Anti-human CX3CR1 antibodies

Abstract

The technical problem of the present invention is to provide an anti-human CX3CR1 antibody or the like. The present invention relates to an antibody against human CX3CR1 having a specific CDR sequence, an antibody fragment thereof, or the like.

Description

Technical Field

[0001] The present invention relates to an antibody or a fragment thereof against human CX3CR1, and its uses. Background Art

[0002] CX3CR1 is a G protein-coupled receptor that functions as a chemokine receptor. CX3CR1 is expressed in cytotoxic lymphocytes such as NK cells and cytotoxic T cells, and immune cells closely related to the clearance of pathogens and abnormal cells such as mature macrophages and mucosal dendritic cells. Its endogenous ligand CX3CL1 (fractalkine (FKN)) is induced to be expressed when vascular endothelial cells are treated with inflammatory cytokines TNF and IL-1, and is expressed on the surface of diseased vascular endothelial cells and smooth muscle cells to mediate leukocyte adhesion. FKN is also released into the circulatory system by proteolytic cleavage and functions as a chemotactic substance.

[0003] As described above, since FKN is induced in vascular endothelial cells during inflammation and CX3CR1 is present in various leukocytes, it has also given strong implications that FKN and CX3CR1 are related to the pathogenesis and progression of inflammatory diseases. For example, it has been taught that it is related to the onset and condition of autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, lupus nephritis, and multiple sclerosis (Non-Patent Document 1).

[0004] In addition, it has been shown that in humans, a CX3CR1 variant (V249I / T280M) with low activity is associated with the risk of cardiovascular disease (Non-Patent Document 2), coronary artery disease (evidence of angiographically narrowed vessels) (Non-Patent Document 3), and carotid occlusive disease (Non-Patent Document 4).

[0005] As described above, as therapeutic agents based on the anti-inflammatory effect of inhibiting the migration and infiltration of CX3CR1-expressing immune cells caused by the binding of FKN and CX3CR1, anti-FKN antibodies (Patent Document 1), antibody-like substances (Patent Document 2), etc. that exert effects by blocking their binding have been developed. In addition, it has been suggested that anti-CX3CR1 antibodies may be effective in the treatment of multiple sclerosis (Patent Document 3).

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: International Publication No. WO2006 / 046739

[0009] Patent Document 2: International Publication No. WO2013 / 130381

[0010] Patent Document 3: International Publication No. WO2018 / 101261

[0011] Non-patent literature

[0012] Non-patent literature 1: Umehara H, Blood Et, Okazaki T, et al.: Fractalkine in Vascular Biology: From Basic Research to Clinical Disease, Arterioscler Thomb Vasc Biol 24:34 - 40, 2004.

[0013] Non-patent literature 2: McDermott, 2001; Circ Res 89:401

[0014] Non-patent literature 3: McDermott, 2003; J.Clin.Invest.111:1241

[0015] Non-patent literature 4: Ghilardi, 2004; Stroke 35:1276 Summary of the invention

[0016] Problems to be solved by the invention

[0017] However, in these literatures, there are few diseases showing significant therapeutic effects.

[0018] Under such circumstances, it is desired to further develop therapeutic agents for diseases related to immune cells expressing CX3CR1. One object of the present invention is to provide a prophylactic agent, progression inhibitor, therapeutic agent, etc. for diseases related to immune cells expressing human CX3CR1 by binding to human CX3CR1. For example, a prophylactic agent, progression inhibitor, therapeutic agent, etc. for progressive immune demyelinating diseases (preferably progressive immune demyelinating diseases of the central nervous system, such as secondary progressive multiple sclerosis (SPMS)) are provided.

[0019] Solutions for solving the problems

[0020] The present invention has been completed in consideration of the above situation, and provides the anti-CX3CR1 antibody and its antibody fragment shown below, and their uses (such as pharmaceutical compositions), etc.

[0021] [1] An antibody against human CX3CR1,

[0022] The amino acid sequences of complementarity determining regions (CDR) 1 (CDR-H1), CDR2 (CDR-H2) and CDR3 (CDR-H3) of the heavy chain variable region (VH) are respectively composed of the amino acid sequences shown by SEQ ID NO: 2, 3 and 4, and

[0023] The amino acid sequences of CDR1 (CDR-L1), CDR2 (CDR-L2), and CDR3 (CDR-L3) of the light chain variable region (VL) are respectively composed of the amino acid sequences shown by SEQ ID NO: 6, 7, and 8, or the amino acid sequences shown by SEQ ID NO: 10, 7, and 8 in sequence.

[0024] [2] The antibody according to [1] above, wherein,

[0025] (a) The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are respectively composed of the amino acid sequences shown by SEQ ID NO: 2, 3, and 4 in sequence, and,

[0026] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are respectively composed of the amino acid sequences shown by SEQ ID NO: 6, 7, and 8 in sequence, or

[0027] (b) The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are respectively composed of the amino acid sequences shown by SEQ ID NO: 2, 3, and 4 in sequence, and,

[0028] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are respectively composed of the amino acid sequences shown by SEQ ID NO: 10, 7, and 8 in sequence.

[0029] [3] An antibody against human CX3CR1,

[0030] The amino acid sequence of the heavy chain variable region (VH) is composed of the amino acid sequence shown by SEQ ID NO: 1, 11, 12, 13, or 14, and,

[0031] The amino acid sequence of the light chain variable region (VL) is composed of the amino acid sequence shown by SEQ ID NO: 5, 9, or 15.

[0032] [4] The antibody according to [3] above, wherein,

[0033] (a) The amino acid sequence of VH is composed of the amino acid sequence shown by SEQ ID NO: 1, and,

[0034] The amino acid sequence of VL is composed of the amino acid sequence shown by SEQ ID NO: 5,

[0035] (b) The amino acid sequence of VH is composed of the amino acid sequence shown by SEQ ID NO: 1, and,

[0036] The amino acid sequence of VL is composed of the amino acid sequence shown by SEQ ID NO: 9,

[0037] (c) The amino acid sequence of VH is composed of the amino acid sequence shown by SEQ ID NO: 11, and,

[0038] The amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 15,

[0039] (d) The amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 12, and

[0040] The amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 15,

[0041] (e) The amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 13, and

[0042] The amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 15, or

[0043] (f) The amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 14, and

[0044] The amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 15.

[0045] [5] The antibody according to any one of [1] to [4] above, wherein the antibody is a humanized antibody.

[0046] [6] The antibody according to any one of [1] to [4] above, which has the activity of blocking or inhibiting the interaction or binding of human CX3CR1 and human CX3CL1 (fractalkine).

[0047] [7] The antibody according to any one of [1] to [4] above, which has the activity of blocking or inhibiting the function of Th cells (helper T cells) expressing Eomes.

[0048] The present invention also includes: the antibody according to any one of [1] to [4] above, which has the activity of blocking or inhibiting the function of Th cells expressing granzyme B (Granzyme B; GZMB), or Th cells expressing Eomes and granzyme B. [8]

[0050] The antibody according to [7] above, which has the activity of blocking or inhibiting the infiltration of Th cells expressing Eomes into the CNS (Central Nervous System).

[0051] The present invention also includes: the antibody according to any one of [1] to [4] above, which has the activity of blocking or inhibiting the infiltration of Th cells expressing granzyme B, or Th cells expressing Eomes and granzyme B into the CNS.

[0052] [9] The antibody according to any one of [1] to [4] above has the function of improving the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of experimental autoimmune encephalomyelitis (EAE).

[0053]

[10] The antibody according to any one of [1] to [4] above is an antibody for the prevention, progression inhibition or treatment of the following diseases: progressive immune demyelinating diseases, preferably progressive immune demyelinating diseases of the central nervous system; autoimmune diseases, preferably rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus or autoimmune myositis; heart diseases including atherosclerosis; chronic kidney diseases; diabetic heart diseases; diabetic nephropathies; interstitial lung diseases; cancers, preferably breast cancer, prostate cancer or colorectal cancer; COVID-19 related central nervous system diseases; or neurodegenerative diseases, preferably ALS, Alzheimer's disease or multiple system atrophy.

[0054]

[11] The antibody according to

[10] above, wherein the progressive immune demyelinating disease is secondary progressive multiple sclerosis (SPMS).

[0055]

[12] An antibody fragment derived from the antibody according to any one of [1] to [4] above.

[0056]

[13] A pharmaceutical composition comprising the antibody according to any one of [1] to [4] above, or the antibody fragment according to

[12] above.

[0057]

[14] The pharmaceutical composition according to

[13] above is a pharmaceutical composition for the prevention, progression inhibition or treatment of the following diseases: progressive immune demyelinating diseases, preferably progressive immune demyelinating diseases of the central nervous system; autoimmune diseases, preferably rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus or autoimmune myositis; heart diseases including atherosclerosis; chronic kidney diseases; diabetic heart diseases; diabetic nephropathies; interstitial lung diseases; cancers, preferably breast cancer, prostate cancer or colorectal cancer; COVID-19 related central nervous system diseases; or neurodegenerative diseases, preferably ALS, Alzheimer's disease or multiple system atrophy.

[0058]

[15] The pharmaceutical composition according to

[14] above, wherein the progressive immune demyelinating disease is secondary progressive multiple sclerosis (SPMS).

[0059]

[16] The pharmaceutical composition according to

[13] above is a pharmaceutical composition for blocking or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine).

[0060]

[17] The pharmaceutical composition described in

[13] above is a pharmaceutical composition for blocking or inhibiting the function of Th cells expressing Eomes.

[0061] The present invention also includes: the pharmaceutical composition described in

[13] above, which is a pharmaceutical composition for blocking or inhibiting the function of Th cells expressing granzyme B, or Th cells expressing Eomes and granzyme B.

[0062]

[18] The pharmaceutical composition described in

[17] above is a pharmaceutical composition for blocking or inhibiting the infiltration of Th cells expressing Eomes into the CNS.

[0063] The present invention also includes: the pharmaceutical composition described in

[13] above, which is a pharmaceutical composition for blocking or inhibiting the infiltration of Th cells expressing granzyme B, or Th cells expressing Eomes and granzyme B into the CNS.

[0064]

[19] The pharmaceutical composition described in

[13] above is a pharmaceutical composition for improving the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of experimental autoimmune encephalomyelitis (EAE).

[0065]

[20] A method for preventing, inhibiting the progression of, or treating the following diseases, which comprises administering to a subject the pharmaceutical composition described in

[13] above, and the diseases are: progressive immune demyelinating diseases, preferably progressive immune demyelinating diseases of the central nervous system; autoimmune diseases, preferably rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus, or autoimmune myositis; heart diseases including atherosclerosis; chronic kidney diseases; diabetic heart diseases; diabetic nephropathies; interstitial lung diseases; cancers, preferably breast cancer, prostate cancer, or colorectal cancer; COVID-19-related central nervous system diseases; or neurodegenerative diseases, preferably ALS, Alzheimer's disease, or multiple system atrophy.

[0066]

[21] According to the method described in

[20] above, wherein the progressive immune demyelinating disease is secondary progressive multiple sclerosis (SPMS).

[0067]

[22] A method for blocking or inhibiting the interaction or binding of human CX3CR1 and human CX3CL1 (fractalkine), which comprises administering to a subject the pharmaceutical composition described in

[13] above.

[0068]

[23] A method for blocking or inhibiting the function of Th cells expressing Eomes, which comprises administering to a subject the pharmaceutical composition described in

[13] above.

[0069] The present invention further includes: a method for blocking or inhibiting the function of Th cells expressing granzyme B or Th cells expressing Eomes and granzyme B, which includes administering the pharmaceutical composition described in

[13] above to a subject.

[0070]

[24] According to the method described in

[23] above, which is a method for blocking or inhibiting the infiltration of Th cells expressing Eomes into the CNS, the method includes administering the pharmaceutical composition described in

[13] above to a subject.

[0071] The present invention further includes: a method for blocking or inhibiting the infiltration of Th cells expressing granzyme B or Th cells expressing Eomes and granzyme B into the CNS, which includes administering the pharmaceutical composition described in

[13] above to a subject.

[0072]

[25] A method for ameliorating the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of experimental autoimmune encephalomyelitis (EAE), which includes administering the pharmaceutical composition described in

[13] above to a subject.

[0073]

[26] A kit for preventing, inhibiting the progression of, treating, or diagnosing the following diseases, which contains the antibody described in any one of [1] to [4] above or the antibody fragment described in

[12] above, and the diseases are: progressive immune demyelinating diseases, preferably progressive immune demyelinating diseases of the central nervous system; autoimmune diseases, preferably rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus, or autoimmune myositis; heart diseases including atherosclerosis; chronic kidney diseases; diabetic heart diseases; diabetic nephropathies; interstitial lung diseases; cancers, preferably breast cancer, prostate cancer, or colorectal cancer; COVID-19 related central nervous system diseases; or neurodegenerative diseases, preferably ALS, Alzheimer's disease, or multiple system atrophy.

[0074]

[27] According to the kit described in

[26] above, wherein the progressive immune demyelinating disease is secondary progressive multiple sclerosis (SPMS).

[0075] Effects of the Invention

[0076] According to the present invention, antibodies that bind to human CX3CR1 and the like can be provided. The anti-human CX3CR1 antibody of the present invention may have, for example, an activity of blocking or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine), and a function of improving the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of the condition of experimental autoimmune encephalomyelitis (EAE). In addition, it can exert an effect as a prophylactic agent, progression inhibitor or therapeutic agent for progressive immune demyelinating diseases (preferably progressive immune demyelinating diseases of the central nervous system, such as secondary progressive multiple sclerosis (SPMS)). BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 A graph showing the results of measuring the binding activity of the m1F8 antibody to human CX3CR1-expressing Ba / F3 cells by flow cytometry.

[0078] Figure 2 A graph showing the results of measuring the blocking activity of the m1F8 antibody on fractalkine-dependent cell migration of human CX3CR1-expressing Ba / F3 cells.

[0079] Figure 3 A schematic diagram of a chimeric CX3CR1 expression construct in which the extracellular domain of CX3CR1 is replaced for epitope analysis. (A) is a diagram showing the construct based on human CX3CR1, and (B) is a diagram showing the construct based on mouse CX3CR1.

[0080] Figure 4 A graph showing the sequences of the heavy chain variable region and the light chain variable region of the m1F8-1 antibody and the m1F8-2 antibody formed by modifying the m1F8 antibody. The underlined regions in each sequence represent the CDR sequences.

[0081] Figure 5 A graph showing the test results of administering anti-CX3CR1-Nanobody (BII00313) to NR4A2 / CX3CR1 gene mutant mice (late EAE model of human CX3CR1-KI mice) induced with monophasic EAE. (A) is a graph showing the amount of free fractalkine (CX3CL1) in the blood, and (B) is a graph showing the EAE score.

[0082] Figure 6 A graph showing the test results of administering the m1F8-1 antibody or the m1F8-2 antibody to a late EAE model of human CX3CR1-KI mice. (A) is a graph showing the amount of free chemokine in the blood, and (B) is a graph showing the EAE score.

[0083] Figure 7Graph showing the EAE scores in an experiment in which m1F8-2 or anti-CX3CR1-Nanobody (BII00313) was administered to a late-stage EAE model of human CX3CR1-KI mice.

[0084] Figure 8 Graph showing the sequences of the heavy-chain variable region and the light-chain variable region of four humanized 1F8 antibodies (h1F8-2-1, h1F8-2-2, h1F8-2-3, h1F8-2-4). The underlined regions in each sequence represent the CDR sequences.

[0085] Figure 9 Graph showing the results of measuring the binding activity of the humanized 1F8 antibody (h1F8-2-2) or 306D-hFc to human, cynomolgus monkey, mouse, or rat CX3CR1-expressing Ba / F3 cells by flow cytometry.

[0086] Figure 10 Graph showing the results of measuring the binding activity of the h1F8-2-2 antibody or 306D-hFc to chimeric CX3CR1-expressing Ba / F3 cells in which the extracellular domains of human and mouse CX3CR1 were replaced by flow cytometry.

[0087] Figure 11 Graph showing the results of measuring the in vitro blocking activity of the h1F8-2-2 antibody or BII00313 in a calcium influx assay using human CX3CR1-expressing CHO-K1 cells. (A) is a graph showing the results of simultaneously measuring the blocking activities of the h1F8-2-2 antibody, BII00313, or Synagis, (B) is a graph showing the results of measuring the concentration-dependent blocking activity of the h1F8-2-2 antibody, and (C) is a graph showing the results of measuring the concentration-dependent blocking activity of BII00313.

[0088] Figure 12 Graph showing the proportion of CD115-positive cells ((A): m1F8-2 staining, (B): 66B02-mFc staining), the proportion of CD115-positive and CX3CR1-positive cells ((C): m1F8-2 staining, (D): 66B02-mFc staining), and the proportion of CX3CR1-positive cells relative to CD115-positive cells ((E): m1F8-2 staining, (F): 66B02-mFc staining) in peripheral blood in human CX3CR1-KI mice administered the h1F8-2-2 antibody or BII00313.

[0089] Figure 13 Graph showing the EAE scores in an experiment in which the h1F8-2-2 antibody was administered to a late-stage EAE model of human CX3CR1-KI mice.

[0090] Figure 14 A graph showing the arthritis scores in the test of administering the m1F8-2 antibody to a human CX3CR1-KI mouse CAIA (collagen antibody-induced arthritis) model. Detailed Description of the Invention

[0091] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and in cases other than those exemplified below, it can be appropriately modified and implemented within the scope not impairing the gist of the present invention.

[0092] In addition, this specification includes the entire content of Japanese Patent Application No. 2022-205800 (filed on December 22, 2022), which is the basis for claiming the priority of this application. In addition, all publications cited in this specification, such as prior art documents, published gazettes, patent gazettes, and other patent documents, are incorporated herein by reference.

[0093] 1. Antibodies against human CX3CR1 (anti-human CX3CR1 antibodies)

[0094] (1) Preparation of Antigen

[0095] Information on the amino acid sequence (SEQ ID NO: 17) of human CX3CR1 (hereinafter also referred to as hCX3CR1) is registered and published, for example, on the Uniprot website (https: / / www.uniprot.org / ) as "Primary (citable) accession number: P49238-1", or on the NCBI (GenBank) website (http: / / www.ncbi.nlm.nih.gov / ) as "Accession number: NP_001328.1". It should be noted that information on the base sequence (SEQ ID NO: 16) encoding the amino acid sequence of hCX3CR1 is registered and published on the NCBI (GenBank) website as "Accession number: NM_001337.3" (it should be noted that the sequence composed of the 93rd to 1160th bases in the registered base sequence corresponds to the base sequence of SEQ ID NO: 16).

[0096] As the antigen, a polypeptide or peptide (hereinafter also simply referred to as a peptide) containing all or a part of the amino acid sequence of hCX3CR1 can be used.

[0097] The method for producing the peptide as the antigen can be chemical synthesis or synthesis using genetic engineering methods such as Escherichia coli, and methods well-known to those skilled in the art can be used.

[0098] When performing chemical synthesis of a peptide, it can be carried out by known methods of peptide synthesis. Additionally, this synthesis can also employ either solid-phase synthesis or liquid-phase synthesis. Commercially available peptide synthesis apparatuses (such as CEM Japan: parallel-type automatic peptide synthesis apparatus MultiPep 2, etc.) can also be used.

[0099] When synthesizing a peptide using genetic engineering, first, a DNA encoding the peptide is designed and synthesized. This design and synthesis can be carried out, for example, by using primers designed to synthesize the desired DNA region with a vector containing the full-length hCX3CR1 gene, etc. as a template, through the PCR method. Additionally, based on the amino acid sequence of the peptide, DNA with a Kozak translation initiation sequence inserted at the 5'-end and a translation termination codon inserted at the 3'-side can be gene-synthesized (using services such as those of GENEWIZ). Then, a recombinant vector for protein expression is obtained by ligating the above DNA with an appropriate vector, and the recombinant vector is introduced into a host in a manner that enables the expression of the target gene, thereby obtaining a transformant (Molecular cloning 4th Ed. Cold Spring Harbor Laboratory Press (2012)).

[0100] As the vector, a phage or plasmid capable of autonomous replication in the host microorganism is used. Furthermore, animal viruses and insect virus vectors can also be used. The production of the recombinant vector can be achieved by simply cutting the purified DNA with an appropriate restriction enzyme, inserting it into the restriction enzyme site of an appropriate vector DNA, etc., and ligating it with the vector. As the host used in transformation, there is no particular limitation as long as it can express the target gene. For example, bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.), insect cells or insects can be cited. Mammals such as goats can also be used as hosts. The method of introducing the recombinant vector into the host is well-known. Then, the above transformant is cultured, and the peptide used as an antigen is collected from the culture. "Culture" refers to any one of (a) culture supernatant, (b) cultured cells or cultured bacteria, or their disrupted products.

[0101] After culturing, when the target peptide is produced inside the bacteria or cells, the peptide is extracted by disrupting the bacteria or cells. Additionally, when the target peptide is produced outside the bacteria or cells, the culture broth is directly used, or the bacteria or cells are removed by centrifugation, etc. Then, the target peptide can be separated and purified by using conventional biochemical methods for peptide separation and purification alone or in appropriate combination, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc.

[0102] The peptide that becomes an antigen can also be obtained by in vitro translation using a cell-free synthesis system. In this case, two methods can be used: the method using RNA as a template and the method using DNA as a template (transcription / translation). As the cell-free synthesis system, commercially available systems can be used, such as the Expressway TM system (Invitrogen), PURESYSTEM (registered trademark; Post Genome Institute), TNT system (registered trademark; Promega), etc.

[0103] The peptide obtained as described above can also be bound to an appropriate carrier protein, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), human thyroglobulin, chicken gamma globulin, etc.

[0104] In addition, the antigen can also be a peptide composed of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of hCX3CR1 (SEQ ID NO: 17) or a partial sequence thereof. For example, a peptide composed of an amino acid sequence in which one or more (preferably one or several (e.g., 1 to 10, more preferably 1 to 5)) amino acids are deleted, one or more (preferably one or several (e.g., 1 to 10, more preferably 1 to 5)) amino acids are substituted with other amino acids, or one or more (preferably one or several (e.g., 1 to 10, more preferably 1 to 5)) other amino acids are added in the amino acid sequence of hCX3CR1 or a partial sequence thereof can also be used.

[0105] As the gene for introducing into cells, etc., genes encoding the hCX3CR1 protein or a partial fragment thereof, or mutant proteins or fragments thereof can be cited. As such genes, for example, genes having the base sequence shown in SEQ ID NO: 16 or a partial sequence thereof can be used.

[0106] In addition, as the gene for introducing into cells, etc., a base sequence that hybridizes under stringent conditions with a sequence complementary to the base sequence shown in SEQ ID NO: 16 and encodes a protein having the same activity and function as hCX3CR1, or a partial sequence thereof can also be used.

[0107] "Stringent conditions" refer to the conditions during washing after hybridization, which are conditions where the salt (sodium) concentration of the buffer is 10 to 500 mM and the temperature is 42°C to 72°C, preferably the salt concentration is 50 to 300 mM and the temperature is 55 to 68°C.

[0108] To introduce mutations into the gene, known methods such as the Kunkel method and the Gapped duplex method can be used. For example, a mutagenesis kit using site-directed mutagenesis, such as GeneArtTM It is carried out using a site-directed mutagenesis system (manufactured by Invitrogen) or a TaKaRa site-directed mutagenesis system (such as Mutan-K and Mutan-Super Express Km: manufactured by Takara Bio).

[0109] (2) Preparation of polyclonal antibodies

[0110] The prepared antigen is administered to a mammal for immunization. The mammal is not particularly limited, and examples include rats, mice, and rabbits, among which mice are preferred.

[0111] The antigen dosage per animal can be appropriately set according to the presence or absence of an adjuvant. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization can mainly be carried out by intravenous injection, plantar injection, subcutaneous injection, intraperitoneal injection, etc. In addition, the interval between immunizations is not particularly limited, and immunization is carried out 1 to 10 times, preferably 2 to 3 times, at intervals of several days to several weeks, preferably at 1-week intervals. Then, 3 to 7 days after the last immunization day, the antibody titer is measured using enzyme immunoassay (ELISA or EIA), radioimmunoassay (RIA), etc., and blood is collected on the day when the desired antibody titer is shown, thereby obtaining antiserum. In the above antibody collection method, when antibody purification is required, it can be purified by appropriately selecting well-known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc., or a combination of them. After that, the reactivity of polyclonal antibodies in the antiserum is measured by ELISA method, etc.

[0112] (3) Preparation of monoclonal antibodies

[0113] (3-1) Collection of antibody-producing cells

[0114] The anti-hCX3CR1 antibody of the present invention is not limited, and a monoclonal antibody is preferred.

[0115] The prepared antigen is administered to mammals such as rats, mice, and rabbits for immunization. The antigen dosage per animal can be appropriately set according to the presence or absence of an adjuvant. The adjuvant is the same as above. The immunization method is also the same as described above. Then, antibody-producing cells are collected 1 to 60 days, preferably 1 to 14 days, after the last immunization day. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, among which lymph node cells and spleen cells are preferred.

[0116] (3-2) Cell fusion

[0117] To obtain hybridomas (antibody-producing cell lines), cell fusion of antibody-producing cells and myeloma cells is performed. As the myeloma cells to be fused with antibody-producing cells, commonly available cell line cells of animals such as mice can be used. As the cell line used, a cell line having the following properties is preferably used: having drug selectivity and being unable to survive in HAT selective medium (including hypoxanthine, aminopterin, and thymidine) in the unfused state, and being able to survive only in the state of being fused with antibody-producing cells in HAT selective medium.

[0118] Examples of myeloma cells include mouse myeloma cell lines such as P3-X63-Ag8.653, P3-X63-Ag8 (X63), P3-X63-Ag8.U1 (P3U1), P3 / NS I / 1-Ag4-1 (NS1), and Sp2 / 0-Ag14 (Sp2 / 0). The selection of myeloma cells can be appropriately carried out considering the compatibility with antibody-producing cells.

[0119] Next, cell fusion of myeloma cells and antibody-producing cells is performed. Cell fusion is carried out in animal cell culture media such as serum-free DMEM and RPMI-1640 medium, where 1×10 6 ~1×10 7 antibody-producing cells per mL and 2×10 5 ~2×10 6 myeloma cells per mL are mixed. The cell ratio of antibody-producing cells to myeloma cells (antibody-producing cells: myeloma cells) is not limited, and is usually preferably 1:1 to 10:1, more preferably 3:1. Then, a fusion reaction is carried out in the presence of a cell fusion promoter. As the cell fusion promoter, for example, polyethylene glycol with an average molecular weight of 1,000 to 6,000 daltons (D) can be used. In addition, a commercially available cell fusion device using electric stimulation (such as electroporation) can also be used to fuse antibody-producing cells and myeloma cells.

[0120] (3-3) Screening and cloning of hybridomas

[0121] The target hybridomas are screened from the cells after cell fusion treatment. As the method, the cell suspension is appropriately diluted in, for example, RPMI-1640 medium containing fetal bovine serum, then inoculated onto a microtiter plate, selective medium is added to each well, and then the selective medium is appropriately changed for culturing. As a result, cells that start to grow around 14 days after the start of culturing in the selective medium can be obtained as hybridomas.

[0122] Next, it is screened whether there is an antibody reactive with hCX3CR1 in the culture supernatant of the proliferated hybridomas. The screening of hybridomas can be carried out by a usual method without particular limitation. For example, a part of the culture supernatant contained in the well where hybridomas grow can be collected and screened by ELISA, EIA, RIA, etc.

[0123] Cloning of the fused cells can be carried out by methods such as the limiting dilution method. Antibodies showing strong reactivity with hCX3CR1 are determined by flow cytometry, etc., hybridomas producing them are selected, and clones are established.

[0124] (3-4) Collection of monoclonal antibodies

[0125] As a method for culturing the established hybridomas and collecting monoclonal antibodies from the obtained culture, conventional cell culture methods or ascites formation methods, etc. can be adopted. "Culturing" means growing hybridomas in a culture dish or culture flask, or proliferating hybridomas in the abdominal cavity of an animal as described below.

[0126] In the cell culture method, hybridomas can be cultured in an animal cell culture medium such as RPMI-1640 medium, MEM medium or serum-free medium containing 10% fetal bovine serum under usual culture conditions (for example, 37 °C, 5% CO2 concentration) for 7 to 14 days, and antibodies are obtained from the culture supernatant.

[0127] In the case of the ascites formation method, about 1×10 7 hybridomas are administered into the abdominal cavity of a mammalian animal of the same strain as the myeloma cells originated from, to proliferate the hybridomas in large numbers. And, it is preferable to collect ascites after 2 to 3 weeks.

[0128] In the above antibody collection methods, when it is necessary to purify the antibody, well-known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography, etc. can be appropriately selected, or they can be combined for purification.

[0129] (3-5) Activity and function of clones

[0130] (3-5-1) The anti-hCX3CR1 antibody of the present invention is preferably an antibody having an activity of blocking or inhibiting the interaction (preferably binding) between human CX3CR1 and human CX3CL1 (fractalkine).

[0131] Here, the blocking or inhibitory activity (%) of the interaction between hCX3CR1 and fractalkine can be calculated by the following formula, for example.

[0132] Blocking or inhibitory activity (%) = 100 - (reactivity when antibody is added, with background correction / reactivity when antibody is not added, with background correction) × 100

[0133] (3-5-2) The anti-hCX3CR1 antibody of the present invention is, for example, preferably an antibody having an activity of blocking or inhibiting the function of Th cells (helper T cells) expressing Eomes. In addition, the anti-hCX3CR1 antibody of the present invention is, for example, also preferably an antibody having an activity of blocking or inhibiting the function of Th cells expressing granzyme B, or an antibody having an activity of blocking or inhibiting the function of Th cells expressing Eomes and granzyme B, or an antibody having an activity of blocking or inhibiting the function of Th cells expressing Eomes and / or IFN-γ. Here, as this function (the function of Th cells expressing Eomes and / or granzyme B, or the function of Th cells expressing Eomes and / or IFN-γ), for example, infiltration into the CNS (central nervous system) can be cited.

[0134] Here, the blocking or inhibitory activity (%) of the function of Th cells expressing Eomes and / or granzyme B or expressing Eomes and / or IFN-γ can be calculated, for example, by the following formula.

[0135] Blocking or inhibitory activity (%) = 100 - (reactivity when the antibody is added, with background correction / reactivity when the antibody is not added, with background correction) × 100

[0136] (3-5-3) The anti-hCX3CR1 antibody of the present invention is, for example, preferably an antibody having a function of improving the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of the condition of experimental autoimmune encephalomyelitis (EAE).

[0137] The EAE condition is the symptom shown by C57BL / 6 mice administered with complete Freund's adjuvant (CFA) containing MOG peptide and heat-killed Mycobacterium tuberculosis H37Ra. The EAE model refers to a model (test system) that reproduces the condition of MS in humans in mice and evaluates its severity according to a numerical benchmark.

[0138] The symptoms and evaluation criteria are specified as follows.

[0139] <EAE evaluation criteria>

[0140] 0: No clinical signs

[0141] 1: Local paralysis of the tail

[0142] 2: Flaccid tail

[0143] 3: Local paralysis of the hind limbs

[0144] 4: Paralysis of the entire hind limbs

[0145] 5: Paralysis of the hindlimbs and forelimbs

[0146] The efficacy of the anti-hCX3CR1 antibody of the present invention in improving or suppressing the progression of MS can be evaluated by the EAE model or the late EAE model described below.

[0147] Herein, the late EAE model used in the present application is a special mouse model that only experimentally reproduces the symptoms of progressive MS and is constructed by using NR4A2-deficient mice. Furthermore, in the present application, in order to confirm the reactivity of 1F8 that binds to hCX3CR1 using this model, mice mated with mice knocked in with hCX3CR1 (human CX3CR1-KI mouse late EAE model) were used.

[0148] More specifically, in the late EAE model, there is no increase in the EAE score that can be observed in the early stage after EAE induction in the EAE test using non-NR4A2-deficient mice, i.e., conventional mice. On the other hand, after a certain period after EAE induction, only an increase in the EAE score (i.e., the EAE condition) caused by progressive MS is observed. Therefore, it can be used in particular to evaluate progressive MS.

[0149] (3-5-4) The anti-hCX3CR1 antibody of the present invention is preferably an antibody that can be used for the prevention, progression inhibition, or treatment of progressive immune demyelinating diseases.

[0150] Herein, as progressive immune demyelinating diseases, progressive immune demyelinating diseases of the central nervous system are preferably listed. As progressive immune demyelinating diseases, progressive multiple sclerosis (MS) can be listed. As progressive MS, primary progressive MS (PPMS), secondary progressive MS (SPMS) in which the RRMS condition turns into a progressive condition after a certain period, and progressive relapsing MS (PRMS) that progresses while relapsing repeatedly are known (see Patent Document 3). However, it is not limited to these diseases. The anti-hCX3CR1 antibody of the present invention can be used, for example, for the prevention, progression inhibition, or treatment of any one or two or more diseases among autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus, and autoimmune myositis, heart diseases including atherosclerosis, chronic kidney disease, diabetic heart disease, diabetic nephropathy, interstitial lung disease, cancer (e.g., breast cancer, prostate cancer, colorectal cancer), COVID-19 (novel coronavirus infection)-related central nervous system diseases, and neurodegenerative diseases such as ALS, Alzheimer's disease, and multiple system atrophy.

[0151] (3-5-5) The anti-hCX3CR1 antibody of the present invention is not limited, and the dissociation constant (Kd value) is preferably 1.0×10 -10M or less, more preferably 1.0×10 -11 M or less, still more preferably 1.0×10 -12 M or less.

[0152] Here, the binding ability (affinity) of the antibody can be measured, for example, by Scatchard analysis or a surface plasmon resonance sensor called Biacore, as the dissociation constant (Kd value), dissociation rate constant (Kdiss [1 / Sec]), and association rate constant (Kass [1 / M.Sec]). Examples of the Biacore device include Biacore3000, Biacore2000, Biacore X, Biacore J, and Biacore Q (all manufactured by Biacore). The smaller the dissociation constant (Kd value) of the antibody, the higher the binding ability (affinity), and thus it is preferred. The Kd value is determined by two parameters, Kdiss and Kass, and can be expressed, for example, by the formula: Kd [M]=Kdiss / Kass.

[0153] (3-6) Epitope of anti-hCX3CR1 antibody

[0154] The epitope (antigenic determinant) of the anti-hCX3CR1 antibody in the present invention may be at least a partial region of hCX3CR1 as an antigen. It is preferably, for example, at least a partial region of the extracellular domain of hCX3CR1, but a region other than the extracellular domain may also be an epitope.

[0155] It should be noted that the present invention also includes an antibody capable of binding to the epitope region bound (recognized) by the anti-hCX3CR1 antibody in the present invention.

[0156] (4) Recombinant antibody and antibody fragment

[0157] (4-1) Recombinant antibody

[0158] As one of the preferred forms of the anti-hCX3CR1 antibody of the present invention, a recombinant antibody can be cited. There is no limitation on the recombinant antibody, and examples include chimeric antibodies, humanized antibodies (humanized antibodies), and human antibodies (fully human antibodies), etc.

[0159] A chimeric antibody (i.e., a humanized chimeric antibody) is an antibody formed by connecting (joining) the variable region of a murine antibody to the constant region of a human (see Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855, (1984), etc.). When making a chimera, such a connected antibody can be easily constructed by genetic recombination technology.

[0160] When producing a humanized antibody, for example, a complementarity-determining region (CDR) is transplanted from the variable region of a murine antibody into a human variable region, thereby producing a reconstructed variable region (so-called CDR Grafting) composed of a sequence in which the framework region (FR) is derived from a human and the CDR is derived from a murine. Then, these humanized reconstructed human variable regions are linked to a human constant region. Such a method for producing a humanized antibody is well-known in the art (see Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); Japanese Patent Application Laid-Open No. Hei 4-502408 (Japanese Patent No. 2828340; Queen et al.), etc.).

[0161] Regarding human antibodies (fully human antibodies), generally, the structures of the hypervariable region, which serves as the antigen-binding site in the V region, the other parts of the V region, and the constant region are the same as those of human antibodies. However, the hypervariable sites can also be derived from other animals. Techniques for producing human antibodies are also well-known, and methods for production by genetic engineering have been established for gene sequences common to humans. Human antibodies can be obtained, for example, by the method of using a human antibody-producing mouse with a human chromosome fragment containing the genes for the H chain and L chain of a human antibody (see Tomizuka, K. et al., Nature Genetics, (1977) 16, 133-143; Kuroiwa, Y. et.al., Nuc. Acids Res., (1998) 26, 3447-3448; Yoshida, H. et.al., Animal Cell Technology: Basic and Applied Aspects, (1999) 10, 69-73 (Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers; Tomizuka, K. et.al., Proc. Natl. Acad. Sci. USA, (2000) 97, 722-727, etc.), the method of obtaining a phage-displayed human antibody selected from a human antibody library (see Wormstone, I.M. et.al, Investigative Ophthalmology & Visual Science., (2002) 43(7), 2301-8; Carmen, S. et.al., Briefings in Functional Genomics and Proteomics, (2002) 1(2), 189-203; Siriwardena, D. et.al., Opthalmology, (2002) 109(3), 427-431, etc.).

[0162] In addition, human antibodies can also be used: antibodies (further purified) produced by clones specifically binding to the desired antigen (hCX3CR1 in the present invention) using the human ADLib technology (the library described in WO2015 / 167011); antibodies in which the amino acid sequences (especially the amino acid sequences of VH and VL, preferably at least one CDR sequence in VH and VL) of the antibody have been modified (preferably antibodies in which amino acid substitutions have been made).

[0163] The above-mentioned chimeric antibody, humanized antibody, and human antibody are not limited. Preferably, the N-glycoside complex sugar chain in the Fc region of the antibody is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain. Specifically, an antibody composed of the following gene recombinant antibody molecule can be cited. The gene recombinant antibody molecule has a sugar chain in which the 1-position of fucose does not form an α-bond with the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside complex sugar chain in the Fc region of the antibody molecule. If it is such an antibody, the ADCC activity can be improved. It should be noted that this point (the characteristics of the N-glycoside complex sugar chain in the Fc region of the antibody) is also preferably applicable to the aforementioned polyclonal antibody and monoclonal antibody.

[0164] As the anti-hCX3CR1 antibody of the present invention, for example, preferably cited are:

[0165] The amino acid sequences of complementarity determining regions (CDR) 1 (CDR-H1), CDR2 (CDR-H2), and CDR3 (CDR-H3) of the heavy chain variable region (VH) are sequentially composed of the amino acid sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and

[0166] An antibody in which the amino acid sequences of CDR1 (CDR-L1), CDR2 (CDR-L2), and CDR3 (CDR-L3) of the light chain variable region (VL) are sequentially composed of the amino acid sequences shown in SEQ ID NOs: 6, 7, and 8, or the amino acid sequences shown in SEQ ID NOs: 10, 7, and 8, etc.

[0167] More specifically, as the anti-hCX3CR1 antibody of the present invention, for example, preferably cited are:

[0168] (a) An antibody in which the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are sequentially composed of the amino acid sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and,

[0169] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are sequentially composed of the amino acid sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and

[0170] (b) An antibody in which the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are sequentially composed of the amino acid sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and,

[0171] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are sequentially composed of the amino acid sequences shown in SEQ ID NOs: 10, 7, and 8, respectively; etc.

[0172] In addition, as a more preferred embodiment of the anti-hCX3CR1 antibody of the present invention, for example, preferably cited are:

[0173] An antibody in which the amino acid sequence of the heavy chain variable region (VH) consists of the amino acid sequence shown in SEQ ID NO: 1, 11, 12, 13 or 14, and the amino acid sequence of the light chain variable region (VL) consists of the amino acid sequence shown in SEQ ID NO: 5, 9 or 15; etc.

[0174] More specifically, as the anti-hCX3CR1 antibody of the present invention, for example, preferably cited are:

[0175] (a) An antibody in which the amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 1, and

[0176] the amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 5;

[0177] (b) An antibody in which the amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 1, and

[0178] the amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 9;

[0179] (c) An antibody in which the amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 11, and

[0180] the amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 15;

[0181] (d) An antibody in which the amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 12, and

[0182] the amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 15;

[0183] (e) An antibody in which the amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 13, and

[0184] the amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 15; and

[0185] (f) An antibody in which the amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 14, and

[0186] the amino acid sequence of VL consists of the amino acid sequence shown in SEQ ID NO: 15; etc.

[0187] In addition, as the anti-hCX3CR1 antibody of the present invention, the VH and VL of each of the m1F8-1 antibody, m1F8-2 antibody, h1F8-2-1 antibody, h1F8-2-2 antibody, h1F8-2-3 antibody, and h1F8-2-4 antibody among the antibodies exemplified in the following examples, and the amino acid sequences of CDR-H1 to CDR-H3 and CDR-L1 to CDR-L3 are antibodies composed of the amino acid sequences represented by the sequence numbers shown in Table A below.

[0188] [Table A]

[0189] Antibody Name VH CDR-H1 CDR-H2 CDR-H3 VL CDR-L1 CDR-L2 CDR-L3 m1F8-1 Sequence Number 1 Sequence Number 2 Sequence Number 3 Sequence Number 4 Sequence Number 5 Sequence Number 6 Sequence Number 7 Sequence Number 8 m1F8-2 Sequence Number 1 Sequence Number 2 Sequence Number 3 Sequence Number 4 Sequence Number 9 Sequence Number 10 Sequence Number 7 Sequence Number 8 h1F8-2-1 Sequence Number 11 Sequence Number 2 Sequence Number 3 Sequence Number 4 Sequence Number 15 Sequence Number 1o Sequence Number 7 Sequence Number 8 h1F8-2-2 Sequence Number 12 Sequence Number 2 Sequence Number 3 Sequence Number 4 Sequence Number 15 Sequence Number 10 Sequence Number 7 Sequence Number 8 h1F8-2-3 Sequence Number 13 Sequence Number 2 Sequence Number 3 Sequence Number 4 Sequence Number 15 Sequence Number 10 Sequence Number 7 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0190] (4-2) Antibody fragment

[0191] Fragments (partial fragments) of the anti-hCX3CR1 antibody of the present invention are also included in the antibodies of the present invention. Here, the antibody fragment of the present invention is an antibody fragment that has the same binding activity to hCX3CR1 as the anti-hCX3CR1 antibody of the present invention (i.e., an antibody fragment capable of binding to hCX3CR1). Preferably, it is an antibody fragment having the activity of blocking or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine), an antibody fragment having the activity of blocking or inhibiting the function of Th cells expressing Eomes and / or granzyme B or Th cells expressing Eomes and / or IFN-γ (such as infiltration into the CNS, etc.), an antibody fragment having the function of improving the condition of EAE or inhibiting the progression of EAE, and an antibody fragment that can be used for the prevention, progression inhibition, or treatment of progressive immune demyelinating diseases (preferably progressive immune demyelinating diseases of the central nervous system, such as secondary progressive multiple sclerosis (SPMS)).

[0192] As a fragment of this antibody, it refers to a partial region of an anti-hCX3CR1 polyclonal antibody or an anti-hCX3CR1 monoclonal antibody (i.e., an antibody fragment derived from the anti-hCX3CR1 antibody of the present invention). Examples include Fab, Fab’, F(ab’)2, Fv (variable fragment of antibody), single-chain antibodies (such as H chain, L chain, VH region of H chain, and VL region of L chain), scFv, diabody (scFv dimer), dsFv (disulfide bond-stabilized V region), and peptides containing at least a part of the complementarity determining region (CDR).

[0193] Fab is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 50,000, in which approximately half of the N-terminal side of the H chain and the entire L chain are bound by a disulfide bond in the fragment obtained by treating an antibody molecule with papain, a proteolytic enzyme. In addition, Fab can also be produced by inserting the DNA encoding the Fab of an antibody into an expression vector for prokaryotes or an expression vector for eukaryotes, and introducing the vector into prokaryotes or eukaryotes to express it.

[0194] F(ab’)2 is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 100,000, which is slightly larger than Fab and is a fragment bound by a disulfide bond in the hinge region in the fragment obtained by treating an antibody molecule with pepsin, a proteolytic enzyme. In addition, it can also be prepared by binding the aforementioned Fab with a thioether bond or a disulfide bond.

[0195] Fab’ is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 50,000, which is obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab’)2. In addition, Fab’ can also be produced by inserting the DNA encoding the Fab’ fragment of an antibody into an expression vector for prokaryotes or an expression vector for eukaryotes, and introducing the vector into prokaryotes or eukaryotes to express it.

[0196] scFv is a VH-P-VL or VL-P-VH polypeptide formed by connecting one heavy-chain variable region (VH) and one light-chain variable region (VL) with an appropriate peptide linker (P), and it is an antibody fragment with an antigen-binding activity. scFv can be produced by obtaining the cDNAs encoding the VH and VL of an antibody, constructing the DNA encoding scFv, inserting the DNA into an expression vector for prokaryotes or an expression vector for eukaryotes, and introducing the expression vector into prokaryotes or eukaryotes to express it.

[0197] Diabody is an antibody fragment formed by the dimerization of scFv, and it is an antibody fragment with a bivalent antigen-binding activity. The bivalent antigen-binding activities can be the same, or one of them can have a different antigen-binding activity. Diabody can be produced by obtaining the cDNAs encoding the VH and VL of an antibody, constructing the DNA encoding scFv in such a way that the amino acid sequence length of the peptide linker (P) is 8 residues or less, inserting the DNA into an expression vector for prokaryotes or an expression vector for eukaryotes, and introducing the expression vector into prokaryotes or eukaryotes to express it.

[0198] dsFv refers to an antibody fragment formed by binding polypeptides in which one amino acid residue each in VH and VL is replaced with a cysteine residue through a disulfide bond between the cysteine residues. The amino acid residues replaced with cysteine residues can be selected based on prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7, 697-704, 1994). dsFv can be produced by obtaining cDNAs encoding VH and VL of an antibody, constructing a DNA encoding dsFv, inserting this DNA into an expression vector for prokaryotes or an expression vector for eukaryotes, and introducing this expression vector into prokaryotes or eukaryotes to cause expression.

[0199] The peptide containing a CDR is composed of at least one region or more among the CDRs of VH (CDR-H1 to CDR-H3) and the CDRs of VL (CDR-L1 to CDR-L3). More preferably, it is a peptide containing all the CDRs of VH and a peptide containing all the CDRs of VL. Particularly preferably, it is a peptide containing all the CDRs of VH and VL (a total of 6 regions). As the amino acid sequences of the CDRs, various amino acid sequences of the above-mentioned CDR-H1 to CDR-H3 and CDR-L1 to CDR-L3 are preferably listed. Peptides containing multiple CDRs can be bound directly or via an appropriate peptide linker. The peptide containing a CDR can be produced by constructing a DNA encoding the CDRs of VH and VL of an antibody, inserting this DNA into an expression vector for prokaryotes or an expression vector for eukaryotes, and introducing this expression vector into prokaryotes or eukaryotes to cause expression. In addition, the peptide containing a CDR can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (tert-butoxycarbonyl method).

[0200] As the antibody fragment of the present invention, it can be an antibody fragment that originally contains an N-glycoside bond complex sugar chain and is a part or all of the antibody Fc region with a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain. Alternatively, it can also be a fusion protein of the above antibody fragment and a part or all of the antibody Fc region with an N-glycoside bond complex sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain. If it is such an antibody fragment, the ADCC activity can be dramatically improved, so it is preferred.

[0201] In the following description in this specification, the above antibody fragment is also included in the anti-hCX3CR1 antibody of the present invention.

[0202] 2. Polynucleotides, recombinant vectors, and transformants

[0203] In the present invention, polynucleotides (genes, DNAs) encoding the anti-hCX3CR1 antibodies and antibody fragments of the present invention described above can also be provided. As such polynucleotides, specifically, polynucleotides comprising base sequences encoding the respective amino acid sequences exemplified as the anti-hCX3CR1 antibodies and antibody fragments of the present invention described above are preferred. In addition, the polynucleotides of the present invention may consist only of polynucleotides encoding the anti-hCX3CR1 antibodies and antibody fragments of the present invention, may partially contain such polynucleotides, and may further contain known base sequences required for gene expression (such as transcriptional promoters, SD sequences, Kozak sequences, terminators, etc.), without limitation.

[0204] As the polynucleotides encoding the anti-hCX3CR1 antibodies and antibody fragments of the present invention, the codons corresponding to the respective translated amino acids are not particularly limited, and may include nucleotide DNAs representing codons commonly used in mammals such as humans (preferably codons with high usage frequencies), and may also include nucleotide DNAs representing codons commonly used in microorganisms such as Escherichia coli and yeast, and plants (preferably codons with high usage frequencies).

[0205] In the present invention, recombinant vectors containing the polynucleotides of the present invention described above and transformants containing such recombinant vectors can also be provided.

[0206] In the polynucleotides (genes, DNAs) integrated into an expression vector used as a recombinant vector, if necessary, a transcriptional promoter, an SD sequence (when the host is a prokaryotic cell), and a Kozak sequence (when the host is a eukaryotic cell) can be ligated upstream in advance, a terminator can be ligated downstream, and in addition, an enhancer, a splicing signal, a PolyA addition signal, a selection marker, etc. can also be ligated. It should be noted that each of the above elements required for gene expression such as the transcriptional promoter can be included in the polynucleotide from the beginning, or can be utilized when originally included in the expression vector, and the usage mode of each element is not particularly limited.

[0207] As a method for integrating the polynucleotide into an expression vector, various methods using known gene recombination techniques can be adopted, such as a method using restriction enzymes, a method using topoisomerases, etc. In addition, as the expression vector, for example, as long as it is a vector capable of carrying the polynucleotide (gene, DNA) encoding the anti-hCX3CR1 antibody and its antibody fragments of the present invention, such as plasmid DNA, phage DNA, retrotransposon DNA, retroviral vector, artificial chromosome DNA, etc., there is no limitation, and a vector suitable for the host cell used can be appropriately selected and used. <�

[0208] Next, the constructed recombinant vector is introduced into a host to obtain a transformant. By culturing the transformant, the anti-hCX3CR1 antibody and antibody fragments of the present invention can be expressed. It should be noted that the "transformant" referred to in the present invention means a transformant into which an exogenous gene has been introduced in a host, for example, including a transformant into which an exogenous gene has been introduced by introducing plasmid DNA, etc. (transformation) into a host, and a transformant into which an exogenous gene has been introduced by infecting a host with various viruses and bacteriophages (transduction).

[0209] As the host, as long as it can express the anti-hCX3CR1 antibody and antibody fragments of the present invention after introducing the above recombinant vector, there is no limitation and it can be appropriately selected. For example, various animal cells such as humans and mice, various plant cells, bacteria, yeast, plant cells, etc., which are well-known hosts, can be listed.

[0210] In the case of using animal cells as the host, for example, human fibroblasts, human fetal kidney cells, HEK293 cells, 293F cells, CHO cells, monkey cells COS-7, Vero, mouse L cells, rat GH3, human FL cells, etc. are used. In addition, insect cells such as Sf9 cells and Sf21 cells can also be used.

[0211] In the case of using bacteria as the host, for example, Escherichia coli, Bacillus subtilis, etc. are used.

[0212] In the case of using yeast as the host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc. are used.

[0213] In the case of using plant cells as the host, for example, tobacco BY-2 cells, etc. are used.

[0214] The method for obtaining the transformant is not limited and can be appropriately selected considering the combination of the types of the host and the expression vector. For example, preferred methods include: electroporation, liposome transfection, heat shock method, PEG method, calcium phosphate method, DEAE dextran method, and methods of infecting various viruses such as DNA viruses and RNA viruses.

[0215] In the obtained transformant, the codon type of the polynucleotide contained in the recombinant vector may be the same as or different from the codon type of the host used, and there is no limitation.

[0216] 3. Pharmaceutical compositions, etc.

[0217] The anti-hCX3CR1 antibody of the present invention (including fragments derived from the antibody) is useful as an active ingredient contained in a pharmaceutical composition.

[0218] The anti-hCX3CR1 antibody of the present invention is, for example, preferably used for the prevention, progression inhibition and / or treatment of progressive immune demyelinating diseases. Therefore, the pharmaceutical composition is useful as a pharmaceutical composition for the prevention, progression inhibition and / or treatment of progressive immune demyelinating diseases. That is, the anti-hCX3CR1 antibody of the present invention is useful as an active ingredient contained in a preventive agent, a progression inhibitor and / or a therapeutic agent for progressive immune demyelinating diseases. The description of the above specific examples and the like can be equally applied to progressive immune demyelinating diseases as the indication of the pharmaceutical composition of the present invention.

[0219] Furthermore, the pharmaceutical composition of the present invention may be a composition for blocking or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine), may also be a composition for blocking or inhibiting the functions (such as infiltration into the CNS, etc.) of Th cells expressing Eomes and / or granzyme B or Th cells expressing Eomes and / or IFN-γ, and may also be a composition for improving the condition of EAE or inhibiting the progression of the EAE condition.

[0220] The pharmaceutical composition of the present invention is preferably provided in the form of a pharmaceutical composition containing the anti-hCX3CR1 antibody of the present invention as an active ingredient and further containing a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition of the present invention can also be used in combination with known medicaments (such as known medicaments for the prevention, progression inhibition and / or treatment of progressive immune demyelinating diseases). The mode of combined use can be, for example, a mode in which the pharmaceutical composition of the present invention further contains the known medicament, or a mode of combined use with the known medicament, without limitation. By such combined use, a higher preventive, progression-inhibiting and / or therapeutic effect can also be obtained.

[0221] Examples of the "pharmaceutically acceptable carrier" include excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, flavoring agents, solubilizers or other additives, etc. By using one or more of such carriers, pharmaceutical compositions in the forms of injections, liquids, capsules, suspensions, emulsions or syrups can be prepared. These pharmaceutical compositions can be administered orally or parenterally. As other forms for parenteral administration, it includes injections and the like prepared by conventional methods containing one or more active substances. In the case of injections, they can be manufactured by dissolving or suspending in a pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection.

[0222] When administering an antibody fragment (especially a low-molecular-weight antibody fragment) of the anti-hCX3CR1 antibody derived from the present invention to a living body, in addition to the above, a colloidal dispersion system can also be used. The colloidal dispersion system can be expected to have the effects of improving the stability of the compound (antibody fragment) in the living body and effectively delivering the compound to specific organs, tissues or cells. The colloidal dispersion system is not limited as long as it is a commonly used colloidal dispersion system, and examples include dispersion systems based on polyethylene glycol, polymer complexes, polymer aggregates, nanocapsules, microspheres, beads, and lipids including oil-in-water emulsifiers, micelles, mixed micelles, and liposomes. Preferred are multiple liposomes and vesicles of artificial membranes (Mannino et al., Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem. et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech., 1995, 6, 698) that have the effect of effectively delivering the compound to specific organs, tissues or cells.

[0223] The dosage of the pharmaceutical composition of the present invention varies depending on the age, sex, weight and symptoms of the patient as the subject, the therapeutic effect, the administration method, the treatment time, or the type of the anti-hCX3CR1 antibody of the present invention contained in the pharmaceutical composition, etc. Generally, for adults, it can be administered once per person in the range of 600 μg - 6000 mg, but it is not limited to this range.

[0224] For example, in the case of administration by injection, for human patients, it can be administered once or several times a day on average in an amount of 100 μg to 100 mg per 1 kg body weight per single administration, and preferably, it can be administered once every 3 days, 1 week, 10 days or 2 weeks, or administered once (the total number of administrations is 1 time). The forms of administration include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection or intraperitoneal injection, etc., and intravenous injection is preferred. In addition, the injection can also be prepared as a non-aqueous diluent (such as polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), a suspension or an emulsion according to circumstances. The sterilization of such an injection can be carried out by filtration sterilization using a filter, adding a bactericide, etc. The injection can be manufactured in a manner of being prepared at the time of use. That is, it can be made into a sterile solid composition by freeze-drying method, etc., and dissolved in sterile distilled water for injection or other solvents before use.

[0225] It should be noted that the present invention also provides the use of the anti-hCX3CR1 antibody of the present invention and / or the complex of the present invention in the preparation of a medicament for treating, preventing and / or diagnosing tumors or inflammatory diseases and / or autoimmune diseases (hereinafter also referred to as tumors, etc.), or blocking or inhibiting the interaction (preferably binding) between hCX3CR1 and human CX3CL1.

[0226] In addition, the present invention also provides the anti-hCX3CR1 antibody of the present invention for the prevention, progression inhibition and / or treatment of progressive immune demyelinating diseases, the anti-hCX3CR1 antibody of the present invention for blocking or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine), the anti-hCX3CR1 antibody of the present invention for blocking or inhibiting the function of Th cells expressing Eomes and / or granzyme B or Th cells expressing Eomes and / or IFN-γ (such as infiltration into the CNS, etc.), or the anti-hCX3CR1 antibody of the present invention for improving the condition of EAE and / or inhibiting the progression of EAE.

[0227] Furthermore, the present invention provides a method for preventing, inhibiting the progression and / or treating progressive immune demyelinating diseases characterized by using (i.e., administering to an object (patient)) the anti-hCX3CR1 antibody of the present invention; a method for blocking and / or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine); a method for blocking and / or inhibiting the function of Th cells expressing Eomes and / or granzyme B or Th cells expressing Eomes and / or IFN-γ (such as infiltration into the CNS, etc.); or a method for improving the condition of EAE and / or inhibiting the progression of EAE. In addition, the present invention provides the use of the anti-hCX3CR1 antibody of the present invention for preventing, inhibiting the progression and / or treating progressive immune demyelinating diseases; the use for blocking and / or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine); the use for blocking and / or inhibiting the function of Th cells expressing Eomes and / or granzyme B or Th cells expressing Eomes and / or IFN-γ (such as infiltration into the CNS, etc.); or the use for improving the condition of EAE and / or inhibiting the progression of EAE.

[0228] 4. Kit

[0229] The anti-hCX3CR1 antibody of the present invention can also be provided in the form of a kit for preventing, inhibiting the progression and / or treating progressive immune demyelinating diseases, a kit for blocking and / or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine), a kit for blocking and / or inhibiting the functions of Th cells expressing Eomes and / or granzyme B or Th cells expressing Eomes and / or IFN-γ (such as infiltration into the CNS, etc.), or a kit for improving and / or inhibiting the progression of EAE. In addition, the anti-hCX3CR1 antibody of the present invention can also be provided in the form of a diagnostic kit capable of diagnosing progressive immune demyelinating diseases. The description of the above specific examples and the like can be equally applied to progressive immune demyelinating diseases.

[0230] In addition to the anti-hCX3CR1 antibody of the present invention, the kit of the present invention may further contain various buffers, sterilized water, various cell culture containers, various reaction containers (such as Eppendorf tubes), blocking agents (serum components such as bovine serum albumin (BSA), skim milk, goat serum), cleaning agents, surfactants, various plates, preservatives such as sodium azide, and an experimental operation manual (instruction manual), etc.

[0231] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[0232] Example 1

[0233] 1. Establishment of monoclonal antibodies against human CX3CR1 (hCX3CR1)

[0234] 1-1. Preparation of hCX3CR1 expression vector and antigen

[0235] Based on the amino acid sequence of the hCX3CR1 protein (Uniprot accession number: P49238-1), a DNA sequence encoding this amino acid sequence was synthesized (using the services of Genscript) and inserted into the pCAGGS vector using the "In-Fusion HD Cloning Kit" (Takara Bio). The DNA was amplified using a forward primer appended with a Kozak translation initiation sequence, a reverse primer appended with a translation stop codon, and "Premix Ex Taq (Ex Taq version 2.0)" (Takara Bio), and inserted into the vector of the "pEF6 / V5-His TOPO TA Expression kit" (Takara Bio) to obtain the hCX3CR1 expression vector (hCX3CR1_pEF6 / V5-His). To prepare antigens for cell immunization and cells for activity evaluation, hCX3CR1_pEF6 / V5-His was introduced into Ba / F3 cells by electroporation and cultured in a selective medium supplemented with blasticidin. hCX3CR1-expressing cells were isolated by flow cytometry and used as stable expressing strains (hCX3CR1-expressing Ba / F3 cells). Additionally, using the same method, based on the amino acid sequence of the mouse CX3CR1 (mCX3CR1) protein (Uniprot accession number: Q9Z0D9), with "pUNO1-mCX3CR1" (invivogen) as a template, the mCX3CR1 expression vector (mCX3CR1_pEF6 / V5-His) and mCX3CR1-expressing Ba / F3 cells were prepared for cross-reactivity tests and epitope analysis tests.

[0236] To prepare the antigen-expressing cell membrane fraction, an expression vector (hCX3CR1_pcDNA3.3) was constructed by introducing the hCX3CR1 gene sequence derived from hCX3CR1_pEF6 / V5-His into pcDNA3.3 (Thermo Fisher Scientifics). Subsequently, hCX3CR1_pcDNA3.3 was used as the transfection plasmid for transient expression using the "Expi293 expression system" (ThermoFisher Scientifics). The membrane fraction was prepared from the cultured cells, suspended in PBS, and used as the hCX3CR1-expressing cell membrane fraction (using the services of SEED Supply) as an antigen.

[0237] In addition, as a recombinant protein antigen, a fusion protein in which GST is bound to the N-terminus of the extracellular domain sequence (DQFPESVTENFEYDDLAEACYIGDIVVFGT (sequence from position 2 to position 31 of the amino acid sequence of SEQ ID NO: 17)) on the N-terminal side of the hCX3CR1 protein produced using Expi293, or a fusion protein bound to the Fc region of a human or mouse antibody, was purified to obtain a purified protein.

[0238] 1-2. Immunization of Animals

[0239] Antibody production was carried out by immunizing ICR mice (Sankyo Labo Service Corporation, CLEA Japan Inc.) etc. with various hCX3CR1 antigens based on the following method.

[0240] The antigen was mixed with the adjuvant "Titer Max Gold" (TiterMax Inc.) and administered to the foot pads at a dose of 200 μg of antigen per mouse. Three days after administration, and then approximately once a week thereafter, a booster immunization was carried out in the absence of adjuvant in the same manner as above at a dose of 10 - 50 μg of antigen per mouse.

[0241] 1-3. Production of Hybridomas

[0242] Monoclonal antibodies against hCX3CR1 were produced using lymph node cells or spleen cells isolated from immunized mice. After the immunization, lymph nodes such as inguinal, iliac, and popliteal were collected from the mice, and after preparing a cell suspension, it was mixed with SP2 / 0-Ag14 myeloma cells, and electrofusion was carried out using an electrocell fusion device "ECFG21" (Nepagene Inc.).

[0243] The fused cells were suspended in "ClonaCell TM -HY Medium D" (STEM CELL Inc.) and inoculated into plastic culture dishes. Colonies formed approximately 10 days after inoculation were isolated in a 96-well plastic plate pre-filled with hybridoma medium, and the culture supernatant was used for in vitro screening of antibodies. It should be noted that as the hybridoma medium, a medium prepared by adding 1 / 50 volume of Nutridoma-CS (Merck & Co., Inc.) and 1 / 50 volume of HAT Supplement (Thermo Fisher Scientifics Inc.) to RPMI 1640 (Thermo Fisher Scientifics Inc.) was used.

[0244] 1-4. Screening of Antibodies Using an In Vitro Test System

[0245] The screening of the obtained hybridoma clones was carried out by the following two methods.

[0246] 1-4-1. Screening using binding activity as an index

[0247] The reactivity of the hybridoma culture supernatant was evaluated by Cell ELISA against plates inoculated with hCX3CR1-expressing rat hematopoietic cells "HTS015C" or hCX3CR1-nonexpressing cells "HTSCHEM-1" (both from Eurofins). For the positive clones in the Cell ELISA evaluation, the reactivity against HTS015C or hCX3CR1-expressing Ba / F3 cells was evaluated by flow cytometry using the culture supernatant or its purified antibody.

[0248] One to three days before the measurement, Eurofins cells were inoculated into a 384-well CellBIND plate (Corning) and cultured at 37°C. Eurofins cells were cultured in a medium containing 10% FBS (HyClone), 1% MEM NEAA (Gibco), 10 mM Hepes (Gibco), and 1% penicillin-streptomycin (Nacalai Tesque) added to DMEM high Glucose (Nacalai Tesque). The Eurofins cell culture supernatant was removed, 20 μL of the hybridoma culture supernatant was added to each well, and the mixture was left standing at 4°C for 1 hour. The cells were washed with phosphate-buffered saline (PBS), 20 μL of HRP-labeled secondary antibody diluted with blocking buffer (PBS containing 1% bovine serum albumin; 1% BSA / PBS) was added to each well, and the mixture was left standing at 4°C for 1 hour. After washing the cells with PBS, TMB (Nakai Tesque) was added as a chromogenic substrate solution for color development. After 15 minutes, 1N sulfuric acid was added to initiate the reaction, and the absorbance at 450 nm was measured.

[0249] For the above Cell ELISA positive clones, the reactivity against HTS015C or hCX3CR1-expressing Ba / F3 cells was further evaluated by flow cytometry using the culture supernatant or its purified antibody. The purified antibody of the positive clones was mainly prepared using rProtein A Sepharose Fast Flow.

[0250] HTS015C (HTSCHEM-1 as a negative control) or hCX3CR1-expressing Ba / F3 cells (Ba / F3 cells transfected with an empty vector gene as a negative control) were cultured at 37.0°C in the presence of 5% CO2. Ba / F3 cells were cultured in RPMI Medium 1640 (Sigma) supplemented with 10% FBS (HyClone), 1 ng / mL murine IL-3 (R&D), and 1% penicillin-streptomycin (Nacalai Tesque). For Eurofins cells, which are adherent cells, cells were detached using a non-enzymatic cell dissociation solution (Thermo Fisher Scientifics). Cells were dispensed into a 96-well plate, and after centrifugation to remove the supernatant, 50 μL of hybridoma culture supernatant or purified antibody diluted with 1% BSA / PBS was added to each well and suspended, and the plate was left standing at 4°C for 30 minutes. Cells were washed with 1% BSA / PBS, 50 μL of PE-labeled secondary antibody diluted with 1% BSA / PBS was added to each well and suspended, and the plate was left standing at 4°C in the dark for 30 minutes. After washing the cells with 1% BSA / PBS, 7-AAD, a dead cell detection reagent diluted with 1% BSA / PBS, was added and suspended, and the reactivity of the hybridoma culture supernatant or its purified antibody against hCX3CR1-expressing cells was measured using FACS Canto II (Becton Dickinson).

[0251] Approximately 30,000 hybridoma clones were screened, and as a result, 46 clones with specific reactivity against hCX3CR1-expressing cells were selected as positive clones.

[0252] 1-4-2. Screening Using Cell Migration (Chemotaxis) Blocking Activity as an Index

[0253] For clones with binding activity against hCX3CR1-expressing cells, the hCX3CR1-expressing Ba / F3 cell migration blocking activity against fractalkine (also known as FKN, CX3CL1), which is a ligand for hCX3CR1, was evaluated.

[0254] 2]Cell chemotaxis was measured using the Boyden chamber method. hCX3CR1-expressing Ba / F3 cells were seeded in the upper chamber of a HTS Transwell 96-well plate with a pore size of 5 μm (Corning), and a medium supplemented with human FKN (PeproTech) was added to the lower chamber. Migrated cells were measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega).

[0255] hCX3CR1-expressing cells were adjusted to 5.33×10 cells per 100 μL with a culture medium containing 1% FBS, cultured at 37 °C for 3 to 6 hours, then a purified antibody was added and the cells were cultured for another 1 hour. A cell suspension (final volume 75 μL, 2×10 cells) was added to the upper chamber, and only the culture medium containing 1% FBS or the culture medium containing 10 ng / mL human fractalkine was added to the lower chamber, and the cells were cultured overnight at 37 °C. After culturing, the upper chamber was gently shaken and removed, 100 μL of the culture medium in the lower chamber containing the migrated cells was mixed with an equal volume of CellTiter-Glo, allowed to stand at room temperature for 10 minutes, and then luminescence was measured using an Enspire Alpha Plate Reader (Perkin Elmer). 5 cells) was added to the upper chamber, and only the culture medium containing 1% FBS or the culture medium containing 10 ng / mL human fractalkine was added to the lower chamber, and the cells were cultured overnight at 37 °C. After culturing, the upper chamber was gently shaken and removed, 100 μL of the culture medium in the lower chamber containing the migrated cells was mixed with an equal volume of CellTiter-Glo, allowed to stand at room temperature for 10 minutes, and then luminescence was measured using an Enspire Alpha Plate Reader (Perkin Elmer). 5 The migration-blocking activity of the above 46 clones was evaluated using hCX3CR1-expressing Ba / F3 cells, and as a result, a clone (m1F8) with a cell migration-blocking activity close to 100% was screened out. As described in 1-4-1 above, this clone is the only functional clone screened from the initial approximately 30,000 hybridoma clones, and it is a clone with extremely high acquisition difficulty that cannot be said to be easily obtained in the trial and error that those skilled in the art can usually perform.

[0256] The migration-blocking activity of the above 46 clones was evaluated using hCX3CR1-expressing Ba / F3 cells, and as a result, a clone (m1F8) with a cell migration-blocking activity close to 100% was screened out. As described in 1-4-1 above, this clone is the only functional clone screened from the initial approximately 30,000 hybridoma clones, and it is a clone with extremely high acquisition difficulty that cannot be said to be easily obtained in the trial and error that those skilled in the art can usually perform.

[0257] Example 2

[0258] 2. Antibody sequence analysis

[0259] Sequence analysis of the variable region of the immunoglobulin gene of the m1F8 antibody was performed.

[0260] The m1F8 hybridoma cells were expanded in culture, mRNA was extracted, reverse transcription based on the 5' RACE method, amplification, cloning of the antibody variable region sequence based on the PCR method, and sequence analysis based on the Sanger method (using the service of GENEWIZ) were performed.

[0261] For the sequence of the obtained antibody (m1F8 antibody), according to the method of Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, US, Department of Health and Human Services, 1991), the amino acid sequences of the CDR regions, the heavy chain variable region (VH), and the light chain variable region (VL) were determined.

[0262] Example 3

[0263] Activity evaluation of m1F8 antibody

[0264] 3-1. Binding activity to Ba / F3 cells expressing hCX3CR1

[0265] The m1F8 antibody or negative antibody was adjusted to a total of 9 concentrations with a common ratio of 5-fold, and the highest concentration was 500 nM. The binding activity of the m1F8 antibody to Ba / F3 cells expressing hCX3CR1 was measured by flow cytometry, and the results are shown in ​ . A mouse IgG2a isotype control antibody was used as the negative antibody. The method of flow cytometry was carried out in the same manner as in 1-4-1 of Example 1. As ​ shown, in the negative antibody, even when the antibody concentration was increased, the fluorescence intensity did not increase, and no binding to Ba / F3 cells expressing hCX3CR1 was found. On the other hand, in the m1F8 antibody, the fluorescence intensity increased depending on the antibody concentration. For Ba / F3 cells expressing hCX3CR1, although the m1F8 antibody was not saturated at the adjusted antibody concentration, it showed obvious reactivity. In addition, there was almost no such reactivity in Ba / F3 cells transfected with the empty vector gene, so it was considered to be a specific reaction dependent on hCX3CR1 expression.

[0266] 3-2. Cell migration blocking activity against Ba / F3 cells expressing hCX3CR1

[0267] The m1F8 antibody or negative antibody was adjusted to a total of 9 concentrations with a common ratio of 5-fold, and the highest concentration was 500 nM. The blocking activity of the fractalkine-dependent cell migration of Ba / F3 cells expressing hCX3CR1 was measured, and the results are shown in ​ . A mouse IgG2a isotype control antibody was used as the negative antibody. The method of the cell migration assay was carried out in the same manner as in 1-4-2 of Example 1. As ​ shown, in the negative antibody, even when the antibody concentration was increased, no blocking of cell migration was observed. On the other hand, for the FKN-dependent cell migration of Ba / F3 cells expressing hCX3CR1, the m1F8 antibody showed more than 95% blocking activity at 500 nM.

[0268] Example 4

[0269] 4. Cross-reactivity of m1F8 antibody with CX3CR1 of other animal species

[0270] The results of the cross-species reactivity of m1F8 and various antibodies with CX3CR1 (binding reactivity with Ba / F3 cells expressing CX3CR1 of human, cynomolgus monkey, mouse, and rat) are shown in Table 1 below. According to the cynomolgus monkey (Uniprot accession number: A0A2K5VJQ4) and rat (P35411), DNA with a Kozak translation initiation sequence inserted at the 5'-end and a translation termination codon inserted at the 3'-side was synthesized (using the service of GENEWIZ). Using the In-Fusion HD Cloning Kit, the synthesized DNA was inserted into the pEF6 / V5-His TOPO vector digested with restriction enzymes (BamHI and NotI) to obtain various CX3CR1 expression vectors (cynomolgus monkey: cCX3CR1_pEF6 / V5-His, rat: rCX3CR1_pEF6 / V5-His). Similar to the production of hCX3CR1-expressing Ba / F3 cells (1-1), various CX3CR1 expression vectors were introduced into Ba / F3 cells by electroporation, cultured in a selective medium containing blasticidin, and CX3CR1-expressing cells were isolated by flow cytometry and used as stable expression strains for the cross-reactivity test. The method of flow cytometry was carried out in the same manner as in 1-4-1 of Example 1. As positive antibodies, an equivalent of 66B02-mFc from Boehringer Ingelheim prepared by the present inventors (mFc fusion protein of anti-human CX3CR1-Nanobody) and the commercially available anti-mouse CX3CR1 antibody SA011 F11 (Biolegend) were used. The reactivity (mean fluorescence intensity) under the condition of only the secondary antibody with cells was subtracted as the background value, and the cross-reactivity was determined based on the reactivity % of the m1F8 antibody at the same concentration when the reactivity of 200 nM of 66B02-mFc with hCX3CR1-expressing Ba / F3 cells or cCX3CR1-expressing Ba / F3 cells was set as 100%, or when the reactivity of the same concentration of SA011F11 with mCX3CR1-expressing Ba / F3 cells or rCX3CR1-expressing Ba / F3 cells was set as 100%. In the results of Table 1, when the relative value to the positive antibody at 200 nM is higher than 70%, it is indicated as "+++", when it is 40 - 70%, it is indicated as "++", when it is 10 - less than 40%, it is indicated as "+", and when it is less than 1%, it is indicated as "-".

[0271] [Table 1]

[0272] Table 1 Cross-species reactivity of m1F8 antibody

[0273]

[0274] Relative value to positive antibody: +++ ; > 70%

[0275] ++; 40 - 70%

[0276] +; 10 - 40%

[0277] -; <10%

[0278] As shown in Table 1, SA011F11 did not respond to humans and cynomolgus monkeys, but showed a response to mice and rats. 66B02-mFc and m1F8 both showed a response to humans and cynomolgus monkeys, but did not show a response to mice and rats. As described above, m1F8 showed cross-reactivity with primate CX3CR1, but did not show cross-reactivity with rodent CX3CR1.

[0279] Example 5

[0280] 5. Epitope analysis of the m1F8 antibody

[0281] 5-1. Analysis of the binding domain of the m1F8 antibody

[0282] The schematic diagram of the chimeric CX3CR1 expression construct that replaces the extracellular domains of human and mouse CX3CR1 for epitope analysis is shown in ​ . Gene sequences in which the 4 extracellular domains present in the CX3CR1 protein sequence are replaced one by one in humans and mice were synthesized (using the service of GENEWIZ). Using the In-Fusion HD Cloning Kit, the synthesized DNA was inserted into the pEF6 / V5-His TOPO vector cut with restriction enzymes (BamHI, NotI) to produce a total of 8 chimeric CX3CR1 expression vectors. Similar to the production of hCX3CR1-expressing Ba / F3 cells (1-1), various CX3CR1 expression vectors were introduced into Ba / F3 cells by electroporation and cultured in a selective medium containing blasticidin. CX3CR1-expressing cells were separated by flow cytometry and used as stable expression strains for binding domain analysis.

[0283] The results of the binding activities of m1F8 and various antibodies to chimeric CX3CR1-expressing Ba / F3 cells are shown in Tables 2 and 3 below. The method of flow cytometry was carried out in the same manner as in 1-4-1 of Example 1. As the positive antibodies, 66B02-mFc equivalent and SA011F11 were used. A mouse IgG2a isotype control antibody was used as the negative antibody. It was confirmed that 66B02-mFc showed significant reactivity to the cell lines expressing the extracellular domain 1 of hCX3CR1 (hCX3CR1, hCX3CR1-2,3,4, mCX3CR1-1), and SA011F11 showed significant reactivity to the cell lines expressing the extracellular domain 1 of mCX3CR1 (mCX3CR1, hCX3CR1-1, mCX3CR1-2,3,4). Based on these results, the reactivity (mean fluorescence intensity) of each cell to the control antibody at 200 nM was subtracted and corrected so that when the reactivity of 66B02-mFc at the same concentration to the cell lines expressing each extracellular domain 1 of hCX3CR1 was set to 100%, or when the reactivity of SA011F11 at the same concentration to the cell lines expressing each extracellular domain 1 of mCX3CR1 was set to 100%, the reactivity % of the m1F8 antibody at the same concentration was used as the basis to determine the binding domain. In the results of Tables 2 and 3, when the relative value to the positive antibody at 200 nM was higher than ⑦0%, it was indicated as "+++", when it was 40-70%, it was indicated as "++", when it was 10- less than 40%, it was indicated as "+", and when it was less than 10%, it was indicated as "-".

[0284] [Table 2]

[0285] Table 2 Epitope analysis

[0286] ​ ​ ​ ​ ​ - +++ +++ ++ ​ +++ - + - ​ - +++ +++ +++ ​ - +++ +++ ++ ​ - +++ +++ +++

[0287] Relative value to positive antibody: +++ ; > 70%

[0288] ++; 40 - 70%

[0289] +; 10 - 40%

[0290] -; < 10%

[0291] [Table 3]

[0292] Table 3 Epitope analysis

[0293] ​ ​ ​ ​ ​ +++ - - - ​ - +++ ++ +++ ​ +++ - - - ​ +++ - - - ​ +++ - + -

[0294] Relative value to positive antibody: +++ ; > 70%

[0295] ++; 40 - 70%

[0296] +; 10 - 40%

[0297] -; < 10%

[0298] As shown in Table 2 and Table 3, m1F8 showed reactivity to the expression cell lines (hCX3CR1, hCX3CR1-2, 3, 4, mCX3CR1-1) with the extracellular domain 1 of hCX3CR1, and also showed a certain reactivity to the expression cell lines (hCX3CR1-1, mCX3CR1-4) without the extracellular domain 1 of hCX3CR1. On the other hand, K0124E1 (Biolegend), another commercially available anti-hCX3CR1 antibody, showed reactivity only to the expression cell lines with the extracellular domain 1 of hCX3CR1, just like 66B02-mFc. The above results strongly suggest the possibility that the m1F8 antibody recognizes the three-dimensional structure of CX3CR1 compared with other antibodies.

[0299] Example 6

[0300] 6. Antibody binning

[0301] 6-1. Binning of 1F8 antibody and control antibodies

[0302] The results of binning the epitopes of human CX3CR1 (hCX3CR1)-expressing Ba / F3 cells using 1F8 and various antibodies are shown in Table 4.

[0303] 10 μM of the competing antibody shown on the left side of Table 4 and 100 nM of the detection antibody shown on the upper side of the table were added to hCX3CR1-expressing Ba / F3 cells and suspended, and left to stand in the dark at 4°C for 30 minutes. The subsequent flow cytometry method was carried out in the same manner as 1-4-1 of Example 1. m / h1F8 represents a chimeric antibody with a mouse-derived Fab and a human-derived Fc. As control antibodies, 66B02-mFc equivalent, 306D-hFc equivalent (hFc fusion protein of anti-hCX3CR1-Nanobody), and K0124E1 were used. As a negative antibody, an equivalent of Synagis (palivizumab, palivizumab), a humanized IgG1 antibody (described as "Synagis" in this specification), was used. The reactivity (mean fluorescence intensity) under the condition of only the secondary antibody with cells was subtracted as the background value, and the binding competitiveness was determined based on the reactivity% of each detection antibody in the presence of each competing antibody when the reactivity of each detection antibody with Synagis as the competing antibody was set to 100%. When the relative value to the competing negative antibody was higher than 70%, it was indicated as "+++", when it was 40 - 70%, it was indicated as "++", when it was 10 - less than 40%, it was indicated as "+", and when it was less than 10%, it was indicated as "-".

[0304] [Table 4]

[0305] Table 4

[0306]

[0307] Relative value compared to negative control antibody: +++; >70%

[0308] ++; 40 - 70%

[0309] +; 10 - 40%

[0310] -; <10%

[0311] As shown in Table 4, it shows that there is very intense competition among 1F8 antibodies, and there is no significant competition with 66B02 - mFc, 306D - hFc, and K0124E1 as control antibodies. On the other hand, the 306D - hFc antibody as a control antibody competes very intensely with 66B02 - mFc and K0124E1 (commercially available antibodies).

[0312] Enhanced activity of 6 - 2.m1F8 antibody

[0313] Based on the results in the previous examples, it is speculated that the m1F8 antibody is very likely to be very different in characteristics, especially in epitope recognition, compared to known anti - CX3CR1 antibodies. On the other hand, its binding activity and blocking activity against CX3CR1 are very low. To overcome this drawback, based on the insight of enhancing the activity of the antibody sequence using ADLib (registered trademark), mutations were artificially introduced into the CDR sequences of the m1F8 antibody to obtain multiple m1F8 antibodies with enhanced activity (m1F8 - 1, m1F8 - 2). The amino acid sequences of the heavy - chain variable region (VH) and light - chain variable region (VL) of the obtained m1F8 - 1 and m1F8 - 2 are as ​ shown. ​ In it, the underlined parts in each amino acid sequence represent CDRs.

[0314] The binding activity (EC50) of m1F8 antibody, two m1F8 antibodies with enhanced activity (m1F8 - 1, m1F8 - 2), and anti - hCX3CR1 - Nanobody to hCX3CR1 - expressing Ba / F3 cells and the blocking activity (IC50) against fractalkine - dependent cell migration are shown in Table 5.

[0315] [Table 5]

[0316] Table 5

[0317]

[0318] As shown in Table 5, as a result of the enhanced activity, the activity-enhanced m1F8 antibodies (m1F8-1 and m1F8-2) achieved an improvement in binding activity of more than 70-fold and blocking activity of more than 15-fold compared to the original m1F8 antibody. However, among these activities, the anti-hCX3CR1-Nanobody was still higher than these activity-enhanced m1F8 antibodies.

[0319] Epitope binning of the 6-3.1F8 activity-enhanced antibody

[0320] The results of epitope binning of the activity-enhanced antibodies of 1F8 (1F8-1, 1F8-2) and anti-hCX3CR1-Nanobody using Ba / F3 cells expressing hCX3CR1 are shown in Table 6. Regarding the method, it was carried out in the same manner as in 6-1 above.

[0321] [Table 6]

[0322] Table 6

[0323]

[0324] Relative value compared to the negative competition antibody: +++ ; > 70%

[0325] ++ ; 40 - 70%

[0326] + ; 10 - 40%

[0327] - ; < 10%

[0328] As shown in Table 6, the competition between 1F8 antibodies or Nanobodies was very intense. On the other hand, no significant competition was shown between 1F8 and Nanobody.

[0329] Based on the results of Tables 4 and 6, it can be considered that the epitopes of 1F8 are different from those of other antibodies (known antibodies).

[0330] Example 7

[0331] 7. Results of the establishment of a late EAE model in human CX3CR1-KI mice and the administration test of BII00313

[0332] 7-1. Preparation of gene mutation mice

[0333] All the mice used were 6 - 8 weeks old and were raised under specific pathogen-free conditions. For the production of NR4A2-deficient mice (NR4A2-cKO), a targeting vector with the NR4A2 gene flanked by loxp sequences was used to establish NR4A2 fl / flMice. That is, NR4A2 flanked by loxp sequences was introduced into C57BL / 6 embryonic stem cells by microinjection. The established strain was crossed with C57BL / 6 FLPe mice (RIKEN BioResource Center), and the strains with the neomycin cassette removed were mated with each other to produce homozygous NR4A2 fl / fl C57BL / 6 mice. By mating the obtained mice with C57BL / 6 CD4-Cre (Taconic), CD4-specific NR4A2-cKO C57BL / 6 mice (C57BL / 6 Cre-CD4 / NR4A2 fl / fl mice = NR4A2-cKO mice) were established. Regarding the production of mice in which mouse CX3CR1 was replaced with human CX3CR1 (human CX3CR1-KI mice), human CX3CR1-KI mice in which the mouse CX3CR1 gene was replaced with the human CX3CR1 gene by genome editing were established. That is, first, a Cas9 expression vector was constructed, and the Cas9 expression vector integrated a guide RNA sequence targeting the protein-coding region of exon 2 of the target gene (Cx3cr1 gene, GeneID: 13051). Next, a homologous recombination vector was constructed, and the homologous recombination vector contained a synthetic sequence containing the knock-in sequence (CX3CR1 gene, Gene ID: 1524, NM_001337.3, CDS region, 1068 bp) and a homologous region cloned from the genome of the RENKA strain derived from C57BL / 6N.

[0334] Next, the guide Cas9 vector, the homologous recombination vector, and the transient drug (Puromycin) resistance gene expression vector were introduced into ES cells (RENKA strain derived from C57BL / 6N) by electroporation, and colonies of ES cell clones formed by drug selection culture were isolated. Next, using ES cell clones confirmed to have homologous recombination, chimeric embryos were produced by the aggregation method using ICR strain as recipient embryos, transplanted into recipient mice, and chimeric mice were produced. The germline chimeric mice were naturally mated with wild-type mice (C57BL / 6N) to produce first-generation mice (KI heterozygous mice), and the KI heterozygous mice were mated with each other to produce KI homozygous mice (human CX3CR1-KI mice). Regarding the production of NR4A2-cKO / human CX3CR1-KI mice, NR4A2-cKO was mated with human CX3CR1-KI mice, and finally CD4-Cre + / - / NR4A2 fl / fl / CX3CR1 human / human mice were used as KI-late EAE test mice.

[0335] 7-2. EAE induction (monophasic EAE)

[0336] A PBS solution containing a peptide equivalent to 100 μg of MOG35-55 residues (synthesized by TORAY RESEARCH CENTER, INC., hereinafter also referred to as "MOG peptide") was mixed in equal amounts with complete Freund's adjuvant (CFA) containing 1 mg of heat-killed Mycobacterium tuberculosis H37Ra (Difco, Kansas, USA), and emulsified using an ultrasonic disruptor to prepare a MOG emulsion. The resulting MOG emulsion was subcutaneously injected into 1 to 2 sites on the back of NR4A2-cKO / human CX3CR1-KI mice to confer immunity. Furthermore, on days 0 and 2 after the administration of immunity, 200 μL of a PBS solution containing 200 ng of pertussis toxin (List Biological Laboratories, USA) was injected into the peritoneal cavity of each mouse.

[0337] Antibody administration test results of the late EAE model in 7-3.KI mice (BII00313)

[0338] For human CX3CR1-KI mice induced with monophasic EAE, PBS was administered 24 days and 28 days after EAE induction ( ​ recorded as the control group and Control in (A) and (B) of ​ respectively). In addition, for NR4A2-cKO / human CX3CR1-KI mice, PBS or 200 μg of anti-CX3CR1-Nanobody (BII00313) was administered 24 days and 28 days after EAE induction ( ​ recorded as NR4A2cKO and BII00313 in (A) and (B) of

[0339] <EAE evaluation criteria>

[0340] 0: No clinical signs

[0341] 1: Local paralysis of the tail

[0342] 2: Flaccid tail

[0343] 3: Local paralysis of the hindlimbs

[0344] 4: Paralysis of the entire hindlimbs

[0345] 5: Paralysis of the hindlimbs and forelimbs

[0346] In addition, to confirm the antibody blockade of ligand-receptor binding, free fractalkine in the blood was quantified. On the last day of the experiment, mice were anesthetized by intraperitoneal administration of a mixture of three anesthetics (medetomidine, midazolam, butorphanol), and serum collected by incising the necks of the mice was used to quantify free fractalkine in the blood using a commercially available ELISA kit (R&D systems, MCX310).

[0347] As ​ shown in (A) of ​ , the amount of free fractalkine in the blood was significantly increased in the BII00313-administered group compared to the control group and the NR4A2 cKO group. It is considered that this change is due to the blockade of the binding of CX3CR1 to fractalkine by the administration of BII00313, resulting in an increase in the amount of free fractalkine in the blood. As

[0348] Example 8

[0349] 8. Results of antibody administration test in a late EAE model of human CX3CR1-KI mice (comparison of m1F8 antibody; comparison of m1F8-1 and m1F8-2)

[0350] NR4A2-cKO / human CX3CR1-KI mice that had induced monophasic EAE in the same manner as in Example 7 were administered 200 μg of anti-hCX3CR1 antibody (m1F8-1 or m1F8-2) or its isotype antibody (mIgG2a) 24 days and 28 days after EAE induction. The EAE condition of the mice was evaluated in the same manner as in Example 7. In addition, fractalkine in the blood was measured.

[0351] As ​ shown in (A) of ​As shown in (B), the EAE condition was significantly improved only when the m1F8-2 antibody was administered. When the m1F8-1 antibody was administered, improvement in several EAE scores was observed after the first administration, but thereafter, it became close to the EAE scores of the isotype antibody administration group. In the case of an antibody such as the m1F8-1 antibody, where good results in in vitro functional activity (increase in the amount of free fractalkine in the blood) do not necessarily clearly correspond to in vivo functional evaluation (improvement in the EAE condition), the m1F8-2 antibody is an antibody with good results in in vitro functional activity and excellent in vivo functional evaluation. An anti-hCX3CR1 antibody that is excellent in function in either in vitro or in vivo, such as the m1F8-2 antibody, is an antibody that is extremely difficult to obtain and cannot be said to be easily obtained in the trial and error that a person skilled in the art can generally perform.

[0352] Example 9

[0353] 9. Antibody Administration Test Results in a Late EAE Model of Human CX3CR1-KI Mice (Comparison of BII00313 and m1F8-2 Antibody)

[0354] 9-1. EAE Score

[0355] For NR4A2-cKO / human CX3CR1-KI mice that induced monophasic EAE in the same manner as in Example 7, 200 μg of BII00313, m1F8-2, or its isotype antibody (mIgG2a) was administered 24 days and 27 days after EAE induction. The EAE condition of the mice was evaluated in the same manner as in Example 7.

[0356] As ​ shown, the EAE condition was particularly strongly improved in the group administered with m1F8-2.

[0357] 9-2. Evaluation of the Number of Infiltrating Cells in the Central Nervous System

[0358] NR4A2-cKO / human CX3CR1-KI mice that were induced with monophasic EAE in the same manner as in Example 7 were administered 200 μg of BII00313, m1F8-2, or their isotype antibodies at 24 days, 28 days, and 31 days after EAE induction. At 32 days after EAE induction, the brain and spinal cord were collected, and the number of cells infiltrating into the central nervous system was evaluated. Specifically, six mice in each antibody administration group were further divided into two groups (Group 1, Group 2). After cutting the tissues into small pieces, they were further digested at 37°C for 40 minutes in RPMI 1640 medium (Invitrogen) containing 1.4 mg / mL of collagenase H and 100 μg / mL of DNase (Roche). The homogenate of the obtained tissues was passed through a 70-μm cell strainer (GE Healthcare Life Science), and leukocytes were concentrated using discontinuous Percol density gradient centrifugation (37% / 80%). Subsequently, using a flow cytometer, CD45-positive TCR β-positive CD4-positive T cells infiltrating into the CNS (Central Nervous System) were separated using FACS Aria II (BD Cytometry Systems). In (A) of Table 7, they were further cultured for 5 hours in the presence of 5 ng / mL of phorbol 12-myristate 13-acetate, 500 ng / mL of ionomycin (Sigma), and GolgiPlug (BD Biosciences), fixed with Foxp3 / Transcription Factor Staining Buffer Set (eBioscience), stained with anti-IL-17 antibody and anti-IFN-γ antibody after membrane permeabilization, and analyzed using FACSCanto II (BD Cytometry Systems). In (B) of Table 7, the isolated CD4-positive T cells were first stained with anti-CX3CR1 antibody, fixed with Foxp3 / Transcription Factor Staining Buffer Set (eBioscience), membrane permeabilized, and then further stained and analyzed with anti-granzyme B antibody. The proportions of IL-17-negative and IFN-γ-positive cells (A) and granzyme B-positive and CX3CR1-positive cells (B) among CD45-positive TCR β-positive CD4-positive cells are shown in Table 7.For the isolation and detection of cells, anti-CD45 antibody (Biolegend), anti-TCRβ antibody (Biolegend), anti-CD4 antibody (Biolegend), anti-IL-17 antibody (Biolegend), anti-IFN-γ antibody (Biolegend), anti-CX3CR1 antibody (Biolegend), and anti-granzyme B antibody (Biolegend) were used.

[0359] [Table 7]

[0360] Table 7. Evaluation of infiltrating cells in the central nervous system (flow cytometry)

[0361] (A)

[0362]

[0363] (B)

[0364]

[0365] As shown in A of Table 7, in the group administered with m1F8-2 antibody, a decrease in IL-17-negative and IFN-γ-positive cells was observed. In addition, as shown in B of Table 7, a decrease in granzyme B (GZMB)-positive cells (upper panel) and GZMB-positive and CX3CR1-positive cells (lower panel) was observed. CD4-positive IFN-γ-positive cells and CD4-positive GZMB-positive cells refer to cytotoxic Th cells (cytotoxic helper T cells). A series of results suggest the possibility that the m1F8-2 antibody acts on CX3CR1 to reduce cytotoxic Th cells in the central nervous system, that is, by blocking or inhibiting the infiltration of cytotoxic Th cells into the CNS, thereby strongly improving the condition of EAE.

[0366] Example 10

[0367] 10. Humanized 1F8 (h1F8-2-1, h1F8-2-2, h1F8-2-3, h1F8-2-4)

[0368] 10-1. Humanization design of 1F8 antibody

[0369] For the m1F8-2 antibody for which the in vitro and in vivo drug effects were confirmed, humanization of the sequence was performed. Humanization was carried out by CDR grafting based on the sequence of the antibody variable region. The design of the humanized sequence was carried out according to the method described in a known paper (Tsurushita et al., Design of humanized antibodies: From anti-Tac to Zenapax. Methods, 36:69-83, 2005).

[0370] First, a three-dimensional molecular model of a mouse antibody is prepared by conventional methods. Then, based on this molecular model, the residues in the amino acid sequence of the framework region that are considered important for the structural formation of the CDR and the residues that are considered necessary for the reaction with the antigen are inferred. In parallel, sequences with high homology to the heavy chain variable region and light chain variable region of the anti-hCX3CR1 antibody are retrieved from the cDNA sequence database of the heavy chain variable region and light chain variable region of the human antibody. Then, a sequence is designed in which the sequence of the framework portion of the retrieved human antibody sequence is connected to the CDR sequence of each hCX3CR1 antibody, and the sequence of the residues that are considered necessary for the formation of the CDR structure or the reaction with the antigen is further transplanted therein to design the humanized antibody sequence. The designed sequence is as follows. ​ The CDR sequences of the humanized antibody sequences are identical to those of the mouse antibody on which they are based, and are identical to the aforementioned sequence numbers (ie, SEQ ID NOs. 2 to 4 and SEQ ID NOs. 10, 7, and 8). ​ In the figure, the underlined portion in each amino acid sequence represents the CDR.

[0371] Since four VH sequences and one VL sequence were designed, four humanized 1F8 antibodies (h1F8-2-1, h1F8-2-2, h1F8-2-3, and h1F8-2-4) were obtained as combinations of humanized sequences. These humanized antibodies were evaluated for their binding activity (EC50) against hCX3CR1-expressing Eurofins cells and their inhibitory activity (IC50) against fractal chemokine-dependent cell migration of human CX3CR1-expressing Ba / F3 cells. The results are shown in Table 8.

[0372] [Table 8]

[0373] Table 8 EC50 and IC50 of humanized 1F8 antibody

[0374]

[0375] As shown in Table 8, humanized 1F8 antibodies (h1F8-2-1, h1F8-2-2, h1F8-2-3, and h1F8-2-4) had binding activity and blocking activity similar to the above-mentioned m1F antibody.

[0376] Example 11

[0377] 11. Cross-reactivity of the modified 1F8 antibody with CX3CR1 from other animal species

[0378] 11-1. Activity Enhancement of m1F8 Antibodies (m1F8-1, m1F8-2) and Cross-reactivity with CX3CR1 in Other Animal Species

[0379] The evaluation results of the cross-species reactivity of the m1F8-1 antibody or m1F8-2 antibody with CX3CR1 (binding reactivity with Ba / F3 cells expressing CX3CR1 of human, cynomolgus monkey, mouse, and rat) are shown in Table 9 below. Using the same method as in Example 4, flow cytometry and quantification were performed to determine the cross-species reactivity. In the results of Table 9, when the relative measurement value relative to the measurement value at 200 nM of the positive antibody is higher than 70%, it is indicated as "+++", when it is 40 - 70%, it is indicated as "++", when it is 10 - less than 40%, it is indicated as "+", and when it is less than 10%, it is indicated as "-".

[0380] As shown in Table 9, the m1F8-1 antibody or m1F8-2 antibody showed reactivity with human and cynomolgus monkey, and did not show reactivity with mouse and rat. As described above, the m1F8-1 antibody and m1F8-2 antibody showed cross-species reactivity with primate CX3CR1, similar to the m1F8 antibody before activity enhancement, and did not show cross-species reactivity with rodent CX3CR1.

[0381] 11-2. Cross-species reactivity of humanized 1F8 antibody (h1F8-2-2) with CX3CR1 of other animal species

[0382] The evaluation results of the cross-species reactivity of the h1F8-2-2 antibody or 306D-hFc with CX3CR1 (binding reactivity with Ba / F3 cells expressing CX3CR1 of human: Human, cynomolgus monkey: Cyno, mouse: Mouse, rat: Rat) are as ​ shown. Flow cytometry was performed using the same method as in Example 4. The relative value of the mean fluorescence intensity relative to Synagis, which is a negative antibody, is shown.

[0383] As ​ shown, the h1F8-2-2 antibody and 306D-hFc showed reactivity with human and cynomolgus monkey, and did not show reactivity with mouse and rat. As described above, the h1F8-2-2 antibody and 306D-hFc showed cross-species reactivity with primate CX3CR1, and did not show cross-species reactivity with rodent CX3CR1.

[0384] [Table 9]

[0385] Table 9

[0386] ​ ​ ​ +++ +++ ​ +++ +++ ​ - - ​ - -

[0387] Relative value to positive antibody: +++; > 70%

[0388] ++; 40 - 70%

[0389] +; 10 - 40%

[0390] -; < 10%

[0391] Example 12

[0392] 12. Epitope analysis of the engineered 1F8 antibody

[0393] 12-1. Binding domain analysis of the activity-enhanced m1F8 antibodies (m1F8-1, m1F8-2)

[0394] The evaluation results of the binding activities of the m1F8-1 antibody or the m1F8-2 antibody to chimeric CX3CR1-expressing Ba / F3 cells are shown in Table 10 below. Using the same method as in Example 5, flow cytometry and quantification were performed to determine the binding domain. In the results of Table 10, when the relative measurement value with respect to the measurement value at 200 nM of the positive antibody is higher than 70%, it is indicated as "+++", when it is 40-70%, it is indicated as "++", when it is 10 - less than 40%, it is indicated as "+", and when it is less than 10%, it is indicated as "-".

[0395] As shown in Table 10, the m1F8-1 antibody and the m1F8-2 antibody showed reactivity to the cell lines expressing the extracellular domain 1 of hCX3CR1 (hCX3CR1, hCX3CR1-2,3,4, mCX3CR1-1). On the other hand, they also showed a certain degree of reactivity to hCX3CR1-1 that does not have the extracellular domain 1 of hCX3CR1. In addition, the m1F8-2 antibody also showed a certain degree of reactivity to mCX3CR1-4 that does not have the extracellular domain 1 of hCX3CR1. The above results strongly suggest the possibility that the m1F8-1 antibody and the m1F8-2 antibody recognize the three-dimensional structure of CX3CR1 in the same way as the m1F8 antibody before the activity enhancement.

[0396] 12-2. Binding domain analysis of the humanized 1F8 antibody (h1F8-2-2)

[0397] The binding domain analysis results of the h1F8-2-2 antibody or 306D-hFc are as ​ shown. Flow cytometry was performed using the same method as in Example 5. The relative value with respect to the mean fluorescence intensity of Synagis as a negative antibody was shown.

[0398] As ​As shown, the h1F8-2-2 antibody showed reactivity against the expression cell lines with the extracellular domain 1 of hCX3CR1 (hCX3CR1, hCX3CR1-2,3,4, mCX3CR1-1), and also showed certain reactivity against the expression cell lines without the extracellular domain 1 of hCX3CR1 (hCX3CR1-1, mCX3CR1-4). On the other hand, 306D-hFc showed reactivity only against the expression cell lines with the extracellular domain 1 of hCX3CR1. The above results strongly suggest the possibility that the h1F8-2-2 antibody also recognizes the three-dimensional structure of CX3CR1 in the same way as other 1F8 antibodies.

[0399] [Table 10]

[0400] Table 10

[0401]

[0402] Relative value to positive antibody: +++; >70%

[0403] ++; 40 - 70%

[0404] +; 10 - 40%

[0405] -; <10%

[0406] Example 13

[0407] 13. Competitive assay using the h1F8-2-2 antibody

[0408] The results of the binding competition assay using the humanized h1F8-2-2 antibody or anti-hCX3CR1-Nanobody with CX3CR1-expressing Ba / F3 cells are shown in Table 11. The method was carried out in the same way as in Example 6.

[0409] 10 μM of the competitive antibody shown on the left side of Table 11 and 100 nM of the detection antibody shown on the upper side of the table were added to hCX3CR1-expressing Ba / F3 cells and suspended, and they were left standing in the dark at 4°C for 30 minutes. The subsequent flow cytometry method was carried out in the same manner as in Example 6. The detection antibody h1F8-2-2 (mFc type) represents a chimeric antibody having the Fab of the h1F8-2-2 antibody, which is a humanized h1F8 antibody, and the mouse Fc. As the negative antibody, Synagis, which is a humanized IgG1 antibody, was used. The reactivity (average fluorescence intensity) under the condition of only the secondary antibody with cells was subtracted as the background value, and the binding competitiveness was determined based on the reactivity % of each detection antibody in the presence of each competitive antibody when the reactivity of each detection antibody using Synagis as the competitive antibody was set to 100% respectively. When the relative value with respect to the competitive negative antibody is higher than 70%, it is indicated as "+++", when it is 40 - 70%, it is indicated as "++", when it is from 10 to less than 40%, it is indicated as "+", and when it is less than 10%, it is indicated as "-".

[0410] As shown in Table 11, the h1F8-2-2 antibodies or the anti-hCX3CR1-Nanobodies compete very fiercely with each other. On the other hand, the h1F8-2-2 antibody and the Nanobody do not show significant competition. From this result, it can be considered that the epitopes of the h1F8-2-2 antibody and the anti-hCX3CR1-Nanobody are different.

[0411] [Table 11]

[0412]

[0413] Relative value with respect to the negative competitive antibody: +++ ; > 70%

[0414] ++ ; 40 - 70%

[0415] + ; 10 - 40%

[0416] - ; < 10%

[0417] Example 14

[0418] 14. Calcium influx assay of the h1F8-2-2 antibody

[0419] The in vitro blocking activity of the h1F8-2-2 antibody against hCX3CR1-expressing cells was evaluated by FKN-induced calcium influx assay.

[0420] Calcium influx assay was performed using hCX3CR1-expressing CHO-K1 cells (AequoZen Human Chemokine CX3CR1 Cell Line, CHO-K1 Frozen Cells, PerkinElmer). After thawing and washing the cells, they were suspended in DMEM / F12 (Gibco) medium containing 5 μM coelenterazine-h (Promega), 0.1% BSA, and 1% penicillin-streptomycin (Nacalai Tesque) and cultured at room temperature for 4 hours. Then, 3 volumes of coelenterazine-h-free culture medium were added, and the cells were suspended and further cultured for 30 minutes. After that, the antibody solution was mixed equally (25 μL) into the wells of a 96-well white plate (ThermoFisher Scientifics) and cultured for 30 minutes. 50 μL of human FKN (PeproTech) was added to each well, and the luminescence over time was measured using a TriStar2SLB942 Multimode Reader (Berthold Technologies) equipped with Instrument Control and Evaluation software. The final cell number was 20,000 cells per well, the FKN concentration was 10 nM, and the antibody concentration was used as shown in ​ . After adding FKN to the cell suspension, the relative luminescence was measured every 1 second for 30 seconds. The relative values of the maximum values during the measurement period under each condition, with the maximum value during the measurement period when FKN was added without antibody set as 100%, are shown in the figure.

[0421] ​ The results of measuring calcium influx in (A) of

[0422] are shown at the final concentration (the state where cells, antibodies, and ligands are all added) in the presence of h1F8-2-2 antibody or BII00313 (anti-hCX3CR1-Nanobody) at 100, 10, and 1 nM. Synagis antibody was used as a negative antibody. Compared with BII00313, the h1F8-2-2 antibody showed low calcium influx blocking activity at low concentrations and high blocking activity at high concentrations.

[0422] In ​ , (B) and (C) show the results of measuring calcium influx in the presence of h1F8-2-2 antibody at a total of 10 concentrations with a maximum concentration of 300 nM and a common ratio of 3 times ( ​ in (B)) or BII00313 ( ​Results of measuring calcium influx in the presence of (C)). In the comparison of IC50, BII00313 was lower than the h1F8-2-2 antibody. On the other hand, in terms of maximum blocking activity, h1F8-2-2 showed high blocking activity. The above results indicate that the differences between the two are not only in epitopes but also in the characteristics of activity.

[0423] Example 15

[0424] 15. Administration test of h1F8-2-2 antibody to human CX3CR1-KI mice (evaluation of CX3CR1-expressing cells in blood)

[0425] For 8- or 9-week-old female human CX3CR1-KI mice, untreated or intraperitoneally administered 200 μg of h1F8-2-2 antibody, BII00313, or Synagis antibody per mouse. Peripheral blood (heparinized whole blood) was collected from the abdominal aorta 1, 3, 5, and 7 days later. 100 μL of peripheral blood was dispensed into a 96-well plate, and a detection antibody mixture was added, followed by reaction at 4 °C for 20 minutes. 1× RBC Lysis / Fixation (Biolegend) was added, and after suspension, the mixture was left standing at room temperature for 20 minutes to lyse red blood cells and fix the stained cells. After washing the cells with 1% FBS / PBS multiple times, analysis was performed using FACSLyric and FlowJo (BD Biosciences). As detection antibodies, labeled anti-CD45 antibody (BD Biosciences), anti-CD11b antibody (Biolegend), and anti-CD115 antibody (Biolegend) were used. In addition, for the anti-hCX3CR1 antibody, two antibodies with different epitopes (m1F8-2 antibody or 66B02-mFc) labeled with Alexa488 were used.

[0426] Labeled cells were visualized by forward scatter light and side scatter light, and the cell population (Singlet) free of cell debris and aggregated cells was set as 100%. The proportions of CD45-positive, CD11b-positive, and CD115-positive cells in Singlet cells are as ​ shown in (A) and (B) (A: m1F8-2 staining, B: 66B02-mFc staining). In the h1F8-2-2 antibody and BII00313 administration groups, a reduction of more than 50% in CD115-positive cells was observed 1 day after administration, and a steady recovery over time was observed 7 days later.

[0427] The proportions of CD45-positive, CD11b-positive, CD115-positive, and CX3CR1-positive cells in Singlet cells are as ​as shown in (C) and (D) (C: m1F8-2 staining, D: 66B02-mFc staining). In the h1F8-2-2 antibody administration group, almost no m1F8-2-positive and 66B02-mFc-positive cells were observed within 5 days after administration. On the other hand, in the BII00313 administration group, almost no 66B02-mFc-positive cells were observed within 7 days after administration, but m1F8-2-positive cells were confirmed 1 day after administration.

[0428] Furthermore, when the proportion of CX3CR1-positive cells with CD115-positive cells set to 100% is as ​ shown in (E) and (F) (E: m1F8-2 staining, F: 66B02-mFc staining). In the h1F8-2-2 antibody administration group, almost no m1F8-2-positive and 66B02-mFc-positive cells on CD115-positive cells were observed within 5 days after administration. On the other hand, in the BII00313 administration group, almost no 66B02-mFc-positive cells on CD115-positive cells were observed within 7 days after administration, but m1F8-2 antibody-positive cells were confirmed 1 day after administration.

[0429] From the above results, it can be seen that both the h1F8-2-2 antibody and BII00313 have the effect of reducing CD115-positive cells. In addition, it gives an indication of the characteristic differences: the h1F8-2-2 antibody has the function of attenuating CX3CR1 (receptor) itself from the CD115-positive cell membrane, while in contrast, BII00313 has the function of occupying the receptor and the function of attenuating the amount of CX3CR1 is weaker than that of the h1F8-2-2 antibody.

[0430] Example 16

[0431] 16. Antibody Administration Test in a Late EAE Model of Human CX3CR1-KI Mice

[0432] 16-1. Evaluation of the Number of Infiltrating Cells in the Central Nervous System Based on m1F8-2 Antibody Administration

[0433] For NR4A2-cKO / human CX3CR1-KI mice that were induced with monophasic EAE in the same manner as in Example 7, 200 μg of m1F8-2 or its isotype-negative antibody (mIgG2a) was administered 24 days and 31 days after EAE induction. Similar to 9-2 in Example 9, 32 days after EAE induction, the brain and spinal cord were collected, and the number of cells infiltrating into the central nervous system was evaluated. Three samples in each antibody administration group were used as one group. Cell concentration, stimulation, staining, fixation, and analysis were performed in the same manner as 9-2 in Example 9. Among CD45-positive TCRβ-positive CD4-positive cells, the ratio of Eomes-positive to IFN-γ-positive cells (A) and the ratio of Eomes-positive to granzyme B (GZMB)-positive cells (B) are shown in Table 12. For cell separation and detection, anti-CD45 antibody (Biolegend), anti-TCRβ antibody (Biolegend), anti-CD4 antibody (Biolegend), anti-IFN-γ antibody (Biolegend), anti-Eomes antibody (eBioscience), and anti-granzyme B antibody (Biolegend) were used.

[0434] As shown in A of Table 12, a decrease in Eomes-positive cells (upper panel) or Eomes-positive and IFN-γ-positive cells (lower panel) was observed in the group administered with the m1F8-2 antibody. In addition, as shown in B of Table 12, a decrease in Eomes-positive cells (upper panel) or Eomes-positive and GZMB-positive cells (lower panel) was observed. CD4-positive Eomes-positive IFN-γ or GZMB-positive cells refer to cytotoxic Th cells. A series of results suggest the possibility that the m1F8-2 antibody acts on CX3CR1, reducing cytotoxic Th cells in the central nervous system, that is, strongly improving the EAE condition by blocking or inhibiting the infiltration of cytotoxic Th cells into the CNS.

[0435] [Table 12]

[0436] Table 12

[0437] (A)

[0438]

[0439] (B)

[0440]

[0441] 16-2. EAE score evaluation based on administration of the h1F8-2-2 antibody

[0442] For NR4A2-cKO / human CX3CR1-KI mice that had been induced with monophasic EAE in the same manner as in Example 7, 200 μg of h1F8-2-2 or its isotype antibody (Synagis) was administered 24 days and 28 days after EAE induction, and the EAE condition of the mice was evaluated.

[0443] As ​ shown, in the group administered with the h1F8-2-2 antibody, the EAE condition was strongly improved.

[0444] 16-3. Evaluation of the number of infiltrating cells in the central nervous system based on administration of the h1F8-2-2 antibody

[0445] For NR4A2-cKO / human CX3CR1-KI mice that had been induced with monophasic EAE in the same manner as in Example 7, 200 μg of h1F8-2-2 or its isotype negative antibody (Synagis) was administered 24 days and 28 days after EAE induction. Similar to 16-1 above, 31 days after EAE induction, the brain and spinal cord were collected, and the number of cells infiltrating into the central nervous system was evaluated. Samples of 7 mice in the negative control antibody administration group or 5 mice in the h1F8-2-2 antibody administration group were used as one group. Cell concentration, stimulation, staining, fixation, and analysis were performed in the same manner as in 16-1 above. The proportions of CX3CR1-positive cells, Eomes-positive cells, and Eomes-positive and CX3CR1-positive cells among CD45-positive TCRβ-positive CD4-positive cells are shown in Table 13. For cell separation and detection, anti-CD45 antibody (Biolegend), anti-TCRβ antibody (Biolegend), anti-CD4 antibody (Biolegend), anti-Eomes antibody (eBioscience), and anti-CX3CR1 antibody (Biolegend) were used.

[0446] As shown in Table 13, in the group administered with the h1F8-..2 antibody, a decrease in CX3CR1-positive cells (upper row), Eomes-positive cells (middle row), or Eomes-positive and CX3CR1-positive cells (lower row) was observed. A series of results suggest the possibility that, similar to the m1F8-2 antibody, the h1F8-2-2 antibody acts on CX3CR1, reducing cytotoxic Th cells in the central nervous system, that is, strongly improving the EAE condition by blocking or inhibiting the infiltration of cytotoxic Th cells into the CNS.

[0447] [Table 13]

[0448] Table 13

[0449]

[0450] Example 17

[0451] 17. Antibody administration test in a human CX3CR1-KI mouse CAIA (collagen antibody mixture-induced arthritis) model

[0452] 17-1. Induction of the CAIA model

[0453] A 5 mg anti-collagen antibody mixture (Chondrex) was intraperitoneally administered to each 7-week-old male human CX3CR1-KI mouse. Three days after administration of the antibody mixture, 50 μg of LPS (Chondrex) was intraperitoneally administered to each mouse to induce the CAIA model. In the untreated group, saline was administered instead of the mixture and LPS.

[0454] 17-2. Arthritis score evaluation based on administration of the m1F8-2 antibody

[0455] To human CX3CR1-KI mice administered with the anti-collagen antibody mixture, 400 μg of the m1F8-2 antibody or mIgG2a, its isotype negative antibody, was intraperitoneally administered 3 days and 10 days after administration of the antibody mixture. As a positive control drug, 6 μg of dexamethasone (Dex, Sigma) was orally administered daily from 3 to 14 days after administration of the antibody mixture. For all animals, the arthritis score of the four limbs was measured daily until 1 to 14 days after administration of the antibody mixture. The score was observed and recorded for three joints, namely the toes, the dorsum of the foot, and the wrist, according to the following evaluation criteria, and the total score of the four limbs was shown.

[0456] <Arthritis evaluation criteria>

[0457] Score 0: Normal (no inflammation was confirmed in any joint)

[0458] Score 1: Slight but obvious redness and swelling of the ankles and wrists, or obvious redness and swelling limited to each toe (inflammation was confirmed in one part of any joint)

[0459] Score 2: Moderate redness and swelling of the ankles or wrists (inflammation was confirmed in two parts of any joint)

[0460] Score 3: Severe redness and swelling of the entire foot including the toes (inflammation was confirmed in all joints)

[0461] Score 4: Severe inflammation of the hands and feet including multiple joints (inflammation was confirmed in all joints, and the entire limb was red and swollen)

[0462] As ​As shown, an increase in the arthritis score was confirmed in the human CX3CR1-KI mice in the CAIA administration group. A stronger arthritis alleviating effect was confirmed in the dexamethasone administration group as a positive control compared to the mIgG2a administration group as a negative control. In addition, an arthritis alleviating effect was also confirmed in the m1F8-2 administration group.

[0463] Industrial Applicability

[0464] According to the present invention, antibodies that bind to human CX3CR1 and the like can be provided. The anti-human CX3CR1 antibody according to the present invention is, for example, an antibody that can have an activity of blocking or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine), and a function of improving the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of the condition of experimental autoimmune encephalomyelitis (EAE). In addition, it is an antibody that can function as a prophylactic agent, progression inhibitor, or therapeutic agent for progressive immune demyelinating diseases (preferably progressive immune demyelinating diseases of the central nervous system, such as secondary progressive multiple sclerosis (SPMS)).

[0465] Sequence Listing Free Text

[0466] Sequence No. 1: Synthetic Peptide

[0467] Sequence No. 3: Synthetic Peptide

[0468] Sequence No. 4: Synthetic Peptide

[0469] Sequence No. 5: Synthetic Peptide Sequence No. 6: Synthetic Peptide Sequence No. 9: Synthetic Peptide Sequence No. 10: Synthetic Peptide Sequence No. 11: Synthetic Peptide Sequence No. 12: Synthetic Peptide Sequence No. 13: Synthetic Peptide Sequence No. 14: Synthetic Peptide Sequence No. 15: Synthetic Peptide

Claims

1. An antibody against human CX3CR1, the amino acid sequences of complementarity-determining regions (CDRs) 1 (CDR-H1), 2 (CDR-H2) and 3 (CDR-H3) of the heavy-chain variable region (VH) are respectively constituted by the amino acid sequences shown in SEQ ID NO: 2, 3 and 4 in sequence, and the amino acid sequences of CDRs 1 (CDR-L1), 2 (CDR-L2) and 3 (CDR-L3) of the light-chain variable region (VL) are respectively constituted by the amino acid sequences shown in SEQ ID NO: 6, 7 and 8 in sequence, or the amino acid sequences shown in SEQ ID NO: 10, 7 and 8 in sequence.

2. The antibody according to claim 1, wherein (a) the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are respectively constituted by the amino acid sequences shown in SEQ ID NO: 2, 3 and 4 in sequence, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are respectively constituted by the amino acid sequences shown in SEQ ID NO: 6, 7 and 8 in sequence, or (b) the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are respectively constituted by the amino acid sequences shown in SEQ ID NO: 2, 3 and 4 in sequence, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are respectively constituted by the amino acid sequences shown in SEQ ID NO: 10, 7 and 8 in sequence.

3. An antibody against human CX3CR1, the amino acid sequence of the heavy-chain variable region (VH) is constituted by the amino acid sequence shown in SEQ ID NO: 1, 11, 12, 13 or 14, and the amino acid sequence of the light-chain variable region (VL) is constituted by the amino acid sequence shown in SEQ ID NO: 5, 9 or 15.

4. The antibody according to claim 3, wherein (a) the amino acid sequence of VH is constituted by the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of VL is constituted by the amino acid sequence shown in SEQ ID NO: 5, (b) the amino acid sequence of VH is constituted by the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of VL is constituted by the amino acid sequence shown in SEQ ID NO: 9, (c) the amino acid sequence of VH is constituted by the amino acid sequence shown in SEQ ID NO: 11, and the amino acid sequence of VL is constituted by the amino acid sequence shown in SEQ ID NO: 15, (d) the amino acid sequence of VH is constituted by the amino acid sequence shown in SEQ ID NO: 12, and the amino acid sequence of VL is constituted by the amino acid sequence shown in SEQ ID NO: 15, (e) the amino acid sequence of VH is constituted by the amino acid sequence shown in SEQ ID NO: 13, and the amino acid sequence of VL is constituted by the amino acid sequence shown in SEQ ID NO: 15, or (f) the amino acid sequence of VH is constituted by the amino acid sequence shown in SEQ ID NO: 14, and the amino acid sequence of VL is constituted by the amino acid sequence shown in SEQ ID NO:

15.

5. The antibody according to any one of claims 1 to 4, wherein, The antibody is a humanized antibody.

6. The antibody according to any one of claims 1 to 4, which has the activity of blocking or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine).

7. The antibody according to any one of claims 1 to 4, which has an activity of blocking or inhibiting the function of Th cells expressing Eomes.

8. The antibody according to claim 7, which has an activity of blocking or inhibiting the infiltration of Th cells expressing Eomes into the CNS.

9. The antibody according to any one of claims 1 to 4, which has a function of improving the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of experimental autoimmune encephalomyelitis (EAE).

10. The antibody according to any one of claims 1 to 4, which is an antibody for the prevention, progression inhibition or treatment of the following diseases: progressive immune demyelinating diseases, preferably progressive immune demyelinating diseases of the central nervous system; autoimmune diseases, preferably rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus or autoimmune myositis; heart diseases including atherosclerosis; chronic kidney diseases; diabetic heart diseases; diabetic nephropathies; interstitial lung diseases; cancers, preferably breast cancer, prostate cancer or colorectal cancer; COVID-19 related central nervous system diseases; or neurodegenerative diseases, preferably ALS, Alzheimer's disease or multiple system atrophy.

11. The antibody according to claim 10, wherein, The progressive immune demyelinating disease is secondary progressive multiple sclerosis (SPMS).

12. An antibody fragment, which is derived from the antibody according to any one of claims 1 to 4.

13. A pharmaceutical composition, which comprises the antibody according to any one of claims 1 to 4, or the antibody fragment according to claim referred to in claim 12.

14. The pharmaceutical composition according to claim 13, which is a pharmaceutical composition for the prevention, progression inhibition or treatment of the following diseases: progressive immune demyelinating diseases, preferably progressive immune demyelinating diseases of the central nervous system; autoimmune diseases, preferably rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus or autoimmune myositis; heart diseases including atherosclerosis; chronic kidney diseases; diabetic heart diseases; diabetic nephropathies; interstitial lung diseases; cancers, preferably breast cancer, prostate cancer or colorectal cancer; COVID-19 related central nervous system diseases; or neurodegenerative diseases, preferably ALS, Alzheimer's disease or multiple system atrophy.

15. The pharmaceutical composition according to claim 14, wherein, The progressive immune demyelinating disease is secondary progressive multiple sclerosis (SPMS).

16. The pharmaceutical composition according to claim 13, which is a pharmaceutical composition for blocking or inhibiting the interaction or binding between human CX3CR1 and human CX3CL1 (fractalkine).

17. The pharmaceutical composition according to claim 13, which is a pharmaceutical composition for blocking or inhibiting the function of Th cells expressing Eomes.

18. The pharmaceutical composition according to claim 17, which is a pharmaceutical composition for blocking or inhibiting the infiltration of Th cells expressing Eomes into the CNS.

19. The pharmaceutical composition according to claim 13, which is a pharmaceutical composition for improving the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of experimental autoimmune encephalomyelitis (EAE).

20. A method for preventing, inhibiting the progression of, or treating the following diseases, which comprises administering to a subject the pharmaceutical composition according to claim 13, the diseases being: progressive immune demyelinating diseases, preferably progressive immune demyelinating diseases of the central nervous system; autoimmune diseases, preferably rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus, or autoimmune myositis; heart diseases including atherosclerosis; chronic kidney diseases; diabetic heart diseases; diabetic nephropathies; interstitial lung diseases; cancers, preferably breast cancer, prostate cancer, or colorectal cancer; COVID-19 related central nervous system diseases; or neurodegenerative diseases, preferably ALS, Alzheimer's disease, or multiple system atrophy.

21. The method according to claim 20, wherein, The progressive immune demyelinating disease is secondary progressive multiple sclerosis (SPMS).

22. A method for blocking or inhibiting the interaction or binding of human CX3CR1 and human CX3CL1 (fractalkine), which comprises administering to a subject the pharmaceutical composition according to claim 13.

23. A method for blocking or inhibiting the function of Th cells expressing Eomes, which comprises administering to a subject the pharmaceutical composition according to claim 13.

24. The method according to claim 23, which is a method for blocking or inhibiting the infiltration of Th cells expressing Eomes into the CNS, and the method comprises administering to a subject the pharmaceutical composition according to claim 13.

25. A method for improving the condition of experimental autoimmune encephalomyelitis (EAE) or inhibiting the progression of experimental autoimmune encephalomyelitis (EAE), which comprises administering to a subject the pharmaceutical composition according to claim 13.

26. A kit for preventing, inhibiting the progression of, or treating, or diagnosing the following diseases, which comprises the antibody according to any one of claims 1 to 4 or the antibody fragment according to claim 12, the diseases being: progressive immune demyelinating diseases, preferably progressive immune demyelinating diseases of the central nervous system; autoimmune diseases, preferably rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, systemic sclerosis, systemic lupus erythematosus, or autoimmune myositis; heart diseases including atherosclerosis; chronic kidney diseases; diabetic heart diseases; diabetic nephropathies; interstitial lung diseases; cancers, preferably breast cancer, prostate cancer, or colorectal cancer; COVID-19 related central nervous system diseases; or neurodegenerative diseases, preferably ALS, Alzheimer's disease, or multiple system atrophy.

27. The kit according to claim 26, wherein, The progressive immune demyelinating disease is secondary progressive multiple sclerosis (SPMS).

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