Antibody targeting cxcr6 and use thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

An antibody targeting CXCR6 and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the priority benefit of Chinese invention patent application No. CN202311766847.6 filed on December 20, 2023, the entire contents of which are hereby incorporated by reference. Technical Field

[0003] The present invention belongs to the field of antibody drugs, and in particular, relates to anti-CXCR6 antibodies and their use in preparing drugs. Background Art

[0004] CXC chemokine ligand 6 (CXCR6) is also known as Bonzo, STRL33, and TYMSTR. CXCR6 is expressed on activated T cells, including CD4+ T cells (Th1 and Th17 cells) and CD8+ T cells. These activated T cells are important drivers of a variety of inflammatory or immune disorders, including autoimmune diseases, graft-versus-host disease (GvHD), and neurodegenerative diseases. For example, abnormally activated T cells have been found to be important pathogenic cells in a variety of autoimmune diseases (such as psoriasis, vitiligo, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, etc.) or graft-versus-host disease.

[0005] Current treatments for inflammatory or immune disorders primarily include broad-spectrum anti-inflammatory drugs, such as glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs); inflammatory cytokine inhibitors, such as TNFα inhibitors or IL23-IL17 pathway inhibitors; JAK inhibitors, such as upadacitinib (JAK1 inhibitor) and ruxolitinib (JAK1 / 2 inhibitor); immune cell migration blockers, such as natalizumab (α4 blocker); and S1PR inhibitors, such as fingolimod. However, none of these therapeutic agents can eliminate pathogenic T cells at the source, requiring continuous medication to control disease progression. Therefore, direct elimination of pathogenic T cells (abnormally activated T cells) is expected to be a new breakthrough in the treatment of inflammatory or immune disorders. CXCR6, a cell surface marker of abnormally activated T cells, has great potential for the treatment of these diseases through clearing antibodies targeting CXCR6.

[0006] CXCR6 belongs to the G protein-coupled receptor (GPCR) family, which has short and segmented extracellular segments. Antibodies targeting members of this family often have a certain degree of nonspecific binding to empty cells, resulting in poor pharmacokinetic properties. CXCR6 is a seven-transmembrane protein, and there is an even greater need to provide an antibody with high specificity for activated T cells expressing CXCR6. In addition, considering the need for antibody molecules with human-monkey cross-reactivity in antibody drug development, especially in preclinical toxicology evaluation, the art also hopes to provide anti-CXCR6 antibodies with human-monkey cross-reactivity. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an anti-CXCR6 antibody that has high affinity, high selectivity and high biological activity for CXCR6-expressing cells; it has no or very low non-specific binding activity; at the same time, the antibody provided is humanized and can be more advantageously used in clinical treatment; and it is expected that the anti-CXCR6 antibody has human-monkey cross-species activity, which is convenient for use in preclinical toxicology evaluation.

[0008] Accordingly, the present invention provides the following technical solutions.

[0009] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof, which can bind to CXC family chemokine receptor 6 (CXCR6), particularly human CXCR6, with high affinity.

[0010] In the context of the present invention, unless otherwise indicated, the term "CXCR6" encompasses any form of CXCR6, such as active and inactive forms or membrane-bound and soluble forms; and encompasses any structural region of CXCR6, such as the extracellular region (ECD) and domains contained therein.

[0011] Specifically, the antibodies or antigen-binding fragments thereof provided herein comprise heavy chain complementarity determining regions (CDRs), i.e., heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), and heavy chain CDR3 (H-CDR3), and light chain complementarity determining regions (CDRs), i.e., light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), and light chain CDR3 (L-CDR3). According to a specific embodiment of the present invention, the heavy chain CDRs comprised by the antibody or antigen-binding fragment thereof are derived from the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3; and / or, the light chain CDRs comprised by the antibody or antigen-binding fragment thereof are derived from the amino acid sequence shown in any one of SEQ ID NO: 4 to SEQ ID NO: 6.

[0012] Any of the amino acid sequences provided above is the amino acid sequence of the heavy chain variable region (VH) or light chain variable region (VL) of the exemplary antibodies provided in the "Specific Embodiments" section of this application, including mouse antibodies, chimeric antibodies, and humanized antibodies. Using tools known in the art to define the complementary determining regions of antibody heavy chains or light chains (e.g., Chothia, Kabat, IMGT, Contact, CCG, etc.), those skilled in the art can easily determine the heavy chain CDRs and light chain CDRs contained therein.

[0013] Preferably, the antibodies or antigen-binding fragments thereof provided by the present invention comprise heavy chain CDRs and light chain CDRs from the heavy chain variable region and light chain variable region shown in the following amino acid sequence pairing:

[0014] (1) SEQ ID NO: 1 + SEQ ID NO: 4;

[0015] (2) SEQ ID NO: 2 + SEQ ID NO: 5; or

[0016] (3)SEQ ID NO:3+SEQ ID NO:6.

[0017] As described above, for example, the CCG numbering system can be used to divide the CDRs in the above amino acid sequence pairings, as shown in the Examples of the present invention.

[0018] Accordingly, in the antibodies or antigen-binding fragments thereof provided by the present invention, the heavy chain CDRs and light chain CDRs are as follows:

[0019] (1) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 7, 8, and 9, in sequence; and L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 13, in sequence;

[0020] (2) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 7, 10, and 9, in sequence; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 14, in sequence; or

[0021] (3) H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively; and L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 15, 16, and 14, respectively.

[0022] Preferably, the antibodies or antigen-binding fragments thereof provided herein specifically target CXCR6, preferably human CXCR6. Alternatively, the antibodies or antigen-binding fragments thereof provided herein may or may not have cross-species binding activity to human, monkey (cyno), or mouse CXCR6. Preferably, the antibodies or antigen-binding fragments thereof provided herein have cross-species binding activity to human and monkey CXCR6.

[0023] Preferably, the antibodies or antigen-binding fragments thereof provided by the present invention comprise a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDRs and a framework region (FR) therebetween, and the arrangement of each region from N-terminus to C-terminus is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0024] Further preferably, in the antibodies or antigen-binding fragments thereof provided by the present invention, the heavy chain variable region may comprise the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3, or an amino acid sequence having at least 75% identity thereto; and / or, the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 4 to SEQ ID NO: 6, or an amino acid sequence having at least 75% identity thereto. In the context of the present invention, the up to 25% difference in amino acid sequence resulting from the term "at least 75% identity" may be present in any framework region in the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibodies or antigen-binding fragments thereof of the present invention. The difference may be caused by deletion, addition or substitution of amino acids at any position, wherein the substitution may be a conservative substitution or a non-conservative substitution. The term "at least 75% identity" encompasses any percentage of identity between at least 75% identity and 100% identity, for example, 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0025] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise one selected from the following amino acid sequence combinations:

[0026] (1) the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 75% identity thereto; and, the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 75% identity thereto;

[0027] (2) the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 75% identity thereto; and, the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 75% identity thereto; or

[0028] (3) the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 75% identity thereto; and, the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 75% identity thereto.

[0029] Preferably, the antibodies provided by the present invention can be mouse antibodies, rabbit antibodies, human antibodies, or mouse antibodies, chimeric antibodies, or fully or partially humanized antibodies. The antibodies can also be called derivatized antibodies, for example, antibodies obtained by CDRs transplantation, affinity maturation, point mutation transformation, chemical modification, etc. on the basis of the initial mouse monoclonal antibody, wherein the chemical modification includes glycosylation, acetylation, pegylation, phosphorylation, amidation, protease cleavage, connection with cell ligands or effector molecules, etc., active reactive group protection and / or blocking, etc. Preferably, the antigen-binding fragment of the antibody can be any form of fragment of the antibody, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

[0030] In addition to the heavy chain and / or light chain variable regions, the antibodies or antigen-binding fragments thereof provided herein further comprise a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably comprising a human or murine heavy chain constant region and / or a light chain constant region. Preferably, the antibodies or fragments thereof comprise a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a kappa or lambda type light chain constant region.

[0031] According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody. According to a specific embodiment of the present invention, the monoclonal antibody comprises an IgG1 heavy chain constant region sequence; and / or comprises a kappa light chain constant region.

[0032] According to a specific embodiment of the present invention, the antibody of the present invention is a monoclonal antibody. Preferably, the anti-CXCR6 antibody provided herein is an immunoglobulin, for example, of the human IgA, IgD, IgE, IgG, or IgM subtype. Further preferably, the antibody is of the human IgG1 or IgG4 subtype. According to a specific embodiment of the present invention, the antibody of the present invention comprises a human IgG1 heavy chain constant region and a human kappa light chain constant region, the specific sequences of which are shown in the Examples section.

[0033] Furthermore, the anti-CXCR6 antibodies or antigen-binding fragments thereof provided by the present invention exhibit antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the anti-CXCR6 antibodies or antigen-binding fragments thereof provided by the present invention have enhanced ADCC activity. For example, the anti-CXCR6 antibodies are defucosylated, for example, monoclonal antibodies modified by removing fucose on N-glycosylation sites. Alternatively, the anti-CXCR6 antibodies provided by the present invention are human IgG1 antibodies and have one or more amino acid residue mutations selected from S298A, E333A, K334A, S239D, I332E, S239D, A330L, and I332E in their heavy chain Fc region, and the amino acid residues are numbered according to the Kabat EU index. According to a specific embodiment of the present invention, the anti-CXCR6 antibodies provided by the present invention have a triple mutation of S298A / E333A / K334A, a double mutation of S239D / I332E, or a triple mutation of S239D / A330L / I332E.

[0034] In another aspect, the present invention further provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0035] The "nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention" refers to a nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable region, heavy chain variable region, heavy chain, and / or light chain contained in the antibody or antigen-binding fragment thereof. For example, the nucleic acid molecule provided by the present invention comprises: a nucleotide sequence encoding each of the heavy chain CDRs and light chain CDRs contained in the above-mentioned antibody or antigen-binding fragment thereof; a nucleotide sequence encoding the heavy chain variable region and light chain variable region contained in the above-mentioned antibody or antigen-binding fragment thereof; or a nucleotide sequence encoding the heavy chain and light chain contained in the above-mentioned antibody or antigen-binding fragment thereof.

[0036] The nucleic acid molecules of the present invention can be cloned into a vector, and then transformed or transfected into a host cell. Therefore, in another aspect, the present invention also provides a vector comprising the nucleic acid molecules of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, and a phage vector, etc. The vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell for purposes such as preservation or antibody expression. Therefore, in another aspect, the present invention provides a host cell comprising the nucleic acid molecules of the present invention and / or the vector, or the host cell is transformed or transfected by the nucleic acid molecules of the present invention and / or the vector. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterium or an insect, fungus, or animal cell.

[0037] The antibodies or antigen-binding fragments thereof provided by the present invention can be obtained using any method known in the art. For example, the host cells provided by the present invention are allowed to express the heavy and light chains of the antibodies, and the host cells are cultured. Optionally, the method further comprises the step of recovering the produced antibodies.

[0038] On the other hand, the antibodies or antigen-binding fragments thereof provided by the present invention may also be directly or indirectly linked to other parts, such as the heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, heavy chains, and light chains of other antibodies; or, such as small molecule compounds, such as cytotoxic compounds used as antibody-drug conjugates; or, such as cell surface receptors, carbohydrates, polymers, etc., that modify the antibodies or antigen-binding fragments thereof. Therefore, accordingly, the present invention provides a conjugate, such as an antibody-drug conjugate, comprising the antibodies or antigen-binding fragments thereof provided by the present invention. The present invention also provides a bispecific or multispecific antibody, comprising the antibodies or antigen-binding fragments thereof provided by the present invention. The present invention also provides a fusion protein, comprising the antibodies or antigen-binding fragments thereof provided by the present invention.

[0039] The antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, conjugates, bispecific or multispecific antibodies, or fusion proteins provided by the present invention can be included in compositions, more particularly in pharmaceutical compositions, such as pharmaceutical preparations, and thus used for various purposes according to actual needs.

[0040] Therefore, in another aspect, the present invention further provides a composition comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, bispecific or multispecific antibody, or fusion protein provided herein, and optional pharmaceutically acceptable excipients. The composition provided herein can be prepared into various dosage forms known in the medical or pharmaceutical fields and administered via an appropriate method.

[0041] On the other hand, the present invention also provides the use of the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, bi- or multi-specific antibody or fusion protein in the preparation of a medicament for preventing, treating and / or improving a disease or condition, wherein the disease or condition is related to CXCR6 expression (including overexpression). In particular, the antibody or its antigen-binding fragment provided by the present invention can exert ADCC effects by binding to CXCR6, but is not limited thereto. For example, the antibody provided by the present invention can be a clearance antibody targeting CXCR6. Further, the disease or condition can be acute graft-versus-host disease, chronic graft-versus-host disease, autoimmune hepatitis, Crohn's disease, ulcerative colitis, contact dermatitis, rheumatoid arthritis, vitiligo, psoriasis, psoriatic arthritis, juvenile rheumatoid arthritis, multiple sclerosis, non-alcoholic steatohepatitis, systemic lupus erythematosus, uveitis, type I diabetes, ankylosing spondylitis, myelin oligodendrocyte glycoprotein-related diseases, Graves' disease, Sjögren's syndrome, etc.

[0042] Accordingly, the present invention also provides a method for preventing, treating and / or improving a disease or condition, comprising administering an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a bispecific or multispecific antibody or a fusion protein of the present invention to a subject in need thereof, wherein the disease or condition is associated with CXCR6 expression (including overexpression). Further, the disease or condition can be acute graft-versus-host disease, chronic graft-versus-host disease, autoimmune hepatitis, Crohn's disease, ulcerative colitis, contact dermatitis, rheumatoid arthritis, vitiligo, psoriasis, psoriatic arthritis, juvenile rheumatoid arthritis, multiple sclerosis, non-alcoholic fatty liver disease, systemic lupus erythematosus, uveitis, type I diabetes, ankylosing spondylitis, myelin oligodendrocyte glycoprotein-related disease, Graves' disease, Sjögren's syndrome, etc. The subject can be a mammal; more preferably, the subject is a human.

[0043] The methods for preventing, treating and / or ameliorating diseases or conditions provided by the present invention depend on a variety of factors when applied, including the specific active ingredients of the pharmaceutical composition administered, the age, weight, sex or physical and medical condition of the patient, the severity of the condition to be treated, the route of administration, etc.

[0044] On the other hand, the present invention also provides the use of the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, bi- or multi-specific antibody of the present invention in the preparation of a reagent for diagnosing a disease or condition, wherein the disease or condition is related to CXCR6 expression (including overexpression). Further, the disease or condition can be acute graft-versus-host disease, chronic graft-versus-host disease, autoimmune hepatitis, Crohn's disease, ulcerative colitis, contact dermatitis, rheumatoid arthritis, vitiligo, psoriasis, psoriatic arthritis, juvenile rheumatoid arthritis, multiple sclerosis, non-alcoholic steatohepatitis, systemic lupus erythematosus, uveitis, type I diabetes, ankylosing spondylitis, myelin oligodendrocyte glycoprotein-related disease, Graves' disease, Sjögren's syndrome, etc.

[0045] In another aspect, the present invention also provides a method for diagnosing a disease or condition, the method comprising contacting an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, bispecific or multispecific antibody or fusion protein of the present invention with a biological sample from a subject, wherein the disease or condition is associated with CXCR6 expression (including overexpression). Further, the disease or condition can be acute graft-versus-host disease, chronic graft-versus-host disease, autoimmune hepatitis, Crohn's disease, ulcerative colitis, contact dermatitis, rheumatoid arthritis, vitiligo, psoriasis, psoriatic arthritis, juvenile rheumatoid arthritis, multiple sclerosis, non-alcoholic steatohepatitis, systemic lupus erythematosus, uveitis, type I diabetes, ankylosing spondylitis, myelin oligodendrocyte glycoprotein-related disease, Graves' disease, Sjögren's syndrome, etc. The subject can be a mammal; more preferably, the subject is a human.

[0046] In another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a bispecific or multispecific antibody of the present invention. The kit can be used for the above-mentioned prevention, treatment and / or improvement, or the above-mentioned diagnosis, or for any detection of the CXCR6. Depending on the intended application, the kit also includes other reagents for prevention, treatment and / or improvement, diagnosis or detection. For example, the kit is a kit for detecting CXCR6 expression (including overexpression) in any biological sample using ELISA.

[0047] Compared with the prior art, the present invention provides a series of anti-CXCR6 mouse antibodies, chimeric antibodies and corresponding humanized sequences and humanized antibody molecules. Experiments have shown that the antibodies provided by the present invention can bind to CXCR6 with high affinity and high specificity by binding to different regions and sites of CXCR6; and the antibodies provided by the present invention also have cross-species binding activity to human and monkey CXCR6. In terms of mechanism of action, without being limited by any theory, the anti-CXCR6 antibodies provided by the present invention can exert their effects through ADCC and are clearance antibodies targeting CXCR6. Therefore, they have great potential for treating inflammatory or immune disorders and have shown significant therapeutic effects in various inflammatory disease models. In addition, compared with existing CXCR6 antibodies, the anti-CXCR6 antibodies provided by the present invention have significantly lower non-specific binding to cells, thereby having lower off-target toxicity and better safety. Of particular importance, the present invention provides humanized anti-CXCR6 antibodies and ADCC-enhanced versions thereof, which can be more conducive to clinical application.

[0048] Therefore, the anti-CXCR6 antibody provided by the present invention has important application potential in the treatment of inflammation or immune disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0050] FIG1 shows the titer responses of mouse sera obtained from mice immunized with human CXCR6-overexpressing cells to human CXCR6 and monkey CXCR6.

[0051] FIG2 shows the amino acid alignment results of the N-terminal region and the extracellular loop region of human CXCR6 and monkey CXCR6, where * indicates identical amino acids.

[0052] FIG3 shows the titer response of mouse sera obtained from mice immunized with human CXCR6 overexpressing cells with amino acids 2 to 13 deleted to human CXCR6 and monkey CXCR6.

[0053] Figure 4 shows the binding activity of the chimeric antibody according to the present invention to different CXCR6-overexpressing cells or blank cells, wherein 4A: HEK293-huCXCR6 cells; 4B: CHOS-huCXCR6-1T48 cells; 4C: HEK293-cynoCXCR6 cells; 4D: CHOS-mCXCR6 cells.

[0054] FIG5 shows the ADCC activity of the chimeric antibody according to the present invention against different CXCR6-overexpressing cells or blank cells, wherein 5A: HEK293-huCXCR6 cells; 5B: CHOS-huCXCR6-1T48 cells; 5C: HEK293-cynoCXCR6 cells.

[0055] FIG6 shows the binding activity of the humanized antibody hz22C6-H3L3 according to the present invention to different CXCR6-overexpressing cells or blank cells, wherein 6A: CHOS-huCXCR6-1T48 cells; 6B: HEK293-cynoCXCR6 cells.

[0056] FIG7 shows the non-specific binding activity of the humanized antibody hz22C6-H3L3 according to the present invention to different blank cells and ssDNA, wherein 7A: CHOS-Blank cells; 7B: HEK293-Blank cells; 7C: ssDNA.

[0057] FIG8 shows the binding activity of the humanized antibody hz22C6-H23L30 according to the present invention to different cells or ssDNA, wherein 8A: HEK293-huCXCR6 cells; 8B: HEK293-cynoCXCR6 cells; 8C: HEK293-Blank cells; 8D: ssDNA.

[0058] Figure 9 shows the ADCC activity of the humanized antibody hz22C6-H23L30 according to the present invention against different CXCR6-overexpressing cells or blank cells, wherein 9A: HEK293-huCXCR6 cells; 9B: HEK293-cynoCXCR6 cells; 9C: HEK293-Blank cells.

[0059] FIG10 shows the activities of different ADCC activity-enhanced versions of the humanized antibody hz22C6-H23L30 according to the present invention, wherein 10A: binding activity to CHOS-huCXCR6-1T48 cells; 10B: ADCC activity in CHOS-huCXCR6-1T48 cells.

[0060] Figure 11 shows the results of epitope analysis of clone 22C6 according to the present invention, wherein 11A: the relative binding strength of the humanized antibody hz22C6-H3L3 to huCXCR6 and its human-mouse chimeric forms in different regions; 11B: the relative binding strength of the humanized antibody hz22C6-H3L3 to huCXCR6 and its ECL2 and ECL3 different point mutation forms.

[0061] FIG12 shows the blood concentration trends of the humanized antibodies hz22C6-H3L3 and hz22C6-H23L30 according to the present invention in mice.

[0062] FIG13 shows the blood concentration trend of the humanized antibody hz22C6-H23L30 according to the present invention in cynomolgus monkeys.

[0063] 14 to 16 show the efficacy of the humanized antibodies hz22C6-H3L3 and hz22C6-H23L30 according to the present invention in a mouse EAE model.

[0064] Figure 17 shows the efficacy of the humanized antibody hz22C6-H23L30 according to the present invention in a mouse CIA model, wherein 17A: CIA clinical score; 17B: mouse body weight trend chart; 17C: CIA pathological score; 17D: typical CIA HE staining pathology image.

[0065] FIG18 shows the efficacy of the humanized antibody hz22C6-H23L30 according to the present invention in a mouse GvHD model.

[0066] Figure 19 shows the efficacy of the humanized antibody hz22C6-H23L30 (Fuc-) according to the present invention in a mouse IBD model, wherein 19A: IBD disease activity clinical score; 19B: statistical graph of the ratio of large intestine weight to length; 19C: photograph of the large intestine at the end of the experiment; 19D: typical large intestine HE staining pathology image; 19E: flow cytometry detection result graph.

[0067] FIG20 shows the efficacy of the humanized antibody hz22C6-H23L30 according to the present invention in a mouse vitiligo model, wherein 20A shows vitiligo scores; 20B shows typical mouse photos.

[0068] FIG21 shows the efficacy of anti-muCXCR6 antibodies in a mouse T1D model, wherein 21A shows the incidence of T1D; 21B shows a trend chart of blood glucose concentration.

[0069] Best Mode for Carrying Out the Invention

[0070] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0071] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0072] In the embodiment:

[0073] Human CXCR6 (huCXCR6 or hCXCR6): See UniProtKB-000574

[0074] Monkey CXCR6 (cynoCXCR6): see UniProtKB-Q9BDS6

[0075] Murine CXCR6 (muCXCR6 or mCXCR6): see UniProtKB-Q9EQ16

[0076] Control antibody Beacon2-G5: From patent publication WO2022251853A1, the variable region sequence is as follows, constructed using the human IgG1 heavy chain constant region and the human kappa light chain constant region:

[0077] Heavy chain variable region [SEQ ID NO: 17]:

[0078] Light chain variable region [SEQ ID NO: 18]:

[0079] Human IgG1 heavy chain constant region [SEQ ID NO: 19]:

[0080] Human kappa light chain constant region [SEQ ID NO: 20]:

[0081] Example 1: Mouse Antibody Screening

[0082] 1.1 Immunization of mice with human CXCR6

[0083] A cell line overexpressing human CXCR6, designated "CHOS-huCXCR6-1T19," was constructed using CHO-S cells (created by Beijing Kono Xincheng Technology Co., Ltd., clone number 1T19). These cells were used to immunize Balb / c mice (female, 6-8 weeks old, purchased from Shanghai Bikekeyi Biotechnology Co., Ltd.). Three immunizations were performed (two weeks apart, with 100 μL of 1E7 cells in PBS injected intraperitoneally each time), followed by orbital blood collection.

[0084] HEK293 cells were used to construct cell lines expressing human CXCR6 and monkey CXCR6, referred to as "HEK293-huCXCR6" and "HEK293-cynoCXCR6" cells, respectively. Serum titers were tested using HEK293-huCXCR6, HEK293-cynoCXCR6, and HEK293-Blank cells. Specifically, a PBS suspension of each cell type was prepared at a density of 4E6 / mL and plated at 50 μl / well in a 96-well plate. Mouse serum was diluted 1:50 in PBS and then added to the 96-well plate at 50 μl / well. The 96-well plate was incubated at 4°C for 1 hour. The supernatant was then discarded by centrifugation, and the plate was washed twice with PBS. A 1:1000 dilution of secondary antibody (APC Goat Anti-Mouse IgG (H+L), Jacksonimmuno, 115-136-146) was added to the 96-well plate. After incubation for 30 minutes, the cells were centrifuged and the supernatant discarded. The cells were then washed twice with PBS. Then, 30 μl of PBS was added to each well and mixed thoroughly. Serum titers were measured using an IQue Screener PLUS (Saorius) flow cytometer.

[0085] The results are shown in Figure 1 (data presented as "FACS signal value of the overexpressing strain / FACS signal value of the blank cell line," indicating target-specific titer responses). The results demonstrated that sera from all four mice exhibited strong specific titer responses to huCXCR6, while the specific titer responses to cynoCXCR6 were relatively weak.

[0086] 1.2 Immunization of mice with human CXCR6 deleted from amino acids 2 to 13

[0087] Preclinical toxicology assessment is a crucial step in drug development, and molecules with cross-species activity between humans and monkeys can greatly facilitate this process. Given the low titer of cynoCXCR6 in sera from mice immunized with CHOS-huCXCR6-1T19, the immunization cell line was redesigned to develop antibodies with strong binding to both human and monkey CXCR6.

[0088] Conservation analysis of the extracellular regions of huCXCR6 and cynoCXCR6 revealed that the two proteins differ primarily in the N-terminal domain (NTD), while they are highly conserved in extracellular loops (ECLs) 1 to 3. The comparison results are shown in Figure 2.

[0089] A cell line overexpressing human CXCR6 with amino acids 2 to 13 deleted (see the gray amino acids in Figure 2) was constructed using NIH / 3T3 cells, referred to as "NIH / 3T3-huCXCR6(δ2-13AA)." NIH / 3T3 cells were used to further reduce immune responses to other irrelevant antigens. Balb / c mice were immunized with NIH / 3T3-huCXCR6(δ2-13AA) cells using the same experimental procedures as above, and titers were determined.

[0090] The results are shown in Figure 3. The results showed that the sera of mice immunized with NIH / 3T3-huCXCR6 (δ2-13AA) cells had strong specific titer responses to both huCXCR6 and cynoCXCR6.

[0091] 1.3 Screening of hybridoma clones

[0092] On the third day after mice were boosted with the corresponding overexpression cell lines, they were killed, and the spleen and lymph nodes of the mice were obtained. After grinding, the obtained cells were pretreated and then electrofused with mouse myeloma cells. After 10 days of culture in HAT medium, the hybridoma supernatant was screened by flow cytometry. The cells used were HEK293-huCXCR6, HEK293-cynoCXCR6 and HEK293-Blank cell lines, and the experimental process was the same as above.

[0093] Finally, more than 100 hybridoma clones with cross-binding activity of huCXCR6 and cynoCXCR6 were obtained.

[0094] Example 2: Preparation and activity characterization of human-mouse chimeric antibodies

[0095] 2.1 Vector construction and antibody production

[0096] The antibody gene sequences of the hybridoma clones were amplified using mouse light and heavy chain degenerate primers. The obtained light and heavy chain amplified product fragments were directly ligated into the PTT5 expression vector containing the human kappa light chain constant region [SEQ ID NO: 20] and the human IgG1 heavy chain constant region [SEQ ID NO: 19]. The ligated product was transformed into competent DH5α cells (Yestern, Cat. No. E607-80VL), plated, and single clones were picked and sequenced.

[0097] After analyzing and obtaining the correct antibody light and heavy chain gene sequences, paired transient expression was performed using suspension-acclimated HEK293 cells. The heavy and light chain plasmids were transfected into HEK293 cells at a density of 2E6 / ml using PEI reagent (Polysciences, Cat. No. 24885) at a ratio of 2:3 (1 μg plasmid: 4 μg PEI). The transfected cells were cultured at 37°C in 5% CO2 for 5-7 days. The supernatant was collected and purified using a purification column filled with rProtein A agarose (GE Healthcare Bio-sciences, Cat. No. 127903). The purified sample was sterilized by filtration using a 0.22 μm filter, and the concentration of the purified antibody was determined using a Nanodrop One (Thermo Fisher Scientific).

[0098] 2.2 Analysis of binding activity of chimeric antibodies

[0099] A series of human-mouse chimeric antibodies were initially screened and identified, and three antibody molecules with better activity were selected and named "ch28N24", "ch22C6" and "ch30N18" respectively; the corresponding clones were named "28N24", "22C6" and "30N18" respectively.

[0100] These antibodies were characterized by FACS binding activity using various cell lines overexpressing huCXCR6, cynoCXCR6, and muCXCR6. Nonspecific binding was also assessed using blank cell lines. Beacon2-G5 was used as a control antibody. Specifically, a PBS suspension of 4E6 cells / mL was prepared and plated at 50 μl / well in a 96-well plate. Each antibody was diluted in PBS to a 2-fold concentration gradient and then added to the plate at 50 μl / well. The plate was incubated at 4°C for 1 hour. The supernatant was then centrifuged and washed twice with PBS. A secondary antibody (APC Goat Anti-human IgG Fcγ, Jacksonimmuno, 109-135-098) was added at a 1:1000 dilution to the plate. After incubation for 30 minutes, the supernatant was centrifuged and washed twice with PBS. Then, 30 μl of PBS was added to each well, mixed thoroughly, and assayed using an IQue Screener PLUS (Saorius) flow cytometer. The FACS binding curves are shown in FIG4 , and the binding EC50 values ​​are shown in Table 1 .

[0101] The results showed that all three antibodies had excellent binding activity to huCXCR6 and cynoCXCR6, with EC50 values ​​ranging from 0.7 to 2.6 μg / mL (Figures 4A-C; Table 1). Furthermore, none of the three antibodies bound to muCXCR6 (Figure 4D). Therefore, the experiments demonstrated that the chimeric antibodies provided by the present invention all had excellent human-monkey cross-binding activity.

[0102] Table 1. Binding activity of chimeric antibodies to target protein-expressing cells

[0103] 2.3 Analysis of ADCC activity of chimeric antibodies

[0104] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the primary mechanism of action of anti-CXCR6 clearance antibodies for the treatment of inflammatory diseases. Therefore, ADCC activity analysis was performed on the chimeric antibody molecules provided by the present invention using ADCC reporter cells. Specifically, overexpressing cell lines or control cell lines were used as target cells, and 2E4 cells / well were plated in a 96-well plate. The initial working concentration of the test antibody was 20 μg / mL, and the cells were diluted 8-fold in 10 gradients. The diluted antibody was added to the cells and mixed thoroughly. The cells were incubated in a 37°C, 5% CO2 incubator for 1 hour. Jurkat-CD16A / NF effector cells (purchased from Jiman Bio) were added to each well at a ratio of 7.5:1. 1.5E5 effector cells were added to each well. The cells were gently mixed to ensure sufficient contact between the effector and target cells. The cells were incubated in a 37°C, 5% CO2 incubator for 5 hours. Bio-Lite™ detection reagent (Vazyme, cat: DD1201) was added to detect the fluorescence signal of the well plate. The ADCC activity is shown in FIG5 .

[0105] The results showed that the three antibody molecules had very strong ADCC activity against huCXCR6 and cynoCXCR6 overexpressing cell lines.

[0106] Example 3 Humanization of mouse antibody from clone 22C6

[0107] 3.1 Humanization

[0108] Mouse antibody 22C6, derived from clone 22C6, was humanized using CDR region grafting, where CDR definitions follow the CCG numbering system. The selected humanization templates are shown in Table 2; the humanized sequences of the different versions of the light and heavy chains are shown in Table 3.

[0109] Table 2. Humanized light and heavy chain templates used for humanization of mouse anti-22C6

[0110] Table 3. Sequences of mouse anti-22C6 and its different humanized versions (the bold and underlined parts are CDRs)

[0111] 3.2 Binding activity analysis of humanized antibody hz22C6-H3L3 (VH: 22C6_VH_hz3; VL: 22C6_VK_hz3)

[0112] The hz22C6-H3L3 humanized antibody was obtained as described in Example 2, and its binding activity was tested by FACS. The FACS binding curve is shown in Figure 6, which shows that hz22C6-H3L3 has very strong binding activity to huCXCR6 and cynoCXCR6.

[0113] Because CXCR6 belongs to the GPCR family of seven transmembrane proteins, the extracellular segments of proteins in this family are relatively short and segmented. Antibodies targeting this family often have a certain degree of nonspecific binding to empty cells and biomacromolecules (proteins, lipids, DNA, etc.). The binding activity of hz22C6-H3L3 was tested using empty cell lines (HEK293-Blank and CHOS-Blank) that do not express CXCR6. The results showed that hz22C6-H3L3 had no nonspecific binding to empty cells, while the control molecule Beacon2-G5 had strong nonspecific binding at high concentrations (Figures 7A and 7B). The nonspecific binding activity of hz22C6-H3L3 to ssDNA was also tested using an ELISA method. Specifically, the coating medium was ssDNA (Sigma, Cat. No. D8899), and the secondary antibody used was Anti-Human IgG, Fcγ fragment specific (jacksononimmuno, 109-135-098). The results showed that at the highest concentration of 30 μg / mL, hz22C6-H3L3 had a low degree of nonspecific binding, while the control molecule Beacon2-G5 was very significant at a concentration of 7.5 μg / mL ( Figure 7C ).

[0114] 3.3 Binding activity analysis of humanized antibody hz22C6-H23L30 (VH: 22C6_VH_hz23; VL: 22C6_VK_hz30)

[0115] To further enhance the humanization of hz22C6-H3L3 and eliminate its ssDNA binding, further mutations were engineered in the light and heavy chains of hz22C6-H3L3, resulting in hz22C6-H23L30. Both the light and heavy chains of hz22C6-H23L30 exhibited enhanced humanization (Table 4).

[0116] Table 4. Degree of humanization of humanized antibodies

[0117] hz22C6-H23L30 was analyzed for binding activity by FACS. The results showed that hz22C6-H23L30 had very strong binding activity to huCXCR6 and cynoCXCR6 (Figures 8A and 8B), while also exhibiting no nonspecific binding to HEK293-blank cells (Figure 8C). An ELISA assay was used to test the nonspecific binding of hz22C6-H23L30 to ssDNA, demonstrating that hz22C6-H23L30 also exhibited no nonspecific binding to ssDNA at a high concentration (30 μg / mL).

[0118] 3.4 Analysis of ADCC activity of hz22C6-H23L30

[0119] hz22C6-H23L30 was analyzed for ADCC activity as described in Example 2. ADCC activity is shown in Figures 9A and 9B. The results demonstrate that hz22C6-H23L30 exhibits very strong ADCC activity against both huCXCR6 and cynoCXCR6-overexpressing cell lines. Furthermore, hz22C6-H23L30 exhibits no ADCC activity against null cells (Figure 9C). In contrast, the control molecule, Beacon2-G5, exhibited ADCC activity at high concentrations, suggesting the potential for off-target activity in vivo.

[0120] Example 4 Activity Detection of ADCC Activity Enhancer Molecules

[0121] According to public information, the ADCC activity of antibodies is affected by Fc receptor glycoforms and amino acid mutations at specific Fc sites. In order to obtain antibody molecules with stronger ADCC activity, the inventors designed and prepared hz22C6-H23L30 to enhance ADCC activity. A total of four different versions of ADCC activity-enhancing molecules were obtained, of which hz22C6-H23L30 (Fuc-) is a molecule without fucose in the N-sugar modification of the Fc region, hz22C6-H23L30 (AAA) is a molecule with a triple mutation of S298A / E333A / K334A introduced into the heavy chain, hz22C6-H23L30 (DE) is a molecule with a double mutation of S239D / I332E introduced into the heavy chain, and hz22C6-H23L30 (DLE) is a molecule with a triple mutation of S239D / A330L / I332E introduced into the heavy chain (wherein the amino acid residues are numbered according to the Kabat EU index).

[0122] The results of FACS and ADCC activity detection showed that the binding activity of the four ADCC-enhanced versions of the molecules to huCXCR6 was consistent with that of hz22C6-H23L30 ( Figure 10A ), and their ADCC activity was significantly improved compared with that of hz22C6-H23L30 ( Figure 10B ), suggesting that they have better clearance ability for CXCR6-positive cells in vivo.

[0123] Example 5 Epitope Analysis of Clone 22C6

[0124] Epitope analysis of clone 22C6 was performed using hz22C6-H3L3.

[0125] 5.1 Combined regional analysis

[0126] Considering that clone 22C6 does not bind to muCXCR6, we first replaced huCXCR6 with the N-terminus and extracellular loops 1–3 of muCXCR6, constructing the pCDNA3.1 transient expression vector for the human-mouse chimeric CXCR6 gene. A 6*his tag was added to the N-terminus of huCXCR6 and each chimeric gene for subsequent data normalization. All vectors were transiently expressed in HEK293 cells. Flow cytometric analysis was performed 48 hours after transfection using a his-tag antibody (Biolegend, Cat. No. 362605) and hz22C6-H3L3, as described above.

[0127] The signal values ​​of the his tag were normalized to those of huCXCR6, and the normalized results are shown in Figure 11 A. The results indicate that the binding epitope of clone 22C6 is mainly located at ECL2, with ECL1 and ECL3 having a minor effect on its binding.

[0128] 5.2 Binding site analysis

[0129] Further mutational analysis of the amino acids that differ between human and mouse on ECL2 and ECL3 was performed to identify key epitopes in clone 22C6. Fourteen His-tagged transient expression vectors containing point mutations (F173Q, D176A, Y182A, H183A, D184A, E185A, R254A, S255A, I256A, W258A, E259A, Y260A, Y261A, and H267K) were constructed and transiently expressed in HEK293 cells and analyzed by flow cytometry. The results are shown in Figure 11B. The results indicate that the key epitopes in clone 22C6 are Y182, H183, D184, E185, Y260, and Y261.

[0130] Example 6 Pharmacokinetic Analysis of Humanized Antibodies

[0131] 6.1 Pharmacokinetic Analysis in Mice

[0132] Mouse PK analysis was performed on different humanized antibody molecules based on clone 22C6.

[0133] Female BALB / c mice aged six weeks or older (purchased from Shanghai Bikeco Biotechnology Co., Ltd.) were used. A single intravenous injection of 200 μg / mouse was administered, with four mice per group. Approximately 50 μl of blood was collected at 1, 4, 8, 24, 48, 96, 120, 144, 192, 240, 288, and 336 hours after administration. Serum was separated and stored at -80°C.

[0134] ELISA was used to measure the plasma concentration of each antibody at various time points. The coating was an anti-huIgG Fab monoclonal antibody (Sigma, Catalog No. I5260-1ML) and the secondary antibody was an anti-human IgG, Fcγ fragment specific (Jackson Immuno, 109-135-098). The plasma concentration trends of each antibody are shown in Figure 12, and the PK analysis results are shown in Table 5. hz22C6-H23L30 exhibited a t½ of 178.06 hours, demonstrating favorable PK properties in mice.

[0135] Table 5. Pharmacokinetic analysis results of humanized antibodies in mice

[0136] 6.2 Pharmacokinetic Analysis in Cynomolgus Monkeys

[0137] PK analysis of hz22C6-H23L30 was performed in cynomolgus monkeys.

[0138] Two crab-eating macaques, one male and one female, were used. The dose was 20 mg / kg and the drug was infused intravenously within 30 minutes. Approximately 500 μl of blood samples were collected immediately after the infusion and at 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 5 days, 7 days, 10 days, 14 days, 21 days, 35 days, 49 days, and 63 days after the infusion. The serum was separated and stored at -80°C.

[0139] Blood drug concentrations at various time points were measured using ELISA. The coating was Goat Anti-Human IgG (H+L), minX Monkey (BERHYL, Catalog No. A80-319A), and the secondary antibody was Goat Anti-Human IgG, Monkey ads-HRP (Southern Biotech, 2049-05). The resulting antibody blood drug concentration trends are shown in Figure 13, and the PK analysis results are shown in Table 6.

[0140] Table 6. Pharmacokinetic analysis results of humanized antibodies in cynomolgus monkeys

[0141] The t1 / 2 of hz22C6-H23L30 is approximately 13 days, and it has good PK metabolic properties in crab-eating monkeys.

[0142] Example 7: Pharmacological efficacy of humanized antibodies

[0143] 7.1 Efficacy in the Experimental Animal Encephalitis Model (EAE Model)

[0144] Because the screened antibody molecules do not bind to muCXCR6, huCXCR6-knock-in-C57BL / 6J mice (Shanghai Model Organisms Co., Ltd.) were used for modeling. The modeling procedure was briefly as follows: after mice were anesthetized with a 1-4% isoflurane and oxygen mixture (flow rate 1-2 L / min), they were immunized subcutaneously with 100 μl of an emulsion (emulsion composition: 2 mg / mL MOG 35-55, complete Freund's adjuvant containing 4 mg / mL Mycobacterium tuberculosis, equal volumes were homogenized) injected subcutaneously on the back. Simultaneously, mice were intraperitoneally injected with pertussis toxin (1 μg / mL, 200 μL). 48 hours later, the same dose of pertussis toxin was injected intraperitoneally again.

[0145] After modeling, mice were weighed and EAE clinical scores were performed daily. The scoring criteria were as follows:

[0146] 0 points: normal, no obvious disease manifestations;

[0147] 1 point: tail weakness or hind limb weakness;

[0148] 2 points: tail weakness and hind limb weakness;

[0149] 3 points: partial hind limb paralysis;

[0150] 4 points: complete paralysis of the hind limbs;

[0151] 5 points: moribund state or death due to EAE.

[0152] When the average EAE score of each mouse reached approximately 1 point, the mice were grouped and intraperitoneally administered with the anti-CXCR6 antibody hz22C6-H3L3 of the present invention and the control hIgG1 (unrelated antibody). Multiple dose groups (15 mg / kg, 5 mg / kg, 2 mg / kg, 0.5 mg / kg) and different dosing times of hz22C6-H3L3 were set up, with 8 mice per group. The results showed that all hz22C6-H3L3 groups showed significant improvement in EAE symptoms compared to the control group, indicating a significant therapeutic effect. At the same time, the therapeutic effect of multiple administration (Figure 14A) was slightly better than that of single administration (Figure 14B), and the therapeutic effects of multiple administration at different doses were comparable. Flow cytometry analysis of blood samples collected on the 24th day after modeling revealed a higher proportion of CXCR6-positive T cells (CXCR6+ T cells) in the peripheral blood of mice in the control group, while the proportion of CXCR6+ T cells in the peripheral blood of each treatment group was significantly reduced ( Figures 15A and 15B ). This indicates that the anti-CXCR6 antibody hz22C6-H3L3 of the present invention can effectively eliminate CXCR6-positive T cells in mice (not limited by any theory, presumably through ADCC and other effects), improve the clinical symptoms of mice, and achieve a therapeutic effect.

[0153] Furthermore, the anti-CXCR6 antibody hz22C6-H23L30 of the present invention was used to verify the effectiveness of preventing and treating EAE in the model, with 8 mice in each group. The results are shown in Figure 16. The results showed that at a dose of 5 mg / kg, three preventive administrations (on days 4, 7, and 10 after modeling) significantly delayed the onset of EAE in mice and reduced the duration and severity of EAE (Figure 16A); four therapeutic administrations (on days 14, 18, 22, and 26 after modeling) almost completely alleviated the symptoms of EAE in mice, showing a good therapeutic effect (Figure 16B).

[0154] The above evidence suggests the therapeutic potential of anti-CXCR6 antibodies in central nervous system inflammatory diseases (such as multiple sclerosis, MOG antibody-related diseases, etc.).

[0155] 7.2 Efficacy in the Rheumatoid Arthritis Model (CIA Model)

[0156] The mouse CIA model is a commonly used rheumatoid arthritis model. HuCXCR6-knock-in C57BL / 6J mice were used for modeling. The procedure is as follows: On days 0 and 21, mice were subcutaneously injected with 100 μl of CII emulsion (4 mg / mL chicken type II collagen (dissolved in 50 mM acetic acid) and equal volumes of Freund's complete adjuvant containing 4 mg / mL Mycobacterium tuberculosis, thoroughly mixed and emulsified) at the base of their tails.

[0157] The CIA clinical scoring criteria are: each foot is scored separately;

[0158] 0 points: normal;

[0159] 1 point: redness and swelling of any joint (ankle joint, tarsometatarsal joint, toe joint, etc.);

[0160] 2 points: redness and swelling of any two joints;

[0161] 3 points: all three types of joints are red and swollen;

[0162] 4 points: The entire foot is completely red and swollen.

[0163] CIA pathological scoring criteria are shown in Table 7:

[0164] Table 7. CIA model pathology scoring criteria *: +1.0 means an additional 1.0 point will be added to the existing score.

[0165] Starting from the 21st day, the mice were scored for CIA clinically. The mice showing clinical symptoms of CIA were divided into two groups according to the scores and intraperitoneally administered (hz22C6-H23L30 and control hIgG1, n=12) at a dose of 10 mg / kg, once every three days for 7 times.

[0166] The results are shown in Figure 17 . The results show that administration of the anti-CXCR6 antibody hz22C6-H23L30 of the present invention significantly improved the clinical symptom scores of CIA in mice ( Figure 17A ). The body weight of mice in the hz22C6-H23L30 group was significantly higher than that in the hIgG1 control group ( Figure 17B ). Furthermore, the pathology score in the hz22C6-H23L30 group was lower than that in the hIgG1 control group ( Figures 17C and 17D , showing the results for the first mouse). These results suggest that anti-CXCR6 antibodies have therapeutic potential in rheumatoid arthritis.

[0167] 7.3 Efficacy in a Mouse Graft-Versus-Host Disease (GvHD) Model

[0168] The modeling process was to inoculate the selected human PBMC (Shanghai Rubai Biotechnology Co., Ltd., catalog number: PBMNC100C) into NCG mice (6-8 weeks old, female mice) through the tail vein at a dose of 1x10 7 Fourteen days after inoculation, the mice were randomly divided into two groups according to their body weight and intraperitoneally administered with hz22C6-H23L30 and control hIgG1 at a dose of 10 mg / kg, once every three days for a total of eight doses.

[0169] The results are shown in Figure 18. The results showed that the survival rate of GvHD model mice was improved after administration of the anti-CXCR6 antibody hz22C6-H23L30 of the present invention, indicating that anti-CXCR6 antibodies have therapeutic potential in GvHD.

[0170] 7.4 Efficacy in the Inflammatory Bowel Disease Model (IBD Model)

[0171] The efficacy of the anti-CXCR6 antibody of the present invention was evaluated using a CD4+CD25-T cell transplantation IBD model. The modeling process is briefly as follows: the donor mice are huCXCR6-knock in-C57BL / 6J transgenic mice and the recipient mice are B6-RAG- / - mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.). The spleen cells of the donor mice are sorted with CD4+CD25-magnetic beads and injected into the recipient mice through the tail vein at 5E6 / mouse after sorting. After injection, the mice were regularly scored for disease activity (twice a week before grouping and once a day thereafter), and scored from three dimensions: body weight, stool characteristics, and fecal occult blood. The scoring criteria are shown in Table 8:

[0172] Table 8. IBD disease activity score

[0173] The disease activity score of mice = (weight loss score + stool characteristics score + stool occult blood score) / 3.

[0174] When the average disease activity score of all mice was approximately 0.3, they were divided into groups (n=4, the day of grouping was designated as D0) and given hz22C6-H23L30 (Fuc-) and control hIgG1 antibodies at a dose of 5 mg / kg twice a week by intraperitoneal injection. The results are shown in Figure 19. The results showed that after administration of the anti-CXCR6 antibody of the present invention, the clinical symptoms of IBD in mice were significantly improved, the disease activity score was significantly reduced, and complete remission was achieved after D16 (Figure 19A). At the end of the experiment, D20, the colon weight / length ratio (the higher the ratio, the more severe the IBD disease) was obtained. The hz22C6-H23L30 (Fuc-) group was comparable to the unmodeled B6 RAG- / - mice and significantly lower than the control hIgG1 group (Figures 19B, 19C). HE pathological results showed that the large intestine of mice in the control hIgG1 group had a large amount of fibrous tissue hyperplasia, neutrophil and lymphocyte infiltration, mucosal necrosis and ulcer foci. The above pathological manifestations were only slightly present or completely absent in the hz22C6-H23L30(Fuc-) group (Figure 19D). At the same time, peripheral blood was collected on D15 and mesenteric lymph nodes were collected at the end of the experiment for flow cytometry analysis. The results showed that a high proportion of CXCR6+CD4+T cells were present in the peripheral blood and mesenteric lymph nodes in the control hIgG1 group, and this group of cells was significantly eliminated in the hz22C6-H23L30(Fuc-) group (Figure 19E). Together, these results indicate that CXCR6 plays an important role in the pathogenesis of the IBD model, suggesting that anti-CXCR6 antibodies have therapeutic potential in inflammatory bowel disease.

[0175] 7.5 Efficacy in Vitiligo Model

[0176] The efficacy of the anti-CXCR6 antibody of the present invention was evaluated using a mouse vitiligo model. The modeling process was briefly as follows: 2E5B16F10 melanoma cells were subcutaneously inoculated on the back of huCXCR6-knock-in-C57BL / 6J transgenic mice on D0. 200 μg of anti-mouse CD4 antibody (BioXcell, Cat. No. BE0003-3-50MG) was injected twice on D4 and D10. Around D21, the subcutaneous tumors were removed from the mice and the mice were randomly divided into groups. Each group of mice (n=7) was given hz22C6-H23L30 and control hIgG1 antibodies at a dose of 10 mg / kg twice weekly via intraperitoneal injection. The mice were continuously observed for the progression of vitiligo. The mice were scored on a scale of 0 to 7 based on the area of ​​white hair. The results showed that administration of the anti-CXCR6 antibody of the present invention improved the progression of vitiligo in the mice (Figures 20A and 20B), suggesting that antibodies targeting CXCR6 have therapeutic potential in vitiligo.

[0177] Example 8 Efficacy of anti-CXCR6 antibodies in type 1 diabetes (T1D)

[0178] Female NOD mice spontaneously develop T1D, but the onset is delayed. Therefore, an accelerated NOD mouse T1D model was used to evaluate the efficacy of antibodies targeting CXCR6. The modeling process was briefly described as follows: NOD mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were injected with 200 μg of anti-mouse PD-1 antibody twice on D0 and D14 to induce T1D. Simultaneously, starting on D0, each group of mice (n=7 or 8) were given a homemade defucosylated anti-mouse CXCR6 antibody and a control mIgG antibody at a dose of 5 mg / kg twice weekly via intraperitoneal injection. Starting on D0, mice were continuously monitored for T1D development (twice weekly). T1D onset was defined as a fasting blood glucose concentration ≥13.9 mmol / L in mice. The results are shown in Figure 21. The results demonstrate that anti-mouse CXCR6 antibodies can completely inhibit the onset of T1D (Figure 21A) and maintain normal blood glucose levels in mice (Figure 21B). This example suggests that antibodies targeting CXCR6 have therapeutic potential in T1D.

[0179] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

Claims

1. An anti-CXCR6 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs, i.e., heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), heavy chain CDR3 (H-CDR3), and light chain CDRs, i.e., light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), light chain CDR3 (L-CDR3), wherein: The heavy chain CDRs and light chain CDRs are shown below: (1) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 9 in sequence; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 11, 12, 13 in sequence; (2) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 7, 10, 9 in sequence; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 11, 12, 14 in sequence; or (3) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 9, in sequence; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 15, 16, 14, in sequence.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof specifically targets CXCR6, preferably human CXCR6; Optionally, the antibody or antigen-binding fragment thereof has species cross-binding activity between human and monkey CXCR6.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise one selected from the following amino acid sequence combinations: (1) the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 75% identity thereto; and, the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 75% identity thereto; (2) the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 75% identity thereto; and, the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 75% identity thereto; or (3) the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence that is at least 75% identical to the amino acid sequence; and, the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence that is at least 75% identical to the amino acid sequence.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antibody is a mouse antibody, a rabbit antibody, a human antibody, or a mouse antibody, a chimeric antibody, a fully or partially humanized antibody, or a derivatized antibody; Alternatively, the antigen-binding fragment of the antibody is a fragment of any form such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv of the antibody; Optionally, the antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably a human or mouse heavy chain constant region and / or a light chain constant region; preferably, the antibody or its fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that The antibody is a monoclonal antibody; preferably, the antibody is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM; preferably, the antibody is human IgG1 or IgG4 subtype; Preferably, the antibody or antigen-binding fragment thereof exhibits antibody-dependent cell-mediated cytotoxicity (ADCC), preferably has enhanced ADCC activity. 6 . A nucleic acid molecule comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof according to claim 1 . A vector comprising the nucleic acid molecule according to claim 6 . A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7 or the host cell according to claim 8, and optional pharmaceutically acceptable excipients.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8 or the composition according to claim 9 in the preparation of a medicament for preventing, treating and / or ameliorating a disease or condition.

11. A method for preventing, treating and / or ameliorating a disease or condition, the method comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8 or the composition of claim 9.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8 or the composition according to claim 9 in the preparation of an agent for diagnosing a disease or condition.

13. A method for diagnosing a disease or condition, the method comprising contacting the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, or the composition of claim 9 with a biological sample from a subject.

14. The use or method according to any one of claims 10 to 13, characterized in that The disease or disorder is associated with CXCR6 expression (including overexpression); Preferably, the disease or disorder is acute graft-versus-host disease, chronic graft-versus-host disease, autoimmune hepatitis, Crohn's disease, ulcerative colitis, contact dermatitis, rheumatoid arthritis, psoriasis, psoriatic arthritis, juvenile rheumatoid arthritis, multiple sclerosis, nonalcoholic steatohepatitis, systemic lupus erythematosus, uveitis, type I diabetes, ankylosing spondylitis, myelin oligodendrocyte glycoprotein-related disease, Graves' disease, Sjögren's syndrome; Preferably, the subject is a mammal; more preferably, the subject is a human.

15. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8 or the composition according to claim 9.