Therapeutic or prophylactic agent for HTLV-1-associated myelopathy (HAM) targeting CCR8

Abstract

To provide a novel therapeutic or prophylactic agent for HAM.SOLUTION: A therapeutic or prophylactic agent for HTLV-1-associated myelopathy (HAM), comprising a substance that removes CCR8-expressing cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic or preventive agent for HTLV-1-associated myelopathy (HAM) that targets CCR8. [Background technology]

[0002] Human T-cell leukemia virus type 1 (HTLV-1) is a virus that infects T cells (mainly CD4-positive T cells), a type of white blood cell in the blood. T cells infected with HTLV-1 cause chronic inflammation in the spinal cord, which results in damage and degeneration of spinal nerve cells, leading to spastic spinal paralysis and other conditions. Spastic spinal paralysis caused by HTLV-1-infected cells is called HTLV-1-associated myelopathy (hereinafter referred to as "HAM"). Symptoms of HAM include paralysis of both legs, pain, and urinary and defecation problems due to nerve tissue damage. As these symptoms progress, patients may become wheelchair-bound or bedridden. HAM is one of the diseases designated as an intractable disease in Japan, but currently, no effective treatment has been established for HAM, and symptomatic treatment is the only treatment available.

[0003] Known treatments for HAM include those using RGMa inhibitors such as anti-RGMa antibodies (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2020 / 017629 Summary of the Invention [Problem to be solved by the invention]

[0005] However, no treatment for HAM has been established. Furthermore, because only minute amounts of HTLV-1-infected cells infiltrating the spinal cord can be collected, comprehensive gene expression analysis is difficult. The present inventors hypothesized that by performing genetic analysis of HTLV-1-infected cells infiltrating the spinal cord using single-cell RNA-seq analysis, targets for treating or preventing HAM could be identified. The problem to be solved by the present invention is to provide a novel agent for treating or preventing HAM. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors discovered that CCR8 is expressed specifically in HTLV-1-infected cells, and that HAM can be treated or prevented by using a substance that removes CCR8-expressing cells, and thus completed the present invention.

[0007] That is, the present invention includes the following embodiments. [1] A therapeutic or preventive agent for HTLV-1 associated myelopathy (HAM), comprising a substance that eliminates CCR8-expressing cells. [2] The substance is (A) an antibody or antibody fragment thereof that binds to CCR8, and (B) Cells genetically engineered to express a chimeric antigen receptor that binds to CCR8. At least one of [1] The therapeutic or preventive agent described in [1]. [3] the substance comprises the antibody (A) or its antibody fragment, and the antibody (A) or its antibody fragment has ADCC activity, CDC activity, or ADCP activity; The therapeutic or preventive agent according to [1] or [2]. [4] The therapeutic or prophylactic agent according to any one of [1] to [3], wherein the cells to be removed are cells expressing both CCR8 and CD4.

[0008] The present invention also includes the following aspects. [A1] A method for treating or preventing HAM, comprising administering to a patient in need thereof a therapeutically effective amount of a substance that eliminates CCR8-expressing cells. [A2] The substance is (A) an antibody or antibody fragment thereof that binds to CCR8, and (B) Cells genetically engineered to express a chimeric antigen receptor that binds to CCR8. At least one of The method described in [A1]. [A3] The method according to [A1] or [A2], wherein the substance comprises the antibody of (A) or an antibody fragment thereof, and the antibody of (A) or an antibody fragment thereof has ADCC activity, CDC activity, or ADCP activity. [A4] The method according to any one of [A1] to [A3], wherein the cells to be removed are cells expressing both CCR8 and CD4.

[0009] [B1] A substance that eliminates CCR8-expressing cells for treating or preventing HAM. [B2] The substance is (A) an antibody or antibody fragment thereof that binds to CCR8, and (B) Cells genetically engineered to express a chimeric antigen receptor that binds to CCR8. The substance according to [B1], which is at least one of the following: [B3] The substance according to [B1] or [B2], wherein the substance comprises the antibody of (A) or its antibody fragment, and the antibody of (A) or its antibody fragment has ADCC activity, CDC activity, or ADCP activity. [B4] The substance according to any one of [B1] to [B3], wherein the cells to be removed are cells expressing both CCR8 and CD4.

[0010] [C1] Use of an agent that eliminates CCR8-expressing cells to treat or prevent HAM. [C2] The substance is (A) an antibody or antibody fragment thereof that binds to CCR8, and (B) Cells genetically engineered to express a chimeric antigen receptor that binds to CCR8. The use according to [C1], wherein the compound is at least one of the following: [C3] The use according to [C1] or [C2], wherein the substance comprises the antibody of (A) or its antibody fragment, and the antibody of (A) or its antibody fragment has ADCC activity, CDC activity, or ADCP activity. [C4] The use according to any one of [C1] to [C3], wherein the cells to be removed are cells expressing both CCR8 and CD4.

[0011] [D1] Use of a substance that eliminates CCR8-expressing cells in the manufacture of an agent for treating or preventing HAM. [D2] The substance is (A) an antibody or antibody fragment thereof that binds to CCR8, and (B) Cells genetically engineered to express a chimeric antigen receptor that binds to CCR8. At least one of Use as described in [D1]. [D3] The use according to [D1] or [D2], wherein the substance comprises the antibody of (A) or its antibody fragment, and the antibody of (A) or its antibody fragment has ADCC activity, CDC activity, or ADCP activity. [D4] The use according to any one of [D1] to [D3], wherein the cells to be removed are cells expressing both CCR8 and CD4.

[0012] [E1] A therapeutic or prophylactic agent for use in treating HTLV-1 associated myelopathy (HAM), comprising a substance that eliminates CCR8 expressing cells. [E2] The substance is (A) an antibody or antibody fragment thereof that binds to CCR8, and (B) Cells genetically engineered to express a chimeric antigen receptor that binds to CCR8. At least one of The therapeutic or prophylactic agent according to [E1]. [E3] the substance comprises the antibody (A) or its antibody fragment, and the antibody (A) or its antibody fragment has ADCC activity, CDC activity, or ADCP activity; The therapeutic or prophylactic agent according to [E1] or [E2]. [E4] The therapeutic or prophylactic agent according to any one of [E1] to [E3], wherein the cells to be removed are cells expressing both CCR8 and CD4.

[0013] In any of the inventions [1] to [4], [A1] to [A4], [B1] to [B4], [C1] to [C4], [D1] to [D4], and [E1] to [E4], the configuration of the invention, such as a substance that removes CCR8-expressing cells, may be any of the preferred embodiments described below as the present embodiments. Furthermore, in any of the inventions [1] to [4], [A1] to [A4], [B1] to [B4], [C1] to [C4], [D1] to [D4], and [E1] to [E4], the configuration of the invention may be any combination of the preferred embodiments described below as the present embodiments. Preferred embodiments include more preferred embodiments, even more preferred embodiments, and even more preferred embodiments. [Effects of the Invention]

[0014] According to the present invention, a novel agent for treating or preventing HAM can be provided. [Brief explanation of the drawings]

[0015] [Figure 1] Cell clustering was performed using UMAP, and the results of cell annotation for each cluster are shown. [Figure 2]This shows the results of a comparative analysis of HTLV-1-infected cells and Th1. The horizontal axis represents the expression level of each gene; values ​​greater than 0 indicate higher expression in HTLV-1-infected cells, and values ​​less than 0 indicate higher expression in HTLV-1-uninfected cells. The vertical axis represents the significance of the difference; values ​​greater than 1.301 are considered to be significant. [Figure 3] The plot displayed in UMAP shows cells expressing the HBZ gene and CCR8 gene. [Figure 4] A schematic diagram of the representation of D, N and P fractions is shown. [Figure 5] The expression rates of CCR8 in HTLV-1-uninfected cells and HTLV-1-infected cells are shown by disease. [Figure 6] Representative plots of CCR8 expression in infected cell fractions (D+N fractions) are shown by disease. [Figure 7] The results of sorting CD4+CCR8+ cells and other cells are shown. [Figure 8] The percentage of HTLV-1 infected cells in each cell population is shown. [Figure 9] The results of measuring cell proliferation rate are shown. [Figure 10] The mean cell proliferation rates are shown. [Figure 11] The results of detecting the CCR8 positivity rate among CD4+Foxp3+ cells by flow cytometry are shown. [Figure 12] The percentages of CD4+Foxp3+CCR8+ cells and CD4+Foxp3+CCR8- cells are shown. [Figure 13] Cell clustering was performed on PBMCs from healthy individuals using UMAP, and the results of cell annotation for each cluster are shown. [Figure 14] The plots displayed in UMAP show cells expressing CCR8, CCR4, GATA3, and FOXP3. [Figure 15] This shows the results of comparing CCR8 expression levels by cell type using single-cell RNA-seq. [Figure 16]CCR8-expressing cells are shown on the plot displayed as UMAP. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following examples. stomach.

[0017] The present disclosure includes the treatment or prevention of HTLV-1 associated myelopathy (HAM) by eliminating CCR8-expressing cells. Hereinafter, CCR8 and substances that eliminate CCR8-expressing cells will be described.

[0018] (Substances that eliminate CCR8-expressing cells) As used herein, CCR8 refers to G protein-coupled seven-transmembrane CC motif chemokine receptor 8. Regarding CCR8 and anti-CCR8 antibodies, for example, Reference Document 1 is known. Furthermore, Reference Documents 2 to 4 disclose methods for treating cancer using anti-CCR8 antibodies that target CCR8 specifically expressed in tumor-infiltrating Tregs. Reference 1: International Publication No. 2020 / 138489 Reference 2: JP 2020-55876 A Reference 3: International Publication No. 2021 / 194942 Reference 4: International Publication No. 2021 / 260210

[0019] As used herein, CCR8-expressing cells refer to cells in which CCR8 is expressed, and are hereinafter referred to as "CCR8-positive cells" and "CCR8 +In this embodiment, the CCR8-expressing cells to be removed are preferably T cells (in this case, the CCR8-expressing cells are also referred to as "CCR8-expressing T cells" or "CCR8-positive T cells"); from the viewpoint of reducing side effects due to hematotoxicity, the CCR8-expressing cells are preferably not endothelial cells, platelets, erythrocytes, granulocytes, or other cells. Furthermore, as shown in the Examples below, the present inventors have found that CCR8 is expressed specifically in HTLV-1-infected cells. That is, in this embodiment, the CCR8-expressing cells to be removed are preferably cells infected with HTLV-1 (hereinafter also referred to as "HTLV-1-infected cells"), and more preferably HTLV-1-infected T cells.

[0020] Furthermore, the present inventors speculate that the reason for the specific expression of CCR8 in HTLV-1-infected cells is as follows, although this is not intended to limit the scope of the present invention. Diseases caused by HTLV-1 infection include HAM and adult T-cell leukemia (ATL). Although both HAM and ATL patients are infected with HTLV-1, it has been found that the viral protein Tax expressed by HTLV-1 is strongly expressed in HAM patients and barely expressed in ATL patients. Furthermore, a correlation has been found between strong Tax expression and strong CCR8 expression. Based on the above, it is believed that the expression of Tax causes the specific expression of CCR8 in HTLV-1-infected cells. The present inventors have discovered that, because CCR8 is specifically expressed in HTLV-1-infected cells, HAM can be treated or prevented by targeting CCR8 and eliminating HTLV-1-infected cells themselves.

[0021] The CCR8-expressing cells to be removed in this embodiment are not particularly limited, but include cells expressing CD4 and / or CD8 in addition to CCR8. Hereinafter, for example, cells expressing CD4 will be referred to as "CD4-positive cells" and "CD4 + Cells that do not express CD4 are called "CD4-negative cells" and "CD4 -As shown in the Examples below, the present inventors have found that HTLV-1-infected cells from HAM patients are concentrated in a fraction of cells expressing both CCR8 and CD4. That is, the CCR8-expressing cells to be removed in this embodiment are preferably cells expressing both CCR8 and CD4. Cells expressing both CCR8 and CD4 are also referred to as "CCR8-expressing and CD4-expressing cells," and when they are T cells, they are also referred to as "CCR8-expressing and CD4-expressing T cells." "CCR8-expressing and CD4-expressing cells" and "CCR8-expressing and CD4-expressing T cells" may also be referred to as "CCR8-positive and CD4-positive cells" and "CCR8-positive and CD4-positive T cells," respectively.

[0022] Examples of T cells include helper T cells (Th cells), cytotoxic T cells (CTLs), and regulatory T cells (Treg cells). In order to reduce side effects, the CCR8-expressing cells to be removed in this embodiment are preferably not Treg cells (CD4-positive Foxp3-positive cells). As shown in the Examples below, Treg cells include both Treg cells that express CCR8 and Treg cells that do not express CCR8. Therefore, even when Treg cells that express CCR8 are removed, Treg cells that do not express CCR8 are not removed. Therefore, the therapeutic or prophylactic agent of this embodiment is considered to have fewer side effects when it comes to removing Treg cells.

[0023] In this embodiment, the CCR8-expressing cells to be removed are preferably HTLV-1-infected cells that express CCR8, more preferably HTLV-1-infected T cells that express CCR8, and particularly preferably HTLV-1-infected T cells that express both CCR8 and CD4.

[0024] As used herein, "removing CCR8-expressing cells" means that the number of CCR8-expressing cells is reduced or eliminated compared to before administration of the therapeutic or prophylactic agent of this embodiment or before implementation of the therapeutic or prophylactic method of this embodiment. CCR8-expressing cells may be removed by physically removing the CCR8-expressing cells, or by damaging or killing the CCR8-expressing cells. CCR8-expressing cells may be removed by cytotoxic activity or by cell-specific chemotherapy using antibody-drug conjugates (ADCs).

[0025] Because CCR8 is specifically expressed in HTLV-1-infected cells, elimination of CCR8-expressing cells can be confirmed by a decrease in the amount of HTLV-1 provirus. The amount of HTLV-1 provirus can be determined, for example, by extracting DNA from a target cell population and calculating the rate of HTLV-1-infected cells in the cell population using quantitative PCR. The calculation method can be the method described in Nakashima M et al., Clin Cancer Res, 2018 (doi: 10.1158 / 1078-0432.CCR-18-0268).

[0026] In this embodiment, a substance that eliminates CCR8-expressing cells refers to a substance that has the activity of eliminating CCR8-expressing cells, and may be a substance that injures, damages, or kills CCR8-expressing cells. Specific examples of substances that eliminate CCR8-expressing cells are described below, including (A) an antibody or antibody fragment thereof that binds to CCR8, and (B) cells that have been genetically modified to express a chimeric antigen receptor that binds to CCR8.

[0027] ((A) Antibody or antibody fragment thereof that binds to CCR8) Herein, an antibody that binds to CCR8 is also referred to as an anti-CCR8 antibody. The description of an anti-CCR8 antibody also applies to an antibody fragment of an anti-CCR8 antibody. Therefore, in this specification, even if no reference is made to an antibody fragment of an anti-CCR8 antibody, the description of an anti-CCR8 antibody is understood to be a description of an antibody fragment of an anti-CCR8 antibody.

[0028] An anti-CCR8 antibody is an antibody that specifically binds to CCR8, and an antibody fragment of an anti-CCR8 antibody also preferably specifically binds to CCR8, and more preferably includes an antigen-binding fragment.

[0029] As used herein, the term "antibody" refers to a molecule having a structure in which two heavy chains (H chains) and two light chains (L chains) are associated and stabilized by a pair of disulfide bonds. Each of the two heavy chains consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, and CH3, and a hinge region located between CH1 and CH2. The light chain consists of a light chain variable region VL and a light chain constant region CL. In an antibody, the variable region fragment (Fv) consisting of VH and VL is the region directly involved in binding to an antigen. The antigen-binding region consisting of VL, CL, VH, and CH1 is called the Fab region, and the region consisting of the hinge region, CH2, and CH3 is called the Fc region.

[0030] Within the variable region, the region that directly contacts the antigen undergoes particularly large changes and is called the complementarity-determining region (CDR). The region other than the CDR, which shows relatively little variation, is called the framework region (FR). The light chain and heavy chain variable regions each contain three CDRs (heavy chain CDRs 1-3 and light chain CDRs 1-3).

[0031] Anti-CCR8 antibodies can be produced using the full-length or partial CCR8 protein as an antigen according to known methods for producing antibodies or antisera. Since anti-CCR8 antibodies preferably bind to CCR8 expressed on the cell surface, the partial protein is preferably the extracellular domain of CCR8. These antigens can be prepared by known protein expression and purification methods. Furthermore, for preparing anti-CCR8 antibodies, for example, cells in which CCR8 is forcibly expressed by an expression vector, a CCR8 expression plasmid vector, a CCR8 expression viral vector (adenovirus vector), or the like may be used as an antigen. CCR8 is preferably derived from a mammal. Mammals are not particularly limited, but include, for example, humans, non-human primates, domestic animals, laboratory animals, and livestock, with humans being preferred. The amino acid sequence of human CCR8 is as set forth in SEQ ID NO: 1. The extracellular domain of human CCR8 corresponds to the N-terminal region consisting of amino acids 1 to 35 of SEQ ID NO: 1, the loop 1 region consisting of amino acids 94 to 107 of SEQ ID NO: 1, the loop 2 region consisting of amino acids 172 to 202 of SEQ ID NO: 1, and the loop 3 region consisting of amino acids 264 to 280 of SEQ ID NO: 1.

[0032] The anti-CCR8 antibody is not particularly limited, but examples thereof include monoclonal antibodies and polyclonal antibodies.

[0033] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, produced, for example, by hybridoma techniques, recombinant DNA techniques, phage display techniques, or techniques utilizing transgenic animals containing all or part of the human immunoglobulin loci, or a combination thereof, without limitation.

[0034] As used herein, the term "polyclonal antibody" refers to a mixture of different antibodies that recognize multiple epitopes on a single antigen. Polyclonal antibodies are produced, but are not limited to, by administering an immunogen containing the antigen of interest to mammals (e.g., rats, mice, rabbits, cows, monkeys, etc.) or birds (e.g., chickens, etc.).

[0035] The anti-CCR8 antibody may be conjugated with another drug, i.e., an antibody-drug conjugate (ADC). Furthermore, the antibody conjugated to the drug is not particularly limited as long as it can bind to CCR8, and may be an anti-CCR8 antibody or an antigen-binding fragment of an anti-CCR8 antibody. The drug to be used to construct the ADC is not particularly limited as long as it has the activity of eliminating CCR8-expressing cells, and various drugs such as anticancer drugs, nucleic acid drugs, and immunomodulatory drugs (IMiDs) can be used.

[0036] As used herein, the term "antibody fragment" refers to a molecule comprising a portion of an antibody and capable of binding to the same antigen as the antibody. Examples of antibody fragments include, but are not limited to, Fab, F(ab'), Fv, Fab', single-chain antibody molecules (e.g., scFv), disulfide-stabilized antibodies (dsFv), dimeric V-region fragments (diabodies), and CDR-containing peptides. Furthermore, the antibody fragment may be a VHH antibody (variable domain of heavy chain of heavy chain antibody) as long as it is capable of binding to CCR8.

[0037] Anti-CCR8 antibodies may have a structure known as an antibody and may be any of the isotypes: IgG, IgM, IgA, IgD, or IgE. IgG is an immunoglobulin with a gamma heavy chain and is produced as part of a secondary immune response to an antigen. IgM is an immunoglobulin with a mu heavy chain and exists as a pentamer in mammals. IgA is an immunoglobulin with an alpha heavy chain, IgD is an immunoglobulin with an epsilon heavy chain, and IgE is an immunoglobulin with a delta heavy chain.

[0038] The anti-CCR8 antibody is not particularly limited, and may be, for example, a chimeric antibody, a humanized antibody, or a human antibody, with a humanized antibody or a human antibody being preferred. The antibody fragment of the anti-CCR8 antibody may be, for example, a chimeric antibody fragment, a humanized antibody fragment, or a human antibody fragment. A chimeric antibody is an antibody in which antibody fragments derived from different species are linked. A humanized antibody is an antibody in which a non-human CDR amino acid sequence has been grafted onto a human antibody. The humanized antibody is not particularly limited, and examples thereof include antibodies having heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of an antibody produced by immunizing a mouse or rat, with all other regions derived from a human antibody. The term "humanized antibody" may also refer to a human chimeric antibody.

[0039] In an anti-CCR8 antibody, regions other than the CDRs when CDRs are defined, and regions other than the variable regions when heavy chain variable regions and / or light chain variable regions are defined, may be selected from regions of conventional antibodies, as long as the anti-CCR8 antibody has the ability to bind to CCR8.

[0040] In this embodiment, the anti-CCR8 antibody or antibody fragment thereof is not particularly limited as long as it is derived from an antibody that binds to CCR8, but is preferably derived from an antibody that binds to the extracellular domain of CCR8. Furthermore, the anti-CCR8 antibody or antibody fragment thereof may be a known or commercially available anti-CCR8 antibody or antibody fragment thereof. For example, the following antibodies are known as anti-CCR8 antibodies: 1) Anti-CCR8 antibodies described in WO 2020 / 138489 2) Anti-CCR8 antibodies described in WO 2021 / 194942 3) Anti-CCR8 antibodies described in WO 2021 / 260210 4) Anti-CCR8 antibodies described in WO 2023 / 219147 5) Anti-CCR8 polyclonal antibody manufactured by Funakoshi Co., Ltd. (Product name: CCR8 antibody, Cat. No.: GTX100343) 6) Anti-CCR8 monoclonal antibodies manufactured by Abcam (product name: Anti-CCR8 antibody, product codes: ab32399, ab259792, ab239814, ab278533 and product name: Alexa Fluor™ 647 Anti-CCR8 antibody, product code: ab300676) 7) Anti-CCR8 polyclonal antibody manufactured by Abcam (product name: Anti-CCR8 antibody, product codes: ab140804, ab140796, ab8019) 8) Anti-CCR8 antibody manufactured by Sigma-Aldrich (product name: Anti-CCR8 antibody produced in rabbit IgG fraction of antiserum, product number: AV09059) 9) Anti-CCR8 polyclonal antibody manufactured by Novus Biologicals (product name: CCR8 Antibody, product code: NBP2-15767) Furthermore, the anti-CCR8 antibody or antibody fragment thereof may comprise an amino acid sequence that is 80% or more, 85% or more, 90% or more, or 95% or more identical to the amino acid sequence of the above-mentioned known anti-CCR8 antibody. Furthermore, when one or more amino acids have been deleted, substituted, or added in the amino acid sequence of the above-mentioned known anti-CCR8 antibody, the deletion, substitution, or addition of the amino acids is preferably in a region other than the CDR or a region other than the variable region. Herein, when one or more amino acids are deleted, substituted, or added in an amino acid sequence, "more than one" may mean, for example, 2 to 15, 2 to 10, 3 to 9, 4 to 8, 5 to 7, 2 to 8, 2 to 6, 2 to 5, or 2 to 4.

[0041] In this embodiment, the anti-CCR8 antibody or antibody fragment thereof is preferably a monoclonal antibody or antibody fragment thereof that recognizes tyrosine at position 17 in the amino acid sequence of human CCR8 represented by SEQ ID NO:1 and binds to CCR8.

[0042] In this embodiment, the anti-CCR8 antibody or antibody fragment thereof is CDR1 consisting of the amino acid sequence of SEQ ID NO: 2; CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 and A light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; CDR2 consisting of the amino acid sequence of SEQ ID NO: 6 and A heavy chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 7 Including, The fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is substituted with glutamine, 11th glycine in the amino acid sequence of SEQ ID NO: 2 is substituted with arginine; Preferably, it is a monoclonal antibody or an antibody fragment thereof that binds to CCR8.

[0043] In this embodiment, the anti-CCR8 antibody or antibody fragment thereof is 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2; CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 and A light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; CDR2 consisting of the amino acid sequence of SEQ ID NO: 6 and A heavy chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 7 (wherein one or more of the following substitutions may be present: A) The 10th asparagine in the amino acid sequence of SEQ ID NO: 2 is substituted with lysine. B) The 11th glycine in the amino acid sequence of SEQ ID NO: 2 is substituted with leucine or arginine C) the fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine; 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2; CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 and A light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 8; CDR2 consisting of the amino acid sequence of SEQ ID NO: 6 and a heavy chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; 3) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2; CDR2 consisting of the amino acid sequence of SEQ ID NO: 9 and A light chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 10; CDR1 consisting of the amino acid sequence of SEQ ID NO: 8; CDR2 consisting of the amino acid sequence of SEQ ID NO: 6 and a heavy chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 11; (wherein one or more of the following substitutions may be present: A) The second serine in the amino acid sequence of SEQ ID NO: 2 is replaced with threonine; B) The third serine in the amino acid sequence of SEQ ID NO: 2 is replaced with threonine; C) The fifth serine in the amino acid sequence of SEQ ID NO: 2 is replaced with threonine; D) The second glutamine in the amino acid sequence of SEQ ID NO: 10 is replaced with asparagine; E) The first threonine in the amino acid sequence of SEQ ID NO: 8 is replaced with serine; F) Alanine at position 14 of the amino acid sequence of SEQ ID NO: 6 is replaced with valine; G) The 18th lysine in the amino acid sequence of SEQ ID NO: 6 is replaced with arginine; H) the 19th aspartic acid in the amino acid sequence of SEQ ID NO: 6 is replaced with glutamic acid; I) the fifth asparagine in the amino acid sequence of SEQ ID NO: 11 is replaced with glutamine; J) tyrosine at position 12 of the amino acid sequence of SEQ ID NO: 11 is replaced with phenylalanine; 4) CDR1 consisting of the amino acid sequence of SEQ ID NO: 12; CDR2 consisting of the amino acid sequence of SEQ ID NO: 13 and A light chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 15; CDR2 consisting of the amino acid sequence of SEQ ID NO: 16 and a heavy chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 17; or 5) CDR1 consisting of the amino acid sequence of SEQ ID NO: 18; CDR2 consisting of the amino acid sequence of SEQ ID NO: 19 and A light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 20; and CDR1 consisting of the amino acid sequence of SEQ ID NO: 21; CDR2 consisting of the amino acid sequence of SEQ ID NO: 22 and A heavy chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 23 Preferably, the antibody is a monoclonal antibody or antibody fragment thereof that binds to CCR8 and comprises any one of the CDRs listed above.

[0044] In this embodiment, the anti-CCR8 antibody or antibody fragment thereof is CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, CDR2 consisting of the amino acid sequence of SEQ ID NO: 25 and A light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 26; and CDR1 consisting of the amino acid sequence of SEQ ID NO: 27, CDR2 consisting of the amino acid sequence of SEQ ID NO: 28 and A heavy chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 29 Preferably, the antibody is a monoclonal antibody or antibody fragment thereof that binds to CCR8, comprising:

[0045] In this embodiment, the anti-CCR8 antibody or antibody fragment thereof is a protein having the amino acid sequence of SEQ ID NO: 1; a protein having one or more selected from the group consisting of the amino acid sequence of positions 1 to 35 of SEQ ID NO: 1, the amino acid sequence of positions 94 to 107 of SEQ ID NO: 1, the amino acid sequence of positions 172 to 202 of SEQ ID NO: 1, and the amino acid sequence of positions 264 to 280 of SEQ ID NO: 1; A protein comprising an amino acid sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity with the amino acid sequence of SEQ ID NO: 1, or one or more amino acid sequences selected from the group consisting of the amino acid sequence of positions 1 to 35 of SEQ ID NO: 1, the amino acid sequence of positions 94 to 107 of SEQ ID NO: 1, the amino acid sequence of positions 172 to 202 of SEQ ID NO: 1, and the amino acid sequence of positions 264 to 280 of SEQ ID NO: 1, and which retains the function of CCR8; or A protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 or one or more amino acid sequences selected from the group consisting of the amino acid sequence of positions 1 to 35 of SEQ ID NO: 1, the amino acid sequence of positions 94 to 107 of SEQ ID NO: 1, the amino acid sequence of positions 172 to 202 of SEQ ID NO: 1, and the amino acid sequence of positions 264 to 280 of SEQ ID NO: 1, and which retains the function of CCR8. Preferably, the antibody or antibody fragment thereof binds to

[0046] In this embodiment, the anti-CCR8 antibody may have neutralizing activity against CCR8, and preferably has cytotoxic activity from the viewpoint of removing CCR8-expressing cells. The cytotoxic activity is preferably antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cellular phagocytosis (ADCP), and more preferably ADCC.

[0047] The presence or absence and strength of ADCC activity, CDC activity, and ADCP activity of an anti-CCR8 antibody can be measured by known methods.

[0048] The method for producing an anti-CCR8 antibody or an antibody fragment thereof is not particularly limited, but in the case of a monoclonal antibody, it may be a method in which antibody-producing cells are isolated from a non-human mammal immunized with CCR8 or a fragment thereof, and these are fused with myeloma cells or the like to produce hybridomas, and the antibodies produced by these hybridomas are purified. Furthermore, polyclonal antibodies may be obtained from the serum of an animal immunized with CCR8 or a fragment thereof.

[0049] When an anti-CCR8 antibody or antibody fragment thereof is produced by genetic recombination, for example, suitable host cells may be transformed with an expression vector containing a nucleic acid encoding the anti-CCR8 antibody or antibody fragment thereof, and the resulting transformant may be cultured under appropriate conditions to express the antibody or antibody fragment. The antibody or antibody fragment may then be isolated and purified by known methods or methods equivalent thereto.

[0050] Methods for isolating and purifying antibodies or antibody fragments include, but are not limited to, column purification using an affinity column using protein A or other chromatography columns, filter filtration, ultrafiltration, salting out, and dialysis, and these may be combined as appropriate.

[0051] When an anti-CCR8 antibody has CDRs consisting of amino acid sequences in which one or more amino acids have been added, substituted, or deleted from the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3, or when the anti-CCR8 antibody has CDRs consisting of amino acid sequences that are 80% or more identical to the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3, the anti-CCR8 antibody may be produced using known methods such as site-directed mutagenesis, random mutagenesis, chain shuffling, and CDR walking.

[0052] It is well known to those skilled in the art that antibodies or antibody fragments thereof having various mutations in their CDRs can be displayed on the surface of phages by phage display and then screened using an antigen to obtain antibodies or antibody fragments thereof containing a set of CDRs with more affinity maturity (e.g., Nature Biotechnology 23, 1105 (2005)). Therefore, anti-CCR8 antibodies may be produced by this method.

[0053] Chimeric antibodies, humanized antibodies, and human antibodies may be prepared by known methods or methods similar thereto. The antibody gene may be cloned from the mRNA of a hybridoma that produces an antibody from a non-human animal, and then linked to a part of a human antibody gene by genetic recombination techniques, to prepare the antibody.

[0054] There are no particular limitations on the method for producing chimeric antibodies. For example, chimeric antibodies may be produced using nucleic acids obtained by replacing the heavy chain variable region (VH) and light chain variable region (LH) with sequences derived from a human antibody by genetic recombination. Alternatively, the heavy and light chain constant regions of human IgG1 may be cloned, and for each of the heavy and light chains, for example, the heavy chain variable region (VH) and light chain variable region (LH) may be linked to a human antibody-derived constant region by PCR using the overlapping hanging method. The amplified DNA may then be inserted into an appropriate vector, which may then be transformed to obtain a chimeric antibody.

[0055] To produce a humanized antibody, the variable region of a human antibody is cloned and then modified to the CDR sequence of an anti-CCR8 antibody by site-directed mutagenesis using the megaprimer method. If humanization of the amino acid sequence constituting the framework region results in a loss of specific binding to the antigen, some amino acids in the framework may be converted from human to rat types.

[0056] CDRs consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of the original CDR, or CDRs consisting of an amino acid sequence that is 80% or more identical to the original sequence, may be prepared using known methods such as site-directed mutagenesis, random mutagenesis, chain shuffling, and CDR walking.

[0057] Antibody fragments of anti-CCR8 antibodies may be expressed by the above-mentioned methods using nucleic acids encoding the fragments, or full-length antibodies may be obtained and then fragmented by treating them with enzymes such as papain and pepsin.

[0058] Whether the anti-CCR8 antibody or antibody fragment thereof obtained by the above production method is an anti-CCR8 antibody can be confirmed, for example, by using as an indicator that the CCR8 protein in a sample can be detected by an immunological assay using the anti-CCR8 antibody or antibody fragment thereof. Immunoassays for detecting a target protein by immunological measurement are well known in the art. Reagents required for each immunoassay are also well known, and immunological detection of CCR8 protein may be carried out using an anti-CCR8 antibody or an antibody fragment thereof as the antibody used in the immunoassay and a conventional immunoassay kit. The immunoassay is not particularly limited, but examples thereof include immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunoblotting, and the like.

[0059] (B) Cells genetically engineered to express a chimeric antigen receptor that binds to CCR8. Herein, a chimeric antigen receptor (CAR) is also referred to as a chimeric antigen receptor (CAR). A CAR is an artificial chimeric protein having, in order from the N-terminus of the CAR, an extracellular domain, a transmembrane domain, and an intracellular domain. Here, the anti-CCR8 antibody may be the same as that described above.

[0060] The extracellular domain preferably comprises, in order from the N-terminus of the CAR, an antigen-binding domain and a hinge region. The antigen-binding domain of the CAR of this embodiment that binds to CCR8 is not particularly limited as long as it can bind to CCR8. However, it preferably comprises the antigen-binding region of the anti-CCR8 antibody, more preferably comprises the heavy chain variable region and / or the light chain variable region of the anti-CCR8 antibody, and particularly preferably comprises the complementarity-determining region of the anti-CCR8 antibody. Furthermore, the antigen-binding domain preferably has a single-chain antibody (scFv) structure comprising the heavy chain variable region and the light chain variable region of the anti-CCR8 antibody. In the scFv, either the heavy chain variable region or the light chain variable region may be located on the N-terminus of the CAR, and a linker peptide consisting of any oligopeptide or polypeptide may be inserted between the heavy chain variable region and the light chain variable region. The linker peptide is not particularly limited, but examples include peptides consisting of 1 to 100 amino acid residues, and preferably a peptide consisting of 10 to 50 amino acid residues.

[0061] The CAR that binds to CCR8 of this embodiment preferably has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 29.

[0062] The hinge region is not particularly limited, but may be, for example, a peptide consisting of 1 to 100 amino acid residues, preferably 10 to 70 amino acid residues. As the hinge region of the CAR, for example, the constant region of an anti-CCR8 antibody may be used as the hinge, or an amino acid sequence derived from a molecule other than an anti-CCR8 antibody (e.g., CD8, CD28, etc.) may be selected.

[0063] The transmembrane domain is a structure that anchors the CAR to the cell membrane. Examples of the transmembrane domain of the CAR include, but are not limited to, polypeptides derived from proteins such as CD8, the α and β chains of the T cell receptor, and CD28.

[0064] The intracellular domain preferably has a costimulatory domain and a signal transduction domain. The costimulatory domain is not particularly limited, but examples thereof include costimulatory factors contained in T cells, such as CD28 and 4-1BB. The signal transduction domain is not particularly limited, but examples thereof include structures derived from CD3ζ and the like.

[0065] A spacer region consisting of any oligopeptide or polypeptide may be provided between the transmembrane domain and the intracellular domain. The spacer region is not particularly limited, but may be, for example, a peptide consisting of 1 to 100 amino acid residues, preferably 10 to 50 amino acid residues.

[0066] Cells genetically modified to express a chimeric antigen receptor that binds to CCR8 include cells genetically modified to express the above-mentioned polynucleotide encoding a chimeric antigen receptor that binds to CCR8.

[0067] From the viewpoint of eliminating CCR8-expressing cells, it is preferable that cells genetically modified to express a chimeric antigen receptor that binds to CCR8 have cytotoxic activity, and examples of such cells include T cells and natural killer cells, with T cells being preferred. T cells genetically modified to express a chimeric antigen receptor are also called CAR-T cells.

[0068] Preferably, when the genetically modified cells express the polynucleotide encoding the chimeric antigen receptor that binds to CCR8, the extracellular domain of the chimeric antigen receptor is exposed to the outside of the cell, and the transmembrane domain and intracellular domain are localized at the cell membrane or within the cell.

[0069] Methods for genetically modifying cells to express a chimeric antigen receptor can be, for example, methods known as methods for producing CAR-T cells.

[0070] One aspect of this embodiment is a chimeric antigen receptor that binds to CCR8. One aspect of this embodiment is a polynucleotide encoding a chimeric antigen receptor that binds to CCR8. One aspect of this embodiment is a cell harboring a polynucleotide encoding a chimeric antigen receptor that binds to CCR8. In one aspect of this embodiment, the cells harboring a polynucleotide encoding a chimeric antigen receptor that binds to CCR8 are chimeric antibody receptor T cells (CAR-T cells).

[0071] (Therapeutic or preventive agent) The therapeutic or preventive agent for HAM of this embodiment contains a substance that eliminates CCR8-expressing cells. Here, the substance that eliminates CCR8-expressing cells may be the same as those described above.

[0072] As used herein, the term "treatment or prophylaxis" refers to therapeutic and / or prophylactic treatment. The term "therapeutic or prophylactic treatment" is art-recognized and may include administration of a therapeutic or prophylactic agent to a subject. Therapeutic treatment may refer to administration after the onset of an undesired condition (e.g., a disease or other undesired condition of interest) and may be intended to attenuate, suppress, reverse, alleviate, or stabilize an existing undesired condition or its side effects. It also includes prevention of attenuation of any direct or indirect pathological effects of the disease. It may also refer to treatment to delay the onset of the disease or slow the progression of the disease. Prophylactic treatment may refer to administration prior to clinical manifestation of an undesired condition and may be intended to inhibit and / or prevent the recurrence of the undesired condition.

[0073] As used herein, the term "therapeutic or prophylactic agent" refers to a pharmaceutical composition for treatment or prevention that contains an active ingredient and additives (such as pharmaceutically acceptable ingredients), and may refer to a pharmaceutical formulation or preparation commonly used in the art. In this embodiment, the therapeutic or prophylactic agent contains at least a substance that removes CCR8-expressing cells as an active ingredient.

[0074] In this embodiment, the effect of treating or preventing HAM is achieved by removing CCR8-expressing cells. Cell removal may mean physically removing, damaging, or killing CCR8-expressing cells. Damaging CCR8-expressing cells is not particularly limited, but includes, for example, damaging CCR8-expressing cells through antibody-dependent cellular cytotoxicity (ADCC activity), complement-dependent cytotoxicity (CDC activity), antibody-dependent cellular phagocytosis (ADCP activity), and the like. ADCC activity is the activity in which, when an antibody binds to a cell expressing a target antigen, the antibody recruits immune cells in the body, such as natural killer (NK) cells and macrophages, to the vicinity, and induces the immune cells to kill the cell to which the antibody is bound. CDC activity is an activity in which an antibody recruits complement to its vicinity and induces damage to the cells to which the antibody is bound through the action of the complement. ADCP activity is an activity in which an antibody recruits macrophages to its vicinity and induces the recruited macrophages to phagocytose cells present in the vicinity of the antibody. The presence or absence of ADCC activity, CDC activity, and ADCP activity, and the strength of these activities can be measured by known methods.

[0075] One aspect of this embodiment is a therapeutic agent for HAM. Treatment of HAM is not particularly limited, but may mean that the number of HTLV-1-infected cells in a subject is reduced, HTLV-1-infected cells are eliminated, or proliferation of HTLV-1-infected cells is suppressed, or various symptoms caused by HAM (e.g., inflammation) are improved, by single-agent administration of the therapeutic agent of this embodiment or combination therapy, and preferably means that the number of HTLV-1-infected cells is reduced or HTLV-1-infected cells are eliminated.

[0076] One aspect of this embodiment is a preventive agent for HAM. Prevention of HAM is not particularly limited, and may mean preventing the onset of HAM in asymptomatic HTLV-1-infected individuals or suppressing the recurrence of HAM by single-drug administration or combination therapy of the preventive agent of this embodiment, and preferably means preventing an increase in the number of HTLV-1-infected cells due to regrowth of reduced HTLV-1-infected cells, preventing the recurrence of various symptoms caused by improved HAM, or preventing the onset of HAM in asymptomatic HTLV-1-infected individuals.

[0077] The therapeutic or prophylactic agent of this embodiment contains a substance that removes CCR8-expressing cells, and may further contain a pharmaceutically acceptable carrier and / or additive. The proportion of the carrier or additive may be appropriately set based on the range commonly used in the pharmaceutical field. The carrier is not particularly limited, but examples thereof include water, physiological saline, other aqueous solvents, and aqueous or oily bases. The additives are not particularly limited, but examples thereof include excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavoring agents, and fragrances.

[0078] The form of the therapeutic or prophylactic agent is not particularly limited, and examples thereof include oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, and parenteral preparations such as injections, infusions, suppositories, ointments, and patches.

[0079] The method of administration of the therapeutic or prophylactic agent is not particularly limited, but is preferably administered as an injection or infusion via a parenteral administration route, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or topically, and more preferably topically. Injectables or infusions containing a substance that removes CCR8-expressing cells can be used as a solution, suspension, or emulsion. Examples of solvents include distilled water for injection, physiological saline, glucose solution, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.). These solvents may be used alone or in combination of two or more.

[0080] Furthermore, the injection or infusion may contain additives such as stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, and pH adjusters. The stabilizer is not particularly limited, but examples thereof include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and dibutylhydroxytoluene. The solubilizing agent is not particularly limited, but examples thereof include alcohols (e.g., ethanol, etc.), polyalcohols (e.g., propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, etc.). The suspending agent is not particularly limited, but examples thereof include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. The emulsifier is not particularly limited, but examples thereof include gum arabic, sodium alginate, and tragacanth. The soothing agent is not particularly limited, but examples thereof include benzyl alcohol, chlorobutanol, and sorbitol. The buffer is not particularly limited, but examples thereof include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, and Tris buffer. The preservative is not particularly limited, but examples thereof include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. The preservative is not particularly limited, but examples thereof include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. The pH adjuster is not particularly limited, but examples thereof include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.

[0081] The dosage and administration interval of the active ingredient of the therapeutic or prophylactic agent of this embodiment can be appropriately selected depending on the subject to be administered, the administration route, the disease, and the age, weight, and symptoms of the subject. For example, in the case of parenteral administration, the dosage of the active ingredient for an adult is, but is not particularly limited to, 0.01 mg to 10 g, or may be 100 mg to 6 g, or may be 50 mg to 500 mg per day. The administration interval of the therapeutic or prophylactic agent may be such that the daily dosage is administered once a day, or may be administered in divided doses several times a day.

[0082] The subject to which the therapeutic or prophylactic agent of this embodiment is administered is a mammal. Mammals are not particularly limited, but include, for example, humans, non-human primates, domestic animals, laboratory animals, and livestock, and are preferably humans.

[0083] The therapeutic or prophylactic agent of this embodiment may be administered in combination with one or more other agents. Examples of other agents include, but are not limited to, known HAM therapeutic agents. Examples of known HAM therapeutic agents include, but are not limited to, interferon α, mogamulizumab, teriflunomide, and humanized anti-RGMa antibodies.

[0084] In the combined administration, the therapeutic or prophylactic agent of this embodiment and the other agent may be administered at different times. When administered simultaneously, two or more active ingredients may be in the same formulation, or may be administered as separate formulations. When administered at separate times, the two or more active ingredients may be administered according to the desired administration regimen, with the dosage and interval of the other active ingredient being in accordance with the prescribed administration regimen.

[0085] It has been found that peripheral blood mononuclear cells (PBMCs) from HAM patients spontaneously proliferate when cultured under unstimulated conditions due to the proliferation of HTLV-1-infected cells and the immune cell response thereto. This phenomenon is known as a useful model reflecting the pathology of HAM. Therefore, in one aspect, the therapeutic or prophylactic agent of this embodiment can inhibit the spontaneous proliferation activity of HTLV-1-infected cells. In one aspect, the therapeutic or prophylactic agent of this embodiment can suppress the spontaneous proliferation activity of HTLV-1-infected cells.

[0086] In any of the above embodiments described herein, the substance that eliminates CCR8-expressing cells may be any of the embodiments described herein, as well as the embodiments described herein as preferred, more preferred, and particularly preferred (all of these embodiments are hereinafter collectively referred to as "preferred embodiments"), or any combination of preferred embodiments.

[0087] The amino acid sequences represented by SEQ ID NOs: 1 to 29 are as follows. [Table 1] [Example]

[0088] Some examples of the above-described embodiments of the present invention are described below. However, the present invention is not limited to these examples.

[0089] [Example 1: Single-cell RNA-seq analysis] Cerebrospinal fluid was extracted from three HAM patients and cells were collected by centrifugation (500 x g, 10 min). Following the protocol of the Chromium Single Cell 5' Reagent Kit (10X GENOMICS), the collected cells were mixed with gel beads (Single Cell VDJ 5' Gel Beads) bearing a unique molecular identifier (UMI) and a barcode (10x Barcode) for cell identification in the wells of a Chromium Next GEM Chip K. Using a Chromium Controller (10X GENOMICS), an oil drop containing only one cell and one gel bead (gel bead-in-emulsion: GEM) was formed. Cells were enzymatically lysed in the GEM, mRNA was reverse transcribed, and the reverse-transcribed cDNA was captured from the base sequence extending from the gel bead. After washing the gel beads, the cDNA containing 10x Barcode and UMI was amplified by polymerase chain reaction (PCR) to generate a cell-level cDNA library. The resulting cDNA library was then adapted using the Chromium Dual Index Kit TT set A (10X GENOMICS) and sequenced using a NovaSeq6000 (Illumina). The resulting sequence data was analyzed (sequence read mapping and alignment) using the Cell Ranger (10X GENOMICS) analysis software pipeline on the SHIROKANE supercomputer (The Institute of Medical Science, The University of Tokyo) to obtain cell-level gene expression information (10,945 cells).Furthermore, we integrated the data from three HAM patient CSF cell cases using Seurat (https: / / satijalab.org / seurat / ), an R package for single-cell RNA-seq analysis. Then, we performed cell clustering using Uniform Manifold Approximation and Projection (UMAP) and performed cell annotation for each cluster (Figure 1). As a result, we identified various cell types contained in the CSF of HAM patients (CD8 T cells, cytotoxic CD4 T cells, CD4 HTLV-1-infected T cells, myeloid cells, B cells, CD4 type 1 helper T cells; CD4-Th1, CD4 CD8 double-positive cells; DP, regulatory T cells; Treg). Independent clusters of HTLV-1-infected cells were detected.

[0090] [Example 2: CCR8 gene expression specific to HTLV-1-infected cells] Using Seurat, we compared the gene expression profiles of HTLV-1-infected cell clusters and Th1 clusters, and plotted all genes as relative differences (horizontal axis) and significant differences (vertical axis) in gene expression levels (Figure 2). Furthermore, in the plots displayed by UMAP, cells expressing the HBZ gene (representing HTLV-1-infected cells) and the CCR8 gene are shown in dark gray (Figure 3). These results demonstrate that the CCR8 gene is specifically expressed in HTLV-1-infected cells. The present inventors focused on CCR8, for which antibody drugs are being developed as a tumor immunotherapy method.

[0091] Example 3: Comparison of CCR8 expression in HTLV-1-infected cells in HTLV-1-associated diseases CCR8 expression was detected in HTLV-1-infected T cells in peripheral blood samples from patients with HTLV-1-associated diseases, HAM (n=3), adult T-cell leukemia-lymphoma (ATL) (n=3), HTLV-1-associated uveitis (HU) (n=2), and asymptomatic HTLV-1-infected individuals (n=3) using flow cytometry (BD Biosciences, BD FACSCelesta). Detection of infected cells was performed using the HAS-Flow method, which combines the expression of CD3, CD4, CD7, and CADM1 (Kobayashi S et al, Clin Cancer Res, 2014). HTLV-1-infected T cells were CD4 + CADM1 + CD7 + The D fraction and CD4 + CADM1 + CD7 - The N fraction is a combined fraction of uninfected T cells, CD4 + CADM1 - CD7 + is indicated as P fraction. A schematic diagram of the indications of D fraction, N fraction, and P fraction is shown in Figure 4.

[0092] The analysis results are shown in Figures 5 and 6. Figure 5 shows the CCR8 expression rate in HTLV-1-uninfected and HTLV-1-infected cells by disease as a bar graph, with the vertical axis representing the CCR8 expression rate. Figure 6 shows representative plots of CCR8 expression in infected cell fractions (D+N fractions) for each disease. ns indicates not significant, and ** indicates P < 0.01. Statistical analysis was performed using the Mann-Whitney U-test to compare two groups: HAM and other diseases. We found that most HTLV-1-infected cells from HAM patients highly expressed CCR8. Approximately half of the HTLV-1-infected cells from asymptomatic HTLV-1-infected cell carriers and HU patients expressed CCR8, while ATL cells tended to have suppressed CCR8 expression.

[0093] The antibodies used are as follows: Anti-CD4 antibody PE-Cy7 (Biolegend, clone:OKT4), Anti-CD3 antibody APC-Cy7 (TONBO, clone: ​​UCHT1), Anti-CD7 antibody (BD Biosciences, clone:M-T701), Anti-CADM1 antibody conjugated with biotin (MBL, clone:3E1), Anti-CCR8 antibody (BD Biosciences, clone:433H), BV421 Streptavidin (Biolegend)

[0094] Example 4: CD4 + CCR8 + Measurement of proviral load in cell populations] Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of HAM patients using Pancoll Human (PAN BIOTECH), and dead cells and doublets were removed using a FACS Aria II (BD Biosciences). + CCR8 + The PBMCs before sorting and the CD4+ SSC-high ... + CCR8 + Cell population, CD4 + CCR8 + DNA was extracted from each cell population after removing the cells, and the percentage of HTLV-1-infected cells in the population was calculated by quantitative PCR (Figure 8). The calculation method is described in the paper by Nakashima M et al., Clin Cancer Res, 2018 (doi: 10.1158 / 1078-0432.CCR-18-0268), and absolute quantification was performed using HTLV-1 plasmid. Figure 8 shows that HTLV-1-infected cells in HAM patients were CD4 + CCR8 +It was found to be concentrated in the fraction.

[0095] The following primers and probes were used: pX-probe(FAM) 5'-CTGTGTACAAGGCGACTGGTGCC-3' (SEQ ID NO: 30) pX2-sense 5'-CGGATACCCAGTCTACGTGTT-3' (SEQ ID NO: 31) pX2-antisense 5'-CAGTAGGGCGTGACGATGTA-3' (SEQ ID NO: 32) Taqman RNase P Control Reagents Kit (ThermoFisher Scientific)

[0096] Example 5: CD4 + CCR8 + Promoting spontaneous cell proliferation by cells] As described in Example 4, CD4 + CCR8 + The cell population was removed and cultured in a 96-well U-bottom dish (Falcon) at 2 x 10 5 The cells were cultured at a cell concentration of 200 μl in RPMI 1640 + 10% Fetal Bovine Serum (FBS) + antibiotics (1% Penicillin-Streptomycin) for 7 days. After 7 days, cell proliferation was measured using the CellTiter-Blue Cell Viability Assay (Promega) on a Varioskan LUX multimode microplate reader (ThermoFisher Scientific) (Figure 9). PBMCs before sorting were measured as a control sample. The average cell proliferation rates for three cases are shown as a bar graph (Figure 10). Statistical significance was assessed by paired t-test. * indicates P < 0.05. From PBMCs of HAM patients, CD4 + CCR8 +It was found that removal of the cells significantly reduced spontaneous cell proliferation, indicating that removal of CCR8-expressing cells can treat or prevent HAM.

[0097] Example 6: CD4 + Foxp3 + CCR8 expression rate in cells] PBMCs from three HAM patients were incubated with anti-CD4 antibody PE-Cy7 (Biolegend, clone OKT4) and anti-CCR8 antibody PE (BD Biosciences, clone 433H) at 4°C for 20 min, followed by cell fixation and permeabilization using eBioscience Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher Scientific), and then incubated with anti-Foxp3 antibody APC (ThermoFisher Scientific, clone 236A / E7) at 4°C for 30 min. + Foxp3 + The CCR8 positivity rate in the cells was detected by flow cytometry (BD Biosciences, BD FACSCelesta). + Foxp3 + The vertical axis of the plot on the left in Figure 11 is 10K. + Foxp3 + CCR8 + Cells and CD4 + Foxp3 + CCR8 - The percentage of CCR8-positive and CCR8-negative Treg cells is shown as a bar graph. These results suggest that Treg cells include CCR8-positive cells and CCR8-negative cells, and that the therapeutic or preventive method of this embodiment, which removes CCR8-expressing cells, has minimal side effects when it comes to removing Treg cells.

[0098] Example 7: Examination of CCR8-expressing cells in PBMCs from healthy individuals CCR8-expressing cells were detected based on single-cell RNA-seq analysis data of PBMCs from healthy individuals provided by 10X GENOMICS. Similar to Example 1, clustering was performed by UMAP using Cell Ranger and Seurat (Figure 13). In the plot displayed by UMAP, cells expressing CCR8, CCR4, GATA3, and FOXP3 are displayed in dark gray (Figure 14). The horizontal axis of Figures 13 and 14 represents UMAP_1, and the vertical axis represents UMAP_2. In single-cell analysis of PBMCs from healthy individuals, CCR8 was expressed in a subset of Th2 (CD4 + CCR4 + GATA3 + ), suggesting that the side effects of the therapeutic or preventive agent of this embodiment are minimal.

[0099] [Example 8: Evaluation of antigen specificity of CCR8] Based on the results of single-cell RNA-seq analysis published in The Human Protein Atlas (https: / / www.proteinatlas.org / ), the normalized CCR8 gene expression levels in various cell types (80 types) are shown in a bar graph (Figure 15). The black arrow indicates the CCR8 expression level in T cells, while the white arrow indicates the expression level in lymphatic endothelial cells, granulocytes, and erythrocytes. Figure 16 shows data obtained from PBMC analysis. Some platelets were detected, and CCR8 expression is shown in dark gray, but no CCR8 expression was observed in platelets. The above results suggest that the therapeutic or prophylactic agent of this embodiment has minimal hematotoxic side effects, since CCR8 expression was not observed in endothelial cells, granulocytes, erythrocytes, or platelets.

Claims

1. A therapeutic or preventive agent for HTLV-1 associated myelopathy (HAM), comprising a substance that eliminates CCR8-expressing cells.

2. The substance is (A) an antibody or antibody fragment thereof that binds to CCR8, and (B) Cells genetically engineered to express a chimeric antigen receptor that binds to CCR8. At least one of The therapeutic or prophylactic agent according to claim 1.

3. the substance comprises the antibody (A) or an antibody fragment thereof, and the antibody (A) or an antibody fragment thereof has ADCC activity, CDC activity, or ADCP activity; The therapeutic or prophylactic agent according to claim 2.

4. The therapeutic or prophylactic agent according to any one of claims 1 to 3, wherein the cells to be removed are cells expressing both CCR8 and CD4.

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