Anti-CX3CL1 antibodies and their applications

Abstract

The present invention belongs to the field of biology and provides an antibody or an antigen-binding fragment thereof that specifically binds to CX3CL1, including a heavy chain variable region and a light chain variable region. The anti-CX3CL1 antibody or the antigen-binding fragment thereof of the present invention has strong binding to human or monkey CX3CL1, can inhibit the migration of cells expressing CX3CR1, block the binding of CX3CL1 to its receptor, and inhibit the activity of cAMP, and has good drug development prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biology and relates to anti-CX3CL1 antibodies. Background Art

[0002] Chemokines are a class of small molecule secreted proteins with related functions. They are named "chemokines" because of their chemotaxis to leukocytes and cytokine activity. According to the different numbers and spaces of the first two cysteines in the N-terminal conserved cysteine ​​structural motif, chemokines can be divided into four subfamilies: C-, CC-, CXC- and CX3C-type chemokines (where C refers to cysteine ​​and X can be any amino acid). Fractalkine (Fkn, CX3CL1) was discovered in 1997 and is currently the only member of the CX3C-type chemokines. Fractalkine has chemotactic function and participates in the migration and activation of leukocytes, especially phagocytes and lymphocytes. At the same time, it exhibits adhesion and mediates cell-to-cell adhesion.

[0003] Inflammatory factors such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interferon (IFN)-γ can induce the expression of membrane-bound Fractlkine on endothelial cells. Secreted Fractalkine contains only the chemoattractant protein functional region and a part of the mucin-like structural region. It has strong chemotactic activity for monocytes, NK cells, and T cells. Its expression in activated endothelial cells can enhance the adhesion of those leukocytes. CX3CL1 can be induced in endothelial cells by proinflammatory cytokines (TNFα, IFNγ, IL-1β) and LPS. CX3CR1 is a specific receptor for Fractalkine, which is mainly expressed on cytotoxic effector lymphocytes (including NK cells, cytotoxic T lymphocytes, and macrophages). It is a highly selective chemokine receptor and surface marker on cytotoxic effector lymphocytes. Soluble Fractalkine can induce NK cells to transmembrane transfer and release particles, thereby enhancing their cytolytic effect. Similar to NK cells, CD8 T cells and CD4 T cells expressing CX3CR1 can terminally differentiate into effector cells with cytotoxic granules, and excessive cytotoxic lymphocyte activation will lead to vascular and tissue damage.

[0004] CX3CL1 and rheumatoid arthritis (RA), Crohn's disease, rheumatoid vasculitis (RV), Fractalkine is associated with the development of various diseases such as SS, systemic lupus erythematosus (SLE), scleroderma, multiple sclerosis and arteriosclerosis. Soluble fractalkine has been found in the cerebrospinal fluid of patients with neuropsychiatric lupus. In the serum of patients with schizophrenia, rheumatoid arthritis and type 2 diabetes, the soluble Fractalkine is higher than that of the control group. The development of anti-CX3CL1 drugs can directly block the relevant pathways, thereby achieving the purpose of inhibiting inflammation. Summary of the invention

[0005] The purpose of the present invention is to provide anti-CX3CL1 antibodies to provide selectivity for the treatment of related diseases.

[0006] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CX3CL1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises three complementarity determining regions (CDRs): LCDR1, LCDR2 and LCDR3; wherein,

[0007] The amino acid sequence of HCDR1 is shown in SEQ ID NO:11;

[0008] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 12, 19, 21, 23 or 43;

[0009] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 13;

[0010] The amino acid sequence of LCDR1 is shown in SEQ ID NO:15;

[0011] The amino acid sequence of LCDR2 is shown in SEQ ID NO:16;

[0012] The amino acid sequence of LCDR3 is shown in SEQ ID NO: 17; or

[0013] The amino acid sequence of HCDR1 is shown in SEQ ID NO:3;

[0014] The amino acid sequence of HCDR2 is shown in SEQ ID NO:4;

[0015] The amino acid sequence of HCDR3 is shown in SEQ ID NO:5;

[0016] The amino acid sequence of LCDR1 is shown in SEQ ID NO:7;

[0017] The amino acid sequence of LCDR2 is shown in SEQ ID NO:8;

[0018] The amino acid sequence of LCDR3 is shown in SEQ ID NO:9.

[0019] The CDRs of the present invention are defined according to the Kabat numbering convention. Those skilled in the art will readily identify the CDRs defined by each numbering system, and the correspondence between different numbering systems is well known to those skilled in the art. In some embodiments, the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

[0020] In some embodiments, the present invention provides an anti-CX3CL1 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a variable heavy chain region and a variable light chain region, wherein:

[0021] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:10, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:10; the light chain variable region sequence is as shown in SEQ ID NO:14, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:14; or,

[0022] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:18, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:18; the light chain variable region sequence is as shown in SEQ ID NO:20, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:20; or,

[0023] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:22, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:22; the light chain variable region sequence is as shown in SEQ ID NO:20, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:20; or,

[0024] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:24, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:24; the light chain variable region sequence is as shown in SEQ ID NO:20, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:20; or,

[0025] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is as shown in any one of SEQ ID NOs: 27 to 35 or SEQ ID NO: 39, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in any one of SEQ ID NOs: 27 to 35 or SEQ ID NO: 39; the light chain variable region sequence is as shown in any one of SEQ ID NOs: 36 to 38, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in any one of SEQ ID NOs: 36 to 38; or

[0026] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:2, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:2; the light chain variable region sequence is as shown in SEQ ID NO:6, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:6.

[0027] In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0028] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 10, and the light chain variable region sequence is shown in SEQ ID NO: 14; or,

[0029] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 18, and the light chain variable region sequence is shown in SEQ ID NO: 20; or,

[0030] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 22, and the light chain variable region sequence is shown in SEQ ID NO: 20; or,

[0031] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 24, and the light chain variable region sequence is shown in SEQ ID NO: 20; or,

[0032] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 39, and the light chain variable region sequence is shown in any one of SEQ ID NOs: 36 to 38; or,

[0033] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is shown in any one of SEQ ID NOs: 27 to 35, and the light chain variable region sequence is shown in any one of SEQ ID NOs: 36 to 38; or

[0034] The heavy chain variable region sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:2, and the light chain variable region sequence is shown in SEQ ID NO:6.

[0035] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof further includes a human or mouse framework region, and / or the light chain variable region of the antibody or antigen-binding fragment thereof further includes a human or mouse framework region.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; the heavy chain constant region and / or the light chain constant region are of human or murine origin.

[0037] In a preferred embodiment, the heavy chain constant region and / or the light chain constant region are of human origin, wherein the sequence of the heavy chain constant region is as shown in SEQ ID NO:25, and the sequence of the light chain constant region is as shown in SEQ ID NO:26.

[0038] In a preferred embodiment, the antibody or antigen-binding fragment thereof is an animal-derived antibody, a chimeric antibody, a humanized antibody or a combination thereof.

[0039] In some embodiments, the anti-CX3CL1 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein:

[0040] The amino acid sequence of the heavy chain is as shown in SEQ ID NO:40 or 42, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:40 or 42, and the amino acid sequence of the light chain is as shown in SEQ ID NO:41, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:41.

[0041] In some embodiments, the anti-CX3CL1 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:40, and the amino acid sequence of the light chain is as shown in SEQ ID NO:41.

[0042] In some embodiments, the anti-CX3CL1 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:42, and the amino acid sequence of the light chain is as shown in SEQ ID NO:41.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof is a full-length antibody protein or an antigen-binding fragment.

[0044] In some embodiments, the antigen binding fragment is selected from Fab, Fab', (Fab') 2 , Fd, Fv, disulfide-linked Fv, scFv and dAb.

[0045] In a second aspect, a biomaterial is provided, which may be:

[0046] (1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention; preferably, the nucleic acid molecule is a DNA molecule or an RNA molecule; a person skilled in the art can infer the DNA or RNA sequence encoding the amino acid sequence based on the amino acid sequence of the antibody disclosed in the present invention, and set a suitable expression element for it so that the DNA or RNA molecule can express the antibody of the present invention;

[0047] (2) a vector containing the nucleic acid molecule as described above; preferably, the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector;

[0048] (3) Host cells containing the above-mentioned nucleic acid molecules or vectors, or cultures such as culture fluids and bacterial suspensions obtained after culturing the above-mentioned host cells.

[0049] In a third aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising:

[0050] 1) Chemical synthesis, based on the amino acid sequence of the antibody or antigen-binding fragment thereof; or,

[0051] 2) Biological preparation, comprising culturing the cells of the second aspect and harvesting the antibody or antigen-binding fragment thereof.

[0052] In a fourth aspect, a composition is provided, comprising the anti-CX3CL1 antibody or antigen-binding fragment thereof, or a biological material of the present invention.

[0053] In some embodiments, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

[0054] In a fifth aspect, a kit is provided, comprising the antibody or antigen-binding fragment thereof, biological material, or antibody composition of the present invention; the kit is used for diagnosing or detecting CX3CL1 protein or cells expressing CX3CL1 protein in an in vitro sample.

[0055] In a sixth aspect, provided is the use of the antibody or antigen-binding fragment thereof, biomaterial or antibody composition of the present invention, wherein the use is for preparing a drug for preventing and / or treating a disease associated with abnormal CX3CL1 expression or function.

[0056] In some embodiments, the disease associated with abnormal CX3CL1 expression or function is selected from: rheumatoid arthritis (RA), Crohn's disease, ulcerative colitis, diabetes, rheumatoid vasculitis (RV), Syndrome (SS), systemic lupus erythematosus (SLE), neuropsychiatric lupus, scleroderma, multiple sclerosis and arteriosclerosis.

[0057] In a seventh aspect, the present invention provides a method for preventing and / or treating a disease associated with abnormal CX3CL1 expression or function, the method comprising administering to the human individual an effective amount of an anti-CX3CL1 antibody or antigen-binding fragment thereof as described in any of the preceding items, or a biomaterial as described above, or a composition as described above.

[0058] The anti-CX3CL1 antibody or antigen-binding fragment thereof described in the present invention has strong binding to human or monkey CX3CL1, can inhibit the migration of cells expressing CX3CR1, block the binding of CX3CL1 to its receptor, and inhibit the activity of cAMP. Therefore, the antibody and antigen-binding fragment thereof of the present invention have good prospects for drug development. DETAILED DESCRIPTION

[0059] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0060] definition

[0061] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operating procedures used herein are all conventional procedures widely used in the corresponding fields.

[0062] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising", "having", "including", etc. should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to". Unless otherwise specified, "comprising" includes "consisting of". For example, for a HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, it explicitly covers the HCDR1 with the amino acid sequence as shown in SEQ ID NO: 2.

[0063] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0064] As used herein, natural "antibodies" refer to immune proteins that are protective and can bind to antigens and are produced by the body due to antigen stimulation, and are secreted by plasma cells (effector B cells); "immunoglobulins" refer to globulins that have antibody (Ab) activity or chemical structure and are similar to antibody molecules. Antibodies are usually composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified into k (kappa) and (lambda) light chains. Heavy chains are divided into five categories, μ, γ, α, δ and ε, according to the different antigenicity of their constant regions, and the corresponding immunoglobulins are IgM, IgG, IgA, IgD and IgE, respectively. And the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of a domain CL. The constant domain does not directly participate in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. In the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in each region or domain can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0065] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). Unless otherwise stated, the variable region and CDR sequences in the embodiments of the present invention are all subject to the "Kabat" numbering rules. Although in a specific embodiment, a numbering system (such as Kabat) is used to define amino acid residues, the corresponding technical solutions of other numbering systems will be regarded as equivalent technical solutions.

[0066] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above. The term "antibody" is not limited to any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0067] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab'), 2 , Fd, Fv, complementary determining region (CDR) fragments, scFv, etc. (technology from Domantis), and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechno1, 23: 1126-1136.

[0068] "Fab" consists of a light chain and the CH1 and variable region of a heavy chain.

[0069] "Fab'", which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0070] "F(ab')2" is a bivalent fragment formed by two Fab' fragments linked by a disulfide bridge in the hinge region.

[0071] "Fd", which has VH and CH1 domains; (iv) an Fd' fragment, which has VH and CH1 domains and has one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0072] "Fv", which has the VL and VH domains of one arm of an antibody.

[0073] "dAbs", which consist of a VH domain.

[0074] "Single-chain antibody molecule (scFv)" means a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker, such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0075] The term "IgG-type antibody" refers to an antibody having a "heavy" chain and a "light" chain cross-linked via intrachain disulfide bonds and interchain disulfide bonds, with a typical "Y" structure. Each heavy chain consists of an N-terminal HCVR and a heavy chain constant region ("HCCR"). Each light chain consists of a LCVR and a light chain constant region ("LCCR").

[0076] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from N to C; and, when the full-length antibody is an IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N to C. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N to C. Two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be from a single species, such as human; or may be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0077] The terms "capable of specific binding", "specific binding" or "binding" refer to the ability of an antibody to bind to an antigen or epitope with a higher affinity than to other antigens or epitopes. -7 M or less (e.g., about 1×10 -8 In some embodiments, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of 10% or less (M or less) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). The KD can be measured using known methods, such as those measured by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope thereof may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologous) such as humans or monkeys, such as Macaca fascicularis (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp) or marmosets (Callithrix jacchus) (common marmoset, marmoset).

[0078] As used herein, the terms "monoclonal antibody", "single antibody", and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, relative to monoclonal antibodies, generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.

[0079] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, e.g., U.S. Patent Application 4,816,567), or phage antibody library technology (see, e.g., Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0080] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, the specific antigen (target molecule recognized by the antibody) or its antigenic epitope can be immobilized on a column and the immunospecific antibody can be purified by immunoaffinity chromatography. The purification of immunoglobulins can be referred to, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc, Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0081] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and the other portion of the light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the binding activity to the target antigen (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies (e.g., human-mouse combined antibodies) in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy chain and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0082] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (acceptor antibody). Typically, at least one or two but usually all three (heavy and / or light immunoglobulin chains) of a humanized antibody are replaced by a donor CDR. The immunoglobulin that provides the CDR is called a "donor", and the immunoglobulin that provides the framework is called an "acceptor". In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the acceptor framework can be a naturally occurring human framework, or a sequence having about 85%, 90%, 95%, 99% or higher identity compared thereto. Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. The donor antibody can be a mouse, rat, rabbit, or non-human primate (eg, cynomolgus monkey) antibody having the desired properties (eg, antigen specificity, affinity, reactivity, etc.).

[0083] The term sequence "identity" refers to the degree (percentage) to which the amino acids / nucleic acids of the two sequences are identical in equivalent positions when the two sequences are optimally aligned. In the alignment process, gaps may be introduced if necessary to obtain the maximum sequence identity percentage, but any conservative substitutions are not considered to constitute a part of the sequence identity. To measure the sequence identity percentage, alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters that are suitable for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequence.

[0084] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. The isolated nucleic acids encoding the antigen-binding molecules refer to one or more nucleic acid molecules encoding antibody heavy chains and light chains (or fragments thereof), including such one or more nucleic acid molecules in a single vector or a separate vector, and such one or more nucleic acid molecules present in one or more positions in a host cell. Unless otherwise indicated, specific nucleic acid sequences also implicitly encompass variants (e.g., degenerate codon substitutions) and complementary sequences of their conservative modifications and sequences explicitly indicated. Specifically, as described in detail below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a degenerate base and / or a deoxyinosine residue.

[0085] The term "pharmaceutical composition" refers to a mixture containing one or more antigen binding molecules or antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0086] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0087] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0088] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0089] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus), sheep, dogs, cows, chickens, amphibians and reptiles. Unless explicitly stated, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cynomolgus" or "cynomolgus" refers to cynomolgus (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

[0090] "Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0091] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.

[0092] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions attempted to be applied to the individual being treated, and may be performed for preventive purposes or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving prognosis. In some embodiments, the molecules of the present disclosure are used to delay the formation of a disease or slow the progression of a disease.

[0093] "Effective amount" is generally enough to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring and / or improve or ameliorate the amount of damage caused by or associated with a disease state. In some embodiments, an effective amount is a therapeutically effective amount or a preventive effective amount. "Therapeutically effective amount" is enough to treat a disease state or symptom, a state or symptom particularly associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. "Preventive effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the onset (or recurrence) possibility of the disease state or related symptoms. A complete treatment or preventive effect may not occur after administering one dose, but may occur after administering a series of doses. Thus, a therapeutically or preventive effective amount may be administered in one or more administrations. A "therapeutically effective amount" and a "prophylactically effective amount" may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0094] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto.

[0095] Unless otherwise specified, the reagents and instruments used in the present invention are conventional reagents and instruments, which can be obtained commercially; the methods used are conventional techniques, and those skilled in the art can undoubtedly complete the experiment and obtain the corresponding results according to the contents described in the instructions.

[0096] Example 1: Screening of anti-CX3CL1 mouse monoclonal antibodies

[0097] The present invention uses hybridoma technology to generate monoclonal antibodies (mAbs) with specificity and high affinity for human and monkey CX3CL1.

[0098] This example describes a method for preparing mouse anti-human CX3CL1 monoclonal antibodies. First, the extracellular region CX3CL1 protein (UniProt number: P78423, SEQ ID NO: 1) was expressed as an immunogen, and a 6xHis (CX3CL1 (Q25-Q341)-6xHis) or human IgG1 Fc (CX3CL1 (Q25-Q341)-IgG1 Fc) tag was added to the C-terminus of the extracellular region CX3CL1 protein amino acid sequence (Q25-Q341), and then the encoding nucleic acid was cloned into an expression vector, and the expression vector was transiently transfected into 293 cells. After 7 days, the cell culture fluid was collected and purified.

[0099] To prepare mouse anti-human CX3CL1 monoclonal antibodies, two tagged CX3CL1 proteins were used to immunize SJL mice (purchased from Ruizhi Chemical) in two groups, and each group was immunized three times. After immunization, the serum immune titer of mice was detected by ELISA. The two tagged CX3CL1 proteins were coated on microplates at 1 μg / mL at 4°C overnight, and then blocked with blocking solution at 37°C for 1 hour. After washing the plates, mouse serum was added to the plates and incubated at 37°C for 1 hour. After washing the plates, HRP-labeled goat anti-mouse IgG (purchased from Thermofisher) was added and reacted at 37°C for 1 hour. After washing the plates, TMB solution was added, reacted at room temperature in the dark for 5 minutes, and then 2N H2SO4 was added to terminate the reaction. The absorbance was detected at a wavelength of 450nm on an enzyme reader.

[0100] Mice with high immune titers were selected, spleen cells were taken and fused with myeloma cells, and two rounds of screening of cell culture supernatants were performed using the ELISA method. After the initial screening, the positive hybridoma clones were transferred to 24-well plates for culture, followed by a second screening. The positive clones obtained by screening were subcloned, and the subclones were tested again using the same primary and secondary screening methods. Positive monoclones were selected for antibody production and frozen for storage. Hybridoma cell lines with monoclonal numbers 36C7-1G2, 15F7B2, and 18H6F1 were selected for amplification culture, and mouse monoclonal antibodies mAb017, mAb022, and mAb032 were produced after supernatant purification.

[0101] Human CX3CL1:

[0102] QHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVVLEPEATGESSSLEPTPSSQEAQRALGTSPELPTGVTGSSGTRLPPTPKAQDGGPVGTE LFRVPPVSTAATWQSSAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVWGQGQSPRPENSLEREEMGPVPAHTDAFQDWGPGSMAHVSVVPVSSEGTPSREPVASGSWTPKAEEPIHATMDPQRLGVLITPVPDAQAATRRQ(SEQ ID NO:1).

[0103] Example 2: Sequencing of mouse monoclonal antibodies and production of chimeric antibodies

[0104] The three hybridoma cell lines 36C7-1G2, 15F7B2, and 18H6F1 corresponding to mAb017, mAb022, and mAb032 were sequenced and three sequence-specific monoclonal antibodies were obtained. The specific sequences are as follows:

[0105] >mAb017-VH

[0106] QIQLVQSGPELKKPGETVKISCKASVYTFT EYAMH WVKQAPGKGFKWMG WINTNSGEPTYADDFKG RFAFSLETSASTAYLQINNLKNEDTATYFCGR GRVVGSFDY WGQGTTLTVSS (SEQ ID NO: 2); wherein,

[0107] HCDR1: EYAMH (SEQ ID NO: 3);

[0108] HCDR2:WINTNSGEPTYADDFKG(SEQ ID NO:4);

[0109] HCDR3: GRVVGSFDY (SEQ ID NO: 5);

[0110] >mAb017-VL

[0111] DIVMSQSPSSLAVSVGEKVTMSC KSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIY WASTRES GVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC QQFYSYPLT FGAGTKLELK(SEQ ID NO:6);

[0112] LCDR1: KSSQSLLYSSNQKNYLA(SEQ ID NO:7);

[0113] LCDR2: WASTRES(SEQ ID NO:8);

[0114] LCDR3: QQFYSYPLT(SEQ ID NO:9);

[0115] >mAb022-VH

[0116] EVQLQQSGPELVKPGASVKISCKASGYTFT DYYMN WVKQSHGKSLEWIG DINPNNGGTNYNQKFKG KATLTVDKSSSTAYMELRSLTSEDSAVFYCAS SGNSYVDY WGQGTTLTVSS(SEQ ID NO:10); wherein,

[0117] HCDR1: DYYMN(SEQ ID NO:11);

[0118] HCDR2: DINPNNGGTNYNQKFKG(SEQ ID NO:12);

[0119] HCDR3: SGNSYVDY(SEQ ID NO:13);

[0120] >mAb022-VL

[0121] DIVMTQSHKFMSTSVGDRVNITC KASQDVSTAVA WYQQKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSSPYT FGGGTKLEIK(SEQ ID NO:14); wherein,

[0122] LCDR1: KASQDVSTAVA(SEQ ID NO:15);

[0123] LCDR2: WASTRHT(SEQ ID NO:16);

[0124] LCDR3: QQHYSSPYT(SEQ ID NO:17);

[0125] >mAb032-VH

[0126] EVQLQQSGPELVKPGASVKISCKASGYTFT DYYMN WVKQSHGKSLEWIG DINPNNGGTSYNQKFKG KATLTVDKSSSTAYMELRRSLTSEDSAVYYCAS SGNSYVDY WGQGTTLTVSS (SEQ ID NO: 18); wherein,

[0127] HCDR1:DYYMN (SEQ ID NO:11);

[0128] HCDR2: DINNPNNGGTSYNQKFKG (SEQ ID NO: 19);

[0129] HCDR3: SGNSYVDY (SEQ ID NO:13);

[0130] >mAb032-VL

[0131] DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQQKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSSPYT FGGGTKLEIK (SEQ ID NO: 20); wherein,

[0132] LCDR1:KASQDVSTAVA(SEQ ID NO:15);

[0133] LCDR2: WASTRHT(SEQ ID NO:16);

[0134] LCDR3: QQHYSSPYT (SEQ ID NO: 17).

[0135] Using DNA recombination technology, the light chain variable regions of mAb022 and mAb032 monoclonal antibodies were connected to the constant regions of human IgG2 antibodies to construct chimeric antibodies C022 and C032. There was an NG hotspot in the CDR2 of mAb032 antibody, so two different mutants were prepared at the same time, C032NA and C032QG.

[0136] The antibody fragment sequences containing the mutation sites are as follows:

[0137] C032NA-HCDR2:DINPNN GTSYNQKFKG (SEQ ID NO: 21);

[0138] C032NA-VH:

[0139] EVQLQQSGPELVKPGASVKISCKASGYTFT DYYMN WVKQSHGKSLEWIG DINPNNAGTSYNQKFKG KATLTVDKSSSTAYMELRRSLTSEDSAVYYCAS SGNSYVDY WGQGTTLTVSS (SEQ ID NO: 22);

[0140] C032QG-HCDR2:DINPN GGTSYNQKFKG (SEQ ID NO: 23);

[0141] C032QG-VH:

[0142] EVQLQQSGPELVKPGASVKISCKASGYTFT DYYMN WVKQSHGKSLEWIG DINPNQGGTSYNQKFKG KATLTVDKSSSTAYMELRRSLTSEDSAVYYCAS SGNSYVDY WGQGTTLTVSS (SEQ ID NO: 24).

[0143] The light and heavy chain variable region genes of the chimeric antibody were connected to the human IgG2 antibody constant region gene, connected into the expression vector pcDNA3.4 using the gene full synthesis technology, and transfected into mammalian cells CHOK1 (purchased from ATCC) to express the chimeric antibody. The anti-human CX3CL1 human-mouse chimeric antibody was isolated from the serum-free culture supernatant containing the target antibody by protein A affinity chromatography. The amino acid sequence of the heavy chain constant region of the chimeric antibody is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 26.

[0144] CH:

[0145] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:25);

[0146] CL:

[0147] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26).

[0148] The obtained chimeric antibody was sequenced and the results were consistent.

[0149] Example 3: Humanization and expression of anti-CX3CL1 mAb

[0150] 3.1 Humanization of C032QG mAb

[0151] The C032QG monoclonal antibody was selected for humanization. The CDR region of the C032QG antibody was transplanted to the matching human variable region gene framework sequence by CDR transplantation, and a back mutation was designed in the framework region, and finally the heavy chain and light chain variable regions of the C032QG humanized antibody were obtained. The C032QG monoclonal antibody was humanized using two human antibody heavy chain variable region templates and one light chain variable region template. The sequences of the heavy chain variable regions of the HumAb032 series of humanized antibodies are as follows:

[0152] HumAb032 VH G1-0

[0153] EVQLVQSGAEVKKPGATVKISCKVSGYTFT DYYMN WVQQAPGKGLEWMG DINPNQGGTSYNQKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCAT SGNSYVDY WGQGTTVTVSS(SEQ ID NO:27);

[0154] HumAb032 VH G1-1

[0155] EVQLVQSGAEVKKPGATVKISCKVSGYTFT DYYMN WVQQAPGKSLEWMG DINPNQGGTSYNQKFKG RVTITADTSTDTAYMELSSLRSEDTAVYYCASS GNSYVDY WGQGTTVTVSS(SEQ ID NO:28);

[0156] HumAb032 VH G1-2

[0157] EVQLVQSGAEVKKPGATVKISCKASGYTFT DYYMN WVQQAPGKSLEWMG DINPNQGGTSYNQKFKG RVTLTVDTSTDTAYMELSSLRSEDTAVYYCAS SGNSYVDY WGQGTTVTVSS(SEQ ID NO:29);

[0158] HumAb032 VH G1-3

[0159] EVQLVQSGAEVKKPGATVKISCKASGYTFT DYYMN WVQQAPGKSLEWMG DINPNQGGTSYNQKFKG RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAS SGNSYVDY WGQGTTVTVSS(SEQ ID NO:30);

[0160] HumAb032 VH G1-4

[0161] EVQLVQSGAEVKKPGATVKISCKASGYTFT DYYMN WVQQAPGKSLEWIG DINPNQGGTSYNQKFKG RATLTVDKSTSTAYMELSSLRSEDTAVYYCAS SGNSYVDY WGQGTTVTVSS(SEQ ID NO:31);

[0162] HumAb032 VH G1-5

[0163] EVQLVQSGAEVKKPGATVKISCKASGYTFT DYYMN WVQQAPGKSLEWIG DINPNQGGTSYNQKFKG KATLTVDKSTSTAYMELRSLRSEDTAVYYCAS SGNSYVDY WGQGTTVTVSS(SEQ ID NO:32);

[0164] HumAb032 VH G2-0

[0165] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYYMN WVRQAPGQGLEWMG DINPNQGGTSYNQKFKG RVTMTRDTSISTAYMELSRLRSDDTAVYYCAS SGNSYVDY WGQGTTVTVSS(SEQ ID NO:33);

[0166] HumAb032 VH G2-1

[0167] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYYMN WVKQAPGQSLEWMG DINPNQGGTSYNQKFKG KVTMTVDKSISTAYMELSRLRSDDTAVYYCAS SGNSYVDY WGQGTTVTVSS(SEQ ID NO:34);

[0168] HumAb032 VH G2-2

[0169] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYYMN WVKQAPGQSLEWIG DINPNQGGTSYNQKFKG KATLTVDKSISTAYMELSRLRSDDTAVYYCAS SGNSYVDY WGQGTTVTVSS(SEQ ID NO:35).

[0170] The sequences of the light chain variable regions are as follows:

[0171] HumAb032 VL g0

[0172] DIQMTQSPSSLSASVGDRVTITC KASQDVSTAVA WYQQKPGKAPKLLLY WASTRHT GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQHYSSPYTFGGGTKVEIK (SEQ ID NO: 36);

[0173] HumAb032 VL g1

[0174] DIQMTQSPSSLSASVGDRVTITC KASQDVSTAVA WYQQKPGKSPKLLLY WASTRHT GVPDRFSGSGSGTDYTLTISSLQPEDFATYYC QQHYSSPYT FGGGTKVEIK (SEQ ID NO: 37);

[0175] HumAb032 VL g2

[0176] DIQMTQSPSSLSASVGDRVTITC KASQDVSTA VAWYQQKPGKSPKLLIY WASTRHT GVPDRFSGSGSGTDYTLTISSLQPEDFATYYC QQHYSSPYT FGGGTKVEIK (SEQ ID NO: 38).

[0177] The variable regions of the obtained humanized antibodies were combined and connected with the heavy chain constant region (such as the heavy chain constant region of IgG2 shown in SEQ ID NO: 25) and the light chain constant region (such as the light chain constant region of kappa shown in SEQ ID NO: 26) of human antibodies to obtain 27 full-length humanized antibodies, as shown in Table 1.

[0178] Table 1. Variable region combinations of humanized antibodies

[0179]

[0180]

[0181] 3.2 Humanization of C022 mAb

[0182] C022 and C032 only differed in one amino acid in the heavy chain CDR region, so the different amino acids between C022 and C032 in the heavy chain variable region CDR2 of HumAb032-4 were directly mutated to the amino acids of C022 to obtain HumAb022-4. The sequences of CDR2, heavy chain variable region and light chain variable region of HumAb022-4 are shown below (wherein the light chain variable region is the same as that of HumAb032-4).

[0183] HCDR2: DINPNQGGTNYNQKFKG (SEQ ID NO: 43);

[0184] HumAb022-4VH

[0185] EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWMGDINPNQGGTNYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTT VTVSS(SEQ ID NO:39);

[0186] HumAb022-4VL: (same as HumAb032VL g0)

[0187] DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLLYWASTRHTG VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIK(SEQ ID NO:36).

[0188] The full-length sequences of exemplary humanized antibodies are as follows:

[0189] 1. HumAb022-4

[0190] Heavy chain:

[0191] EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWMGDINPNQGGTNYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:40);

[0192] Light chain:

[0193] DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLLYWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:41)

[0194] 2、HumAb032-4

[0195] Heavy chain:

[0196] EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWMGDINPNQGGTSYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:42);

[0197] Light chain (same as the light chain of HumAb022-4):

[0198] DIQMTQSPSSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLLYWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:41).

[0199] All humanized antibodies were expressed using the chimeric antibody production method in Example 2, and the sequences of the humanized antibodies were detected, and the results were consistent.

[0200] The sequence numbers of the murine antibodies, chimeric antibodies, humanized antibodies and fragments thereof involved in the above examples are shown in Table 2.

[0201] Table 2: Sequence numbers of antibodies and their fragments

[0202]

[0203]

[0204] Example 4: Preparation of positive control antibody

[0205] A CX3CL1 positive control antibody was produced based on the Quetmolimab antibody sequence published in the Tabs database and named Tab#1.

[0206] Tab#1 Heavy Chain

[0207] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVKQAPGQGLEWIGWIYPGDGSPKFNERFKGRTTLTADKSTNTAYMLLSSLRSEDTAVYFCATGPTDGDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:44);

[0208] Tab#1 Light chain

[0209] DIQMTQSPSSLSASVGDRVTITCRASGNIHNFLAWYQQKPGKAPKLLIYNEKTLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQFWSTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:45).

[0210] Test Example 1: ELISA Binding of Antibody to Human CX3CL1 and Monkey CX3CL1

[0211] Dilute two antigen proteins with PBS: human CX3CL1 extracellular region protein (huCX3CL1-His, i.e., CX3CL1 (Q25-Q341) -6xHis in Example 1) and monkey CX3CL1 extracellular region protein (cynoCX3CL1-His, purchased from Bio-Bio B23523001) to 1 μg / mL, add 100 μL / well to ELISA microplate, and incubate overnight at 4°C. Block with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4, the percentage is mass percentage) at 37°C for two hours. Add gradient diluted antibodies, a total of 8 concentration points (starting concentration is 100nM, 10-fold gradient dilution, including 0 point). 100 μL / well, incubate at 37°C for 1 hour. Wash the plate 3 times, add the secondary antibody (purchased from Thermofisher): anti-mouse IgG-HRP (1:5000 dilution), 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate 3 times, add TMB colorimetric solution, 100 μL / well, incubate at 37°C for 10 minutes, and then add 2N H2SO4 to terminate the colorimetric reaction. Read the OD450nm reading using an enzyme reader.

[0212] The results are shown in Tables 3-1 to 3-4. The tested monoclonal antibodies have good binding to the extracellular domain proteins of human CX3CL1 and monkey CX3CL1.

[0213] Table 3-1. Binding of mouse antibodies to human CX3CL1 and monkey CX3CL1

[0214]

[0215] Table 3-2. Binding of chimeric antibodies to human CX3CL1 and monkey CX3CL1

[0216]

[0217] Table 3-3. Binding of humanized antibodies to human CX3CL1 and monkey CX3CL1

[0218]

[0219]

[0220] Table 3-4. Binding of humanized antibodies to human CX3CL1 and monkey CX3CL1

[0221]

[0222] Test Example 2: Detection of monoclonal antibody blocking the binding activity of human CX3CL1 and receptor

[0223] Antigen huCX3CL1-His was diluted to 0.2 μg / mL with PBS, and monoclonal antibodies were diluted in gradients (starting concentration was 15 μg / mL, 2-fold gradient dilution, 7 concentration points). 60 μL of antigen was mixed with 60 μL of monoclonal antibody and incubated at 4°C for 30 minutes. Human CX3CR1 overexpressing cells (CHOK1-hCX3CR1, human CX3CR1, whose Uniprot number is P49238, was transferred into CHOK1 cells by lentivirus to obtain a stable cell line) were digested with TrypLE (purchased from Gibco), the cells were collected by centrifugation, and resuspended in PBS buffer to a cell density of 2E6 cells / ml. The cell suspension was added to a 96-well cell plate at 100 μL per well, and the supernatant was discarded after centrifugation. The cells were resuspended by adding the antigen and monoclonal antibody mixture at 100 μl / well and incubated at 4°C for 1 hour. The cells were washed twice with PBS, 100 μl of 0.5 μg / mL (1:1000) anti-His-647 fluorescent secondary antibody (cat: A01802-100) was added to each well, the cells were resuspended, and incubated at 4°C in the dark for 1 hour. The cells were washed twice with PBS, and the cells were resuspended in PBS for FACS analysis.

[0224] The results are shown in Tables 4-1 to 4-4 below. The tested monoclonal antibodies can effectively block the binding of human CX3CL1 to the receptor.

[0225] Table 4-1. Blocking activity of mouse antibodies

[0226] Antibody IC50(nM) mAb017 6.1 mAb022 7.6 mAb032 11.5

[0227] Table 4-2. Blocking activity of chimeric antibodies

[0228] Antibody IC50(nM) Antibody IC50(nM) C022 0.885 C032NA 0.865 C032 0.731 C032QG 0.811

[0229] Table 4-3. Blocking activity of humanized antibodies

[0230] Antibody IC50(nM) Antibody IC50(nM) HumAb032-1 2.396 HumAb032-15 1.366 HumAb032-2 1.525 HumAb032-16 1.303 HumAb032-3 1.283 HumAb032-17 1.396 HumAb032-4 1.223 HumAb032-18 1.324 HumAb032-5 1.086 HumAb032-19 1.267 HumAb032-6 1.251 HumAb032-20 1.068 HumAb032-7 2.665 HumAb032-21 1.159 HumAb032-8 1.495 HumAb032-22 2.779 HumAb032-9 1.281 HumAb032-23 1.5 HumAb032-10 1.267 HumAb032-24 1.366 HumAb032-11 1.068 HumAb032-25 1.303 HumAb032-12 1.159 HumAb032-26 1.369 HumAb032-13 2.779 HumAb032-27 1.324 HumAb032-14 1.5 C032QG 1.492

[0231] Table 4-4. Blocking activity of humanized antibodies

[0232] Antibody IC50(nM) HumAb022-4 1.275 C022 1.702 Tab#1 1.957

[0233] Test Example 3: Detection of the monoclonal antibody's activity in inhibiting cell secretion of cAMP

[0234] Digest, centrifuge, and collect CHOK1-hCX3CR1 cells according to standard procedures, resuspend the cells in F12K (Hyclone, Cat#SH30526.01) and incubate at room temperature for 30-40 minutes. Mix 10 μL 0.4 μg / ml huCX3CL1-His (antigen concentration in mouse monoclonal antibody experiments is 0.2 μg / ml) with 10 μL monoclonal antibody (starting concentration is 15 μg / mL, 2-fold gradient dilution, 8 gradients) and incubate at room temperature for 30 minutes. Collect the cells by centrifugation, resuspend in F12K containing 0.5 mM IBMX (Sigma) to a cell concentration of 2.5E5 / ml (cell concentration in mouse monoclonal antibody experiments is halved), and add the cells to a new assay plate (Corning#3365) at 10 μL / well. Add the antigen-antibody mixture to the assay plate at 10 μL / well and incubate the cells at room temperature for 10 minutes. Add 9.9μM Forskolin (MCE, CAS#66575-29-9) to the assay plate at 5μL / well and incubate at 37°C for 30 minutes. Then transfer 10μl of the mixture to the detection plate (OptiPlate-384), add 5μL cAMP-Cryptate working solution (Cisbio, 62AM9PEC Lot#02E) and mix thoroughly, then add 5μL anti-cAMP-d2 working solution (Cisbio, 62AM9PECLot#02E). Incubate at room temperature for 1 hour. Read the fluorescence values ​​at two different wavelengths (665nm and 620nm) on Envision.

[0235] The results are shown in Tables 5-1 to 5-3 below. The tested monoclonal antibodies can effectively block the binding of human CX3CL1 to the receptor, thereby inhibiting the production of cAMP by cells, and are superior to the control antibody Tab#1.

[0236] Table 5-1. Inhibition of cAMP activity by mouse antibodies

[0237] Antibody IC50(nM) MAX mAb017 0.70 100.0 mAb022 0.80 105.2 mAb032 1.07 114.9

[0238] Table 5-2. Inhibitory cAMP activity of chimeric antibodies

[0239] Antibody IC50(nM) MAX C022 3.284 92.88 C032 3.404 95.82 C032NA 3.633 92.51 C032QG 3.963 96.95 Tab#1 5.686 94.04

[0240] Table 5-3. Antibodies inhibiting cAMP activity

[0241] Antibody IC50(nM) MAX HumAb022-4 2.69 100.5 C022 2.41 100.1 Tab#1 4.07 92.5

[0242] Test Example 4: Migration Activity Detection of Monoclonal Antibodies

[0243] CHOK1-CX3CR1 cells were washed with PBS and then starved overnight with F12K medium only. CHOK1-hCX3CR1 cells were digested, centrifuged, and collected according to standard procedures. Cells were resuspended in F12K and adjusted to a concentration of 3E6 / ml (the cell concentration was halved in chimeric antibody experiments). 60 μL of cells (3E6 / ml) and 60 μL of gradient dilutions of monoclonal antibodies (starting concentration 30 μg / ml, 2-fold gradient dilution, 5 concentration points) were mixed. The chamber (Corning, 3422) was transferred to a 24-well plate, 100 μL of the cell and antibody mixture was added, and 300 μL of 1 μg / ml concentration of huCX3CL1-His and 300 μL of the above-diluted mouse monoclonal antibodies were added to the lower wells of the 24-well plate, and several wells without antigen were set up at the same time. Migrate at 37°C for 4 hours. Do not move the culture plate during cell migration. Migrated cells were then collected and the chamber was gently moved to a new 24-well plate containing 600 μL PBS. The cells in the lower layer of the chamber membrane and the lower layer of the original 24-well plate were collected and transferred to a U-bottom 96-well plate (Corning, #3799), and the supernatant was discarded after centrifugation. 120 μL CellTiter-Glo was added to each well and 120 μL of cell lysate was transferred to the detection plate (Corning, #3917), and the plate was left to stand for 10 minutes, and the luminescence was recorded by Thermo VarioSkan Lux (1 second / well).

[0244] The results are shown in Tables 6-1 to 6-3 below. The tested monoclonal antibodies can effectively block the binding of human CX3CL1 to the receptor, thereby inhibiting cell migration, and are superior to the control Tab#1.

[0245] Table 6-1. Cell migration inhibition activity of mouse antibodies

[0246] Antibody IC50(nM) mAb017 7.55 mAb022 7.19 mAb032 8.72

[0247] Table 6-2. Cell migration inhibitory activity of chimeric antibodies

[0248] Antibody IC50(nM) Antibody IC50(nM) C022 2.892 C032QG 2.265 C032 2.491 Tab#1 19.010 C032NA 4.005

[0249] Table 6-3. Antibody cell migration inhibitory activity

[0250] Antibody IC50(nM) HumAb022-4 0.299 C022 0.579

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CX3CL1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions (CDRs): HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises three complementary determining regions (CDRs): LCDR1, LCDR2 and LCDR3; wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 11; The amino acid sequence of HCDR2 is shown in SEQ ID NO: 12, 19, 21, 23 or 43; The amino acid sequence of HCDR3 is shown in SEQ ID NO: 13; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 15; The amino acid sequence of LCDR2 is shown in SEQ ID NO: 16; The amino acid sequence of LCDR3 is shown in SEQ ID NO: 17; or The amino acid sequence of HCDR1 is shown in SEQ ID NO: 3; The amino acid sequence of HCDR2 is shown in SEQ ID NO: 4; The amino acid sequence of HCDR3 is shown in SEQ ID NO: 5; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 7; The amino acid sequence of LCDR2 is shown in SEQ ID NO: 8; The amino acid sequence of LCDR3 is shown in SEQ ID NO:

9.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region sequence is as shown in SEQ ID NO: 10, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 10; the light chain variable region sequence is as shown in SEQ ID NO: 14, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 14; or, The heavy chain variable region sequence is as shown in SEQ ID NO: 18, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 18; the light chain variable region sequence is as shown in SEQ ID NO: 20, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 20; or, The heavy chain variable region sequence is as shown in SEQ ID NO: 22, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 22; the light chain variable region sequence is as shown in SEQ ID NO: 20, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 20; or, The heavy chain variable region sequence is as shown in SEQ ID NO: 24, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 24; the light chain variable region sequence is as shown in SEQ ID NO: 20, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 20; or, The heavy chain variable region sequence is as shown in any one of SEQ ID NOs: 27 to 35 or SEQ ID NO: 39, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in any one of SEQ ID NOs: 27 to 35 or SEQ ID NO: 39; the light chain variable region sequence is as shown in any one of SEQ ID NOs: 36 to 38, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in any one of SEQ ID NOs: 36 to 38; or The heavy chain variable region sequence is as shown in SEQ ID NO: 2, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 2; the light chain variable region sequence is as shown in SEQ ID NO: 6, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region sequence is shown in SEQ ID NO: 10, and the light chain variable region sequence is shown in SEQ ID NO: 14; or, The heavy chain variable region sequence is shown in SEQ ID NO: 18, and the light chain variable region sequence is shown in SEQ ID NO: 20; or, The heavy chain variable region sequence is shown in SEQ ID NO: 22, and the light chain variable region sequence is shown in SEQ ID NO: 20; or, The heavy chain variable region sequence is shown in SEQ ID NO: 24, and the light chain variable region sequence is shown in SEQ ID NO: 20; or, The heavy chain variable region sequence is shown in SEQ ID NO: 39, and the light chain variable region sequence is shown in any one of SEQ ID NOs: 36 to 38; or, The heavy chain variable region sequence is shown in any one of SEQ ID NOs: 27 to 35, and the light chain variable region sequence is shown in any one of SEQ ID NOs: 36 to 38; or The heavy chain variable region sequence is shown in SEQ ID NO: 2, and the light chain variable region sequence is shown in SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof according to claim 2, further comprising a heavy chain constant region and / or a light chain constant region; the heavy chain constant region and / or the light chain constant region are of human or mouse origin.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein: The sequence of the heavy chain constant region is shown in SEQ ID NO: 25, and the sequence of the light chain constant region is shown in SEQ ID NO:

26.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody comprises a heavy chain and a light chain, wherein: The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 40 or 42, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 40 or 42, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 41, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:

41.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody comprises a heavy chain and a light chain, wherein: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 40, and the amino acid sequence of the light chain is shown in SEQ ID NO: 41; or, The amino acid sequence of the heavy chain is shown in SEQ ID NO: 42, and the amino acid sequence of the light chain is shown in SEQ ID NO:

41.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein The antigen binding fragment is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv and scFv.

9. A biomaterial, the biomaterial being: (1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; (2) a vector containing the nucleic acid molecule described in (1); (3) A host cell containing the nucleic acid molecule described in (1) or the vector described in (2); (4) Culture fluid or bacterial suspension obtained by culturing the host cells described in (3).

10. The biomaterial according to claim 9, wherein The nucleic acid molecule is a DNA molecule or an RNA molecule.

11. The biomaterial according to claim 9, wherein The vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or other vectors.

12. The biomaterial according to claim 11, wherein The mammalian cell virus is an adenovirus or a retrovirus.

13. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising: 1) Chemical synthesis, based on the amino acid sequence of the antibody or antigen-binding fragment thereof; or, 2) Biological preparation, comprising culturing the host cell of claim 9 and harvesting the antibody or antigen-binding fragment thereof.

14. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the biomaterial according to any one of claims 9 to 12.

15. The composition according to claim 14, wherein The composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

16. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the biomaterial according to any one of claims 9 to 12, or the composition according to claim 14 or 15.

17. The kit according to claim 16, wherein The kit is used for diagnosing or detecting CX3CL1 protein or cells expressing CX3CL1 protein in an in vitro sample.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the biomaterial according to any one of claims 9 to 12, or the composition according to claim 14 or 15, for preparing a drug for preventing and / or treating a disease associated with abnormal expression or function of CX3CL1; in, The disease associated with abnormal CX3CL1 expression or function is selected from: rheumatoid arthritis (RA), Crohn's disease, ulcerative colitis, diabetes, rheumatoid vasculitis (RV), Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), neuropsychiatric lupus, scleroderma, multiple sclerosis and arteriosclerosis.

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