CXCR6 targeting antibody and application thereof

Abstract

The invention provides an anti-CXCR6 antibody and an application thereof. The anti-CXCR6 antibody provided by the invention can bind to CXCR6 with high affinity and high specificity, has species cross binding activity of human and monkey CXCR6, and is a CXCR6-targeted clearance antibody, so that the anti-CXCR6 antibody has great potential to be used for treating inflammation or immune disorder diseases; moreover, compared with the existing CXCR6 antibody, the anti-CXCR6 antibody provided by the invention has obviously lower cell nonspecific binding, so that the anti-CXCR6 antibody provided by the invention has lower off-target toxicity and better safety.

Description

[0001] Cross - reference to related applications

[0002] This patent application claims the priority of the Chinese invention patent application with the application number CN202311766847.6 filed on December 20, 2023, and the entire content thereof is incorporated herein by reference. Technical Field

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

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

[0005] Currently, the therapeutic drugs for inflammatory or immune dysregulation diseases are mainly broad-spectrum anti-inflammatory drugs, such as glucocorticoids and non-steroidal anti-inflammatory drugs; 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); S1PR inhibitors, such as fingolimod; and so on. However, these therapeutic drugs cannot completely eliminate the pathogenic T cells at the root, and continuous medication is required to control the disease progression. Therefore, the treatment method of directly eliminating pathogenic T cells (abnormally activated T cells) is expected to become a new breakthrough direction for the treatment of inflammatory or immune dysregulation diseases. As a cell surface marker of abnormally activated T cells, clearance antibodies targeting CXCR6 have great potential for application in the treatment of the above diseases.

[0006] CXCR6 belongs to the G protein-coupled receptor (GPCR) family. Proteins in this family have relatively short and segmented extracellular domains. Antibodies targeting members of this family often have a certain degree of non-specific binding to empty cells, resulting in poor pharmacokinetic properties. CXCR6 is a seven-transmembrane protein, and there is a 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 assessments, there is also a desire in the art 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 with high affinity, high selectivity, and high biological activity for CXCR6-expressing cells; it has no or extremely low non-specific binding activity; at the same time, the provided antibody is humanized and can be more advantageously applied to clinical treatment; and, it is expected that the anti-CXCR6 antibody has human-monkey cross-reactivity for convenient application in preclinical toxicology assessments.

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

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

[0010] In the context of the present invention, unless otherwise specified, 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 domain (ECD) and the domains it contains.

[0011] Specifically, the antibody or antigen-binding fragment provided by the present invention contains the complementarity-determining regions (CDRs) of the heavy chain, namely heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), heavy chain CDR3 (H-CDR3), and the complementarity-determining regions (CDRs) of the light chain, namely light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), light chain CDR3 (L-CDR3). According to the specific embodiments of the present invention, the heavy chain CDRs contained in the antibody or antigen-binding fragment are derived from the amino acid sequences shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3; and / or, the light chain CDRs contained in the antibody or antigen-binding fragment are derived from the amino acid sequences 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 the light chain variable region (VL) of the exemplary antibodies provided in the "Specific Embodiments" section of this application. The exemplary antibodies include murine antibodies, chimeric antibodies, and humanized antibodies. Using the well-known definition tools for antibody heavy chain or light chain complementarity-determining regions in the art (such as 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 antibody or its antigen-binding fragment provided by the present invention comprises the heavy chain CDRs and light chain CDRs of the heavy chain variable region and the light chain variable region shown in the following amino acid sequence pairs:

[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 pairs. See the examples shown in the present invention.

[0018] Accordingly, in the antibody or its antigen-binding fragment 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 sequentially comprising the amino acid sequences shown in SEQ ID NO:7, 8, 9; and, L-CDR1, L-CDR2, and L-CDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO:11, 12, 13;

[0020] (2) H-CDR1, H-CDR2, and H-CDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO:7, 10, 9; and, L-CDR1, L-CDR2, and L-CDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO:11, 12, 14; or

[0021] (3) H-CDR1, H-CDR2, and H-CDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO:7, 8, 9; and, L-CDR1, L-CDR2, and L-CDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO:15, 16, 14.

[0022] Preferably, the antibody or antigen-binding fragment thereof provided by the present invention specifically targets CXCR6, preferably human CXCR6. Optionally, the antibody or antigen-binding fragment thereof provided by the present invention has or does not have cross-species binding activity to human, cynomolgus monkey, and mouse CXCR6. Preferably, the antibody or antigen-binding fragment thereof provided by the present invention has cross-species binding activity to human and cynomolgus monkey CXCR6.

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

[0024] More preferably, in the antibody or antigen-binding fragment thereof provided by the present invention, the heavy-chain variable region may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3, or an amino acid sequence having at least 75% identity with the amino acid sequence; and / or, the light-chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6, or an amino acid sequence having at least 75% identity with the amino acid sequence. In the context of the present invention, the term "at least 75% identity" allows for up to 25% differences in the amino acid sequence to 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 antibody or antigen-binding fragment thereof of the present invention. The differences may be caused by amino acid deletions, additions, or substitutions at any position, where the substitutions may be conservative substitutions or non-conservative substitutions. The "at least 75% identity" encompasses any percentage of identity between at least 75% identity and 100% identity, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0025] According to the specific embodiments 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, and the heavy-chain variable region and the light-chain variable region comprise one of the following combinations of amino acid sequences:

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

[0027] (2) The amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 75% identity with said amino acid sequence; or

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

[0029] Preferably, the antibody provided by the present invention can be murine, rabbit, human, or can be murine-derived antibody, chimeric antibody or fully or partially humanized antibody. The antibody can also be referred to as a derivatized antibody, for example, an antibody obtained by CDR transplantation, affinity maturation, point mutation modification, chemical modification, etc. on the basis of an initial murine monoclonal antibody, wherein the chemical modification includes glycosylation, acetylation, polyethylene glycolylation, phosphorylation, amidation, protease cleavage, ligation with cell ligands or effector molecules, protection and / or blocking of reactive groups, etc. Preferably, the antigen-binding fragment of the antibody can be any form of fragment such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv of the antibody.

[0030] In addition to the heavy chain and / or light chain variable regions, the antibody or its antigen-binding fragment provided by the present invention further comprises 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 antibody or its fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a kappa or lambda 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 a heavy chain constant region sequence of IgG1; 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 by the present invention is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM. Further preferably, the antibody is a 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, and the specific sequences are shown in the example part.

[0033] Furthermore, the anti-CXCR6 antibody or its antigen-binding fragment provided by the present invention exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the anti-CXCR6 antibody or its antigen-binding fragment provided by the present invention has enhanced ADCC activity. For example, the anti-CXCR6 antibody is defucosylated, such as a monoclonal antibody with fucose modification removed from the N-glycosylation site. Alternatively, the anti-CXCR6 antibody provided by the present invention is a human IgG1 antibody and has one or more amino acid residue mutations selected from S298A, E333A, K334A, S239D, I332E, S239D, A330L, I332E in its heavy-chain Fc region, and the amino acid residue numbering is based on the Kabat EU index numbering. According to the specific embodiments of the present invention, the anti-CXCR6 antibody provided by the present invention has a triple mutation of S298A / E333A / K334A, a double mutation of S239D / I332E, or a triple mutation of S239D / A330L / I332E.

[0034] On the other hand, the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or its antigen-binding fragment of the present invention.

[0035] The "nucleotide sequence encoding the antibody or its antigen-binding fragment of the present invention" refers to the 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 its antigen-binding fragment. 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 antibody or its antigen-binding fragment; a nucleotide sequence encoding the heavy-chain variable region and light-chain variable region contained in the above antibody or its antigen-binding fragment; or, a nucleotide sequence encoding the heavy chain and light chain contained in the above antibody or its antigen-binding fragment.

[0036] The nucleic acid molecule of the present invention can be cloned into a vector and then transformed or transfected into a host cell. Thus, on the other hand, the present invention also provides a vector, and the vector comprises the nucleic acid molecule of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, 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, on the other hand, the present invention provides a host cell, and the host cell comprises the nucleic acid molecule and / or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and / or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal or animal cell.

[0037] The antibody or antigen-binding fragment thereof provided by the present invention can be obtained by any method known in the art. For example, the host cells provided by the present invention are cultured under conditions that allow the expression of the heavy and light chains of the antibody. Optionally, the method further includes the step of recovering the produced antibody.

[0038] On the other hand, the antibody or antigen-binding fragment thereof provided by the present invention can also be directly or indirectly linked to other moieties, such as heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, heavy chains, light chains of other antibodies; or, the other moieties are, for example, small molecule compounds, such as cytotoxic compounds used in antibody-drug conjugates; or, the other moieties are, for example, cell surface receptors, sugars, polymers and other moieties that modify the antibody or antigen-binding fragment thereof. Accordingly, the present invention provides a conjugate, such as an antibody-drug conjugate, which comprises the antibody or antigen-binding fragment thereof provided by the present invention. The present invention also provides a bispecific or multispecific antibody, which comprises the antibody or antigen-binding fragment thereof provided by the present invention. The present invention also provides a fusion protein, which comprises the antibody or antigen-binding fragment thereof provided by the present invention.

[0039] The antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, bispecific or multispecific antibody or fusion protein provided by the present invention can be included in a composition, more particularly included in a pharmaceutical composition, such as a pharmaceutical preparation, for various purposes according to actual needs.

[0040] Therefore, in yet another aspect, the present invention also provides a composition, which comprises the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, bispecific or multispecific antibody or fusion protein provided by the present invention, and optionally a pharmaceutically acceptable excipient. The composition provided by the present invention can be formulated into various dosage forms known in the medical or pharmaceutical field and administered by an appropriate route.

[0041] In another aspect, the present invention also provides the use of the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, bispecific or multispecific antibody or fusion protein in the preparation of a medicament for preventing, treating and / or ameliorating a disease or disorder associated with CXCR6 expression (including overexpression). In particular, the antibody or its antigen-binding fragment provided by the present invention can exert ADCC effect by binding to CXCR6, but is not limited thereto. For example, the antibody provided by the present invention can be a depleting antibody targeting CXCR6. Further, the disease or disorder 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, Sjogren's syndrome and the like.

[0042] Accordingly, the present invention also provides a method for preventing, treating and / or ameliorating a disease or disorder, the method comprising administering to a subject in need thereof the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, bispecific or multispecific antibody or fusion protein of the present invention, the disease or disorder being associated with CXCR6 expression (including overexpression). Further, the disease or disorder 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, Sjogren's syndrome and the like. The subject can be a mammal; more preferably, the subject is a human.

[0043] The above method for preventing, treating and / or ameliorating a disease or disorder provided by the present invention depends on various factors when applied, including the specific active ingredient 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] In another aspect, the present invention also provides the use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, bispecific or multispecific antibody of the present invention in the preparation of a reagent for diagnosing a disease or disorder associated with CXCR6 expression (including overexpression). Further, the disease or disorder may 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, Sjogren's syndrome, and the like.

[0045] In still another aspect, the present invention also provides a method for diagnosing a disease or disorder, the method comprising contacting the 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, the disease or disorder being associated with CXCR6 expression (including overexpression). Further, the disease or disorder may 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, Sjogren's syndrome, and the like. The subject may be a mammal; more preferably, the subject is a human.

[0046] In yet another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, bispecific or multispecific antibody of the present invention. The kit can be used in the above-mentioned prevention, treatment and / or improvement, or in the above-mentioned diagnosis, or in any detection of CXCR6. Depending on the intended mode of application, the kit further contains 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 murine antibodies, chimeric antibodies, as well as corresponding humanized sequences and humanized antibody molecules. Experiments have demonstrated 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 species cross-binding activity with human and monkey CXCR6. In terms of the mechanism of action, without being limited to any theory, the anti-CXCR6 antibodies provided by the present invention can exert their effects through ADCC, and are clearance antibodies targeting CXCR6, so they have great potential for treating inflammatory or immune dysregulation diseases, and have shown significant therapeutic effects in various inflammatory disease models. In addition, compared with existing CXCR6 antibodies, the non-specific binding of the anti-CXCR6 antibodies provided by the present invention to cells is significantly lower, thus having lower off-target toxicity and better safety. Particularly importantly, the humanized antibodies against CXCR6 and their ADCC-enhanced forms provided by the present invention are more conducive to clinical application.

[0048] Therefore, the anti-CXCR6 antibodies provided by the present invention have important application potential in the treatment of inflammatory or immune dysregulation diseases and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Hereinafter, the embodiments of the present invention will be described in detail in conjunction with the drawings, wherein:

[0050] Figure 1 Shows the titer responses of mouse sera obtained by immunizing mice with human CXCR6-overexpressing cells against human CXCR6 and monkey CXCR6.

[0051] Figure 2 Shows the amino acid alignment results of the N-terminal region and extracellular loop region of human CXCR6 and monkey CXCR6, where * represents the same amino acid.

[0052] Figure 3 Shows the titer responses of mouse sera obtained by immunizing mice with human CXCR6-overexpressing cells from which amino acids at positions 2-13 have been removed against human CXCR6 and monkey CXCR6.

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

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

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

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

[0057] Figure 8 Shows the binding activity of the humanized antibody hz22C6-H23L30 according to the present invention against different cells or ssDNA, where 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, where 9A: HEK293-huCXCR6 cells; 9B: HEK293-cynoCXCR6 cells; 9C: HEK293-Blank cells.

[0059] Figure 10 Shows the activity of different ADCC activity-enhanced versions of the humanized antibody hz22C6-H23L30 according to the present invention, where 10A: Binding activity against CHOS-huCXCR6-1T48 cells; 10B: ADCC activity in CHOS-huCXCR6-1T48 cells.

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

[0061] Figure 12 shows the blood drug concentration trends of the humanized antibodies hz22C6-H3L3 and hz22C6-H23L30 according to the present invention in mice.

[0062] Figure 13 Shows the blood drug concentration trend of the humanized antibody hz22C6-H23L30 according to the present invention in cynomolgus monkeys.

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

[0064] Figure 17 Shows the drug effect of the humanized antibody hz22C6-H23L30 according to the present invention in the mouse CIA model, where 17A: CIA clinical score; 17B: mouse body weight trend chart; 17C: CIA pathological score; 17D: typical CIA HE staining pathological picture.

[0065] Figure 18 Shows the drug effect of the humanized antibody hz22C6-H23L30 according to the present invention in the mouse GvHD model.

[0066] Figure 19 Shows the drug effect of the humanized antibody hz22C6-H23L30 (Fuc-) according to the present invention in the mouse IBD model, where 19A: IBD disease activity clinical score; 19B: statistical chart 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 pathological picture; 19E: flow cytometry detection result picture.

[0067] Figure 20 Shows the drug effect of the humanized antibody hz22C6-H23L30 according to the present invention in the mouse vitiligo model, where 20A: vitiligo score; 20B: typical mouse photograph.

[0068] Figure 21 Shows the drug effect of the anti-muCXCR6 antibody in the mouse T1D model, where 21A: T1D incidence; 21B: blood glucose concentration trend chart. Detailed implementation manners

[0069] The present invention will be described below with reference to specific embodiments. Those skilled in the art can understand that these embodiments are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0070] In the following examples, the experimental methods are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0071] In the examples:

[0072] Human CXCR6 (huCXCR6 or hCXCR6): See UniProtKB - O00574

[0073] Monkey CXCR6 (cynoCXCR6): See UniProtKB - Q9BDS6

[0074] Mouse CXCR6 (muCXCR6 or mCXCR6): See UniProtKB - Q9EQ16

[0075] Control antibody Beacon2 - G5: From patent publication WO2022251853A1, with the variable region sequences as follows, constructed with human IgG1 heavy chain constant region and human kappa light chain constant region:

[0076] Heavy chain variable region [SEQ ID NO:17]:

[0077] EVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISSRGSYTYYPDSVKGRLTISRENAKNTLSLQMSSLKSEDTAMYYCARRVFPYWFFDVWGAGTTVTVSS

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

[0079] DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKSGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYTTPYTFGGGTKLEIK

[0080] Human IgG1 heavy chain constant region [SEQ ID NO:19]:

[0081] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0082] Human kappa light chain constant region [SEQ ID NO:20]:

[0083] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0084] Example 1: Mouse antibody screening

[0085] 1.1 Immunization of mice with human CXCR6

[0086] A cell line overexpressing human CXCR6 was constructed using CHO-S cells, abbreviated as "CHOS-huCXCR6-1T19" cells (constructed by Beijing Konoxin Technology Co., Ltd., clone number 1T19). Balb / c mice (female, 6 - 8 weeks old, purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd.) were immunized with these cells. After three immunizations (immunized once every 2 weeks, and each immunization was an intraperitoneal injection of 100 uL of PBS suspension containing 1E7 cells), orbital blood was collected from the mice.

[0087] HEK293 cell lines expressing human CXCR6 and cynomolgus CXCR6 were constructed, abbreviated as "HEK293-huCXCR6" and "HEK293-cynoCXCR6" cells. Serum titration tests were performed using HEK293-huCXCR6, HEK293-cynoCXCR6 and HEK293-Blank cells. Specifically, PBS suspensions of the three cell lines with a density of 4E6 / mL were prepared and seeded into 96-well plates at 50 μL / well; mouse serum was diluted with PBS at a dilution factor of 1:50 and then added to the 96-well plates at 50 μL / well; the 96-well plates were incubated at 4 °C for 1 h. After that, the supernatant was discarded by centrifugation and then washed twice with PBS. A secondary antibody diluted 1:1000 (APC Goat Anti-Mouse IgG(H+L), jacksonimmmuno, 115-136-146) was added to the 96-well plates, and after incubation for 30 min, the supernatant was discarded by centrifugation and washed twice with PBS. Then, 30 μL of PBS was added to each well and mixed thoroughly, and the serum titer was detected using an IQue Screener PLUS (Saorius) flow cytometer.

[0088] The results are shown in Figure 1 (The data presentation method is "FACS signal value of the overexpression strain / FACS signal value of the Blank cell strain", which is used to indicate the titer response of target specificity). The results showed that the sera of all 4 mice had a strong specific titer response to huCXCR6, while the specific titer response to cynoCXCR6 was relatively weak.

[0089] 1.2 Mice were immunized with human CXCR6 with amino acids 2-13 removed

[0090] Preclinical toxicological evaluation is a very important part of drug development. Molecules with human-macaque species cross-reactivity can bring great convenience to toxicological evaluation. Given that the serum of mice immunized with CHOS-huCXCR6-1T19 had a weak titer against cynoCXCR6, the immunized cell line was redesigned to develop antibodies with strong binding ability to both human and cynomolgus CXCR6.

[0091] Through the conservative analysis of the extracellular regions of huCXCR6 and cynoCXCR6, it was found that huCXCR6 and cynoCXCR6 were mainly different in the N-terminal region (N-Terminal domain, NTD), while the conservation of extracellular loop (ECL) 1-3 was relatively high. The alignment results are shown in Figure 2 。

[0092] An NIH / 3T3 cell line overexpressing human CXCR6 with amino acids 2 - 13 removed (see the amino acids in the gray part in Figure 2 ) was constructed, abbreviated as "NIH / 3T3-huCXCR6(δ2-13AA)" cells. The NIH / 3T3 cells were used to further reduce the immune response to other irrelevant antigens of the cells. Using the same experimental procedure as above, the NIH / 3T3-huCXCR6(δ2-13AA) cells were used to immunize Balb / c mice and detect the titer.

[0093] The results are shown in Figure 3 . The results showed that the sera of the mice immunized with NIH / 3T3-huCXCR6(δ2-13AA) cells had strong specific titer responses to both huCXCR6 and cynoCXCR6.

[0094] 1.3 Screening of hybridoma clones

[0095] On the third day after the mice were boosted with the corresponding overexpressing cell line, the mice were sacrificed, and the spleens and lymph nodes of the mice were taken. After grinding, the obtained cells were pretreated and electrofused with mouse myeloma cells. After culturing in HAT medium for 10 days, flow cytometry screening of the hybridoma supernatant was performed. The cell lines used were HEK293-huCXCR6, HEK293-cynoCXCR6, and HEK293-Blank cell lines, and the experimental procedure was the same as above.

[0096] Finally, more than 100 hybridoma clones with cross-binding activity to huCXCR6 and cynoCXCR6 were obtained.

[0097] Example 2: Preparation and activity characterization of human-mouse chimeric antibodies

[0098] 2.1 Vector construction and antibody production

[0099] The antibody gene sequences of the hybridoma clones were amplified using mouse light and heavy chain degenerate primers. The obtained light and heavy chain amplification product fragments were directly ligated to 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 ligation product was transformed into competent cells DH5α (Yestern, catalog number E607-80VL), plated, and single clones were picked and sequenced.

[0100] After obtaining the correct antibody heavy and light chain gene sequences through analysis, 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 at a ratio of 2:3 using PEI reagent (Polysciences, catalog number 24885) (1 μg plasmid: 4 μg PEI), and the transfected cells were cultured at 37 °C and 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, catalog number 127903). The purified sample was filtered and sterilized using a 0.22 μm filter, and the concentration of the purified antibody was measured using Nanodrop One (Thermofisher Scientific).

[0101] 2.2 Analysis of the binding activity of chimeric antibodies

[0102] A series of human-mouse chimeric antibodies were initially screened and identified, and three antibody molecules with better activities were selected and named "ch28N24", "ch22C6", and "ch30N18" respectively; the corresponding clones were named "28N24", "22C6", and "30N18" respectively.

[0103] These antibody molecules were identified for their FACS binding activities using various overexpressing cell lines of huCXCR6, cynoCXCR6, and muCXCR6, and non-specific binding tests were performed using blank cell lines. In the experiment, Beacon2-G5 was used as a control antibody molecule. Specifically, PBS suspensions with a density of 4E6 / mL of each cell were prepared and plated in 96-well plates at 50 μl / well; each antibody was diluted to 2-fold the specified gradient concentration with PBS and then added to the 96-well plates at 50 μl / well; the 96-well plates were incubated at 4 °C for 1 h. After that, the supernatant was discarded by centrifugation, and then washed twice with PBS. A secondary antibody diluted 1:1000 (APC Goat Anti-human IgG Fcγ, jacksonimmmuno, 109-135-098) was added to the 96-well plates, and after incubation for 30 min, the supernatant was discarded by centrifugation and washed twice with PBS. Then, 30 μl of PBS was added to each well and mixed thoroughly, and detection was performed using an IQue Screener PLUS (Saorius) flow cytometer. The FACS binding curves are shown in Figure 4 , and the binding EC50 values are shown in Table 1.

[0104] The results showed that all three antibody molecules had very good binding activities to huCXCR6 and cynoCXCR6, and the EC50 values were between 0.7 - 2.6 μg / mL ( Figure 4A-C; Table 1), and at the same time, none of the three antibody molecules binds to muCXCR6 ( Figure 4 D). Therefore, the experiments prove that several chimeric antibodies provided by the present invention all have good human-monkey cross-binding activity.

[0105] Table 1. Binding activity of chimeric antibodies to target protein-expressing cells

[0106]

[0107] 2.3 Analysis of ADCC activity of chimeric antibodies

[0108] Antibody dependent cell-mediated cytotoxicity (ADCC) is the main mechanism of action of anti-CXCR6 clearance antibodies in the treatment of inflammatory diseases. Therefore, the ADCC activity of the chimeric antibody molecules provided by the present invention was analyzed using ADCC reporter gene cells. Specifically, an overexpressing cell line or a control cell line was used as the target cell, and 96-well plates were seeded at 2E4 / well. The initial working concentration of the antibody to be tested was 20 μg / mL, and it was diluted 8-fold for a total of 10 gradients. The diluted antibody was added to the cells and mixed well, and then cultured in a 37 °C, 5% CO2 incubator for 1 h. The effector cells jurkat-CD16A / NF (purchased from GeneCopoeia) were added to each well at a ratio of effector to target of 7.5:1, with 1.5E5 effector cells added per well, and gently mixed to allow the effector and target cells to come into full contact. Then, the cells were cultured in a 37 °C, 5% CO2 incubator for 5 hours, and the Bio-LiteTM detection reagent (Vazyme, cat: DD1201) was added to detect the fluorescence signal of the well plate. The ADCC activity is shown in Figure 5 .

[0109] The results show that all three antibody molecules have very strong ADCC activity against huCXCR6 and cynoCXCR6 overexpressing cell lines.

[0110] Example 3 Humanization of the murine antibody from clone 22C6

[0111] 3.1 Humanization

[0112] The murine antibody 22C6 from clone 22C6 was humanized by the method of CDR region grafting, and the CDRs were defined with reference to the CCG numbering system. The selected humanization templates are shown in Table 2; the humanized sequences of different versions of the heavy and light chains are shown in Table 3.

[0113] Table 2. Heavy and light chain humanization templates used for humanizing the murine antibody 22C6

[0114] Master heavy chain V - region template J - region template Master light chain V - region template J - region template 22C6_VH IGHV3 - 23*01 IGHJ6 - 1 22C6_VK IGKV1 - 12*01 IGKJ4 - 1

[0115] Table 3. Sequences of murine anti-22C6 and its different humanized versions (the bold and underlined parts are CDRs)

[0116]

[0117]

[0118] 3.2 Binding activity analysis of humanized antibody hz22C6-H3L3 (VH: 22C6_VH_hz3; VL: 22C6_VK_hz3)

[0119] As described in Example 2, the humanized antibody molecule hz22C6-H3L3 was obtained, and its binding activity was detected by FACS. The FACS binding curve is shown in Figure 6 , and the results showed that hz22C6-H3L3 had very strong binding activities to huCXCR6 and cynoCXCR6.

[0120] Since CXCR6 belongs to the seven-transmembrane proteins of the GPCR family, and the extracellular segments of proteins in this family are all short and fragmented, antibodies targeting this family often have a certain degree of non-specific binding to empty cells and biological macromolecules (proteins, lipids, DNA, etc.). The binding activities of hz22C6-H3L3 were tested using empty cell lines that do not express CXCR6 (HEK293-Blank and CHOS-Blank), and the results showed that hz22C6-H3L3 had no non-specific binding to empty cells, while the control molecule Beacon2-G5 had strong non-specific binding at high concentrations ( Figure 7 A, 7B). At the same time, the ELISA method was used to detect the non-specific binding activity of hz22C6-H3L3 to ssDNA. Specifically, the coating was ssDNA (Sigma, catalog number D8899), and the secondary antibody used was Anti-Human IgG, Fcγ fragment specific (jacksonimmmuno, 109-135-098). The results showed that at the highest concentration of 30 μg / mL, hz22C6-H3L3 had a low degree of non-specific binding, while the control molecule Beacon2-G5 was very significant at a concentration of 7.5 μg / mL ( Figure 7 C).

[0121] 3.3 Binding activity analysis of humanized antibody hz22C6-H23L30 (VH: 22C6_VH_hz23; VL: 22C6_VK_hz30)

[0122] Furthermore, to enhance the humanization level of hz22C6-H3L3 and simultaneously eliminate its binding to ssDNA, further mutagenesis designs were made on the heavy and light chains of hz22C6-H3L3, resulting in the molecule hz22C6-H23L30. The humanization levels of both the heavy and light chains of hz22C6-H23L30 were enhanced (Table 4).

[0123] Table 4. Humanization level of humanized antibodies

[0124]

[0125] FACS binding activity analysis was performed on hz22C6-H23L30. The results showed that hz22C6-H23L30 had very strong huCXCR6 and cynoCXCR6 binding activities ( Figure 8 A, 8B), and at the same time had no non-specific binding to the empty cell HEK293-blank ( Figure 8 C). ELISA was used to test the non-specific binding of hz22C6-H23L30 to ssDNA. The results showed that hz22C6-H23L30 also had no non-specific binding to ssDNA at high concentration (30 μg / mL).

[0126] 3.4 ADCC activity analysis of hz22C6-H23L30

[0127] According to Example 2, ADCC activity analysis was performed on hz22C6-H23L30. The ADCC activity is shown in Figure 9 A, 9B. The results showed that hz22C6-H23L30 had very strong ADCC activities against both huCXCR6 and cynoCXCR6 overexpressing cell lines; and hz22C6-H23L30 had no ADCC activity against empty cells ( Figure 9 C), while the control molecule Beacon2-G5 had an ADCC signal at high concentration, indicating that it may have off-target killing activity in vivo.

[0128] Example 4 Activity detection of ADCC activity enhancing molecules

[0129] According to the public information, the ADCC activity of antibodies is affected by the glycoform of Fc receptor and amino acid mutations at specific Fc sites. To obtain antibody molecules with stronger ADCC activity, the inventors designed the enhancement of ADCC activity for hz22C6-H23L30 and prepared antibody molecules. A total of four different versions of ADCC activity-enhanced molecules were obtained. Among them, hz22C6-H23L30(Fuc-) is a molecule without fucose in the N-glycosylation of the Fc region, hz22C6-H23L30(AAA) is a molecule with triple mutations of S298A / E333A / K334A introduced into the heavy chain, hz22C6-H23L30(DE) is a molecule with double mutations of S239D / I332E introduced into the heavy chain, and hz22C6-H23L30(DLE) is a molecule with triple mutations of S239D / A330L / I332E introduced into the heavy chain (where the amino acid residue numbers are based on the Kabat EU index number).

[0130] The results of FACS and ADCC activity assays showed that the four ADCC-enhanced versions of the molecules had the same binding activity to huCXCR6 as hz22C6-H23L30( Figure 10 A), and at the same time, their ADCC activity was significantly improved compared to hz22C6-H23L30( Figure 10 B), suggesting that they have better clearance ability for CXCR6-positive cells in vivo.

[0131] Example 5 Epitope analysis of clone 22C6

[0132] Epitope analysis of clone 22C6 was performed using hz22C6-H3L3

[0133] 5.1 Binding region analysis

[0134] Considering that clone 22C6 does not bind to muCXCR6, the N-terminal and extracellular loop segments 1-3 of muCXCR6 were used to replace huCXCR6 one by one, and a transient expression vector of the human-mouse chimeric CXCR6 gene, pCDNA3.1, was constructed. At the same time, a 6*his tag was added to the N-terminus of huCXCR6 and each chimeric gene for subsequent standardization of experimental data. All vectors were transiently expressed in HEK293 cells. After 48 hours of transfection, flow cytometry analysis was performed using a his-tag antibody (Biolegend, catalog number 362605) and hz22C6-H3L3, and the method was the same as above.

[0135] By normalizing the signal value of the his tag relative to the signal value of huCXCR6, the normalized results are shown in Figure 11A. The results showed that the binding epitope of clone 22C6 was mainly in ECL2, and ECL1 and ECL3 had secondary effects on its binding.

[0136] 5.2 Binding site analysis

[0137] Furthermore, mutation analysis was performed on the human-mouse differential amino acids on ECL2 and ECL3 to clarify the key epitopes of clone 22C6. A total of 14 point mutation transient expression vectors with his tags, namely F173Q, D176A, Y182A, H183A, D184A, E185A, R254A, S255A, I256A, W258A, E259A, Y260A, Y261A, H267K, were constructed and subjected to HEK293 transient expression and flow cytometry analysis. The results are shown in Figure 11 B. The results showed that the key epitopes of clone 22C6 were Y182, H183, D184, E185, Y260, and Y261.

[0138] Example 6 Pharmacokinetic analysis of humanized antibodies

[0139] 6.1 Pharmacokinetic analysis in mice

[0140] Mouse PK analysis was performed on different humanized antibody molecules based on clone 22C6.

[0141] Balb / c mice, female, older than six weeks of age (purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd.) were used. The dosing regimen was a single intravenous injection at a dose of 200 μg / mouse, with 4 mice in each group. Then, approximately 50 μl of blood was collected at 1 h, 4 h, 8 h, 24 h, 48 h, 96 h, 120 h, 144 h, 192 h, 240 h, 288 h, and 336 h after dosing, the serum was separated and stored at -80 °C.

[0142] The blood drug concentration of each antibody molecule at each time point was detected by ELISA. The coating agent was anti-huIgG Fab monoclonal antibody (Sigma, catalog number I5260-1ML), and the secondary antibody was Anti-HumanIgG, Fcγ fragment specific (jacksonimmmuno, 109-135-098). The blood drug concentration trends of each antibody finally detected are shown in Figure 12 , and the PK analysis results are shown in Table 5. Among them, the t1 / 2 of hz22C6-H23L30 was 178.06 hours, showing good mouse PK metabolic properties.

[0143] Table 5. Pharmacokinetic analysis results of humanized antibodies in mice

[0144] PK parameters (unit) hz22C6 - H3L3 hz22C6 - H23L30 t1 / 2 (hours) 153.37 178.06 Cmax (μg / ml) 256.48 255.53 AUC - 192 (μg / ml*hours) 23435.82 24936.78

[0145] 6.2 Pharmacokinetic Analysis in Cynomolgus Monkeys

[0146] PK analysis of hz22C6-H23L30 was performed in cynomolgus monkeys.

[0147] Two cynomolgus monkeys (one male and one female) were used. The dosing dose was 20 mg / kg, and the drug was intravenously infused within 30 min. Approximately 500 μl of blood samples were collected immediately after the infusion, and at 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, on the 5th day, 7th day, 10th day, 14th day, 21st day, 35th day, 49th day, and 63rd day after the infusion. The serum was separated and stored at -80 °C.

[0148] The blood drug concentration at each time point was detected by ELISA. The coating agent was Goat Anti-HumanIgG(H+L), minXMonkey (BERHYL, catalog number A80-319A), and the secondary antibody was GoatAnti-Human IgG, Monkey ads-HRP (southernbiotech, 2049-05). The trend of the blood drug concentration of the finally detected antibody is shown in Figure 13 , and the PK analysis results are shown in Table 6.

[0149] Table 6. Pharmacokinetic Analysis Results of Humanized Antibody in Cynomolgus Monkeys

[0150] PK parameters (unit) hz22C6 - H23L30 (male monkey) hz22C6 - H23L30 (female monkey) t1 / 2 (days) 12.37 13.65 Cmax (μg / ml) 1212.00 1082.00 AUC - D63 (μg / ml*days) 8521.56 7725.94

[0151] The t1 / 2 of hz22C6-H23L30 was approximately 13 days, showing good PK metabolic properties in cynomolgus monkeys.

[0152] Example 7 Efficacy of Humanized Antibody

[0153] 7.1 Efficacy in Experimental Animal Encephalitis Model (EAE Model)

[0154] Since the screened antibody molecules do not bind to muCXCR6, the model mice used were huCXCR6-knock in-C57BL / 6J mice (Shanghai Model Organisms Center, Inc.). The modeling process is briefly as follows: After the mice were anesthetized by inhalation of a mixed gas of 1-4% isoflurane and oxygen (flow rate 1-2 L / min), they were immunized by subcutaneous injection of 100 ul of emulsion (composition of the emulsion: 2 mg / mL of MOG 35-55, complete Freund's adjuvant containing 4 mg / mL of Mycobacterium tuberculosis, homogenized at high speed in equal volume), and at the same time, the mice were intraperitoneally injected with pertussis toxin (1 ug / mL, 200 uL). 48 hours later, the same dose of pertussis toxin was intraperitoneally injected again.

[0155] After modeling, the mice were weighed and clinically scored for EAE daily. The scoring criteria were as follows:

[0156] Score 0: Normal, without obvious disease manifestations;

[0157] Score 1: Weak tail or weak hind limbs;

[0158] Score 2: Weak tail and weak hind limbs at the same time;

[0159] Score 3: Partial hind limb paralysis;

[0160] Score 4: Complete hind limb paralysis;

[0161] Score 5: Near death state or death due to EAE.

[0162] When the average score of EAE in each mouse reached about 1, the mice were grouped and intraperitoneally administered drugs. The drugs used were the anti-CXCR6 antibody hz22C6-H3L3 of the present invention and the control hIgG1 (irrelevant antibody). Multiple dose groups (15 mg / kg, 5 mg / kg, 2 mg / kg, 0.5 mg / kg) and tests with different administration frequencies were set for hz22C6-H3L3, with 8 mice in each group. The results showed that significant improvement in the symptoms of EAE mice was observed in all hz22C6-H3L3 groups compared with the control group, that is, a significant therapeutic effect was achieved; at the same time, the therapeutic effect of multiple administrations ( Figure 14 A) was slightly better than that of single administration ( Figure 14 B), and the therapeutic effects of multiple administrations at different doses were comparable. By collecting blood on the 24th day after modeling for flow cytometry analysis, it was found that there was a higher proportion of CXCR6-positive T cells (CXCR6+ T cells) in the peripheral blood of the control group mice, while the proportion of CXCR6+ T cells in the peripheral blood of each treatment group was significantly reduced ( Figure 15A, 15B), indicating that the anti-CXCR6 antibody hz22C6-H3L3 of the present invention can effectively eliminate CXCR6-positive T cells in mice (without being limited to any theory, presumably through effects such as ADCC), improve the clinical symptoms of mice, and achieve a therapeutic effect.

[0163] Furthermore, the anti-CXCR6 antibody hz22C6-H23L30 of the present invention was used to verify the preventive onset and therapeutic effects on the EAE 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, 3 times of preventive administration (on the 4th, 7th, and 10th days after modeling) could significantly delay the onset of EAE mice and at the same time reduce the duration and severity of EAE onset ( Figure 16 A); 4 times of therapeutic administration (on the 14th, 18th, 22nd, and 26th days after modeling) could almost achieve a complete remission of the EAE symptoms in mice, showing good therapeutic effects ( Figure 16 B).

[0164] 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.).

[0165] 7.2 Pharmacodynamic effects in the rheumatoid arthritis model (CIA model)

[0166] The mouse CIA model is a commonly used rheumatoid arthritis model. The mice used for modeling were also huCXCR6-knock in-C57BL / 6J mice. The modeling process was briefly as follows: On the 0th day and the 21st day, 100 μL of CII emulsion (the emulsion composition was 4 mg / mL chicken type II collagen (dissolved in 50 mM acetic acid solution) and an equal volume of Freund's complete adjuvant containing 4 mg / mL Mycobacterium tuberculosis was fully mixed and emulsified) was subcutaneously injected at the root of the mouse tail.

[0167] The CIA clinical scoring criteria were as follows: Each foot was scored separately;

[0168] 0 points: Normal;

[0169] 1 point: Redness and swelling of any joint (ankle joint, tarsometatarsal joint, toe joint, the same below);

[0170] 2 points: Redness and swelling of any two joints;

[0171] 3 points: Redness and swelling of all three types of joints;

[0172] 4 points: The entire foot was completely red and swollen.

[0173] The CIA pathological scoring criteria are shown in Table 7:

[0174] Table 7. CIA model pathological scoring criteria

[0175]

[0176]

[0177] *: +1.0 means adding an additional 1.0 point to the existing score.

[0178] On the 21st day, CIA clinical scoring was started for the mice. The mice showing CIA clinical symptoms were evenly divided into two groups according to the score for intraperitoneal administration (hz22C6-H23L30 and control hIgG1, n = 12), with a dose of 10 mg / kg, once every three days, for 7 administrations.

[0179] The results are shown in Figure 17 ... The results showed that after administration of the anti-CXCR6 antibody hz22C6-H23L30 of the present invention, the CIA clinical symptom score of the mice was significantly improved ( Figure 17 A), and the body weight of the mice in the hz22C6-H23L30 group was significantly higher than that in the control hIgG1 group ( Figure 17 B). At the same time, the pathological score of the hz22C6-H23L30 group was lower than that of the control hIgG1 group ( Figure 17 C, 17D, showing the results of the first mouse). These results indicate that the anti-CXCR6 antibody has therapeutic potential in rheumatoid arthritis.

[0180] 7.3 Pharmacodynamic effect in a mouse graft-versus-host disease model (GvHD model)

[0181] The modeling process was to inoculate the screened human PBMC (Shanghai Rubai Biotechnology Co., Ltd., product number: PBMNC100C) into NCG mice (6 - 8 weeks old, female mice) through the tail vein, with an inoculation dose of 1x10 7 / 200 ul. 14 days after inoculation, the mice were randomly divided into two groups according to their body weight for intraperitoneal administration (hz22C6-H23L30 and control hIgG1), with a dose of 10 mg / kg, once every three days, for 8 administrations.

[0182] The results are shown in Figure 18 ... The results showed that after administration of the anti-CXCR6 antibody hz22C6-H23L30 of the present invention, the survival rate of the GvHD model mice was improved, indicating that the anti-CXCR6 antibody has therapeutic potential in GvHD.

[0183] 7.4 Pharmacodynamic effect in an inflammatory bowel disease model (IBD model)

[0184] The efficacy of the anti-CXCR6 antibody of the present invention was evaluated using a CD4+CD25-T cell transplanted IBD model. The modeling process is briefly as follows: The donor mice were huCXCR6-knock in-C57BL / 6J transgenic mice and the recipient mice were B6-RAG- / - mice (Jiangsu Jicui Yakang Biotechnology Co., Ltd.). The spleen cells of the donor mice were sorted using CD4+CD25- magnetic beads and then injected into the recipient mice via the tail vein at a dose of 5E6 per mouse. After injection, the mice were regularly scored for disease activity (twice a week for one week before grouping and once a day after grouping). The scoring was performed from three dimensions: body weight, stool characteristics, and fecal occult blood. The scoring criteria are shown in Table 8:

[0185] Table 8. IBD disease activity scoring table

[0186] Score Weight loss (%) Stool consistency Stool occult blood 0 - Normal Negative (-) 1 1-5 Loose but not sticky around anus Positive (±) 2 5-10 Loose and sticky around anus Positive (+) 3 10-15 Diarrhea Positive (++) 4 >15 Blood - streaked loose stool Gross bloody stool (>++)

[0187] The disease activity score of the mice = (body weight loss score + stool characteristics score + fecal occult blood score) / 3.

[0188] When the average value of the disease activity scores of all the mice was approximately 0.3, the mice were grouped for drug administration (n = 4, the grouping day was recorded as D0), and hz22C6-H23L30 (Fuc-) and the control hIgG1 antibody were respectively administered 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 murine IBD were significantly improved, the disease activity score was significantly reduced and completely relieved after D16( Figure 19 A). At the end point of the experiment D20, the large intestine weight / length ratio (the higher this ratio, the more severe the IBD disease) was measured. The hz22C6-H23L30 (Fuc-) group was comparable to the non-modeled B6RAG- / - mice and was significantly lower than the control hIgG1 group( Figure 19 B, 19C). The HE pathological results showed that there was a large amount of fibrous tissue hyperplasia, infiltration of neutrophils and lymphocytes, mucosal layer necrosis and ulcer lesions in the large intestine of the mice in the control hIgG1 group, and these pathological manifestations were only slightly present or completely absent in the hz22C6-H23L30 (Fuc-) group( Figure 19 D). At the same time, peripheral blood was collected at D15 and mesenteric lymph nodes were collected at the end point of the experiment for flow cytometry analysis. The results showed that there was a relatively high proportion of CXCR6+CD4+T cells in the peripheral blood and mesenteric lymph nodes in the control hIgG1 group, and this group of cells was significantly cleared in the hz22C6-H23L30 (Fuc-) group( Figure 19 E). These results together indicate the important role of CXCR6 in the pathogenic process of the IBD model, suggesting that the anti-CXCR6 antibody has therapeutic potential in inflammatory bowel disease.

[0189] 7.5 Pharmacodynamic effect in the vitiligo model

[0190] The pharmacodynamic effect of the anti-CXCR6 antibody of the present invention was evaluated using a murine vitiligo model. The modeling process was briefly as follows: On D0, 2E5 B16F10 melanoma cells were subcutaneously inoculated into the back of huCXCR6-knock in-C57BL / 6J transgenic mice. On D4 and D10, 200 μg of anti-mouse CD4 antibody (BioXcell, catalog number BE0003-3-50MG) was injected twice. Around D21, the subcutaneous tumors of the mice were removed and randomly grouped. Each group of mice (n = 7) was respectively given hz22C6-H23L30 and control hIgG1 antibody at a dose of 10 mg / kg, twice a week, by intraperitoneal injection. The progression of vitiligo in the mice was continuously observed. The white hair area of the mice was scored from 0 to 7 points. The results showed that after administration of the anti-CXCR6 antibody of the present invention, the progression of vitiligo in the mice was improved ( Figure 20 A, 20B), indicating that the antibody targeting CXCR6 has therapeutic potential in vitiligo.

[0191] Example 8: Efficacy of anti - CXCR6 antibody in type 1 diabetes (T1D)

[0192] NOD female mice will spontaneously develop T1D, but the onset time is relatively late. Therefore, an accelerated NOD mouse T1D model was used to evaluate the pharmacodynamic effect of the antibody targeting CXCR6. The modeling process was briefly as follows: On D0 and D14, NOD mice (Jiangsu Jicui Yakang Biotech Co., Ltd.) were injected with 200 μg of anti-mouse PD1 antibody twice to induce the onset of T1D. At the same time, starting from D0, each group of mice (n = 7 or 8) was respectively given the fucose-depleted anti-mouse CXCR6 antibody prepared by ourselves and control mIgG antibody at a dose of 5 mg / kg, twice a week, by intraperitoneal injection. Starting from D0, the blood glucose changes of the mice were continuously monitored (measured twice a week), and the onset of T1D in the mice was observed. The criteria for the onset of T1D: The fasting blood glucose concentration of the mice ≥ 13.9 mmol / L was defined as the onset of T1D. The results are shown in Figure 21 . The results showed that the anti-mouse CXCR6 antibody could completely inhibit the onset of T1D ( Figure 21 A), maintaining the normal blood glucose level of the mice ( Figure 21 B). This example indicates that the antibody targeting CXCR6 has therapeutic potential in T1D.

[0193] 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 deformations according to the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims of 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 of SEQ ID NO:3, or an amino acid sequence having at least 75% identity thereto; and, the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 75% identity thereto; Preferably, the antibody is mouse antibody, rabbit antibody, human antibody, or mouse antibody, chimeric antibody, fully or partially humanized antibody, or 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.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, 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. 5 . A nucleic acid molecule comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 . A vector comprising the nucleic acid molecule according to claim 5 .

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6 or the host cell according to claim 7, and optional pharmaceutically acceptable excipients.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7 or the composition according to claim 8 in the preparation of a medicament for preventing, treating and / or ameliorating a disease or condition or an agent for diagnosing a disease or condition.

10. The use according to claim 9, 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.

11. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7 or the composition according to claim 8.

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