shown, the H96-L80IgG1 antibody of the present invention is relatively stable, with an average half-life of 205 hours at a dose of 1 mg / kg and an average half-life of 371 hours at a dose of 10 mg / kg. No anti-H96-L80 anti-antibody was produced.

[0348] Example 15 Anti-OX40 Antibodies that Enhance ADCC Effects

[0349] The present application further provides an anti-OX40 antibody with enhanced ADCC effect. This example illustrates that the anti-OX40 antibody provided by the present application has enhanced ADCC effect.

[0350] For example, the heavy chain constant region of IgG1 in the antibody with enhanced ADCC in the present application comprises mutations of S239D, I332E and / or A330L.

[0351] Compared with the above H96-L80 IgG1, the antibody with L at position 106 on the light chain variable region has the following amino acid sequence in the heavy chain variable region:

[0352] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSVSWDWIRQSPSRGLEWLGRTYYRSK WYNEYAVSVESRITINPDTSKNQFSLQLNSVTPEDTAIYFCVRNDYFFDLWGRGTLVTVSS (SEQ ID NO: 79);

[0353] The amino acid sequence of its light chain variable region is:

[0354] EIVLTQSPATLSLSPGERATLSCRASQSVGSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPGYTFGQGTKLEIK (SEQ ID NO: 81).

[0355] The antibodies having the above heavy chain variable region and light chain variable region, and the antibodies after mutation of the heavy chain constant region of IgG1 (IgG1 VHS239D+I332E, SEQ ID NO: 83; IgG1 VH S239D+I332E+A330L, SEQ ID NO: 84) were compared with the antibodies without mutation of the heavy chain constant region of IgG1 (SEQ ID NO: 82), and the ADCC effect strength was tested.

[0356] 50ul of 20,000 Jurkat-V158 cells and 50ul of 10,000 HEK293-OX40 cells were inoculated into a 96-well plate. The antibodies were diluted to 10, 1, 0.1, 0.01, and 0.001ug / ml, respectively, and added to the 96-well plate inoculated with cells. The cells were incubated for 6 hours, and chemiluminescence was detected using 100ul / well Promega one-glo E6110.

[0357] The results showed that the signals of the two IgG1 mutation forms at an antibody concentration of 0.01ug / ml were greater than the signal of IgG1 without mutation at an antibody concentration of 0.1ug / ml, indicating that the IgG1 mutated antibody has an enhanced ADCC effect; the ADCC effect of the antibody after IgG1 S239D+I332E mutation is stronger than that of IgG1 S239D+I332E+A330L.

[0358] Example 16 Therapeutic effect of anti-OX40 antibody on GVHD in mice

[0359] This example shows that the anti-OX40 antibody provided in the present application has a therapeutic effect on GVHD.

[0360] The ADCC-enhanced anti-OX40 antibody after the IgG1 heavy chain variable region S239D+I332E mutation in Example 15 was used for the experiment. 24 M-NSG hIL15 mice (6-8w, female) were irradiated and recorded as day 0. After irradiation, the mice were randomly divided into 3 groups according to body weight, including Group 1 PBS, ip, Q2D group (hereinafter referred to as Group 1), Group 2 Abatacept, ip, 10 mpk, Q2D group (hereinafter referred to as Group 2), Group 3 ADCC-enhanced anti-OX40 antibody, iv, 10 mpk, Q2D group (hereinafter referred to as Group 3) and Group 4 Benchmark, iv, 10 mpk, Q2D group (hereinafter referred to as Group 4), with 6 mice in each group (such as Fig.18 As shown). The day of grouping is PG-D0, and the drug was administered on the day of grouping. All drugs were administered by intravenous injection, and the administration volume was 10mL / kg. Before administration, PBMC cells (XW0801070W) (number of cells inoculated: 1E7 / mouse) were injected into the tail vein. The weight of mice was recorded three times a week. The life and death and general survival status of mice (hair loss, piloerection, reduced activity, lower body temperature, etc.) were observed and recorded every day on weekdays, and the survival curve was drawn.

[0361] The data show that Fig.19A -C, after treatment with the anti-OX40 antibody of the present application, the proportion of hCD4 in mice was significantly reduced compared with that in the Abacept group, and the survival period of mice was significantly prolonged compared with that in the PBS group. The data show that the anti-OX40 antibody of the present application can reduce the proportion of CD4 in T cells and alleviate the GVHD effect in mice.

Claims

1. Use of an anti-OX40 antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating an autoimmune disease, wherein the anti-OX40 antibody or the antigen-binding fragment thereof comprises a CDR region selected from any one of the following groups: 1) The sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 5-7, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 14-16, respectively; 2) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 23-25, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 32-34, respectively; 3) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 41-43, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 50-52, respectively; 4) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 63, 42 and 43, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 50-52, respectively; 5) the sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 41, 67, and 43, respectively, and the sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 50-52, respectively; 6) the sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 41, 42, and 70, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 50-52, respectively; 7) the sequences of heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 63, 67, and 70, respectively, and the sequences of light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 75-77, respectively; 8) The sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 41, 42 and 70, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NOs: 75-77, respectively.

2. The use according to claim 1, wherein the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of the following group, whose sequences are shown in SEQ ID NOs: 4, 22, 40, 62, 66, 72 and 79.

3. The use according to any one of claims 1-2, wherein the anti-OX40 antibody or antigen-binding fragment thereof comprises the following group of light chain variable regions, whose sequences are shown in SEQ ID NOs: 13, 31, 49, 74 and 81.

4. The use according to any one of claims 1 to 3, wherein the anti-OX40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 79 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:

81.

5. The use according to any one of claims 1 to 4, wherein the anti-OX40 antibody or antigen-binding fragment thereof is a whole antibody, a bispecific antibody, a scFv, a Fab, a Fab', a F(ab')2 or a Fv.

6. The use according to any one of claims 1 to 5, wherein when the anti-OX40 antibody or antigen-binding fragment thereof is scFv, a connecting peptide may be contained between the heavy chain and light chain variable regions.

7. The use according to any one of claims 1 to 6, wherein the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof is selected from IgG, IgM, IgE, IgD and IgA.

8. The use according to any one of claims 1 to 7, wherein the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof is selected from IgG1 or IgG4.

9. The use according to any one of claims 1-8, wherein the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof is selected from IgG1, and the heavy chain constant region of the IgG1 comprises mutations of S239D, I332E and / or A330L.

10. The use according to any one of claims 1 to 9, wherein the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 82 to 84.

11. The use according to any one of claims 1 to 10, wherein the light chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof is κ or λ.

12. The use according to any one of claims 1 to 11, wherein the light chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

85.

13. The use according to any one of claims 1 to 12, wherein the heavy chain constant region of the anti-OX40 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 83, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in NO:

85.

14. The use according to any one of claims 1 to 13, wherein the medicament comprises a nucleic acid molecule encoding the anti-OX40 antibody or antigen-binding fragment thereof according to any one of the preceding claims.

15. The use according to any one of claims 14, wherein the medicament comprises a vector comprising the nucleic acid molecule.

16. The use according to any one of claims 14-15, wherein the medicament comprises a cell comprising the nucleic acid molecule or the vector.

17. The use according to any one of claims 1 to 16, wherein the medicament comprises a conjugate comprising the anti-OX40 antibody or antigen-binding fragment thereof.

18. The use according to any one of claims 1 to 17, wherein the medicament comprises a pharmaceutical composition comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of the preceding claims, the nucleic acid molecule according to the preceding claim, the vector according to the preceding claim, the cell according to the preceding claim and / or the conjugate according to the preceding claim, and a pharmaceutically acceptable carrier.

19. The use according to any one of claims 1-18, wherein the autoimmune disease comprises an OX40-mediated autoimmune disease.

20. The use according to any one of claims 1-19, wherein the autoimmune disease comprises a T cell-mediated autoimmune disease.

21. The use according to any one of claims 1-20, wherein the autoimmune disease comprises graft-versus-host disease.

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