Antigen binding proteins targeting cxcr3 and uses thereof

Abstract

The present application relates to an antigen binding protein targeting CXCR3 and its use, the antigen binding protein can specifically bind CXCR3, comprising a heavy chain variable region and a light chain variable region. The present application also provides a preparation method of the antigen binding protein and its use.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an antigen-binding protein that targets CXCR3. Background Technology

[0002] Chemokines are a class of cell-secreted cytokines or signaling proteins that induce targeted chemotaxis in nearby responding cells, hence the name chemokines. Chemokines and their receptors have been shown to influence many cellular functions, including survival, adhesion, invasion, and proliferation, and to regulate chemokine levels. Most malignant tumors express one or more chemokine receptors. The chemokine receptor CXCR3 is a G protein-coupled seven-transmembrane domain receptor that selectively binds to CXC chemokines (CXCL9, CXCL10, and CXCL11). It is primarily expressed on the surface of activated T cells, plasma cells, memory B cells, and NK cells, and induces targeted migration and immune responses by binding to specific receptors on target cell membranes. CXCR3 and its ligands play important roles in infection, autoimmune diseases, and tumor immunity. Current research indicates that CXCR3 and its ligands CXCL9, CXCL10, and CXCL11 are closely related to the development and progression of many diseases. For example, upregulation of CXCR3 is involved in a range of autoimmune disorders. CXCR3 expression is essentially absent in naive T cells but is upregulated upon activation with antigens. CXCR3 recruits these cells (including T helper cells type 1 (Th1)) to sites of tissue inflammation in response to their major ligands.

[0003] Therefore, there is a need in the art for improved antigen-binding proteins that target CXCR3 and can be used as therapeutic agents. Summary of the Invention

[0004] This application provides an antigen-binding protein targeting CXCR3, which specifically binds to CXCR3 and blocks the binding of CXCR3 to its ligand. The antigen-binding protein targeting CXCR3 provided in this application has strong ADCC activity, thereby facilitating the killing or clearance of CXCR3-positive cells by effector cells. Furthermore, the anti-CXCR3 antibody of this application can effectively inhibit CXCR3-mediated cell migration. In chemokine-induced Transwell migration assays, the antibody of this application can block the migration of CXCR3-expressing cells induced by CXCL9, CXCL10, and / or CXCL11, indicating that the antibody of this application can interfere with the cell migration process mediated by CXCR3-related chemokine signals.

[0005] Since CXCR3 is involved in the chemotactic migration of activated T cells, B cells, and other immune cells to sites of inflammation, the antibody in this application, by mediating ADCC in CXCR3-positive cells and inhibiting the migration of CXCR3-expressing cells to inflammation-related chemokines, can potentially reduce the aggregation of immune cells in inflammatory areas and / or decrease the number of CXCR3-positive immune cells in inflammatory areas, thereby helping to alleviate local immune responses. Therefore, the antibody in this application has potential application in the prevention and / or treatment of T-cell and / or B-cell-related diseases, especially autoimmune or inflammatory diseases.

[0006] On one hand, this application provides an antigen-binding protein capable of specifically binding to CXCR3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0007] a) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 1-3, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4-6, respectively; or

[0008] b) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 8, SEQ ID NO: 5 and SEQ ID NO: 9, respectively.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13.

[0011] In some embodiments, it includes an antibody or an antigen-binding fragment thereof.

[0012] In some embodiments, the antigen-binding fragment includes Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv and / or di-scFv.

[0013] In some embodiments, the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, and humanized antibodies.

[0014] In some embodiments, the antibody comprises a heavy chain constant region and a light chain constant region.

[0015] In some embodiments, the antibody heavy chain constant region is derived from the human IgG constant region, and the antibody light chain constant region is derived from human κ or λ.

[0016] In some embodiments, the antibody heavy chain constant region is derived from the human IgG1 constant region.

[0017] In some embodiments, the amino acid sequence of the human IgG1 constant region is as shown in SEQ ID NO: 14.

[0018] In some embodiments, the antibody comprises an antibody heavy chain and an antibody light chain, wherein the amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 15 and the amino acid sequence of the antibody light chain is shown in SEQ ID NO: 16.

[0019] In some embodiments, the antibody comprises an antibody heavy chain and an antibody light chain, wherein the amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 17 and the amino acid sequence of the antibody light chain is shown in SEQ ID NO: 18.

[0020] On the other hand, this application provides a nucleic acid molecule that encodes the antigen-binding protein described in this application.

[0021] On the other hand, this application provides a carrier containing the nucleic acid molecules described in this application.

[0022] On the other hand, this application provides a cell that contains the nucleic acid molecules or vectors described in this application.

[0023] On the other hand, this application provides a pharmaceutical composition comprising the antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, as well as a pharmaceutically acceptable carrier.

[0024] On the other hand, this application provides the use of the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the pharmaceutical composition described in this application in the preparation of a drug.

[0025] On the other hand, this application provides a kit comprising the antigen-binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, and / or the cells described in this application.

[0026] On the other hand, this application provides the use of the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application in the preparation of a drug for the prevention and / or treatment of a disease.

[0027] In some embodiments, the disease and / or condition includes diseases and / or conditions related to CXCR3.

[0028] In some implementations, the disease and / or condition includes autoimmune diseases associated with CXCR3.

[0029] In some embodiments, the disease and / or condition includes allergic diseases associated with CXCR3.

[0030] In some embodiments, the disease and / or condition includes: systemic lupus erythematosus, lupus nephritis, and / or rheumatoid arthritis.

[0031] On the other hand, this application provides a kit comprising the antigen-binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, and / or the cells described in this application.

[0032] In some implementations, the kit is used to detect CXCR3 in a sample.

[0033] On the other hand, this application provides a method for detecting CXCR3 in a sample, the method comprising using the antigen-binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and / or the kit described in this application.

[0034] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0035] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0036] Figure 1The diagram shown is a structural schematic of an antigen-binding protein targeting CXCR3 as described in this application.

[0037] Figure 2 The image shown is a pTT5 plasmid map used to express the antigen-binding protein targeting CXCR3 described in this application.

[0038] Figure 3 The results shown are SDS-PAGE analysis of the antigen-binding protein targeting CXCR3 described in this application, where R represents the reducing condition and NR represents the non-reducing condition.

[0039] Figure 4 The results shown are the affinity of the antigen-binding protein targeting CXCR3 described in this application to CXCR3 as assessed by ELISA.

[0040] Figure 5 The results show that the antigen-binding protein targeting CXCR3 described in this application can block the migration of human CXCR3-expressing cells.

[0041] Figure 6 The results shown are the assessment results of the ADCC effect of the antigen-binding protein targeting CXCR3 described in this application.

[0042] Figure 7 This demonstrates that the antigen-binding protein targeting CXCR3 described in this application can specifically bind to cells overexpressing CXCR3.

[0043] Figure 8 The results show the therapeutic effect of antigen-binding protein targeting CXCR3 on a bovine collagen-induced type II arthritis (CIA) mouse model.

[0044] Figure 9 The results show the therapeutic effect of an antigen-binding protein targeting CXCR3 on systemic lupus erythematosus (SLE) simulated by a mouse MRL / LPR model. Detailed Implementation

[0045] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0046] Terminology Definition

[0047] In this application, the term "CXCR3," also known as "GPR9" or "CD183," generally refers to a G protein-coupled seven-transmembrane receptor, a chemokine receptor expressed in various cell types. CXCR3 is primarily expressed on the surface of activated T cells, plasma cells, memory B cells, and NK cells, and is largely not expressed on naive T cells, naive B cells, and monocytes. In this application, CXCR3 can include all its subtypes. For example, mice have a single subtype of the CXCR3 receptor, while humans have three subtypes: CXCR3A, CXCR3B, and CXCR3-alt. CXCR3-A binds to CXC chemokines CXCL9 (interferon-induced monocytokine, MIG), CXCL10 (interferon-induced protein 10, IP-10), and CXCL11 (I-TAC), while CXCR3-B, in addition to CXCL9, CXCL10, and CXCL11, can also bind to CXCL4. In this application, the CXCR3 may include any naturally occurring CXCR3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. In this application, the CXCR3 may include "full-length," unprocessed CXCR3, and any form of CXCR3 derived from cell processing; it may also include naturally occurring variants of CXCR3, such as splice variants or allelic variants. For example, the accession number for the complete amino acid sequence of human CXCR3 is NP_001495. In this application, the "CXCR3 ligand" may include, but is not limited to, CXCL9, CXCL10, and / or CXCL11. In this application, the "cell expressing CXCR3" may include, but is not limited to, immune cells expressing CXCR3. For example, cells expressing CXCR3 may include CXCR3-expressing T cells, B cells, NK cells, and / or DC cells. For example, cells expressing CXCR3 include, but are not limited to, CXCR3+ / CD4+ T cells, CXCR3+ / CD8+ T cells, and CXCR3+ / CD19+ B cell subsets.

[0048] In this application, the term "immune cell" generally refers to all cells involved in or related to the immune response. Based on function, immune cells can be classified into non-specific immune cells, specific immune cells, and antigen-presenting cells. Non-specific immune cells may include macrophages, neutrophils, natural killer cells (NK cells), mast cells, etc.; specific immune cells may include T cells and B cells; antigen-presenting cells may include dendritic cells (DC cells), macrophages, and B cells, etc.

[0049] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. If a substance or component is found in nature as an "isolated" substance, it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0050] In this application, the term "antigen-binding protein" generally refers to a protein with antigen-binding ability. For example, an antigen-binding protein may include an isolated antigen-binding protein. In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability that has been removed from its naturally occurring state. This "isolated antigen-binding protein" may include an antigen-binding portion and optionally, allow the antigen-binding portion to employ a framework or structural portion that promotes the antigen-binding portion's conformation for binding antigens. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic framework regions containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Examples of antigen-binding proteins include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof.

[0051] In this application, the term "CDR," also known as "complementarity-determining region," generally refers to a region within the variable structural domain of an antibody whose sequence is highly variable and / or forms a structurally defining loop. Typically, an antibody comprises six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel antibodies consisting only of the heavy chain can function normally and stably even in the absence of the light chain. 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 using various coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art, and for example, see www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence number of the antigen-binding protein can be in accordance with the IMGT numbering scheme (IMGT, the international ImMunoGeneTics informationsystem@imgt.cines.fr; 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, DevComp 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, Department of Health and Human Services, NIH Publication No. 91-3242).

[0052] In this application, the term "FR" generally refers to the more conserved portion of the antibody variable domain, which is referred to as the frame region. Typically, the variable domains of the natural heavy and light chains each contain four FR regions: four in VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0053] In this application, the terms "variable domain" and "variable region" are used interchangeably, generally referring to a portion of the antibody heavy chain and / or light chain. The variable domains of the heavy and light chains can be respectively referred to as "V..." H " and "V L (or referred to as "VH" and "VL" respectively). These domains are usually the most varied parts of an antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0054] In this application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ significantly in sequence between antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire variable domain. It is typically concentrated in three segments within the light and heavy chain variable domains, known as hypervariable regions (CDRs or HVRs). The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration, linked by three CDRs forming a ring link, and in some cases forming part of a β-sheet structure. The CDRs in each chain are held together closely by the FR regions, and 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)).

[0055] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof or a derivative thereof, encompassing any polypeptide that includes an antigen-binding site, whether it is produced in vitro or in vivo. This term includes, but is not limited to, polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "complete," such as in "complete antibody," for the purposes of this 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., capable of specifically binding CXCR3). 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 five basic heterotetrameric units and another polypeptide chain called the J chain, containing 10 antigen-binding sites. IgA antibodies consist of 2-5 basic tetrameric units that can bind to the J chain and polymerize to form multivalent combinations. For IgG, a tetrameric unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds depending on the H chain isoform. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for α and γ chains, and four CH domains for μ and ε isoforms. Each L chain has a variable domain (VL) at its 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 thought to form interfaces between the variable domains of the light and heavy chains. 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. L chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of their heavy chain (CH) constant domains. Currently, there are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named α, δ, ε, γ, and μ, respectively.

[0056] In this application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind an antigen (e.g., CXCR3). In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb.

[0057] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, an intact antibody can be digested using papain. After papain digestion, the antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as above). The Fab fragment can consist of a complete L chain with a variable region of a heavy chain and the H chain (V... H The first constant region (C) H 1) Composition.

[0058] In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is slightly larger than the Fab fragment. For example, the Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, the Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0059] In this application, the term "F(ab')2" generally refers to an antibody fragment produced by digesting an intact antibody with pepsin. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. The F(ab')2 fragment has bivalent antigen-binding activity and is capable of cross-linking antigens.

[0060] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy chain variable region and light chain variable region, and lacking the heavy chain constant region and light chain constant region. The heavy chain variable region and light chain variable region include, for example, CDRs. For example, an Fv fragment comprises all or part of the amino-terminal variable region of about 110 amino acids of the heavy and light chains.

[0061] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment including a variable region comprising a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used in this application, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0062] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation consisting of a single molecule. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of antibodies through any particular method. For example, the monoclonal antibodies used in this application can be prepared in hybridoma cells or through recombinant DNA methods.

[0063] 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 from a laboratory animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to elicit an adverse immune response in human individuals compared to parental (e.g., mouse-derived) antibodies.

[0064] In this 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., a murine antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids within the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of the constant region of human immunoglobulins. "Humanized antibodies" retain antigen specificity similar to the original antibody. "Humanized" forms of non-human (e.g., alpaca) antibodies may minimally contain chimeric antibodies with sequences derived from non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues from a non-human species (donor antibody) (such as an alpaca, mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications not found in receptor antibodies or in donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0065] In this application, the term "affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a peptide or antibody) and its binding partner (e.g., a target or antigen). The antigen-binding proteins described in this application can acquire a higher affinity for CXCR3 through affinity maturation. Affinity can be measured using methods commonly known in the art, including but not limited to surface plasmon resonance-based assays, enzyme-linked immunosorbent assays, competitive assays, etc. As used herein, in the context of antigen-binding molecules (e.g., antibodies), the term "affinity maturation" means, for example, that an antigen-binding molecule derived from a reference antigen-binding molecule through mutation binds to the same antigen, preferably to the same epitope, and has a higher affinity for the antigen than for the reference antigen-binding molecule. Affinity maturation generally involves the modification of one or more amino acid residues in one or more CDRs of an antigen-binding molecule. Typically, the affinity-matured antigen-binding molecule binds to the same epitope as the initial reference antigen-binding molecule.

[0066] In this application, the terms "antibody-dependent cytotoxicity" or "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., NK cells, neutrophils, macrophages, etc.) recognize the Fc receptor (FcR) of such cytotoxic cells that mediate ADCC. ADCC can be measured by methods commonly known in the art and can be determined in in vivo or in vitro cell killing assays. In this application, "enhanced antibody-dependent cytotoxicity" generally refers to an increase in the number of target cells lysed within a given time period by means of an antibody at a given concentration in the target cell pericellular medium via the ADCC mechanism defined above, and / or a decrease in the antibody concentration in the target cell pericellular medium required to achieve the lysis of a given number of target cells within a given time period via the ADCC mechanism.

[0067] In this application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue isolated from its natural environment or synthesized artificially.

[0068] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein is inserted, thereby enabling the protein to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material elements they carry to be expressed within the host cells. For example, vectors can include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. The carrier may also include components that help it enter the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0069] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this invention. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). Cells may include cells transfected in vitro using the vectors described in this application. Cells may be bacterial cells (e.g., *E. 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, and NSO cells. Cells may also include engineered cells.

[0070] In this application, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0071] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions such as sodium; and / or nonionic surfactants.

[0072] In this application, the term "kit" generally refers to a product or combination comprising one or more components and suited to achieve a particular purpose. The kit may include one or more active ingredients, detection reagents, auxiliary reagents, buffers, controls, diluents, washing solutions, chromogenic substrates, labels, containers, packaging materials, instructions, or any combination thereof. In some embodiments, the kit includes the CXCR3-targeting antigen-binding protein described in this application. The CXCR3-targeting antigen-binding protein may be labeled or may be used in conjunction with detectable secondary antibodies, avidin-biotin systems, enzymes, fluorescent groups, chemiluminescent groups, radioisotopes, colloidal gold, magnetic beads, or other detectable labels.

[0073] In this application, the terms "specific binding" or "specific" generally refer to measurable and reproducible interactions, such as binding between a target and an antibody, where the presence of the target is determined 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, strength, ease, and / or duration than it binds to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins of different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding.

[0074] In this application, when describing the binding relationship between antigen-binding proteins and CXCR3, the expressions "targeting CXCR3", "against CXCR3", "binding to CXCR3", "specifically binding to CXCR3" or similar expressions can be used interchangeably. Generally, it means that the antibody can specifically recognize and / or bind to CXCR3, including but not limited to binding to one or more epitopes on CXCR3.

[0075] In this application, the term "CXCR3-related diseases and / or conditions" generally refers to any disease and / or condition mediated by or associated with CXCR3 and / or its ligands. For example, CXCR3-related diseases and / or conditions may include diseases and / or conditions associated with the expression of CXCR3 and / or its ligands, or diseases and / or conditions associated with cells expressing CXCR3 and / or its ligands. For example, CXCR3-related diseases and / or conditions may include diseases and / or conditions associated with abnormal CXCR3 and / or its ligand activity. For example, CXCR3-related diseases and / or conditions may include diseases and / or conditions in which the expression levels of CXCR3 and / or its ligands are upregulated relative to those of healthy individuals. For example, CXCR3-related diseases and / or conditions may include allergic diseases and / or autoimmune diseases.

[0076] In this application, the term "allergic disease" generally includes diseases characterized by an allergic and / or atopic immunological response to an antigen, which results in allergic and / or atopic symptoms in patients with the allergic disease. Allergic reactions can include type I, type II, type III, and / or type IV allergic reactions. Type I allergic reactions (immediate anaphylaxis) are mediated by IgE. The antigen (allergen) binds to specific IgE (which binds to tissue mast cells and blood basophils), triggering the release of inflammatory mediators, including pre-existing inflammatory mediators (such as histamine, proteases, and chemokines) and newly synthesized mediators (such as prostaglandins, leukotrienes, platelet-activating factor, and interleukins). These inflammatory mediators can lead to vasodilation, increased capillary permeability, increased mucus secretion, smooth muscle contraction, and infiltration of eosinophils, helper T cells (TH2), and other inflammatory cells into tissues. Type II allergic reactions (antibody-dependent cytotoxic anaphylaxis) are triggered by the binding of antibodies to cell surface antigens or haptens. Surface-bound antigen-antibody structures (as opposed to circulating antigen-antibody complexes in type III hypersensitivity reactions) activate cells involved in antibody-dependent cell-mediated cytotoxicity (such as natural killer cells, eosinophils, and macrophages), complement, or both. This reaction can cause tissue and cellular damage. Type III hypersensitivity reactions are acute inflammatory responses caused by the deposition of circulating soluble antigen-antibody immune complexes in blood vessels or tissues. These immune complexes can activate the complement system or bind to and activate certain immune cells, thereby triggering the release of inflammatory mediators. Type IV hypersensitivity reactions do not involve antibodies but are mediated by T cells.

[0077] In this application, the term "autoimmune disease" generally refers to a disorder or condition resulting from an autoimmune response mediated by antibodies against self-antigens. Autoimmune diseases result in the inappropriate production and / or excessive production of autoantibodies against self-antigens or self-antigens. Autoimmune diseases are systemic diseases that can occur in virtually all parts of the body, including the nervous system, gastrointestinal system, endocrine system, skin, skeletal system, and vascular tissue. "Self-antigen" refers to an endogenous antigen that stimulates an autoimmune response, such as the production of autoantibodies. Self-antigens also include autoantigens or antigens from normal tissues that are targets of cell-mediated or antibody-mediated immune responses that can lead to the development of autoimmune diseases. For example, "autoimmune disease" as described in this application can include autoimmune diseases mediated by immune cells. For example, said autoimmune diseases can include autoimmune diseases mediated by immune cells expressing CXCR3.

[0078] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0079] In this application, homology generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence homology percentage" can be calculated by comparing two sequences to be aligned within a comparison window, determining the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence homology percentage. Alignments performed to determine the sequence homology percentage can be performed in various 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 suitable parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. Homology can also be determined using FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Pearson and D. J. Lipman, “An Improved Tool for Biological Sequence Alignment,” Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and D. J. Lipman and W. R. Pearson, “A Fast and Sensitive Search for Protein Similarity,” 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 Tool for Local Alignment Search,” Journal of Molecular Biology, 215: 403-410, 1990.

[0080] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0081] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 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. Invention Details

[0083] antigen-binding proteins

[0084] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at 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 antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR, including amino acid sequences with substitutions, deletions, and / or additions of one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; this also includes homologs, which can be amino acid sequences having at least about 85% (e.g., having 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 higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the antigen-binding protein described in this application can be defined by the IMGT coding system.

[0085] On one hand, this application provides an antigen-binding protein capable of specifically binding to CXCR3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. For example, the CXCR3 includes human CXCR3 or cells expressing human CXCR3.

[0086] In some embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO: 21, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO: 22, SEQ ID NO: 5, and SEQ ID NO: 23, respectively.

[0087] In some embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO: 1-3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 4-6, respectively.

[0088] In some embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO: 7, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO: 8, SEQ ID NO: 5, and SEQ ID NO: 9, respectively.

[0089] The CDR sequences of antigen-binding proteins 6-8 and 6-10 targeting CXCR3 in this application, as defined by the IMGT coding system, are shown in Table 1.

[0090] Table 1

[0091]

[0092] In some embodiments, the heavy chain variable region of the antigen-binding protein described in this application may contain an amino acid sequence as shown in SEQ ID NO: 10 or 12, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10 or 12.

[0093] In some embodiments, the light chain variable region of the antigen-binding protein described in this application may contain an amino acid sequence as shown in SEQ ID NO: 11 or 13, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11 or 13.

[0094] In some embodiments, the antigen-binding protein described in this application may include a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 11.

[0095] In some embodiments, the antigen-binding protein described in this application may include a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 12 and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 13.

[0096] In this application, the antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.

[0097] In some embodiments, the antigen-binding fragment may include Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv and / or di-scFv.

[0098] In some embodiments, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0099] In this application, the antigen-binding protein may include an antibody heavy chain constant region. In some embodiments, the antibody heavy chain constant region may be derived from the human IgG heavy chain constant region. In some embodiments, the isolated antigen-binding protein may be derived from the human IgG1 heavy chain constant region. For example, the human IgG1 heavy chain constant region may contain the amino acid sequence shown in SEQ ID NO: 14.

[0100] In this application, the isolated antigen-binding protein may comprise an antibody heavy chain (HC). In some embodiments, the HC may comprise the amino acid sequence shown in SEQ ID NO: 15 or 17.

[0101] In this application, the antigen-binding protein may include an antibody light chain constant region. In some embodiments, the antibody light chain constant region may be derived from human κ or λ.

[0102] In some embodiments, the isolated antigen-binding protein may comprise an antibody light chain (LC). In some embodiments, the LC may comprise the amino acid sequence shown in SEQ ID NO: 16 or 18.

[0103] In some embodiments, the antigen-binding protein may include an antigen-binding protein with enhanced effector function. The enhanced effector function may include a measurable increase in any one or more of the following: antibody-dependent cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and / or antibody-dependent cell-mediated phagocytosis (ADCP) against suitable target cells.

[0104] In some embodiments, the antigen-binding protein described herein may comprise a variant of the human IgG1 Fc region. A human IgG1 Fc region variant generally refers to a human IgG1 Fc region that has been engineered or modified to include one or more amino acid mutations or modifications compared to the wild-type human IgG1 Fc.

[0105] In some cases, the antigen-binding protein described in this application may have a reduced fucose content or be defucosylated. In some cases, the antigen-binding protein described in this application may include an antigen-binding protein that is substantially free of fucose. In some cases, the antigen-binding protein described in this application may include an antigen-binding protein that is substantially free of ribofucose or has ribofucose removed from the N-glycan attached to the CH2 domain of Fc. In some cases, having a reduced fucose content or being defucosylated can result in enhanced antibody-dependent cytotoxicity (ADCC). For example, the antigen-binding protein described in this application may be expressed or produced by engineered cells that can provide altered glycosylation examples of antibodies expressed therefrom, such as non-fucosylated cell lines or FUT8 knockout CHO cell lines.

[0106] In this application, the antigen-binding protein specifically binds to CXCR3. In some embodiments, the binding of the antigen-binding protein to CXCR3 can be detected by an ELISA method. For example, the antigen-binding protein of this application can bind to CXCR3 with an EC50 value of less than or equal to about 0.10 μg / mL, less than or equal to about 0.09 μg / mL, less than or equal to about 0.08 μg / mL, less than or equal to about 0.07 μg / mL, less than or equal to about 0.06 μg / mL, or less than or equal to about 0.05 μg / mL.

[0107] The CXCR3 antigen-binding protein described in this application 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 this application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0108] The antigen-binding protein provided in this application can be used to antagonize CXCR3 activity. In this application, the antigen-binding protein is capable of preventing and / or treating diseases and / or conditions. The antigen-binding protein described in this application can possess one or more of the following functions to exert a therapeutic effect on diseases and / or conditions: 1) inhibiting the binding of CXCR3 to one or more ligands (e.g., CXCL9, CXCL10, and / or CXCL11); 2) inhibiting CXCR3-mediated signal transduction; 3) inhibiting the migration, accumulation, recruitment, and / or infiltration (e.g., to sites of inflammation) of cells expressing CXCR3; 4) mediating the killing effect on cells expressing CXCR3; 5) inducing endocytosis of CXCR3 receptors on the cell surface, thereby reducing the activation of immune cells.

[0109] Nucleic acid molecules, vectors, cells, drug compositions and reagent kits

[0110] On the other hand, this application provides nucleic acid molecules that can encode the antigen-binding protein described in this application or contain a nucleotide sequence encoding the antigen-binding protein described in this application. For example, it can be produced or synthesized by: (i) in vitro amplification, for example by polymerase chain reaction (PCR); (ii) by clonal recombination; (iii) purification, for example by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, for example by chemical synthesis.

[0111] On the other hand, this application provides a vector that may contain the nucleic acid molecule described in this application. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain 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 may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells to express the genetic material elements it carries within the host cells. The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. Furthermore, the vector may also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but not only these substances.

[0112] On the other hand, this application provides a cell that may contain the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain one or more of the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more (e.g., two or more) nucleic acid molecules or vectors described in this application. For example, the vectors described in this application may be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi, or yeast cells. In some embodiments, the cell may be a bacterial cell (e.g., 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, NSO cells, or myeloma cells. The vectors described in this application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0113] The cells described in this application may also include engineered cells. For example, the cells may provide examples of altered glycosylation by antibodies expressed therein, such as non-fucosylated cell lines or FUT8 knockout CHO cell lines. Alternatively, the cells may lack one or more glycosidases required for early N-glycan treatment and / or the culture conditions may inhibit the activity of one or more of these glycosidases. For example, the cells may lack one or more glycosidases, such as α-glucosidase I, α-glucosidase II, and α-mannosidase I. Furthermore, or alternatively, engineered cells may be contacted with inhibitors of one or more glycosidases (such as α-glucosidase I, α-glucosidase II, and α-mannosidase I). In some embodiments, the inhibitor is an inhibitor of α-mannosidase I, such as the α-mannosidase I-specific inhibitor, kifbase.

[0114] On the other hand, this application also provides pharmaceutical compositions that may comprise the antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable carrier.

[0115] In some embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0116] In some embodiments, the pharmaceutical composition may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0117] In some embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration and are generally safe and non-toxic.

[0118] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously. This continuous (or uninterrupted) administration may be achieved using a small pump system worn by the patient to measure the amount of therapeutic agent flowing into the patient, as described in WO2015 / 036583.

[0119] On the other hand, this application also provides a kit which may contain the antigen-binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, and / or the cell described in this application.

[0120] The kit described in this application can be used to detect, identify, quantify, and / or analyze CXCR3 in samples, or to detect, sort, or analyze cells expressing CXCR3. The samples may include biological samples, cell samples, tissue samples, blood samples, serum samples, plasma samples, body fluid samples, cell lysates, tissue sections, cell suspensions, cultured cells, or any combination thereof. The detection can be performed by flow cytometry, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, cell sorting, or other detection methods based on antigen-antibody specific binding.

[0121] Preparation method

[0122] On the other hand, this application provides a method for preparing the antigen-binding protein. The method may include culturing the cells of this application under conditions that allow the antigen-binding protein to be expressed. For example, this can be achieved by using appropriate culture media, appropriate temperatures, and culture times, methods known to those skilled in the art.

[0123] In some embodiments, the cell contains a nucleic acid molecule encoding the antigen-binding protein described in this application, or contains the expression vector described in this application. The host cell may be a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a mammalian cell, such as CHO cells, HEK293 cells, NSO cells, SP2 / 0 cells, or other cells suitable for expressing antibodies or antigen-binding fragments.

[0124] In some embodiments, the host cells can be cultured using appropriate culture media, culture temperature, culture time, pH, dissolved oxygen conditions, feeding methods, and / or induction conditions to induce expression of the antigen-binding protein. The culture conditions can be adjusted according to the host cell type, expression vector type, target protein properties, and production scale, and these conditions can be determined by those skilled in the art using conventional methods. In some embodiments, the antigen-binding protein can be secreted into the culture medium or present within the host cells, on the cell membrane, or in cell lysates. The method may further include the steps of harvesting the culture supernatant, harvesting cells, lysing cells, and / or obtaining a sample containing the antigen-binding protein.

[0125] In some embodiments, the antigen-binding protein described in this application is an isolated antigen-binding protein. The preparation method further includes the step of isolating and / or purifying the antigen-binding protein from the culture system, culture supernatant, cells, cell lysates, or other samples containing the antigen-binding protein. The isolation and / or purification can be performed using methods known in the art, including but not limited to affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, protein A chromatography, protein G chromatography, antigen affinity chromatography, filtration, ultrafiltration, dialysis, centrifugation, precipitation, or any combination thereof.

[0126] Methods and uses

[0127] On the other hand, this application provides methods for preventing and / or treating diseases and / or conditions, which may include administering the antigen-binding protein, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition described in this application to a subject in need.

[0128] On the other hand, this application also provides the use of the antigen-binding protein, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the prevention and / or treatment of diseases.

[0129] On the other hand, this application also provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the cell and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0130] In this application, the disease and / or condition may include CXCR3-related diseases and / or conditions. In some cases, the disease and / or condition may include allergic diseases. In some cases, the disease and / or condition may include CXCR3-related allergic diseases. In some cases, the disease and / or condition may include autoimmune diseases. In some cases, the disease and / or condition may include CXCR3-related autoimmune diseases. In some cases, the disease and / or condition may include diseases and / or conditions involved or mediated by cells expressing CXCR3. In some cases, the disease and / or condition may include, but is not limited to: systemic lupus erythematosus, lupus nephritis, and / or rheumatoid arthritis.

[0131] In this application, the use can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration. Subjects to be treated by the methods provided herein may include humans or other mammals.

[0132] In this application, the subject in need may include those who have been diagnosed with CXCR3-related diseases and / or conditions, or who are predisposed to developing CXCR3-related diseases and / or conditions.

[0133] In some implementations, subjects may receive preventative treatment or treatment after the onset of disease and / or symptoms. In some implementations, subjects may receive preventative treatment using the methods provided herein before the onset or worsening of CXCR3-related disease and / or symptoms.

[0134] In some embodiments, the subject in need may include mammals. In some embodiments, the subject in need may include humans.

[0135] On the other hand, this application also provides a method for detecting CXCR3 in a sample, the method comprising administering the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cells, and / or the kit.

[0136] In some cases, the method for detecting CXCR3 in a sample may be an in vitro method. For example, the isolated antigen-binding protein described in this application is brought into contact with an in vitro sample to detect the presence and / or content of CXCR3 in the sample. In some cases, the method for detecting CXCR3 in a sample is for non-therapeutic purposes. In some cases, the method for detecting CXCR3 in a sample is not a diagnostic method.

[0137] On the other hand, this application also provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the vector, and / or the cells in the preparation of a kit. The kit can be used to detect the presence and / or content of CXCR3 in a sample.

[0138] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the antigen-binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0139] Example

[0140] The present application is further illustrated by the following specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods not specifically described in the following embodiments are performed according to conventional conditions in the art, such as those described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (Third Edition) (2001), CSHL Press, or according to the manufacturer's recommendations. Unless otherwise stated, all experimental materials and reagents used in the following embodiments are commercially available.

[0141] Table 2. Reagents used in the embodiments of this application.

[0142]

[0143] Table 3. Instruments used in the embodiments of this application

[0144]

[0145] Example 1: Preparation of anti-CXCR3 antibody

[0146] 1.1. Construction of anti-CXCR3 antibody plasmid

[0147] The heavy and light chain gene sequences of the anti-CXCR3 antibody (antibody structure as shown in the image) were obtained. Figure 1 As shown, the Fc domain is derived from human IgG1 antibody), and is inserted into the pTT5 vector (e.g. Figure 2 The HindIII-NotI expression cassette (shown) was used to obtain the recombinant plasmid. The constructed plasmid was sequenced to obtain the correctly sequenced recombinant plasmid, which was then extracted using the Qiagen Midi plasmid extraction kit.

[0148] 1.2. Transient expression of anti-CXCR3 antibody

[0149] In this embodiment, anti-CXCR3 antibody was produced using the ExpiCHO expression system. Frozen ExpiCHO-S cells were revived and cultured (using ExpiCHO Expression Medium). After the cells reached normal growth rate and underwent at least two passages, transfection was performed.

[0150] The cell density was adjusted to 3×10⁻⁶ the day before transfection. 6 - 4×10 6 cells / ml, and the cell density increased to 7-10 × 10⁻⁶ on the second day. 6 cells / ml.

[0151] On the day of transfection, the cell density was diluted to 6 × 10⁶ cells using fresh ExpiCHO Expression Medium preheated to 37°C. 6 cells / ml. ExpiFectamin CHO Reagent and plasmid DNA (heavy chain:light chain = 1:1) were diluted separately using cold OptiPR medium. The diluted ExpiFectamin CHO Reagent was added to the diluted plasmid, and the mixture was inverted to mix thoroughly, preparing an ExpiFectamin CHO / plasmid DNA mixture. After incubating at room temperature for 5 min, the mixture was slowly added to ExpiCHO-S cell suspension, mixed well, and incubated at 37°C in an 8% CO2 incubator. One day after transfection, 120 μL of ExpiCHO Enhancer and 4.8 mL of ExpiCHO Feed were added to the cell suspension, and expression was continued at 37°C.

[0152] Seven days after transfection, the supernatant was collected by centrifugation and used for antibody purification.

[0153] 1.3. Purification and Analysis of Anti-CXCR3 Antibody

[0154] The antibody-cell expression supernatant obtained in the previous step was purified using a Mabselect SuRe affinity chromatography column. After supernatant capture, the sample was washed with 50 mM Tris + 150 mM NaCl, and then eluted with 0.1 M Glycine pH 3. The eluted protein was then adjusted to pH 8 by adding 1 M Tris HCl. The purified protein was then analyzed using SDS-Page, as shown in the image. Figure 3 .

[0155] Example 2: Determination of anti-CXCR3 antibody affinity

[0156] This embodiment assesses the affinity of anti-CXCR3 antibodies for human CXCR3 VLP (virus-like particle) using ELISA.

[0157] Experimental methods:

[0158] Prepare the experimental solutions according to the preparation method shown in Table 4.

[0159] Table 4

[0160]

[0161] Sample dilution: The Human CXCR3 antibody was first diluted to 4 μg / ml using Dilution buffer, and then further diluted 1:2.5 for 10 gradients, for a total of 11 concentrations.

[0162] The specific operating steps are as follows:

[0163] (1) Coating antibody with Stripwell Microplate according to sample quantity: Dilute Human CXCR3 Full Length Protein-VLP to 5 μg / ml using Coating buffer, 50 μl / well, and incubate overnight at 4°C in the dark;

[0164] (2) Remove the coated microplate and wash it 4 times with 300 μl / well of washing buffer.

[0165] (3) Add 100 μl of Blocking buffer per well, incubate at 37°C in the dark for 1.5 h, then wash 4 times with 300 μl of Washing buffer per well, shake off and pat dry.

[0166] (4) Add 50 μl of the diluted standard and sample per well and incubate at 37°C in the dark for 1 h. Then wash 4 times with 300 μl of washing buffer per well and shake off the excess water.

[0167] (5) Add 50 μL of the diluted secondary antibody to each well and incubate at 37°C in the dark for 1 hour. Then wash 4 times with 300 μL of washing buffer per well and shake off the excess water.

[0168] (6) Add 75 μl of chromogenic solution to each well, incubate at 37°C for 15 min, then remove and add 75 μl of stop solution to each well. Place in a microplate reader and set the OD450 reading.

[0169] The control antibody 53hu37 is also an antibody targeting CXCR3. Its heavy chain variable region amino acid sequence is shown in SEQ ID NO: 19, and its light chain variable region amino acid sequence is shown in SEQ ID NO: 20. The control antibody was prepared using the same method as in Example 1. The results are as follows: Figure 4 As shown, anti-CXCR3 antibodies 6-8 (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 11) and 6-10 (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 13) can effectively bind to human CXCR3. The EC50 value of 6-8 is 0.03423 μg / mL, and the EC50 value of 6-10 is 0.02431 μg / mL; the EC50 value of the control antibody binding to human CXCR3 is 0.04303 μg / mL.

[0170] Example 3: Anti-CXCR3 antibody blocks chemokine-induced migration assay

[0171] In this embodiment, the inhibitory effect of the antibody described in this application on the migration of human CXCR3-expressing cells was evaluated by a chemokine-induced migration assay.

[0172] In this embodiment, the number of cells migrating from the upper chamber of the Transwell (containing Jurkat cells and antibodies overexpressing human CXCR3) to the lower chamber of the Transwell (containing ligands CXCL9, CXCL10, or CXCL11) was detected by flow cytometry, and the ability of the antibody to block CXCR3-mediated cell migration was determined.

[0173] Experimental methods:

[0174] Prepare the experimental solutions according to the preparation method shown in Table 5.

[0175] Table 5

[0176]

[0177] The specific operating steps are as follows:

[0178] (1) Cell and antibody treatment: The total volume of each well is 100 μL, with cell suspension and antibody each accounting for 50% of the total volume. The antibody dosage is calculated at 50 μL / well, and the antibody concentration is 50 μg / mL. Add 50 μL of antibody to the cells at a rate of 50 μL / well, mix well, and then incubate the cells at 37°C for 30 min.

[0179] (2) Ligand preparation: Calculate the amount of ligands working solution to be used based on 300 μL / well volume. Dilute ligands to the corresponding concentrations using migration buffer (the concentrations in this experiment are CXCL9: 250 ng / mL, CXCL10: 20 ng / mL, CXCL11: 40 ng / mL).

[0180] (3) Add 300 μL / well to the lower chamber of the tanswell. Set up the no Ab no ligand group and add 300 μL migration buffer to the lower chamber of the tanswell.

[0181] (4) After incubating the cells for 30 min, mix the cell suspension and transfer the cell suspension to the upper chamber of the transwell. Migrate at 37℃ for 4 h.

[0182] (5) Prepare reference cells: Jurkat-WT cell counting, according to the number of samples, at 3×10⁻⁶. 4 / well prepares cells and prepares them in MACS solution.

[0183] (6) After migration, transfer the cells from the lower chamber of the transwell to flow cytometry tubes, adding 0.5 ml of 3 × 10⁻⁶ cells to each tube. 4 The reference cells.

[0184] (7) Centrifuge at 500g for 5 minutes. After centrifugation, resuspend the cells and mix well before loading them onto the machine.

[0185] (8) Use Cytek to select GFP (the fluorescent tag carried by CXCR3 expressing cells), circle single cells and GFP+ / - cells, and record 10,000-20,000 cells / samples.

[0186] (9) During FlowJo analysis, single cells are circled, and the ratio of GFP+ to GFP- in the single cells is calculated. The number of GFP+ cells is then calculated as (GFP+ / GFP-) × 3 × 10. 4 This refers to the number of cells that migrated to the lower chamber of the transwell.

[0187] (10) Migration Index = Number of cells in the experimental group / Number of cells in the no Ab no ligand group.

[0188] Experimental results are as follows Figure 5As shown, the anti-CXCR3 antibodies 6-8 and 6-10 provided in this application can inhibit the migration of CXCR3-expressing cells induced by CXCL9, CXCL10, and CXCL11, and their inhibitory effect on the migration of CXCR3-expressing cells is superior to that of the control antibody. CXCR3 plays a crucial role in the migration of activated immune cells, mediating the chemotaxis of T cells and B cells to inflammatory sites. Therefore, by inhibiting the migration of CXCR3-expressing cells induced by CXCL9, CXCL10, and / or CXCL11, the antibodies in this application can interfere with the cell migration process mediated by CXCR3-related chemotactic signals, thereby potentially reducing the recruitment and aggregation of CXCR3-positive immune cells to inflammatory areas, and thus helping to alleviate local immune responses.

[0189] Example 4: Evaluation of ADCC effect of anti-CXCR3 antibody

[0190] This embodiment illustrates that the anti-CXCR3 antibody described in this application can recognize target cells expressing CXCR3. Simultaneously, the Fc fragment of the antibody binds to the Fc receptor CD16a (158V) on the surface of effector cells NK-MI-CD16a (158V), mediating the release of cytotoxic molecules from effector cells NK-MI-CD16a (158V) to kill target cells expressing CXCR3. Lactate dehydrogenase (LDH) from the killed target cells is released into the culture medium; the killing status of the target cells is confirmed by detecting the amount of LDH.

[0191] Experimental methods:

[0192] Prepare the experimental solutions according to the preparation method shown in Table 6.

[0193] Table 6

[0194]

[0195] The specific operating steps are as follows:

[0196] (1) Wash the target cells 1-2 times with Assay medium, discard the supernatant, resuspend the cells with Assay medium and count them, 25,000 cells / well, 25 μl / well.

[0197] (2) Antibody preparation: Dilute the antibody with MACS buffer to the corresponding concentration 4 times, add 25 μl / well to the cells, mix well, and incubate at 37°C for 30 min.

[0198] (3) Prepare effector cells: Take NK92-CD16a (158V) cells and add 50 μl of cell suspension to each well of a 96-well plate, 75,000 cells / well, and mix well.

[0199] (4) Incubation: Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 4 hours. For High Control, add 5 μl of Lysis solution to each well 15 minutes before the end of incubation and mix well.

[0200] (5) Color development: Add 100 μl of fresh reaction mixture (blue cap: red cap = 1:45, freshly prepared, cytotoxicity test kit) to each well, mix well, and develop color at room temperature for 25 min in the dark.

[0201] (6) Termination: Add 50 μl of stop solution (green cap) to each well and mix well.

[0202] (7) Reading: The absorbance of the microplate reader is measured at wavelengths of 492 nm and 690 nm (reference wavelength).

[0203] (8) Data processing: Subtract the background control from all holes, then substitute it into the formula to calculate the kill percentage.

[0204] Cytotoxicity (%) = (well to be tested - effector control - low control) / (high control - low control) * 100

[0205] Experimental results are as follows Figure 6 As shown, the anti-CXCR3 antibodies 6-8 and 6-10 provided in this application can effectively mediate ADCC effects. The EC50 value of 6-8 is 7.896 ng / mL, and the EC50 value of 6-10 is 5.776 ng / mL. The EC50 value of the control antibody mediating ADCC effects is 13.18 ng / mL. The antibodies 6-8 and 6-10 of this application have stronger ADCC effect mediating ability.

[0206] Example 5: Specific binding activity of anti-CXCR3 antibody

[0207] In this embodiment, the ability of the antibody to specifically bind to CXCR3 was determined by detecting the binding ability of the antibody to cells overexpressing CXCR3, cells overexpressing other chemokines, and wild-type cells.

[0208] Experimental methods:

[0209] Prepare the experimental solutions according to the preparation method shown in Table 7.

[0210] Table 7

[0211]

[0212] The specific operating steps are as follows:

[0213] (1) Cell counting: Mix the cell suspension, take 10 μl of cell suspension and spot it on the spotting stage of the cell counter. Repeat three times and calculate the average value.

[0214] (2) Add 1×10 to each hole 5 1 cell, 25 μl / well, 500g, centrifuged at 4℃ for 5 min, discard supernatant.

[0215] (3) Antibody preparation: Dilute the antibody with MACS buffer to twice the corresponding concentration, add 25 μl / well to the cells, mix well, and incubate at 4°C for 30 min.

[0216] (4) After incubation, add 200 μl of MACS buffer per well for washing: centrifuge at 500 g for 5 min at 4 °C and discard the supernatant. Wash twice.

[0217] (5) Secondary antibody preparation: First, prepare DAPI working solution, then dilute the secondary antibody with DAPI working solution to a final concentration of 0.5 μg / mL, add 50 μl / well to the cells, mix well, and incubate at 4℃ for 30 min.

[0218] (6) After incubation, add 200 μl of MACS buffer per well for washing: centrifuge at 500 g for 5 min at 4 °C and discard the supernatant. Wash once or twice.

[0219] (7) After discarding the supernatant, add 100 μl / well of MACS buffer to resuspend the cells for flow cytometry detection.

[0220] (8) Statistical analysis of gMFI of secondary antibody fluorescence in live cells.

[0221] In this embodiment, the binding ability of the antibody (10 μg / mL) of this application to (1) CHO cells overexpressing human CXCR3, (2) CHO cells overexpressing human CXCR5, (3) CHO cells overexpressing human CCR7, (4) CHO cells overexpressing human CCR9, and (5) WT CHO cells was detected by flow cytometry.

[0222] Experimental results are as follows Figure 7 As shown, the antibody provided in this application only exhibits significant fluorescence signal in CXCR3-overexpressing cells, while showing virtually no signal in other cell groups. This experimental result demonstrates that the antibody described in this application has good specificity for CXCR3.

[0223] Example 6: In vivo study of anti-CXCR3 antibody in an arthritis model

[0224] In this embodiment, the therapeutic effect of the antibody described in this application on rheumatoid arthritis was explored by using a bovine collagen-induced arthritis (CIA) mouse model to simulate rheumatoid arthritis in humans.

[0225] Experimental methods:

[0226] The mice used in this experiment were 10-week-old male DBA1 mice. Thirty-two DBA1 mice were used to establish an arthritis model induced by bovine collagen type II (CII) and were randomly divided into four groups according to the severity of the disease at 1-2 minutes after the onset of the disease. The negative control group received an intraperitoneal injection of 500 μg mouse-IgG each time, while the experimental group received an intraperitoneal injection of 500 μg CXCR3-173-ADCC (Surrogate with ADCC, an anti-mouse CXCR3-173 monoclonal antibody used as a surrogate antibody in preclinical experiments; CXCR3-173 is described as a blocking antibody that does not deplete CD4+ T cells in vivo (see Uppaluri et al., Transplantation 86: 137-47 (2008), for information on using CXCR3-173 as a surrogate antibody see WO2018119288A1; CXCR3-173-ADCC is a molecule of CXCR3-173 with mouse Fc and defucosylated). Another experimental group received an intraperitoneal injection of 500 μg CXCR3-173-LALA (Surrogate no ADCC). It is also a mouse-derived Fc, whose Fc region contains L234A / L235A mutations), and the positive control group was injected intraperitoneally with 200 μg of anti-TNFa (trade name: InVivoMAb anti-mouse). TNFα (brand name: bioxcell / 100mg, catalog number: BE0058) was administered three times a week. After starting administration, the degree of joint swelling in mice was scored two to three times a week. The scoring criteria were as follows: no joint redness and swelling = 0 points; inflammation and swelling of the toes = 1 point; inflammation and swelling of the toes and dorsum of the foot = 2 points; inflammation and swelling of the toes, dorsum of the foot, and ankle joints = 3 points; severe swelling, with severe inflammation and swelling of the toes, dorsum of the foot, and ankle joints, = 4 points. The scores for joint swelling of all four limbs were added together to obtain the total score for arthritis. Simultaneously, mouse joint sections were stained with hematoxylin and eosin to assess pathological conditions, including the degree of synovial cell proliferation, cell erosion, pannus, inflammation, and bone erosion. The level of anti-CII-IgG autoantibodies in mouse serum was detected using ELISA.

[0227] Experimental results are as follows Figure 8As shown, administration of CXCR3-173-ADCC significantly alleviated the disease progression of arthritis (degree of joint swelling), with clinical scores significantly lower than the negative control group and similar to the positive control group. In contrast, administration of CXCR3-173-LALA failed to alleviate the disease progression due to its lack of cell-killing properties. The pathological scores of the joint portion of CXCR3-173-ADCC and the antibody levels against the autoantigen were lower than the negative control group and comparable to the positive control group. These results demonstrate that the antibody described in this application has a good therapeutic effect on CIA in mice, suggesting that antibodies targeting CXCR3 have a therapeutic effect on rheumatoid arthritis in humans.

[0228] Example 7: In vivo study of anti-CXCR3 antibody in a systemic lupus erythematosus model

[0229] In this embodiment, the therapeutic effect of the antibody described in this application on systemic lupus erythematosus (SLE) was explored by using a mouse MRL / LPR model to simulate SLE in humans.

[0230] Experimental methods:

[0231] The mice used in this experiment were 8-week-old male MRL / LPR female mice. Forty MRL / LPR mice were randomly divided into four groups based on body weight. The negative control group received an intraperitoneal injection of 1 mg mouse-IgG each time. The experimental groups received an intraperitoneal injection of 1 mg CXCR3-173-ADCC (Surrogate with ADCC, same as in Example 7) each time. Another experimental group received an intraperitoneal injection of 1 mg CXCR3-173-LALA (Surrogate no ADCC, same as in Example 7) each time. The positive control group received an intraperitoneal injection of 1 mg anti-CD20 (trade name: InVivoMAb anti-mouse CD20, brand specification: bioxcell / 100mg, catalog number: BE0356). All injections were administered once a week. The presence of skin ulceration and lymph node enlargement was observed in the mice to determine the proportion of these conditions within each group. Flow cytometry was used to detect the number of inflammatory cells in the kidneys to assess their infiltration level, and ELISA was used to detect the level of anti-dsDNA-IgG autoantibodies in the mice.

[0232] Experimental results are as follows Figure 9 As shown, administration of CXCR3-173-ADCC significantly reduced the proportion of skin ulceration and lymphadenopathy. Mice treated with CXCR3-173-ADCC also showed reduced renal inflammatory cell infiltration and antibody levels against autoantigens compared to the negative control group. These results demonstrate the therapeutic efficacy of the CXCR3-targeting antibody in a mouse model, suggesting its potential therapeutic effect on human systemic lupus erythematosus.

Claims

1. An antigen-binding protein capable of specifically binding to CXCR3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein: a) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 1-3, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 4-6, respectively; or b) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 8, SEQ ID NO: 5 and SEQ ID NO: 9, respectively.

2. The antigen-binding protein according to claim 1, wherein: a) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11; or b) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

13.

3. The antigen-binding protein according to any one of claims 1-2, comprising an antibody or an antigen-binding fragment thereof.

4. The antigen-binding protein according to claim 3, wherein the antigen-binding fragment comprises Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv and / or di-scFv.

5. The isolated antigen-binding protein according to claim 3, wherein the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, and humanized antibodies.

6. The antigen-binding protein according to claim 3, wherein the antibody comprises a heavy chain constant region and a light chain constant region.

7. The antigen-binding protein according to claim 6, wherein the antibody heavy chain constant region is derived from the human IgG constant region, and the antibody light chain constant region is derived from human κ or λ.

8. The antigen-binding protein according to claim 3, wherein the antibody comprises an antibody heavy chain and an antibody light chain, wherein: a) The amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 15, and the amino acid sequence of the antibody light chain is shown in SEQ ID NO: 16; or b) The amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the antibody light chain is shown in SEQ ID NO:

18.

9. A nucleic acid molecule encoding an antigen-binding protein as described in any one of claims 1-8.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

12. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-8, the nucleic acid molecule of claim 9, the carrier of claim 10, and / or the cell of claim 11, and a pharmaceutically acceptable carrier.

13. Use of the isolated antigen-binding protein of any one of claims 1-8, the nucleic acid molecule of claim 9, the carrier of claim 10, and / or the cell of claim 11 in the preparation of a medicament.

14. The use according to claim 13, wherein the drug is used to treat systemic lupus erythematosus, lupus nephritis and / or rheumatoid arthritis.

15. A kit comprising the antigen-binding protein of any one of claims 1-8, the nucleic acid molecule of claim 9, the vector of claim 10, and / or the cell of claim 11.

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