Antigen binding protein targeting CXCR3 and use thereof

Abstract

An antigen binding protein targeting CXCR3 and the use thereof. The antigen binding protein can specifically bind to CXCR3. The antigen binding protein contains a heavy chain variable region, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3.

Description

Antigen binding proteins targeting CXCR3 and their applications Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting CXCR3. Background Art

[0002] Chemokines are a class of cell-secreted cytokines or signaling proteins known as chemokines because they can induce targeted chemotaxis of nearby responding cells. Chemokines and chemokine receptors have been shown to influence numerous 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, NK cells, mast cells, and dendritic 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. Studies have shown that CXCR3 and its ligands, CXCL9, CXCL10, and CXCL11, are closely associated with the development and progression of numerous diseases. For example, upregulation of CXCR3 is implicated in a range of autoimmune disorders. CXCR3 expression is essentially absent in naive T cells and is upregulated following activation with antigen. CXCR3 recruits these cells, including T helper type 1 (Th1) cells, to sites of tissue inflammation in response to its primary ligand.

[0003] Therefore, there is a need in the art for improved antigen binding proteins targeting CXCR3 that are useful as therapeutic agents. Summary of the Invention

[0004] The present application provides an antigen binding protein capable of targeting CXCR3, which can inhibit the binding of the receptor and its ligand by targeting CXCR3, thereby preventing the migration of immune cells, or eliminate activated immune cells by mediating the killing effect on cells expressing CXCR3, thereby treating diseases and / or conditions related to excessive activation of immune cells.

[0005] The antigen binding protein targeting CXCR3 provided in the present application may have one or more of the following properties: 1) can specifically bind to CXCR3 and / or cells expressing CXCR3; 2) can inhibit the binding of CXCR3 to at least one of its ligands; 3) can inhibit CXCR3-mediated signal transduction; 4) can inhibit the migration, accumulation, recruitment and / or infiltration of cells expressing CXCR3; 5) can mediate the killing effect on cells expressing CXCR3; 6) can induce the endocytosis of CXCR3 receptors on the cell surface, thereby reducing the activation of immune cells; 7) can be used to treat CXCR3-related diseases and / or conditions; 8) can be used for the detection of CXCR3 and / or cells expressing CXCR3.

[0006] The present application also provides nucleic acid molecules encoding the isolated antigen-binding protein, expression vectors, host cells, immunoconjugates comprising the antigen-binding protein, pharmaceutical compositions, methods for preparing the isolated antigen-binding protein, and uses of the isolated antigen-binding protein described herein.

[0007] On the one hand, the present application provides an isolated antigen-binding protein that can specifically bind to CXCR3, wherein the antigen-binding protein comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14.

[0008] In certain embodiments, the HCDR1, HCDR2, and HCDR3 are selected from any one of the following groups:

[0009] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15;

[0010] (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16;

[0011] (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16;

[0012] (4) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.

[0013] In certain embodiments, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-2.

[0014] In certain embodiments, the HCDR2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 4-7.

[0015] In certain embodiments, the HCDR3 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 11-13.

[0016] In certain embodiments, the HCDR1, HCDR2, and HCDR3 are selected from any one of the following groups:

[0017] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0018] (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0019] (3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12;

[0020] (4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0021] (5) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0022] (6) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13;

[0023] (7) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0024] (8) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13;

[0025] (9) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13.

[0026] In certain embodiments, the isolated antigen-binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 19.

[0027] In certain embodiments, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0028] In certain embodiments, the isolated antigen-binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 25.

[0029] In certain embodiments, the H-FR2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 20-24.

[0030] In certain embodiments, the isolated antigen-binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 38.

[0031] In certain embodiments, the H-FR3 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29-37.

[0032] In certain embodiments, the isolated antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 44.

[0033] In certain embodiments, the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 42 or SEQ ID NO: 43.

[0034] In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4.

[0035] In certain embodiments, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 25, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 38, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 44.

[0036] In certain embodiments, the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from any one of the following groups:

[0037] (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 39, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 44;

[0038] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 40, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0039] (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 41, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0040] (4) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 43.

[0041] In certain embodiments, the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from any one of the following groups:

[0042] (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 29, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 42;

[0043] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0044] (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0045] (4) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0046] (5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0047] (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0048] (7) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 22, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0049] (8) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 43;

[0050] (9) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 36, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0051] (10) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 24, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 37, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 43.

[0052] In certain embodiments, the isolated antigen-binding protein heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:63 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:63.

[0053] In certain embodiments, the isolated antigen-binding protein heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 64-67.

[0054] In certain embodiments, the isolated antigen-binding protein heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 64-67.

[0055] In certain embodiments, the isolated antigen-binding protein heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 45-62.

[0056] In certain embodiments, the heavy chain variable region of the isolated antigen-binding protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 45-62.

[0057] In certain embodiments, the isolated antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0058] In certain embodiments, the antibody comprises a single domain antibody.

[0059] In certain embodiments, the antibody is selected from one or more of the following groups: a monoclonal antibody, a chimeric antibody, and a humanized antibody.

[0060] In certain embodiments, the isolated antigen binding protein comprises an Fc domain.

[0061] In certain embodiments, the N-terminus of the Fc domain is directly or indirectly linked to the C-terminus of the heavy chain variable region.

[0062] In certain embodiments, the Fc domain is derived from the Fc domain of human IgG.

[0063] In certain embodiments, the Fc domain comprises an Fc domain derived from any one of the following immunoglobulins: IgG1, IgG2, IgG3, and IgG4.

[0064] In certain embodiments, wherein the Fc domain comprises an Fc domain variant, the Fc domain variant has at least one amino acid mutation relative to a wild-type Fc domain.

[0065] In certain embodiments, the Fc domain variant has at least one amino acid mutation selected from the group consisting of H285D, L309D, L309G, M252Y, N434A, Q295R, Q311R, T250Q, T256D, T307A, T307Q, V308P, A330V, A378V, E380A, H285D, H435R, I332Y, L328W, M428E, M428L, N434A, N434W, P331V, Q311H, Q311V, T256D, T307Q, T307R and T307W according to the Kabat EU numbering system.

[0066] In certain embodiments, the Fc domain variant has a mutation or combination of mutations selected from the group consisting of: a) Q311R and M428L; b) L309G and M428L; c) Q311R, M428E and N434W; d) T250Q and M428L; e) T307A, E380A and N434A; f) V308P; g) H285D, T307Q and A378V; h) N434A and E380A; i) T250R and M428L; j) Q295R, L328W, A330V, P331V and I332Y; k) )N434A; l)N434W; m)T256D and T307W; n)M252Y and T256D; o)T307Q, Q311V and A378V; p)T256D, H285D, T307R, Q311V and A378V; q)L309D, Q311H and N434S; r)H435R; s)T307Q, Q311V and A378V; t)T256D, H285D, T307R, Q311V and A378V; u)L309D, Q311H and N434S; and v)H435R.

[0067] In certain embodiments, the Fc domain variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 68-86.

[0068] In certain embodiments, the antigen binding protein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 87-428.

[0069] In certain embodiments, the isolated antigen binding protein comprises a defucosylated antibody.

[0070] In another aspect, the present application provides a polypeptide molecule comprising the isolated antigen-binding protein described herein.

[0071] In certain embodiments, the polypeptide molecules described herein comprise fusion proteins.

[0072] In another aspect, the present application provides an immunoconjugate comprising the isolated antigen-binding protein described herein.

[0073] In another aspect, the present application provides a nucleic acid molecule encoding the isolated antigen-binding protein or the polypeptide molecule described in the present application.

[0074] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0075] In another aspect, the present application provides a cell comprising the nucleic acid molecule or the vector described in the present application.

[0076] In another aspect, the present application provides a method for preparing the isolated antigen-binding protein described herein.

[0077] In certain embodiments, the method of preparing the isolated antigen-binding protein described herein comprises culturing the cells described herein under conditions that allow for expression of the antigen-binding protein.

[0078] On the other hand, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0079] On the other hand, the present application provides a method for preventing and / or treating a disease, which comprises administering to a subject in need thereof the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein and / or the pharmaceutical composition described herein.

[0080] On the other hand, the present application provides the use of the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein and / or the pharmaceutical composition described herein in preventing and / or treating diseases.

[0081] On the other hand, the present application provides a use of the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein and / or the pharmaceutical composition described herein in the preparation of a drug, wherein the drug is used to prevent and / or treat a disease.

[0082] In certain embodiments, the disease and / or condition comprises a disease and / or condition associated with CXCR3.

[0083] In certain embodiments, the disease and / or disorder comprises an autoimmune disease associated with CXCR3.

[0084] In certain embodiments, the disease and / or condition comprises an allergic disease associated with CXCR3.

[0085] In certain embodiments, the diseases and / or conditions include systemic lupus erythematosus, lupus nephritis, and / or rheumatoid arthritis.

[0086] On the other hand, the present application provides a reagent or kit for detecting CXCR3 in a sample, which comprises the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the immunoconjugate 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 pharmaceutical composition described in the present application.

[0087] On the other hand, the present application provides a method for detecting CXCR3 in a sample, which method comprises using the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the immunoconjugate 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 pharmaceutical composition described in the present application.

[0088] Those skilled in the art can easily discern 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 this 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 this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0090] FIG1 is a schematic diagram showing a structure of the antigen binding protein targeting CXCR3 described in the present application.

[0091] FIG2 shows a map of the pTT5 plasmid used to express the antigen binding protein targeting CXCR3 described in the present application.

[0092] FIG3 shows the purification results of the CXCR3-targeting antigen-binding protein described in the present application using the MabSelect SuRe affinity chromatography column.

[0093] FIG4 shows the SDS-PAGE analysis results of the antigen binding protein targeting CXCR3 described in the present application, wherein R represents reducing conditions and NR represents non-reducing conditions.

[0094] FIG5 shows the binding of the antigen binding protein targeting CXCR3 described in the present application to CXCR3-positive cells assessed by flow cytometry.

[0095] FIG6 shows the evaluation results of the ADCC effect of the antigen binding protein targeting CXCR3 described in the present application.

[0096] FIG7 shows the evaluation results of the CXCR3-targeting antigen binding protein described in the present application for inhibiting downstream signals.

[0097] FIG8 shows that the antigen binding protein targeting CXCR3 described in the present application can specifically bind to cells overexpressing CXCR3.

[0098] FIG9 shows that the antigen binding protein targeting CXCR3 described in the present application can block the migration of cells expressing human CXCR3.

[0099] FIG10 shows the therapeutic effect of the antigen binding protein targeting CXCR3 on the type II bovine collagen-induced arthritis (CIA) model in mice.

[0100] FIG11 shows the therapeutic effect of the antigen binding protein targeting CXCR3 on systemic lupus erythematosus (SLE) simulated in the mouse MRL / LPR model. DETAILED DESCRIPTION

[0101] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0102] Definition of terms

[0103] In this application, the term "CXCR3" is also called "GPR9" and "CD183", which generally refers to a seven-transmembrane domain receptor coupled to a G protein. It is a chemokine receptor that is expressed on a variety of cell types. CXCR3 is mainly expressed on the surface of activated T cells, NK cells, mast cells and dendritic cells. It is also expressed in small amounts on some epithelial cells and endothelial cells, and is basically not expressed on resting T cells and monocytes. In this application, the CXCR3 may 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 the CXC chemokines CXCL9 (interferon-induced mononuclear cytokine, MIG), CXCL10 (interferon-induced protein 10, IP-10) and CXCL11 (I-TAC), while CXCR3-B can bind to CXCL4 in addition to CXCL9, CXCL10 and CXCL11. In the present application, the CXCR3 may include any natural CXCR3 of any vertebrate origin, 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 the present application, the CXCR3 may include "full-length," unprocessed CXCR3, and any form of CXCR3 derived from cell processing; naturally occurring variants of CXCR3, such as splice variants or allelic variants, may also be included. For example, the accession number for the complete amino acid sequence of human CXCR3 is NP_001495. In the present application, the "CXCR3 ligand" may include, but is not limited to, CXCL9, CXCL10, and / or CXCL11. In the present application, the "CXCR3-expressing cell" may include, but is not limited to, an immune cell expressing CXCR3. For example, a CXCR3-expressing cell may include a T cell, a B cell, a NK cell, and / or a DC cell expressing CXCR3. 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.

[0104] In this application, the term "immune cell" generally refers to all cells involved in or associated with an immune response. According to their function, immune cells can be divided into nonspecific immune cells, specific immune cells, and antigen-presenting cells. Nonspecific immune cells can include macrophages, neutrophils, natural killer cells (NK cells), mast cells, etc.; specific immune cells can include T cells and B cells; antigen-presenting cells can include dendritic cells (DC cells), macrophages, and B cells, etc.

[0105] In this application, the term "isolated" generally refers to a substance obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been altered, or that the substance has been separated from its natural environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal and has not been separated is considered isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0106] In the present application, the term "antigen binding protein" generally refers to a protein with antigen binding ability. For example, an antigen binding protein can include an isolated antigen binding protein. In the present application, the term "isolated antigen binding protein" generally refers to a protein with antigen binding ability that has been separated from its naturally occurring state. The "isolated antigen binding protein" can include a portion that binds to an antigen and, optionally, allows the antigen binding portion to adopt a framework or framework portion that promotes the conformation of the antigen binding portion to bind to the antigen. The antigen binding protein can include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or an artificial framework region with a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions that include, for example, mutations introduced into the three-dimensional structure of the antigen binding protein and fully synthetic framework regions that include, 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 fragment, F(ab')2, F(ab)2, scFv, di-scFv, dAb, VHH, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody, and fragments thereof.

[0107] In this application, the term "CDR" is also referred to as "complementarity determining region", which generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structure-defining loop. Typically, three CDRs (HCDR1, HCDR2, and HCDR3) are included in the heavy chain variable region (variable domain of heavy chain, VH) of an antibody. An antibody consisting only of a heavy chain can function normally and stably in the absence of a light chain, for example, naturally occurring camel antibodies, 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 specifically can be found at, for example, www.bioinf.org.uk / abs / index.html#kabatnum. For 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; 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 CDRs of the antigen binding protein can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann N Y 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).

[0108] 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 evenly distributed across the entire variable domain. It is typically concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light or heavy chain variable domain. The more highly conserved portions of the variable domain are called framework regions (FRs).

[0109] In this application, the term "FR" generally refers to the more highly conserved portion of the antibody variable domain, which is called the framework region. Typically, a natural heavy chain variable domain comprises four FR regions, namely H-FR1, H-FR2, H-FR3 and H-FR4.

[0110] In this application, the term "antibody" generally refers to an immunoglobulin or its fragment or derivative, encompassing any polypeptide comprising an antigen binding site, whether 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, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "complete," as in "complete antibody," for the purposes of this application, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, VHH, dAb, and other antibody fragments that retain antigen binding function (e.g., capable of specific binding to CXCR3).

[0111] As used herein, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind to an antigen (e.g., CXCR3). As used herein, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0112] In this 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, as described, for example, in Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11): 484-490, and in other published patent applications such as WO 06 / 030220, WO 06 / 003388, and Domantis Ltd. The term "dAb" generally includes sdAbs. The term "sdAb" generally refers to a single-domain antibody.

[0113] In this application, the term "single domain antibody" generally refers to an antibody that lacks a light chain. The single domain antibody described herein may include a heavy chain antibody (HcAb), which includes a heavy chain variable region and conventional heavy chain CH2 and CH3 regions. The single domain antibody described herein may include the minimum binding unit of an antigen binding protein. The single domain antibody described herein may include an antibody fragment consisting only of an antibody heavy chain variable region.

[0114] In the present application, the term "VHH (variable domain of heavy chain of heavy chain antibody, VHH)" generally refers to the variable region antigen binding domain of a heavy chain antibody (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review by Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH may also be referred to as a nanobody (Nanobody, Nb). In the present application, a single domain antibody may include a nanobody.

[0115] In this application, the term "Fc domain" or "Fc region" generally refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The Fc region of an immunoglobulin generally contains two constant domains - a CH2 domain and a CH3 domain, and optionally a CH4 domain. For example, the Fc region may not contain a CH1 domain. Amino acid residue substitutions in the Fc portion that alter antibody effector functions are known in the art (Winter et al. U.S. Patent Nos. 5,648,260; 5,624,821). The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and half-life / clearance rate of antibodies and antigen-antibody complexes. Depending on the therapeutic purpose, these effector functions are desirable for therapeutic antibodies in some cases, but may be unnecessary or even harmful in other cases.

[0116] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation of single molecule composition. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which generally have different antibodies for different determinants), each monoclonal antibody is for a single determinant on the 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 characteristic 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 herein can be prepared by recombinant DNA methods.

[0117] 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 camelid ("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., camelid) antibody.

[0118] 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 camel antibody) are replaced by corresponding amino acids derived from human immunoglobulins. In the CDR region, small additions, deletions, insertions, replacements, 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. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., camel) antibody may minimally comprise a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, the CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as camel, alpaca, mouse, rat, rabbit, or non-human primate) with desired properties, affinity, and / or ability. In some cases, the 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.

[0119] 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, then the fully human antibody may contain mouse sugar chains. Similarly, a "camelid antibody" generally refers to an antibody that contains only camel immunoglobulin sequences. Fully human antibodies can be generated in humans and in transgenic animals with human immunoglobulin germline sequences by phage display or other molecular biology 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.

[0120] In the present application, the term "affinity" generally refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., a polypeptide or an antibody) and its binding partner (e.g., a target or an antigen). The antigen-binding proteins described herein can have a higher affinity for binding to CXCR3 by affinity maturation. Affinity can be measured by conventional methods known in the art, including but not limited to assays based on surface plasmon resonance, enzyme-linked immunosorbent assays, competitive assays, etc. As used herein, in the context of antigen-binding molecules (e.g., antibodies), the term "affinity maturation" refers to, for example, an antigen-binding molecule derived from a reference antigen-binding molecule by mutation and a reference antigen-binding protein that binds to the same antigen, preferably in conjunction with the same epitope; and there is 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, affinity-matured antigen-binding molecules bind to the same epitope as the initial reference antigen-binding molecule.

[0121] In this application, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., NK cells, neutrophils, macrophages, etc.) recognize that such cytotoxic cells that mediate ADCC typically express Fc receptors (FcRs). ADCC can be measured by conventional methods known in the art and can be measured in vivo or in standard in vitro cell killing assays. In this application, "enhanced antibody-dependent cellular cytotoxicity" generally refers to an increase in the number of target cells that are lysed within a given time at a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and / or 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 by the ADCC mechanism.

[0122] Throughout this application, the terms "polypeptide molecule," "polypeptide," and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked moieties. Each moiety can be a polypeptide with a distinct property. This property can be a biological property, such as in vitro or in vivo activity. It can also be a simple chemical or physical property, such as binding to a target molecule or catalysis of a reaction. The two moieties can be directly linked by a single peptide bond or through a peptide linker.

[0123] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from their natural environment or artificially synthesized.

[0124] In this 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. A vector can be used to transform, transduce, or transfect a host cell, allowing the genetic material elements it carries to be expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. 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, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0125] In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that can be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules described herein or vectors described herein. Cells can include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original mother cell (in the form of total DNA complement or on the genome). Cells can include cells transfected in vitro with the vectors described herein. Cells can 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. Cells can also include cells after engineered transformation.

[0126] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (for example, covalently linked via a linker molecule) other agents (for example, chemotherapeutic agents, radioactive elements, cytostatic agents and cytotoxic agents, etc.) to the antibody or its antigen-binding fragment, which can deliver the other agents to target cells (for example, tumor cells) through the specific binding of the antibody or its antigen-binding fragment to the antigen on the target cell.

[0127] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise 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 non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition described herein includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0128] 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 thereto at the dosages 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.

[0129] 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, that 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 one that binds to that 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.

[0130] In the present application, the term "CXCR3-related disease and / or condition" generally refers to any disease and / or condition mediated by CXCR3 and / or its ligands, 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 increased relative to individuals in a healthy state. For example, CXCR3-related diseases and / or conditions may include allergic diseases and / or autoimmune diseases.

[0131] In this application, the term "allergic disease" generally includes diseases characterized by allergic and / or atopic immunological reactions to antigens, which cause allergic and / or atopic symptoms in patients with allergic diseases. Allergic reactions can include type I, type II, type III and / or type IV hypersensitivity reactions. Type I hypersensitivity reactions (immediate anaphylaxis) are mediated by IgE. Antigens (allergens) bind 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, chemokines) and newly synthesized other mediators (such as prostaglandins, leukotrienes, platelet-activating factor, interleukins). These inflammatory mediators can lead to vasodilation, increased capillary permeability, hypersecretion of mucus, contraction of smooth muscle, and infiltration of eosinophils, helper T cells (TH2) and other inflammatory cells in tissues. Type II hypersensitivity reactions (antibody-dependent cytotoxicity) are triggered by the binding of antibodies to cell surface antigens or haptens. Surface-bound antigen-antibody complexes (as opposed to circulating antigen-antibody complexes in type III hypersensitivity reactions) activate cells involved in antibody-dependent cell-mediated cytotoxicity (e.g., natural killer cells, eosinophils, 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, triggering the release of inflammatory mediators. Type IV hypersensitivity reactions do not involve antibodies but are mediated by T cells.

[0132] In the present application, the term "autoimmune disease" generally refers to an obstacle or condition caused by an autoimmune response mediated by antibodies to self-antigens. Autoimmune diseases result in inappropriate and / or excessive production of autoantibodies to self-antigens or self-antigens. 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). "Autoantigen" means an endogenous antigen that stimulates the production of an autoimmune response (such as the production of autoantibodies). Autoantigens also include self-antigens or antigens from normal tissues, which are targets of cell-mediated or antibody-mediated immune responses, which can lead to the development of autoimmune diseases. For example, "autoimmune diseases" described herein can include autoimmune diseases mediated by immune cells. For example, the autoimmune diseases can include autoimmune diseases mediated by immune cells expressing CXCR3.

[0133] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0134] 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 that have the same or similar functions as the proteins or polypeptides.

[0135] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that can specifically bind to CXCR3). 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.

[0136] In the present application, the homolog can 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 can specifically bind to CXCR3).

[0137] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in 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 in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use 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 interest. 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 D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," 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," J. Mol. Biol., 215: 403-410, 1990.

[0138] In this application, the term "comprising" generally means including, encompassing, containing or encompassing. In some cases, it also means "being", "consisting of...".

[0139] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, 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 the specified value.

[0140] Detailed Description of the Invention

[0141] Isolated antigen binding protein

[0142] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see, for example, www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20, or 1-10, and further 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 can 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 to the amino acid sequence of the CDR. In certain embodiments, the antigen-binding proteins described herein can be defined by the IMGT coding system.

[0143] In one aspect, the present application provides an isolated antigen binding protein that can specifically bind to CXCR3.

[0144] In certain embodiments, the isolated antigen binding proteins described herein comprise a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3.

[0145] In certain embodiments, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 14.

[0146] In certain embodiments, the HCDR1, HCDR2, and HCDR3 may be selected from any one of the following groups:

[0147] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15;

[0148] (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16;

[0149] (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16;

[0150] (4) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.

[0151] In certain embodiments, the HCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 1-2.

[0152] In certain embodiments, the HCDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 4-7.

[0153] In certain embodiments, the HCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 11-13.

[0154] In certain embodiments, the HCDR1, HCDR2, and HCDR3 may be selected from any one of the following groups:

[0155] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0156] (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0157] (3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12;

[0158] (4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0159] (5) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0160] (6) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13;

[0161] (7) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0162] (8) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13;

[0163] (9) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13.

[0164] In certain embodiments, the antigen-binding proteins described herein comprise H-FR1, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1. In certain embodiments, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

[0165] In certain embodiments, the antigen-binding proteins described herein comprise H-FR2, which is located between the HCDR1 and HCDR2. In certain embodiments, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the H-FR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 20-24.

[0166] In certain embodiments, the antigen-binding proteins described herein comprise an H-FR3 positioned between the HCDR2 and the HCDR3. In certain embodiments, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the H-FR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 29-37.

[0167] In certain embodiments, the antigen-binding proteins described herein comprise an H-FR4, wherein the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3. In certain embodiments, the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43.

[0168] In certain embodiments, the antigen binding proteins described herein comprise H-FR1, H-FR2, H-FR3, and H-FR4.

[0169] In certain embodiments, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 19, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 25, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 38, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 44.

[0170] In certain embodiments, the H-FR1, H-FR2, H-FR3 and H-FR4 may be selected from any one of the following groups:

[0171] (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 39, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 44;

[0172] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 40, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0173] (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 41, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0174] (4) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 43.

[0175] 18. The isolated antigen-binding protein according to any one of claims 1 to 16, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from any one of the following groups:

[0176] (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 29, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 42;

[0177] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0178] (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0179] (4) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0180] (5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0181] (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0182] (7) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 22, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0183] (8) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 43;

[0184] (9) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 36, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43;

[0185] (10) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 24, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 37, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 43.

[0186] In certain embodiments, the heavy chain variable region of the antigen binding protein described herein may comprise the amino acid sequence as shown in SEQ ID NO: 63, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO: 63.

[0187] In certain embodiments, the heavy chain variable region of the antigen binding protein described herein may comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 64-67, or an amino acid sequence shown in any one of SEQ ID NOs: 64-67.

[0188] In certain embodiments, the heavy chain variable region of the antigen binding protein described herein may comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 45-62, or an amino acid sequence shown in any one of SEQ ID NOs: 45-62.

[0189] In the present application, the isolated antigen-binding protein may comprise at least one CDR in the amino acid sequence set forth in any one of SEQ ID NOs: 45-62, and the CDR may comprise a CDR delineated in any manner. CDRs delineated in any manner, if their sequences are identical to those in the amino acid sequence set forth in any one of SEQ ID NOs: 45-62, fall within the scope of protection of the claims of the present application.

[0190] In certain embodiments, the isolated antigen-binding protein described herein comprises an antigen-binding protein having a HCDR1 with an amino acid sequence as set forth in SEQ ID NO: 1, a HCDR2 with an amino acid sequence as set forth in SEQ ID NO: 4, and a HCDR3 with an amino acid sequence as set forth in SEQ ID NO: 11, as well as humanized antigen-binding proteins thereof. In certain embodiments, the HCDR1 of the humanized antigen-binding protein may have one or more amino acid mutations, the HCDR2 of the humanized antigen-binding protein may have one or more amino acid mutations, and / or the HCDR3 of the humanized antigen-binding protein may have one or more amino acid mutations, and the humanized antigen-binding protein still has the ability to bind to CXCR3.

[0191] In certain embodiments, the isolated antigen-binding protein described herein comprises a variable region having an amino acid sequence as shown in SEQ ID NO: 45, and a humanized antigen-binding protein thereof.

[0192] In the present application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof. In certain embodiments, the isolated antigen-binding protein described in the present application may be an antibody or an antigen-binding fragment thereof.

[0193] In certain embodiments, the isolated antigen-binding proteins described herein may comprise single domain antibodies or antigen-binding fragments thereof. In certain embodiments, the isolated antigen-binding proteins described herein may comprise heavy chain antibodies or antigen-binding fragments thereof. In certain embodiments, the isolated antigen-binding proteins described herein may comprise nanobodies.

[0194] In certain embodiments, the isolated antigen-binding protein may comprise a heavy chain-only antibody. In certain embodiments, the heavy chain-only antibody consists of a variable region antigen-binding domain, which consists of Framework 1, CDR1, Framework 2, CDR2, Framework 3, CDR3, and Framework 4. In certain embodiments, the heavy chain-only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In certain embodiments, the heavy chain-only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In certain embodiments, the heavy chain-only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In further embodiments, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but lacks a hinge region. Heavy chain-only antibodies may be in the form of dimers, in which the two heavy chains are bonded by disulfide bonds, but are otherwise covalently or non-covalently linked to each other. Only heavy chain antibodies can belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclasses, are also included herein. In specific embodiments, the heavy chain antibody can be IgG1, IgG2, IgG3 or IgG4 subtype, particularly IgG1 subtype.

[0195] In certain embodiments, the antibody may be selected from one or more of the following groups: a monoclonal antibody, a chimeric antibody, and a humanized antibody.

[0196] In the present application, the isolated antigen-binding protein may further comprise an Fc domain. In certain embodiments, the N-terminus of the Fc domain is directly or indirectly connected to the C segment of the heavy chain variable region. In certain embodiments, the N-terminus of the Fc domain may be connected to the C segment of the heavy chain variable region via a hinge region. In certain embodiments, the Fc domain may be derived from the Fc domain of human IgG. In certain embodiments, the antigen-binding protein described herein comprises a wild-type Fc domain derived from human IgG. The amino acid sequence of an Fc domain derived from human IgG is known in the art. In certain embodiments, the Fc domain may include an Fc domain derived from any immunoglobulin in the following group: IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antigen-binding protein described herein comprises a wild-type Fc domain derived from any immunoglobulin in the following group: IgG1, IgG2, IgG3, and IgG4. The amino acid sequence of an Fc domain derived from IgG1, IgG2, IgG3, or IgG4 is known in the art. For example, the Fc domain may comprise an Fc domain derived from human IgG1, the Fc domain derived from human IgG1 may comprise Glu99-Lys330 of human IgG1, and the amino acid sequence of human IgG1 can be found in Uniprot Accession No. P01857. For example, the Fc domain may comprise an Fc domain derived from human IgG3, the Fc domain derived from human IgG3 may comprise Glu99-Lys377 of human IgG3, and the amino acid sequence of human IgG3 can be found in Uniprot Accession No. P01860. For example, the Fc domain may comprise an Fc domain derived from human IgG4, the Fc domain derived from human IgG4 may comprise Glu99-Lys327 of human IgG4, and the amino acid sequence of human IgG4 can be found in Uniprot Accession No. P01861.

[0197] In certain embodiments, the antigen-binding proteins described herein comprise Fc domain variants having one or more amino acid mutations relative to the wild-type Fc domain. The half-life of the antigen-binding protein can be prolonged and / or the effector function of the antigen-binding protein can be enhanced by optimizing the Fc domain sequence.

[0198] In certain embodiments, the antigen binding proteins described herein comprise an Fc domain variant derived from a human antibody, wherein the Fc domain variant has at least one amino acid mutation selected from the group consisting of H285D, L309D, L309G, M252Y, N434A, Q295R, Q311R, T250Q, T256D, T307A, T307Q, V308P, A330V, A378V, E380A, H285D, H435R, I332Y, L328W, M428E, M428L, N434A, N434W, P331V, Q311H, Q311V, T256D, T307Q, T307R, and T307W according to the Kabat EU numbering system, relative to the Fc domain of a wild-type human antibody. In certain embodiments, the antigen-binding proteins described herein comprise an Fc domain variant derived from a human antibody, wherein the Fc domain variant has a mutation or combination of mutations selected from the following relative to the Fc domain of a wild-type human antibody: a) Q311R and M428L; b) L309G and M428L; c) Q311R, M428E and N434W; d) T250Q and M428L; e) T307A, E380A and N434A; f) V308P; g) H285D, T307Q and A378V; h) N434A and E380A; i) T250R and M428L; j) Q295R, L328W, A330V, P331V and I332Y; k) N434A; l) N434W; m) T256D and T307W; n) M252Y and T256D; o) T307Q, Q311V and A378V; p) T256D, H285D, T307R, Q311V and A378V; q) L309D, Q311H and N434S; r) H435R; s) T307Q, Q311V and A378V; t) T256D, H285D, T307R, Q311V and A378V; u) L309D, Q311H and N434S; and v) H435R, the mutation sites are numbered according to the Kabat EU numbering system. For example, the human antibody Fc domain variant can be a variant of the human IgG antibody Fc domain. In certain embodiments, the antigen binding protein described herein comprises an Fc domain variant, which can comprise the amino acid sequence shown in any one of SEQ ID NOs: 68-86.

[0199] In certain embodiments, the antigen-binding protein described herein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 87-428, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 87-428.

[0200] In the present application, the isolated antigen-binding proteins may also include isolated antigen-binding proteins that have been further functionally optimized or modified. In certain embodiments, the antigen-binding proteins described herein may include antigen-binding proteins with enhanced affinity for binding to CXCR3. In certain embodiments, the antigen-binding proteins described herein may include antigen-binding proteins with enhanced ability to inhibit the binding of CXCR3 to its ligand. In certain embodiments, the antigen-binding proteins described herein may include antigen-binding proteins with enhanced ability to inhibit CXCR3-mediated signal transduction. In certain embodiments, the antigen-binding proteins described herein may include antigen-binding proteins with enhanced ability to inhibit the migration, accumulation, recruitment, and / or infiltration of cells expressing CXCR3. In certain embodiments, the antigen-binding proteins described herein may include antigen-binding proteins with enhanced ability to mediate cytotoxicity against cells expressing CXCR3. In certain embodiments, the antigen-binding proteins described herein may include antigen-binding proteins with enhanced ability to induce endocytosis of cell surface CXCR3 receptors. In certain embodiments, the enhanced ability may be relative to the antigen-binding protein before functional optimization or modification.

[0201] Antigen-binding proteins described herein can have enhanced binding affinity to CXCR3. Antigen-binding proteins described herein can have enhanced effector functions. The enhanced effector functions can include any one or more measurable increases in the antibody-dependent cellular toxicity (ADCC), complement-mediated cytotoxicity (CDC) and / or antibody-dependent cellular-mediated phagocytosis (ADCP) of suitable target cells. Antigen-binding proteins described herein can have increased half-life.

[0202] The isolated antigen-binding proteins described herein may include heavy chain sequences that have one or more conservative sequence modifications thereto. So-called "conservative sequence modifications" refer to amino acid modifications that do 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 isolated antigen-binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are the replacement of an amino acid residue with an amino acid residue having a similar side chain. 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), beta-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 isolated antigen-binding protein described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not abolish 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; Komissrov 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.

[0203] In certain embodiments, the antigen-binding proteins described herein may have a reduced fucose content or be defucosylated. In certain embodiments, the antigen-binding proteins described herein may include antigen-binding proteins that are substantially free of fucose. In certain embodiments, the antigen-binding proteins described herein may include antigen-binding proteins that are substantially free of nuclear fucose or that have had nuclear fucose removed from the N-glycans attached to the CH2 domain of Fc. In certain embodiments, the reduced fucose content or defucosylation may result in enhanced antibody-dependent cellular cytotoxicity (ADCC). For example, the antigen-binding proteins described herein may be expressed or produced by engineered cells that may provide altered glycosylation of the antibodies expressed therein, such as a non-fucosylated cell line or a FUT8 knockout CHO cell line.

[0204] In the present application, the antigen-binding protein is capable of specifically binding to CXCR3. In certain embodiments, the binding affinity of the antigen-binding protein to CXCR3 can be detected by detecting the binding ability of the antigen-binding protein to cells expressing CXCR3. For example, it can be detected by FACS. In certain embodiments, the binding of the antigen-binding protein to CXCR3 can be detected by ELISA. For example, the antigen-binding protein described herein 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, less than or equal to about 0.05 μg / mL, less than or equal to about 0.04 μg / mL, less than or equal to about 0.03 μg / mL, less than or equal to about 0.02 μg / mL, or less than or equal to about 0.01 μg / mL. For example, the antigen binding proteins described herein can bind to CXCR3 with an IC50 value of less than or equal to 50 nM, less than or equal to about 40 nM, less than or equal to 30 nM, less than or equal to 20 nM, or less than or equal to 10 nM.

[0205] The CXCR3 antigen-binding proteins described herein can be determined, identified, or characterized by various methods known in the art. For example, the antigen-binding activity of the antigen-binding proteins 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.

[0206] The antigen binding proteins provided herein can be used to antagonize CXCR3 activity. In the present application, the antigen binding proteins can prevent and / or treat diseases and / or conditions. The antigen binding proteins described herein can have one or more of the following functions to exert therapeutic effects 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 of cells expressing CXCR3 (e.g., to sites of inflammation); 4) mediating a killing effect on cells expressing CXCR3; 5) inducing endocytosis of CXCR3 receptors on the cell surface, thereby reducing the activation of immune cells.

[0207] Polypeptide molecules, nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions

[0208] In another aspect, the present application provides a polypeptide molecule, which may comprise the isolated antigen-binding protein described herein.

[0209] In certain embodiments, the polypeptide molecule may comprise a fusion protein. In certain embodiments, the polypeptide molecule may be a fusion protein.

[0210] In another aspect, the present application provides isolated nucleic acid molecules that can encode the isolated antigen-binding proteins described herein. For example, they can be produced or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification; (ii) cloning and recombination; (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, such as by chemical synthesis.

[0211] On the other hand, the present application provides a vector that can contain the nucleic acid molecules described herein. In addition, the vector can also contain other genes, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector can also 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 can include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells so that the genetic material elements it carries are expressed in the host cells. The vector can include, for example, a plasmid, a cosmid, a virus, a bacteriophage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector can also include components that assist its entry into cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0212] 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 certain embodiments, each kind or each host cell can comprise one or a kind of nucleic acid molecule or vector described in the application.In certain embodiments, each kind or each host cell can comprise multiple (for example, 2 or more) or multiple (for example, 2 or more) nucleic acid molecules or vector described in the application.For example, the vector described in the application can be introduced into the host cell, for example, eukaryotic cell, such as cell, fungus or yeast cell from plant etc.In certain embodiments, the cell can be bacterial cell (for example, Escherichia coli), yeast cell or other eukaryotic cell, for example COS cell, Chinese hamster ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, 293T cell, COS-1 cell, SP2 / 0 cell, NS0 cell or myeloma cell.The vector described in the application can be introduced into the host cell by methods known in the art, for example, electroporation, lipofectine transfection, lipofectamin transfection etc.

[0213] On the other hand, the present application also provides an immunoconjugate, which may comprise the isolated antigen-binding protein described in the present application.

[0214] In certain embodiments, the antigen-binding proteins or fragments thereof separated as described herein can be connected to another reagent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, etc. The connection can be through one or more covalent bonds, or non-covalent interactions, and can include chelation. A variety of linkers (the linkers can be known in the art) can be used to form immunoconjugates. In addition, immunoconjugates can be provided in the form of fusion proteins, which can be expressed by the polynucleotide encoding the immunoconjugates. The immunoconjugates can also include, for example, antibody-drug conjugates (ADCs). Suitable drugs can include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, antibodies and therapeutic agents can be cross-linked by a linker that can be cut, such as a peptide linker, a disulfide linker, or a hydrazone linker.

[0215] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0216] 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 described herein includes but is not limited to liquid, frozen and lyophilized compositions.

[0217] In certain embodiments, the pharmaceutical composition may also contain more than one active compound, generally those having complementary activities that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, and the clinical parameters of the subject.

[0218] In certain embodiments, the pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe and non-toxic.

[0219] In certain embodiments, the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, intrathecal and / or intranasal administration or direct injection into tissue.For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered uninterruptedly. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015 / 036583.

[0220] Preparation method

[0221] In another aspect, the present application provides a method for preparing the isolated antigen-binding protein. The method may include culturing the host cell described herein under conditions that allow expression of the antigen-binding protein. For example, the method may be performed using an appropriate culture medium, at an appropriate temperature and for an appropriate time, as is known to those skilled in the art.

[0222] Any method suitable for producing monoclonal antibodies can be used to produce the antigen-binding proteins described herein. For example, animals can be immunized with linked or naturally occurring CXCR3 or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes of administration. In certain embodiments, isolated antigen-binding proteins against CXCR3 can be isolated by extracting peripheral blood lymphocytes from immunized alpacas, cloning nucleic acid fragments from the cells into vectors, and screening and enrichment using a phage display system.

[0223] Any suitable form of CXCR3 can be used as an immunogen (antigen) to generate non-human antibodies specific for CXCR3 and screen for their biological activity. For example, the stimulating immunogen can be full-length CXCR3, including native homodimers, or peptides containing single or multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0224] Methods and uses

[0225] On the other hand, the present application provides a method for preventing and / or treating a disease and / or condition, which may comprise administering the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein to a subject in need thereof.

[0226] On the other hand, the present application also provides uses of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition in preventing and / or treating diseases.

[0227] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease and / or condition.

[0228] In the present application, the disease and / or condition may include a disease and / or condition associated with CXCR3. In certain embodiments, the disease and / or condition may include an allergic disease. In certain embodiments, the disease and / or condition may include an allergic disease associated with CXCR. In certain embodiments, the disease and / or condition may include an autoimmune disease. In certain embodiments, the disease and / or condition may include an autoimmune disease associated with CXCR3. In certain embodiments, the disease and / or condition may include a disease and / or condition involved in or mediated by cells expressing CXCR3. In certain embodiments, the disease and / or condition may include, but is not limited to, systemic lupus erythematosus, lupus nephritis, and / or rheumatoid arthritis.

[0229] In the present application, the use can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. The subject to be treated by the methods provided herein may include humans or other mammals.

[0230] In the present application, the subject in need thereof may include a subject who has been diagnosed with a CXCR3-related disease and / or disorder, or who is prone to developing a CXCR3-related disease and / or disorder.

[0231] In certain embodiments, the subject can be treated prophylactically or after the onset of a disease and / or condition. In certain embodiments, the subject can be treated prophylactically using the methods provided herein before the onset or worsening of a CXCR3-related disease and / or condition.

[0232] In certain embodiments, the subject in need thereof may include a mammal. In certain embodiments, the subject in need thereof may include a human.

[0233] On the other hand, the present application also provides a method for detecting CXCR3 in a sample, which comprises administering the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition.

[0234] In certain embodiments, the method for detecting CXCR3 in a sample can be an in vitro method. For example, the isolated antigen-binding protein described herein is contacted with an ex vivo sample to detect the presence and / or amount of CXCR3 in the sample. In certain embodiments, the method for detecting CXCR3 in a sample is for non-therapeutic purposes. In certain embodiments, the method for detecting CXCR3 in a sample is not a diagnostic method.

[0235] On the other hand, the present application also provides a reagent or kit for detecting CXCR3 in a sample, which comprises the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition.

[0236] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition in preparing a kit for detecting the presence and / or content of CXCR3 in a sample.

[0237] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0238] Example

[0239] The present application will be further described by the following specific examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are based on conventional conditions in this area, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are commercially available.

[0240] Example 1 Preparation of anti-CXCR3 antibodies

[0241] 1.1. Anti-CXCR3 plasmid construction

[0242] The gene sequence of the anti-CXCR3 antibody (the antibody structure is shown in Figure 1; the Fc domain can be derived from an IgG antibody) was inserted into the HindIII-NotI expression cassette of the pTT5 vector (shown in Figure 2) to generate a recombinant plasmid. The constructed plasmid was sequenced to obtain a correctly sequenced recombinant plasmid, which was then extracted using the Qiagen Midi Plasmid Extraction Kit.

[0243] 1.2. Transient Expression of Anti-CXCR3 Antibodies

[0244] In this example, anti-CXCR3 antibody was produced by ExpiCHO expression system. TM Cells were revived and cultured (ExpiCHO culture medium TM Expression Medium), and transfection can be performed after the cells have grown at a normal rate and have been passaged for more than 2 times.

[0245] The day before transfection, the cell density was adjusted to 3 × 10 6 -4×10 6 cells / ml, and the cell density grew to 7-10×10 6 cells / ml.

[0246] On the day of transfection, use fresh ExpiCHO preheated at 37°C TM Dilute the cells to a density of 6 × 10 6 cells / ml. Use cold OptiPRO TM Dilute ExpiFectamine TM CHO Reagent and plasmid DNA, dilute ExpiFectamine TMAdd CHO Reagent to the diluted plasmid and mix it upside down to make ExpiFectamine TM CHO / plasmid DNA mixture. After incubation at room temperature for 5 minutes, the mixture was slowly added to the ExpiCHO-S cell suspension, mixed well, and placed in a 37°C, 8% CO2 incubator for culture. One day after transfection, 120 μL of ExpiCHO was added to the cell suspension. TM Enhancer, 4.8mL ExpiCHO TM Feed and continue expression at 37°C.

[0247] Seven days after transfection, the supernatant was collected by centrifugation and used for antibody purification.

[0248] Purification and Analysis of Anti-CXCR3 Antibodies

[0249] The antibody cell expression supernatant obtained in the previous step was purified using a Mabselect SuRe affinity chromatography column. After capture, the supernatant was washed with 50mM Tris + 150mM NaCl and eluted with 0.1M Glycine pH 3. The loading and elution results are shown in Figure 3. After elution, the pH of the sample was adjusted to 8 by adding 1M Tris HCl. The purified protein was analyzed using SDS-PAGE, as shown in Figure 4.

[0250] Example 2 Anti-CXCR3 Antibody Affinity Determination

[0251] In this example, an anti-CXCR3 antibody (primary antibody) was incubated and bound to target cells expressing CXCR3. After incubation and binding of a fluorescently labeled secondary antibody to the primary antibody, the intensity of the fluorescent labeling of the cells was detected by flow cytometry to evaluate the specific binding ability of the antibody to cells expressing CXCR3. This example and subsequent examples will use the 53hu37 antibody (its sequence can be found in CN110300763A) as a benchmark for comparison with the CXCR3 antibody in this application. The binding of the anti-CXCR3 antibody to the target cells was evaluated, and the results are shown in Figure 5. The antibodies of the present application all have the ability to specifically bind to cells expressing CXCR3.

[0252] Experimental methods:

[0253] Prepare the experimental solutions according to the preparation method shown in the table below.

[0254] The specific steps are as follows:

[0255] (1) Count cells. Mix the cell suspension and spot 10 μl of the cell suspension on the spotting platform of the cell counter. Repeat three times and calculate the average value.

[0256] (2) Add 1×10 5 cells, 25 μl / well, centrifuge at 500 g, 4°C for 5 min, and discard the supernatant.

[0257] (3) Antibody preparation: Antibodies were diluted to twice the corresponding concentration using MACS buffer, added to cells at a rate of 25 μl / well, mixed, and incubated at 4°C for 30 min.

[0258] (4) After incubation, add 200 μl / well of MACS buffer and wash twice: centrifuge at 500g, 4°C for 5 min, and discard the supernatant.

[0259] (5) Preparation of secondary antibody: 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°C for 30 min.

[0260] (6) After incubation, add 200 μl / well of MACS buffer and wash: centrifuge at 500g for 5 min at 4°C and discard the supernatant. Wash once or twice.

[0261] (7) After discarding the supernatant, add 100 μl / well MACS buffer to resuspend the cells and perform flow cytometry.

[0262] (8) Calculate the gMFI of secondary antibody fluorescence in living cells.

[0263] Example 3 Evaluation of ADCC Effect of Anti-CXCR3 Antibodies

[0264] This embodiment illustrates that the anti-CXCR3 antibody described in the present application can recognize target cells expressing CXCR3. At the same time, the Fc segment of the antibody binds to the Fc receptor CD16a (158V) on the surface of the effector cell NK-MI-CD16a (158V), mediating the effector cell NK-MI-CD16a (158V) to release cytotoxic molecules to kill target cells expressing CXCR3. Lactate dehydrogenase (LDH) in the killed target cells will be released into the culture medium, and the killing of the target cells can be confirmed by detecting the amount of LDH. IgG was used as a control, and 53hu37 was used as a benchmark. As shown in Figure 6, the antibodies of the present application all have the ability to mediate ADCC.

[0265] Experimental methods:

[0266] Prepare the experimental solutions according to the preparation method shown in the table below.

[0267] The specific steps are as follows:

[0268] (1) Wash the target cells 1-2 times with Assay medium, discard the supernatant, resuspend and count the cells with Assay medium, 25,000 cells / well, and plate 25 μl / well.

[0269] (2) Antibody preparation: The antibody was diluted to 4 times the corresponding concentration with MACS buffer, added to the cells at 25 μl / well, mixed, and incubated at 37°C for 30 min.

[0270] (3) Preparation of effector cells: Take NK92-CD16a (158V) cells, add 50 μl of cell suspension to each well of a 96-well plate, 75,000 cells / well, and mix well.

[0271] (4) Incubation: Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 4 h. For the High Control, add 5 μl of Lysis solution to each well 15 min before the end of incubation and mix thoroughly.

[0272] (5) Color development: Add 100 μl of fresh reaction mixture (blue cap: red cap = 1:45, prepared immediately before use, cytotoxicity detection kit) to each well, mix well, and develop color at room temperature for 25 min, protected from light.

[0273] (6) Stop: Add 50 μl of stop solution (green cap) to each well and mix well.

[0274] (7) Reading: Detect absorbance using a microplate reader at a wavelength of 492 nm and a wavelength of 690 nm (reference wavelength).

[0275] (8) Data processing: Subtract the background control from all wells and then substitute it into the formula to calculate the killing percentage.

[0276] Cytotoxicity (%)=(well to be tested-effector control-low control) / (high control-low control)*100

[0277] Example 4 Inhibition of Downstream Signaling by Anti-CXCR3 Antibodies

[0278] In this example, the ability of the antibodies described herein to inhibit CXCR3-mediated cAMP reduction was assessed by measuring cAMP signals in CXCR3-overexpressing 293T cells (which also overexpressed the GloSensor-22F biosensor). The higher the cAMP (luminescence) level, the greater the antibody's inhibitory effect. IgG was used as a control, and 53hu37 was used as a benchmark.

[0279] The experimental results are shown in Figure 7. 3F11, 3F11hu1, 3F11hu8 and their subsequent optimized antibodies, 3F11hu9, and 3F11hu13 increased the level of cAMP, showing an increase comparable to or greater than that of the benchmark, indicating that the above antibodies can inhibit the decrease in cAMP in CXCR3-expressing cells induced by CXL10 and CXCL11.

[0280] Experimental methods:

[0281] Prepare the experimental solutions according to the preparation method shown in the table below.

[0282] (1) Day 1: Count the 293T cells in a 10 cm dish, prepare 15,000 cells / well, and seed them with 100 μl / well of complete medium. Culture in an incubator overnight.

[0283] (2) Day 2: Centrifuge at 500g for 5 minutes. After centrifugation, discard the liquid by shaking the plate. Add equilibration medium (100 μl / well). Incubate at room temperature (do not place in incubator) for 2 hours in the dark.

[0284] (3) Use Equilibration medium to prepare antibodies, etc.: Note that there is already 100 μL of solution in the system. Prepare antibodies (128 nM is used in this experiment), forskolin (forskolin can increase the background cAMP level, so the final concentration is 10 μM in this experiment), and ligand (CXCL10: 1000 ng / ml, CXCL11: 200 ng / ml are used in this experiment. CXCL9 cannot stimulate the decrease of cAMP and is therefore not included in this experiment). Add 5 μL of each, and the final total system will be 115 μL. This can be used to calculate the required working solution concentrations of antibodies, forskolin, and ligand.

[0285] (4) After incubation, add the following in the following order: 1. Antibodies, incubate at room temperature for 15 minutes 2. Ligands 3. Forskolin

[0286] (5) After mixing with a pipette tip, read the luminescence value using a Tecan multi-function plate reader at 3, 6, 9, and 12 minutes after sample addition. Use the result at 9 minutes for statistical plotting.

[0287] Example 5 Specific Binding Activity of Anti-CXCR3 Antibodies

[0288] In this example, the ability of the antibodies of the present application to specifically bind to CXCR3 was determined by detecting their ability to bind to cells overexpressing CXCR3, cells overexpressing other chemokines, and wild-type cells.

[0289] Experimental methods:

[0290] In this example, the binding ability of the antibodies of the present 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. IgG was used as a control, and 53hu37 was used as a benchmark.

[0291] The experimental results are shown in Figure 8. Significant fluorescence signals were observed only on CXCR3-overexpressing cells, while no signals were observed on cells in other groups. This experimental result demonstrates that the antibodies described herein have good specificity for CXCR3 and exhibit no nonspecific binding to the other chemokines used in the experiment and WT cells.

[0292] Example 6 Inhibition of cell migration by anti-CXCR3 antibodies

[0293] In this example, the blocking effect of the antibodies described herein on the migration of cells expressing human CXCR3 (hCXCR3) was evaluated using a chemokine-induced migration assay.

[0294] In this example, the ability of the antibodies to block CXCR3-mediated cell migration was determined by flow cytometry, measuring the number of cells that migrated from the upper Transwell chamber (containing CXCR3-overexpressing Jurkat cells and antibodies) to the lower Transwell chamber (containing ligands CXCL9, CXL10, or CXCL11). IgG was used as a control, and 53hu37 was used as a benchmark.

[0295] The experimental results, shown in Figure 9, show that 3F11, 3F11hu1, 3F11hu8, and their subsequent optimized antibodies, including 3F11hu9, can inhibit the migration of CXCR3-expressing cells induced by CXCL9, CXL10, and CXCL11. CXCR3 plays a crucial role in the migration of activated immune cells, mediating the chemotaxis of T and B cells to sites of inflammation. Antibodies inhibit the migration of CXCR3-expressing cells, a process known as chemotaxis, potentially reducing the accumulation of immune cells in inflamed areas and alleviating local immune responses. This inhibitory effect may be associated with slowing disease progression and alleviating symptoms, and has potential for treating T and B cell-related autoimmune diseases.

[0296] Experimental methods:

[0297] Prepare the experimental solutions according to the preparation method shown in the table below.

[0298] (1) Cell and Antibody Treatment: Each well contains 100 μl of the total system, with the cell suspension and antibody each accounting for 50% of the total volume. Calculate the amount of antibody based on a volume of 50 μl / well. Add 50 μl / well of antibody to the cells, mix thoroughly, and incubate the cells in a 37°C incubator for 30 min.

[0299] (2) Ligand preparation: Calculate the amount of ligand working solution based on a volume of 300 μl / well. Dilute the ligands to the corresponding concentrations using migration buffer (in this experiment, the experimental concentrations are CXCL9: 250 ng / ml, CXCL10: 20 ng / ml, and CXCL11: 40 ng / ml).

[0300] (3) Add 300 μl / well to the lower chamber of the tanswell. Set up the no Ab no ligand group and add 300 μl of ligation buffer to the lower chamber of the tanswell.

[0301] (4) After incubating the cells for 30 min, mix the cell suspension and transfer the cell suspension to the upper chamber of the transwell. Incubate the cells at 37°C for 4 h.

[0302] (5) Preparation of reference cells: Count Jurkat-WT cells and prepare cells at 3 × 10 4 / well according to the number of samples, and mix them in MACS solution.

[0303] (6) After migration, the cells in the lower chamber of the transwell were transferred to a flow cytometry tube, and 0.5 ml of 3 × 104 reference cells were added to each tube.

[0304] (7) Centrifuge at 500 g for 5 min. Resuspend the cells and mix thoroughly before loading onto the analyzer.

[0305] (8) Cytek was used to analyze the fluorescence of GFP (fluorescent label of CXCR3-expressing cells), single cells and GFP+ / - cells were circled, and 10,000-20,000 cells / sample were recorded.

[0306] (9) During FlowJo analysis, single cells were circled and the ratio of GFP+ to GFP- in single cells was calculated. The number of GFP+ cells was then calculated as (GFP+ / GFP-) × 3 × 104, which was the number of cells that migrated to the lower chamber of the transwell.

[0307] (10) Migration Index = number of cells in the experimental group / number of cells in the no Ab no ligand group.

[0308] Example 7 In vivo study of anti-CXCR3 antibodies in an arthritis model

[0309] In this example, the therapeutic effect of the antibodies described in the present application on rheumatoid arthritis was explored by using the type II bovine collagen-induced arthritis (CIA) model in mice to simulate rheumatoid arthritis in humans.

[0310] Experimental methods:

[0311] The mice used in this experiment were 10-week-old male DBA1 mice. 32 DBA1 mice were modeled with type II bovine collagen (CII)-induced arthritis and randomly divided into 4 groups according to the severity of the disease at 1-2 minutes after onset. The negative control group was intraperitoneally injected with 500 μg mouse-IgG each time, the experimental group was intraperitoneally injected with 500 μg CXCR3-173-ADCC (Surrogate with ADCC, anti-mouse CXCR3-173 monoclonal antibody was 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 the use of CXCR3-173 as a surrogate antibody, see WO2018119288A1; CXCR3-173-ADCC is a molecule of CXCR3-173 with a mouse Fc and defucosylated), and the other experimental group was intraperitoneally injected with 500 μg CXCR3-173-LALA (Surrogate no ADCC, also a mouse Fc, whose Fc region contains L234A / L235A mutations), and the positive control group was intraperitoneally injected with 200 μg anti-TNFa (trade name: InVivoMAb anti-mouse TNFα, brand specification: bioxcell / 100mg, article number: BE0058), frequency is 1 week 3 times.After starting administration, with the frequency of weekly 2-3 times, the joint swelling degree of mice is scored, and scoring standard is that no joint is red, swelling is 0 point, and toe inflammation and swelling is 1 point, toe and instep inflammation and swelling are 2 points, toe, instep and ankle joint are all inflamed and swollen and are 3 points, severe swelling, toe, instep and ankle joint are all severely inflamed and swollen and are 4 points, and the limb joint swelling score adds up to the total score of arthritis. Meanwhile, mouse joint section is scored to pathological condition by hematoxylin eosin staining method, and scoring item comprises synovial cell proliferation degree, cell erosion degree, pannus degree, inflammation degree and bone erosion degree. Anti-CII-IgG self-antibody level is detected with ELISA method in mouse serum.

[0312] The experimental results are shown in Figure 10. The administration of CXCR3-173-ADCC significantly alleviated the progression of arthritis (degree of joint swelling), with clinical scores significantly lower than those in the negative control group and similar to those in the positive control group. However, the administration of CXCR3-173-LALA was unable to alleviate the disease progression due to its lack of killer cell properties. The pathological scores of the CXCR3-173-ADCC joints and the antibody levels against autoantigens were both reduced compared to the negative control group, and comparable to those in the positive control group. These results demonstrate that the antibodies described herein have a good therapeutic effect on CIA in mice, suggesting that the antibodies have a therapeutic effect on human rheumatoid arthritis.

[0313] Example 8 In vivo study of anti-CXCR3 antibodies in a systemic lupus erythematosus model

[0314] In this example, the therapeutic effect of the antibodies described in this application on systemic lupus erythematosus (SLE) was explored by using the mouse MRL / LPR model to simulate systemic lupus erythematosus (SLE) in humans.

[0315] Experimental methods:

[0316] This experiment used 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 of mouse-IgG each time. The experimental group received an intraperitoneal injection of 1 mg of CXCR3-173-ADCC (Surrogate with ADCC, as in Example 7). Another experimental group received an intraperitoneal injection of 1 mg of CXCR3-173-LALA (Surrogate without ADCC, as in Example 7). The positive control group received an intraperitoneal injection of 1 mg of anti-CD20 (trade name: InVivoMAb anti-mouse CD20, brand specification: bioxcell / 100 mg, catalog number: BE0356). All injections were administered once a week. Mice were photographed to observe skin ulcers and lymphadenopathy to determine the incidence rate within each group. Flow cytometry was used to assess the number of inflammatory cells in the kidneys to assess their infiltration level, and ELISA was used to measure the level of anti-dsDNA-IgG antibodies in the mice.

[0317] As shown in Figure 11, administration of CXCR3-173-ADCC significantly reduced skin ulceration and lymphadenopathy. In mice treated with CXCR3-173-ADCC, renal inflammatory cell infiltration and antibody levels against autoantigens were also reduced compared to the negative control group. These results demonstrate the therapeutic efficacy of the antibodies described herein in mouse models and suggest that they have therapeutic efficacy for human systemic lupus erythematosus.

[0318] Example 9 Safety and Pharmacokinetic Studies of Anti-CXCR3 Antibodies

[0319] In this example, the pharmacokinetic characteristics of the antibody of the present application were tested in primate experimental animals.

[0320] Experimental methods:

[0321] In this study, 6 cynomolgus macaques were randomly divided into a vehicle control group and a low-, mid-, mid-, high-, and high-dose group of the presently disclosed antibody (e.g., 3F11, 3F11hu1, 3F11hu8, and subsequent optimized antibodies, 3F11hu9) (dosages were 0, 30, 75, 150, 225, and 300 mg / kg, respectively), for a total of 6 groups, 1 monkey per group, for a total of 6 monkeys. Each group received an intravenous infusion of the vehicle control or the test substance at the corresponding concentration at a volume of 5 mL / kg, once a week (a total of 5 times in the first group, and 3 times in each test substance group). The drug was discontinued for 1 week after the last dose, and the day of the first dose was defined as the first day of the trial (D1).

[0322] During the trial, the general condition of all surviving animals was observed every day, and all surviving animals were subjected to detailed clinical observation once a week; body weight and food intake were measured once a week, and body temperature (rectal temperature) was measured before the first and last administration and about 24 hours after administration; blood pressure and electrocardiogram were measured before the first administration, on D2 and D16; hematology and blood biochemistry were tested before the first administration, on D2, before D8, on D9, D16, and on D22; blood coagulation was tested before the first administration, on D2, D9, D16, and on D22; 11. Lymphocyte subtypes (accompanied by RO) were detected on D15 before administration, D16, D18, and D22. Cytokines were detected before the first administration, 4 hours after administration on D1, and D2, and 4 hours after administration on D15 and D16. Flow cytometry was performed at the end of the administration period of Groups 2-6 (D22) and Group 1 (D36). The animals planned for dissection were euthanized for gross anatomical observation and histopathological examination of the heart, liver, spleen, lungs, kidneys, uterus, ovaries, testicles, thymus, submandibular lymph nodes, and administration site.

[0323] The results are shown in the table below. These results indicate that the antibodies described in this application have ideal safety and pharmacokinetic properties.

Claims

1. An isolated antigen-binding protein that can specifically bind to CXCR3, the antigen-binding protein comprising a heavy chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

14.

2. The isolated antigen-binding protein according to claim 1, wherein the HCDR1, HCDR2, and HCDR3 are selected from any one of the following groups: (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15; (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16; (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16; (4) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

16.

3. The isolated antigen-binding protein according to any one of claims 1-2, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-2.

4. The isolated antigen-binding protein according to any one of claims 1 to 3, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 4 to 7.

5. The isolated antigen-binding protein according to any one of claims 1 to 4, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 11 to 13.

6. The isolated antigen-binding protein according to any one of claims 1 to 5, wherein the HCDR1, HCDR2, and HCDR3 are selected from any one of the following groups: (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; (3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12; (4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; (5) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; (6) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13; (7) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; (8) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13; (9) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

13.

7. The isolated antigen-binding protein according to any one of claims 1 to 6, comprising H-FR1, wherein the C-terminus of said H-FR1 is directly or indirectly connected to the N-terminus of said HCDR1, and said H-FR1 comprises the amino acid sequence shown in SEQ ID NO:

19.

8. The isolated antigen-binding protein according to claim 7, wherein the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO:

18.

9. The isolated antigen-binding protein according to any one of claims 1 to 8, comprising H-FR2, wherein the H-FR2 is located between the HCDR1 and HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:

25.

10. The isolated antigen-binding protein of claim 9, wherein the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 20-24.

11. The isolated antigen-binding protein according to any one of claims 1 to 11, comprising H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:

38.

12. The isolated antigen-binding protein of claim 11, wherein the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 29-37.

13. The isolated antigen-binding protein according to any one of claims 1 to 12, comprising H-FR4, wherein the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

44.

14. The isolated antigen-binding protein of claim 13, wherein the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 42 or SEQ ID NO:

43.

15. The isolated antigen-binding protein according to any one of claims 1 to 14, comprising H-FR1, H-FR2, H- FR3 and H-FR4.

16. The isolated antigen-binding protein according to any one of claims 1 to 15, wherein the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 25, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 38, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

44.

17. The isolated antigen-binding protein according to any one of claims 1 to 16, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from any one of the following groups: (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 39, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 44; (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 40, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 41, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (4) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

43.

18. The isolated antigen binding protein according to any one of claims 1 to 17, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from any one of the following groups: (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 29, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 42; (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (4) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (7) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 22, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (8) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 21, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 43; (9) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 36, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 43; (10) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 24, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 37, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

43.

19. The isolated antigen-binding protein of any one of claims 1-18, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:

63.

20. The isolated antigen-binding protein of any one of claims 1-19, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 64-67.

21. The isolated antigen-binding protein according to any one of claims 1 to 20, wherein the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 64 to 67.

22. The isolated antigen-binding protein of any one of claims 1-21, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 45-62.

23. The isolated antigen-binding protein according to any one of claims 1 to 22, wherein the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 45 to 62.

24. The isolated antigen binding protein of any one of claims 1-23, which comprises an antibody or antigen binding fragment thereof.

25. The isolated antigen binding protein of claim 24, wherein the antibody comprises a single domain antibody.

26. The isolated antigen binding protein of any one of claims 1-25, wherein the antibody is selected from one or more of the following groups: a monoclonal antibody, a chimeric antibody, and a humanized antibody.

27. The isolated antigen binding protein of any one of claims 1-26, comprising an Fc domain.

28. The isolated antigen binding protein of claim 27, wherein the N-terminus of the Fc domain is directly or indirectly linked to the C-terminus of the heavy chain variable region.

29. The isolated antigen binding protein according to any one of claims 27-28, wherein the Fc domain is derived from the Fc domain of human IgG.

30. The isolated antigen binding protein of any one of claims 27-29, wherein the Fc domain comprises an Fc domain derived from any one of the following immunoglobulins: IgGl, IgG2, IgG3, and IgG4.

31. The isolated antigen binding protein of any one of claims 27-30, wherein the Fc domain comprises an Fc domain variant having at least one amino acid mutation relative to a wild-type Fc domain.

32. The isolated antigen binding protein of claim 31 , wherein the Fc domain variant has at least one amino acid mutation selected from the group consisting of H285D, L309D, L309G, M252Y, N434A, Q295R, Q311R, T250Q, T256D, T307A, T307Q, V308P, A330V, A378V, E380A, H285D, H435R, I332Y, L328W, M428E, M428L, N434A, N434W, P331V, Q311H, Q311V, T256D, T307Q, T307R, and T307W according to the Kabat EU numbering system.

33. The isolated antigen binding protein of any one of claims 31-32, wherein the Fc domain variant has a mutation or combination of mutations selected from the group consisting of: a) Q311R and M428L; b) L309G and M428L; c) Q311R, M428E and N434W; d) T250Q and M428L; e) T307A, E380A and N434A; f) V308P; g) H285D, T307Q and A378V; h) N434A and E380A; i) T250R and M428L; j) Q295R, L328W, A330V, P3 31V and I332Y; k) N434A; l) N434W; m) T256D and T307W; n) M252Y and T256D; o) T307Q, Q311V and A378V; p) T256D, H285D, T307R, Q311V and A378V; q) L309D, Q311H and N434S; r) H435R; s) T307Q, Q311V and A378V; t) T256D, H285D, T307R, Q311V and A378V; u) L309D, Q311H and N434S; and v) H435R.

34. The isolated antigen binding protein of any one of claims 31-33, wherein the Fc domain variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 68-86.

35. The isolated antigen binding protein of any one of claims 1-34, comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 87-428.

36. The isolated antigen binding protein of any one of claims 1-35, comprising a defucosylated antibody.

37. A polypeptide molecule comprising the isolated antigen binding protein of any one of claims 1-36.

38. The polypeptide molecule of claim 37, comprising a fusion protein.

39. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-36.

40. A nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-36 or the polypeptide molecule of any one of claims 37-38.

41. A vector comprising the nucleic acid molecule of claim 40.

42. A cell comprising the nucleic acid molecule of claim 40 or the vector of claim 41.

43. A method of making the isolated antigen binding protein of any one of claims 1-36.

44. The method of claim 43, comprising culturing the cell of claim 42 under conditions such that the antigen binding protein is expressed.

45. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1-36, the polypeptide molecule of any one of claims 37-38, the immunoconjugate of claim 39, the nucleic acid molecule of claim 40, the vector of claim 41 and / or the cell of claim 42, and optionally a pharmaceutically acceptable carrier.

46. ​​A method for preventing and / or treating a disease, the method comprising administering to a subject in need thereof the isolated antigen-binding protein of any one of claims 1-36, the polypeptide molecule of any one of claims 37-38, the immunoconjugate of claim 39, the nucleic acid molecule of claim 40, the vector of claim 41, the cell of claim 42 and / or the pharmaceutical composition of claim 45.

47. Use of the isolated antigen-binding protein of any one of claims 1-36, the polypeptide molecule of any one of claims 37-38, the immunoconjugate of claim 39, the nucleic acid molecule of claim 40, the vector of claim 41, the cell of claim 42 and / or the pharmaceutical composition of claim 45 in preventing and / or treating a disease.

48. Use of the isolated antigen-binding protein of any one of claims 1-36, the polypeptide molecule of any one of claims 37-38, the immunoconjugate of claim 39, the nucleic acid molecule of claim 40, the vector of claim 41, the cell of claim 42 and / or the pharmaceutical composition of claim 45 in the preparation of a medicament for preventing and / or treating a disease.

49. The method of claim 46 or the use of any one of claims 47-48, wherein the disease and / or condition comprises a disease and / or condition associated with CXCR3.

50. The method of claim 46 or the use of any one of claims 47-48, wherein the disease and / or disorder comprises an autoimmune disease associated with CXCR3.

51. The method of claim 46 or the use of any one of claims 47-48, wherein the disease and / or disorder comprises an allergic disease associated with CXCR3.

52. The method of claim 46 or the use of any one of claims 47-48, wherein the disease and / or condition comprises: Systemic lupus erythematosus, lupus nephritis, and / or rheumatoid arthritis.

53. A reagent or kit for detecting CXCR3 in a sample, comprising the isolated antigen-binding protein of any one of claims 1-36, the polypeptide molecule of any one of claims 37-38, the immunoconjugate of claim 39, the nucleic acid molecule of claim 40, the vector of claim 41, the cell of claim 42 and / or the pharmaceutical composition of claim 45.

54. A method for detecting CXCR3 in a sample, the method comprising using the isolated antigen binding protein of any one of claims 1-36, the polypeptide molecule of any one of claims 37-38, the immunoconjugate of claim 39, the nucleic acid molecule of claim 40, the vector of claim 41, the cell of claim 42 and / or the pharmaceutical composition of claim 45.