TW202423987A - Anti-ccr8 antibody and application thereof - Google Patents

Abstract

The present disclosure relates to the technical field of biotechnology, and provides an anti-human CCR8 antibody and application thereof. The anti-human CCR8 antibody provided in the present disclosure includes a heavy chain complementary determining region and a light chain complementary determining region. The anti-CCR8 antibody can specifically bind to human CCR8 antigen and can be used to treat tumors.

Description

Anti-CCR8 antibodies and their applications

[Priority Claim]

This application claims the priority and benefits of the Chinese patent application numbered 202211578659.6, filed with the National Intellectual Property Administration of China on December 7, 2022, entitled "Anti-CCR8 Antibodies and Their Applications", and the entire text of which is incorporated into this application by reference.

The present disclosure relates to the field of biotechnology, and specifically, to an anti-CCR8 antibody and its application.

As people's understanding of the tumor immune microenvironment continues to improve, the role of Treg (regulatory T cell) cells in inhibiting the body's anti-tumor immune response has become increasingly important.

Chemokine receptor 8 (CCR8) is a seven-transmembrane G protein-coupled receptor that belongs to the CC subfamily of chemokine receptors. Some studies in recent years have shown that in various mouse tumor models and human tumor tissues, only tumor tissue-infiltrating Treg cells widely and highly express CCR8, while Treg cells in peripheral blood and other PBMCs (Peripheral Blood Mononuclear Cells) do not express CCR8. CCR8 has been regarded as a surface marker of tumor-infiltrating Treg. Targeting CCR8 is a promising cancer immunotherapy method. However, CCR8 is a seven-transmembrane GPCR (G protein-coupled receptor) receptor with a very short and discontinuous extracellular end. Compared with general single-transmembrane proteins, it is more difficult to prepare antibodies.

The present invention discloses an anti-CCR8 antibody. In some embodiments, the anti-CCR8 antibody can enhance the body's anti-tumor immune response and improve the survival rate of tumor patients.

In some embodiments, the present disclosure discloses an anti-human CCR8 antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, 3, 5, 7, 9 or 11, and/or the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.2, 4, 6, 8, 10 or 12.

In some embodiments, wherein, A. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.2; B. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.3, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.4; C. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.5, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.6; D. the heavy chain variable region of the antibody includes SEQ ID E. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.7, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.8; E. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.9, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.10; or F. The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.11, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.12.

In some embodiments, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system; in some embodiments, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system. In some embodiments, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the IMGT numbering system.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items, wherein, a. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, HCDR2 comprises the amino acid sequence of SEQ ID NO.14, and HCDR3 comprises the amino acid sequence of SEQ ID NO.15; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18; b. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.19, HCDR2 comprises the amino acid sequence of SEQ ID NO.20, and HCDR3 comprises the amino acid sequence of SEQ ID NO.21; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18; c. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.22, HCDR2 comprises the amino acid sequence of SEQ ID NO.23, and HCDR3 comprises the amino acid sequence of SEQ ID NO.24; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.25, LCDR2 comprises the amino acid sequence of SEQ ID NO.26, and LCDR3 comprises the amino acid sequence of SEQ ID NO.27; d. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, HCDR2 comprises the amino acid sequence of SEQ ID NO.28, and HCDR3 comprises the amino acid sequence of SEQ ID NO.29; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.30, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18; e. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31, HCDR2 comprises the amino acid sequence of SEQ ID NO. ID NO.32, and HCDR3 comprises the amino acid sequence of SEQ ID NO.33; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34, LCDR2 comprises the amino acid sequence of SEQ ID NO.35, and LCDR3 comprises the amino acid sequence of SEQ ID NO.36; or f. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, HCDR2 comprises the amino acid sequence of SEQ ID NO.37, and HCDR3 comprises the amino acid sequence of SEQ ID NO.38; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO. LCDR1, LCDR2 and LCDR3 shown in NO.27.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items is a murine antibody, a chimeric antibody or a humanized antibody.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No.1, 3, 5, 7, 9 or 11, and/or the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No.2, 4, 6, 8, 10 or 12.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No.1, 3, 5, 7, 9 or 11, and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No.2, 4, 6, 8, 10 or 12.

In some embodiments, the present disclosure provides an anti-human CCR8 antibody, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID No. 2, 4, 6, 8, 10 or 12.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No.1, 3, 5, 7, 9 or 11, and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No.2, 4, 6, 8, 10 or 12.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items, (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.2; (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.4; (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.6; (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.8; (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.10; (vi) the heavy chain variable region comprises SEQ The amino acid sequence of SEQ ID NO.11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.12.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items further comprises a constant region; in some embodiments, the heavy chain constant region of the antibody is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is selected from the κ or λ chain constant region; in some embodiments, the species origin of the constant region is mouse or human; in some embodiments, the heavy chain constant region is mouse IgG2a constant region, mouse IgG1 constant region, human IgG1 constant region; in some embodiments, the heavy chain constant region is human IgG1 (DLE) or human IgG1 (DE) constant region; and/or the light chain constant region is mouse κ constant region or human κ constant region.

In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO.39, 41 or 42, and the light chain constant region comprises the amino acid sequence of SEQ ID NO.40 or 43.

In some embodiments, the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.44, and the light chain comprises the amino acid sequence of SEQ ID NO.45; or the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.46, and the light chain comprises the amino acid sequence of SEQ ID NO.47; or the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.48, and the light chain comprises the amino acid sequence of SEQ ID NO.49.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items is a full-length antibody or any antigen-binding fragment selected from F(ab')2, Fab'-SH, Fab', Fab, scFab, dsFv, (dsFv)2, Fv and scFv.

In some embodiments, the invention discloses an antibody that competitively binds to human CCR8 with any of the anti-human CCR8 antibodies described above, or an antibody that binds to the same epitope as any of the anti-human CCR8 antibodies described above.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items, wherein the anti-human CCR8 antibody has at least one of the following properties: A. The anti-human CCR8 antibody can specifically bind to human CCR8; In some embodiments, the anti-human CCR8 antibody can bind to human CCR8 with an EC50 value (Concentration for 50% of Maximal Effect) binds to Raji cells expressing human CCR8, wherein the EC50 value is determined by flow cytometry; in some embodiments, the EC50 value is determined by the method of Example 2 of this application; B. The anti-human CCR8 antibody has the function of inhibiting tumor growth; in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is greater than or equal to 30% (for example, greater than 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or greater); in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is determined by the method of Example 5 of this disclosure; C. The anti-human CCR8 antibody has ADCC (Antibody-dependent cellular cytotoxicity) activity; in some embodiments, the anti-human CCR8 antibody ADCC activity is determined by the method of Example 4 of this disclosure.

In some embodiments, the anti-human CCR8 antibody as described in any of the above items, wherein the anti-human CCR8 antibody can specifically bind to human CCR8; in some embodiments, the anti-human CCR8 antibody can bind to Raji cells expressing human CCR8 with an EC50 value of ≤5nM (e.g., ≤4.50 nM, ≤3.00 nM, ≤2.00 nM, ≤1.00 nM, ≤0.9nM, ≤0.8nM, ≤0.7nM, ≤0.6nM, ≤0.5nM, ≤0.4nM, ≤0.3nM, ≤0.2nM, ≤0.1nM, ≤0.09nM, ≤0.08nM or less), wherein the EC50 value is determined by flow cytometry.

In some embodiments, the EC50 value is determined by the method of Example 2 of this application.

In some embodiments, the anti-human CCR8 antibody described in any of the above items has the function of inhibiting tumor growth.

In some embodiments, the anti-human CCR8 antibody tumor inhibition rate is greater than or equal to 30% (e.g., greater than 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more).

In some embodiments, the anti-human CCR8 antibody tumor inhibition rate is determined by the method of Example 5 of the present disclosure.

In some embodiments, the anti-human CCR8 antibody described in any of the above items has ADCC activity.

In some embodiments, the anti-human CCR8 antibody ADCC activity is determined by the method of Example 4 of the present disclosure.

The present disclosure also provides a multispecific antibody, which comprises the anti-human CCR8 antibody described in any of the above items.

In some embodiments, the antibody is a bispecific antibody.

The present disclosure also provides an antibody conjugate, which comprises the anti-human CCR8 antibody described in any of the above items.

In some embodiments, the antibody conjugate further comprises a therapeutic agent or an imaging agent conjugated to the antibody.

In some embodiments, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

In some embodiments, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

In some embodiments, the antibody conjugate further comprises a label conjugated to the antibody.

In some embodiments, the label is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

In some embodiments, the label is colloidal gold.

The present disclosure also provides a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain, and the antigen binding domain comprises the anti-human CCR8 antibody described in any of the above items.

The present disclosure also provides a CAR-immune cell, which expresses the chimeric antigen receptor described above.

The present disclosure also provides an isolated nucleic acid encoding the anti-human CCR8 antibody described in any of the above items.

The present disclosure also provides a cell comprising the aforementioned nucleic acid.

The present disclosure also provides a pharmaceutical composition comprising: the anti-human CCR8 antibody, multispecific antibody, antibody conjugate, cell or nucleic acid described in any one of the above items.

In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

The present disclosure also provides the use of any of the above-mentioned anti-human CCR8 antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids or drug compositions in the preparation of products having at least one of the following uses: diagnosing diseases related to abnormal human CCR8 expression, treating diseases related to abnormal human CCR8 expression or detecting human CCR8 expression.

In some embodiments, the disease related to abnormal human CCR8 expression is a tumor. The tumor with abnormal human CCR8 expression is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.

In some embodiments, the present disclosure provides a method for treating a disease, comprising the step of administering to a subject in need thereof a therapeutically effective amount of any of the above anti-human CCR8 antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids or drug compositions.

In some embodiments, the disease is a disease related to abnormal expression of human CCR8.

In some embodiments, the disease is a tumor. The tumor in which human CCR8 expresses abnormally is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.

In another aspect, the present disclosure also provides any of the aforementioned anti-human CCR8 antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids or drug compositions for use as a drug.

In some embodiments, the drug is used to treat a disease. In some embodiments, the disease is a disease related to abnormal human CCR8 expression. In some embodiments, the disease related to abnormal human CCR8 expression is a tumor. The tumor with abnormal human CCR8 expression is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.

In some embodiments, the treatment described in any of the above items further comprises administering an additional therapeutic drug to the subject.

In some embodiments, the additional therapeutic agent is a chemotherapy agent, such as a platinum complex, a taxane, pemetrexed, gemcitabine, fluorouracil, irinotecan, etanercept, or doxorubicin.

The present disclosure also provides a method for detecting or measuring human CCR8, which comprises the step of detecting or measuring human CCR8 using the anti-human CCR8 antibody or antibody conjugate as described in any of the preceding items.

The present disclosure also provides a kit comprising the anti-human CCR8 antibody or antibody conjugate as described in any of the preceding items; in some embodiments, the kit is used to detect or measure human CCR8.

The present disclosure also provides a method for preparing the anti-human CCR8 antibody as described in any of the preceding items, which comprises the steps of culturing the cells as described in any of the preceding items, and then isolating and purifying the anti-human CCR8 antibody.

The anti-human CCR8 antibody disclosed in the present disclosure can specifically bind to human CCR8; in some embodiments, the antibody has significant anti-tumor effects both in vitro and in vivo; in some embodiments, it also has good ADCC activity.

In order to make the purpose, technical scheme and advantages of the disclosed embodiments clearer, the technical scheme in the disclosed embodiments is described clearly and completely below, but the description and embodiments should not be interpreted as limiting the scope of the disclosure. If no specific conditions are specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the field to which the disclosure belongs.

In this disclosure, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. In addition, the terms "first", "second", and "first" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. The term "plurality" means at least two, such as 2, 3, etc., unless otherwise clearly and specifically limited.

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

The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs or amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine; common natural amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamate (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure (i.e., alpha carbon bound to hydrogen, carboxyl, amino, and R groups) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfonate, and methionine methyl sulfonate. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

In the present disclosure, the term "antibody" is used in the broadest sense, and the term covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, chimeric antibodies, humanized antibodies, full-length antibodies or antigen-binding fragments thereof (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

"Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains. From N to C terminus, each heavy chain has a heavy chain variable region (VH, also called a variable heavy domain), followed by a heavy chain constant region, and the natural IgG heavy chain constant region usually includes three constant domains (CH1, CH2, and CH3). Similarly, from N to C terminus, each light chain has a light chain variable region (VL, also called a variable light domain), followed by a light chain constant region (CL, also called a light chain constant domain).

The term "full-length antibody" or "intact antibody" refers to an antibody that comprises a structure substantially similar to a native antibody structure, or an antibody whose rechain comprises an Fc region.

The term antibody "variable region" or "variable domain" refers to the domain of the antibody heavy chain or light chain involved in the antibody binding to the antigen. Herein, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved framework regions (FR) and three complementary determining regions (CDR). The term "complementary determining region" or "CDR" refers to the region in the variable region that mainly contributes to the specific binding to the antigen; "framework" or "FR" refers to the variable domain residues in the variable region excluding the CDR residues. VH contains 3 CDR regions: HCDR1 (heavy chain complementation determining region 1), HCDR2 (heavy chain complementation determining region 2) and HCDR3 (heavy chain complementation determining region 3); VL contains 3 CDR regions: LCDR1 (light chain complementation determining region 1), LCDR2 (light chain complementation determining region 2) and LCDR3 (light chain complementation determining region 3). Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order from the amino terminus (also called the N terminus) to the carboxyl terminus (also called the C terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In the specific embodiments of the present disclosure, CDRs refer to more than 2 CDRs in the heavy chain and light chain of the antibody. The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and the ImMunoGenTics (IMGT) numbering rule (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc.; the correspondence between various numbering systems is well known to those skilled in the art.

In some embodiments, the present invention discloses an anti-human CCR8 antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, 3, 5, 7, 9 or 11, and/or the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.2, 4, 6, 8, 10 or 12.

In some embodiments, the present invention discloses an anti-human CCR8 antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, 3, 5, 7, 9 or 11, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.2, 4, 6, 8, 10 or 12.

In some embodiments, the present disclosure discloses an anti-human CCR8 antibody, wherein: A. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.2; B. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.3, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.4; C. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.5, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.6; D. the heavy chain variable region of the antibody includes SEQ ID No.7, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.8; E. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.9, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.10; or F. The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.11, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.12.

In some embodiments, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.

In some embodiments, the anti-human CCR8 antibodies disclosed herein, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

In some embodiments, the present disclosure discloses an anti-human CCR8 antibody, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the IMGT numbering system.

In some embodiments, the anti-human CCR8 antibody disclosed herein, wherein: a. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, HCDR2 comprises the amino acid sequence of SEQ ID NO.14, and HCDR3 comprises the amino acid sequence of SEQ ID NO.15; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18; b. HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.19, HCDR2 comprises the amino acid sequence of SEQ ID NO.20, and HCDR3 comprises the amino acid sequence of SEQ ID NO.21; and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18; c. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.22, HCDR2 comprises the amino acid sequence of SEQ ID NO.23, and HCDR3 comprises the amino acid sequence of SEQ ID NO.24; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.25, LCDR2 comprises the amino acid sequence of SEQ ID NO.26, and LCDR3 comprises the amino acid sequence of SEQ ID NO.27; d. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, HCDR2 comprises the amino acid sequence of SEQ ID NO.28, and HCDR3 comprises the amino acid sequence of SEQ ID NO.29; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.30, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18; e. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31, HCDR2 comprises the amino acid sequence of SEQ ID NO.32, and HCDR3 comprises the amino acid sequence of SEQ ID NO.33; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34, LCDR2 comprises the amino acid sequence of SEQ ID NO.35, and LCDR3 comprises the amino acid sequence of SEQ ID NO.36; or f. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, HCDR2 comprises the amino acid sequence of SEQ ID NO.37, and HCDR3 comprises the amino acid sequence of SEQ ID NO.38; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18.

In some embodiments, the anti-human CCR8 antibody described in the present disclosure comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO. NO.27; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.28 and SEQ ID NO.29, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.30 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.33, SEQ ID NO.34 and SEQ ID NO.35, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.36 and SEQ ID NO.37, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.31 and SEQ ID NO.32, respectively. LCDR1, LCDR2 and LCDR3 shown in NO.17 and SEQ ID NO.18.

The term "antigen-binding fragment" or "antigen-binding domain" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that is capable of specifically binding to the antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fv (composed of VH and VL), Fab (composed of one light chain and one heavy chain constant region 1 (CH1) and a heavy chain variable region), Fab' (composed of a Fab region and a hinge region), Fab'-SH (the cysteine residue in the hinge region of the Fab' fragment carries a free thiol group), (Fab')2 (dimeric Fab'), scFv (single-chain antibody molecule, in which a light chain variable region is directly linked to a heavy chain variable region or is linked via a linker), scFab (single-chain Fab), dsFv (disulfide-stabilized Fv fragment), (dsFv)2 (dimeric dsFv), and multispecific antibodies formed from antibody fragments.

The term "Fc region" is used to define the C-terminal region of the antibody rechain, including native Fc regions and modified Fc regions. In some embodiments, the Fc region used in the antibodies described herein includes the Fc region of human IgG1, IgG2 (IgG2a, IgG2b), IgG3 and IgG4. In some embodiments, the Fc region is a human IgG1 comprising a DLE mutation (i.e., the Fc of human IgG1 undergoes S239D/A330L/I332E mutations, and the sequence is shown in SEQ ID No. 42). In some embodiments, the Fc region is a human IgG1 comprising a DE mutation (i.e., the Fc of human IgG1 undergoes S239D/I332E mutations). In some embodiments, the boundaries of the Fc region may also be altered, such as by deleting the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or deleting the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise indicated, the numbering convention for the Fc region is the EU numbering system, also known as the EU index.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and/or light chain is derived from one species, while the other portion of the heavy chain and/or light chain is derived from another species.

The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having reduced immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the rest with human antibody counterparts (i.e., the constant region and the framework region portion of the variable region).

The terms "human antibody", "humanized antibody", "fully human antibody" and "completely human antibody" are used interchangeably to refer to antibodies whose variable and constant regions are human sequences.

The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise indicated, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art. The term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to a dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as molar concentration (M). The KD value of an antibody can be determined using methods known in the art, for example, using a biosensor system such as a system measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution by solution equilibrium titration (SET).

The term "effector function" refers to those biological activities attributable to an antibody Fc region (either a native sequence Fc region or an Fc region with amino acid sequence mutations). Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the population are identical, except for possible natural mutations that may be present in small amounts. In contrast, a polyclonal antibody preparation typically contains a plurality of different antibodies having different amino acid sequences in their variable domains, which are typically specific for different epitopes. "Monoclonal" refers to the characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of an antibody by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

The term "antigen" refers to a molecule that can be selectively bound by an antigen binding protein (e.g., an antibody). An antigen may have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

The term "epitope" refers to an area or region on an antigen that is capable of specific binding to an antibody. An epitope may be formed by a string of consecutive amino acids (linear epitope) or comprise non-consecutive amino acids (conformational epitope), for example brought into spatial proximity by folding of the antigen (i.e., by tertiary folding of proteinaceous antigens). Conformational epitopes differ from linear epitopes in that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. A conformational epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

An antibody that binds to the same epitope as a reference antibody or an antibody that competes for binding with a reference antibody means an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, or an antibody whose binding to the antigen is blocked by the reference antibody by 50% or more. For example, to determine whether the test antibody binds to the same epitope as the reference antibody, the reference antibody is allowed to bind to the antigen under saturated conditions, and then after removing excess reference antibody, the ability of the test antibody to bind to the antigen is assessed; if the test antibody is able to bind to the antigen after saturated binding of the reference antibody, then it can be concluded that the test antibody binds to a different epitope than the reference antibody; and if the test antibody is unable to bind to the antigen after saturated binding of the reference antibody, then the test antibody can bind to the same epitope as the reference antibody. To confirm whether the test antibody binds to the same epitope, conventional experiments (e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art) can be used. In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if an excess (e.g., a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amount) of one antibody inhibits binding of the other antibody to the antigen by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

The term "capable of specific binding", "specific binding" or "binding" means that the antibody is able to bind to an antigen or an epitope within the antigen with a higher affinity than other antigens or epitopes. Typically, the antibody binds to the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of about 100 nM or less (e.g., about 10 nM, 1 nM, 0.1 nM, 0.01 nM or less). In some embodiments, the KD of the antibody binding to the antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, including but not limited to Biacore assays, Octet methods, microthermophoresis, HPLC-MS methods, and flow cytometry fluorescence sorting techniques; for example, as measured by BIACORE® surface plasmon resonance assays.

The binding of the anti-human CCR8 antibody provided in the present disclosure to human CCR8 can also be expressed by the "half-maximal effective concentration" (EC50) value. Generally, the smaller the EC50, the better the affinity, indicating that it can bind to the antigen at a lower concentration. The EC50 value can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometry and other binding assays).

The term "antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages and neutrophils) via Fcγ receptors (FcγRs) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII and FcγRIIIa. The ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of a marker (e.g., a radioactive substrate, a fluorescent dye, or a native intracellular protein) from the lysed cells.

The term "antibody-dependent cellular phagocytosis" (ADCP) refers to the mechanism by which antibody-coated target cells are eliminated by internalization by phagocytic cells such as macrophages or dendritic cells.

The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of cell death induction in which the Fc effector domain of a target-binding antibody binds and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

"CAR" herein is a chimeric antigen receptor, which is an artificially modified receptor, for example, it is a receptor that can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens or viral antigens on immune cells (such as T cells), allowing immune cells to recognize tumor antigens or viral antigens and kill tumor cells or viruses. T cells expressing CAR are called CAR-T (or CART). Generally, chimeric antigen receptors contain an extracellular domain, a transmembrane region, and an intracellular signaling domain in sequence. The transmembrane region and intracellular signaling domain known in the art for constructing CAR can be used to construct the chimeric antigen receptor disclosed herein. When the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal is transmitted to the cell through the hinge region and the transmembrane region. The intracellular signal domain then converts the signal into an activation signal to activate the effector cells. The effector cells kill the tumor cells by secreting perforins or producing cytokines. At the same time, the effector cells themselves also expand, further expanding the immune killing effect. The extracellular domain can contain an antigen binding domain (also called the antibody part that specifically binds to the antigen), and the antigen binding domain can be a single-chain variable fragment (scFv) that specifically binds to the antigen. The single-chain variable fragment can serve as the extracellular domain of CAR, which determines the specificity of CAR-immune cells. The hinge region is the extracellular domain of CAR that connects the single-chain antibody unit and the transmembrane domain. It usually maintains the stability required for robust CAR expression and activity in effector cells. The hinge region of most CARs is derived from the hinge of IgG or the extracellular region of CD8α/CD28. The type and length of the hinge region have an important influence on the functional activity of CAR. The transmembrane domain connects the extracellular domain of CAR to the intracellular signal transduction domain. Commonly used transmembrane domains are derived from CD4, CD8α, CD28 and CD3ζ. The choice of transmembrane domain affects the degree of activation of the CAR structure in cell function. The intracellular domain is composed of a co-stimulatory domain and a signal transduction domain. In an optional embodiment, the CAR is a chimeric antigen receptor, and its main components may also include a transmembrane domain and an intracellular domain. In an optional embodiment, the CAR may also include a co-stimulatory molecule. "Co-stimulatory molecules" refer to molecules that exist on the surface of antigen-presenting cells and can bind to co-stimulatory molecule receptors on Th cells to produce synergistic stimulation signals. The proliferation of T lymphocytes requires not only the binding of antigens, but also the acceptance of co-stimulatory molecule signals. The co-stimulatory signal is transmitted to T cells mainly through the binding of co-stimulatory molecules CD80 and CD86 expressed on the surface of antigen-presenting cells to CD28 molecules on the surface of T cells. B cells can receive costimulatory signals through general pathogen components such as LPS, or through complement components, or through CD40L on the surface of activated antigen-specific Th cells. Costimulatory molecules known in the art for constructing CARs can be used, including but not limited to CD27, CD28, 4-1BB, OX40, ICOS, B7-H3 and/or any combination thereof.

The term "CAR-immune cell" refers to an immune cell expressing CAR or a CAR-modified immune cell, wherein the immune cell includes but is not limited to T cells, natural killer (NK cells), macrophages (M cells), and Treg cells.

The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing backbone residues or linkages of known nucleotide analogs or modifications, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present in one or more positions in a host cell. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. Specifically, as described in detail below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed alkali and/or deoxyinosine residues.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term sequence "identity" refers to the degree (percentage) to which the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned (introducing gaps, if necessary, to obtain the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity). To determine the percentage of sequence identity, alignment can be achieved by techniques known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.

In the present disclosure, "antibody conjugates" include conjugates formed by coupling anti-human CCR8 antibodies to therapeutic agents or imaging agents. In an optional embodiment, the antibody conjugate is formed by coupling anti-human CCR8 antibodies to therapeutic agents via a linker; in an optional embodiment, the therapeutic agent is a cytotoxic agent; in an optional embodiment, the therapeutic agent can be selected from toxins, chemotherapeutic agents, antibiotics, radioactive isotopes and nucleolytic enzymes.

In this disclosure, "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction; "toxin" refers to a substance that can have a harmful effect on the growth or proliferation of cells (such as maytansine, calicheamicin, taxanes, vincristine, colchicine, auristatin, etc.). "Chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer.

In an alternative embodiment, the antibody conjugate disclosed herein further comprises biotin or a biotin derivative conjugated to the antibody.

In an alternative embodiment, the antibody conjugate further comprises a label conjugated to the antibody.

In an optional embodiment, the above-mentioned marker refers to a type of substance having properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target object can be achieved.

In an optional embodiment, the marker is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle markers. In actual use, those skilled in the art can select other suitable markers according to detection conditions or actual needs. No matter what marker is used, it belongs to the protection scope of this disclosure.

In an optional embodiment, the fluorescent dye includes but is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.3A, Cy3.3A, Cy3.3A, Cy3.3B ... y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

In an alternative embodiment, the enzyme includes but is not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphoglucose deoxidase. In an alternative embodiment, the radioactive isotope includes but is not limited to ²¹² Bi, ¹³ 1I, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ²¹¹ At, ¹²⁵ I, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹³ Bi, ³² P, ⁹⁴ mTc, ⁹⁹ mTc, ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁷ Sc, ¹¹⁰ mIn, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ¹⁷² Lu and ¹⁸ F.

In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, luminol, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, lophanol and its derivatives, and peroxyoxalate and its derivatives. In an optional embodiment, the nanoparticle marker includes but is not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In an optional embodiment, the colloid includes but is not limited to colloidal metal, colloidal selenium, disperse dye, dye-labeled microspheres and latex. In an optional embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver. In an optional embodiment, the colloidal metal is colloidal gold.

In an alternative embodiment, the antibody conjugate may further include a solid phase carrier coupled to the antibody. In the antibody conjugate, the antibody is coupled to a solid phase carrier. In an alternative embodiment, the solid phase carrier is selected from microspheres, plates and membranes. In an alternative embodiment, the solid phase includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microporous plates, glass, capillaries, nylon and cellulose nitrate membranes. In an alternative embodiment, the solid phase carrier is a cellulose nitrate membrane.

The term "linker", "linker" or "connector" refers to a connecting unit that connects two polypeptide fragments, which usually has a certain degree of flexibility, and the use of the linker will not cause the original function of the protein domain to be lost. The linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. In some embodiments, the linker is selected from (GxS)y linkers, wherein x is selected from integers of 1-5 and y is selected from integers of 0-6. In some embodiments, the linker is selected from (GxS)y linkers, wherein x is selected from integers of 1-5 and y is selected from integers of 1-6.

On the other hand, the disclosed embodiments also provide a method for detecting CCR8, comprising: mixing the antibody as described in any of the preceding items, or the antibody conjugate as described in any of the preceding items, or the reagent or reagent kit as described in any of the preceding items with a sample to be tested, so that the antibody contacts CCR8 in the sample to be tested to form an immune complex. In a preferred embodiment, the immune complex further comprises a second antibody, and the second antibody binds to the antibody. In a preferred embodiment, the immune complex further comprises a second antibody, and the second antibody binds to CCR8.

On the other hand, the disclosed embodiments also provide a method for preparing the antibody as described in any of the preceding items, comprising: culturing the cells as described in any of the preceding items, and further comprising a step of purifying and recovering the antibody.

Based on the amino acid sequence of the antibody disclosed in the present disclosure, a person skilled in the art can prepare the antibody using genetic engineering technology or other technology (chemical synthesis, recombinant expression), for example, the antibody can be separated and purified from the culture product of recombinant cells that can recombinantly express the antibody as described in any of the above items. This is easy to achieve for a person skilled in the art. Based on this, no matter what technology is used to prepare the antibody disclosed in the present disclosure, it is within the scope of protection of the present disclosure.

The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector that can transform a host cell and contains a nucleic acid sequence that directs and/or controls (together with the host cell) the expression of one or more heterologous coding regions operably linked thereto. The expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of the coding region operably linked thereto.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the initial transformed cell are included herein. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastorii, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia cactus (Pichia thermotolerans), Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp. methanolica), Bisaccharomyces spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Bifidobacterium, any brewer's yeast, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Scyphozoite, Yarrowia lipolytica, and Neurospora crassa.

As used in this application, "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. The terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It is also understood that not all progeny have exactly the same DNA content, due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as the original transformed cell from which they were selected are included.

The term "pharmaceutical composition" refers to a mixture containing one or more active ingredients such as the antibodies described herein and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.

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

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

"Administer" or "administer," as applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, means the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid.

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

"Treatment" refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or reducing the disease state, and regression or improved prognosis. In some embodiments, the antibodies disclosed herein are used to delay the development of a disease or slow the progression of a disease.

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

In order to make the purpose, technical solution and advantages of the disclosed embodiment clearer, the technical solution in the disclosed embodiment is described clearly and completely below. If no specific conditions are specified in the embodiment, the experiment is carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise specified, the techniques used or considered herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

Unless otherwise indicated, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); and Current Protocols in Molecular Biology (Ed., 1996). Biology" (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

Embodiment 1. anti- CCR8 Hybridoma Antibody preparation

Balb/c mice were used, and CHO (Chinese hamster ovary cells) cells expressing human CCR8 (CHO-hCCR8) and a fusion protein of the N-terminal 1-35 amino acids of human CCR8 and mouse Fc (CCR8N-term-mFc) were used as immunogens for multiple immunizations. This was done once every one to two weeks, for a total of 6-10 times. After the fourth immunization, the mice were bled from the eye sockets, and the serum titer of anti-human CCR8 antibodies was detected by ELISA and flow cytometry. The mouse serum was diluted in multiples and co-cultured with Raji cell lines (human BURKITT'S lymphoma cells) and Raji cell lines expressing human CCR8 (Raji-hCCR8), and stained with PE-labeled goat anti-mouse Fc antibodies (Biolegend), and the serum titer was detected by flow cytometry. At the same time, CCR8N-term-hFc protein (a fusion protein of the N-terminal 1-35 amino acids of human CCR8 and human Fc) was coated into a 96-well plate, and the serum was incubated with multiple dilutions, followed by the addition of HRP-labeled goat anti-mouse Fc antibody (Sigma), and the serum titer was tested by ELISA. The mice with the highest titer were determined based on the results of flow cytometry and ELISA, and the mice were euthanized, spleen cells were collected, and fused with the SP20 cell line.

Monoclonal cells with strong binding activity to Raji-hCCR8 and no binding to Raji cell line were selected for expansion culture. After one week, the supernatant was collected and the antibodies in the supernatant were purified.

Embodiment 2. anti- CCR8 Antibody Binding Activity Screening

Count Raji-hCCR8 and Raji cells, adjust the density to 2E6/ml, add 100μL/well cell suspension to a 96-well plate, centrifuge at 400×g for 5 minutes, and discard the supernatant. Prepare hybridoma-purified antibodies at a concentration of 200nM and perform a 3-fold gradient dilution. Resuspend the cells with 100μL of diluted hybridoma-purified antibodies and incubate at 4℃ for 30 minutes. After washing twice with PBS, add 100μL of PE-labeled goat anti-mouse Fc antibodies diluted 1:500 and resuspend the cells, and incubate at 4℃ for 30 minutes. After washing once with PBS, 100 μL/well PBS was added to resuspend the cells, and the PE fluorescence intensity was analyzed by flow cytometry.

The binding test results of anti-CCR8 antibodies to Raji-hCCR8 are shown in Figure 1. The experimental results show that the six hybridoma-purified antibodies F022-7, F022-59, F022-63, F022-66, F022-68, and F022-75 exhibited strong binding activity to Raji-hCCR8.

Embodiment 3. Recombinant Antibodies CCR8 Antibody preparation

The sequences of F022-7, F022-59, F022-63, F022-66, F022-68, and F022-75 hybridoma-purified antibodies were retrieved, and the sequencing results showed that the light chain constant regions of all hybridoma-purified antibodies were of mouse κ type (as shown in SEQ ID No.40), the heavy chain constant regions of F022-7, F022-63, and F022-75 were of mIgG2a type (as shown in SEQ ID No.41), and the heavy chain constant regions of F022-59, F022-66, and F022-68 were of mIgG1 type (as shown in SEQ ID No.39). The heavy chain constant region of the hybridoma purified antibody was replaced with mouse mIgG2a, and other sequences remained unchanged. The antibody was cloned into pCDNA3.4A expression plasmid, and the plasmid was transfected into EXPI293 cell line. After 7 days, the supernatant was collected and purified with protein A to obtain recombinant antibodies. The recombinant antibodies were named F022-7-mIgG2a, F022-59-mIgG2a, F022-63-mIgG2a, F022-66-mIgG2a, F022-68-mIgG2a, and F022-75-mIgG2a. For example, the recombinant antibody "F022-59-mIgG2a" means that the heavy chain constant region of the hybridoma purified antibody "F022-59" was replaced with "mIgG2a", and the others are similar. The heavy chain constant region of the hybridoma purified antibody was replaced with human hIgG1Fc (DLE) (i.e., human IgG1 Fc was mutated with S239D/A330L/I332E, the sequence is shown in SEQ ID No.42), and the light chain constant region was replaced with human κ light chain constant region (as shown in SEQ ID No.43), and the other sequences were kept unchanged. ... of the hybridoma purified antibody was replaced with human κ light chain constant region (as shown in SEQ ID No.43), and the other sequences were kept unchanged. The heavy chain constant region of the hybridoma purified antibody was replaced with human hIgG1Fc (DLE), and the light chain constant region of the hybridoma purified antibody was replaced with human hIgG1Fc (DLE), and the light chain constant region of the hybridoma purified antibody was replaced with human hIgG1Fc (DLE After purification, recombinant antibodies were obtained. The recombinant antibodies were named F022-7-hIgG1 (DLE), F022-59-hIgG1 (DLE), F022-63-hIgG1 (DLE), F022-66-hIgG1 (DLE), F022-68-hIgG1 (DLE), and F022-75-hIgG1 (DLE). For example, the recombinant antibody "F022-59-hIgG1 (DLE)" means that the heavy chain constant region of the hybridoma purified antibody "F022-59" is replaced by human hIgG1 (DLE) and the light chain constant region is replaced by human κ light chain constant region, and the others are similar. The variable region sequences of the antibodies are shown in Table 1, and the CDRs sequences are shown in Table 2.

Table 1. Anti-CCR8 antibody variable region sequence list antibody VH V L F022-7 EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARVRSKSNFYATYYADSVKDRFTISRDDSQNMLSLQMNNLKTEDTAIYYCVRGKDAKGIYAMDFWGQGTSVTVSS (SEQ ID No. 1) DIVMTQAAPSVVVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTILEIK (SEQ ID No. 2) F022-59 EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSRSLLYLQMNTLKTEDSAMYYCVRGGYGYDVRSGMEYWGQGTSVTVSS (SEQ ID No. 3) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTVFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELK (SEQ ID No. 4) F022-63 EVQLVESGGGLVQPKGSLKLSCAASGFTFNPYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWVRRDYAMDYWGQGTSVTVSS (SEQ ID No. 5) DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIK (SEQ ID No. 6) F022-66 EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVGRIRTKSNSYATYYADSVKDRFTISRDDSESMVYLQMNNLKTEDGAMYYCVRGGWGYRNFVYFDVWGTGTTVTVSS (SEQ ID No. 7) DIVMTQDAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRTSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID No. 8) F022-68 EVQLVESGGGLVQPKGSLKLSCAASGFSFNIYAMNWVRQAPGKGLEWVARIRSKSNKYATFYADSVKDRFTISRDDSESKLYLHMNNLKTEDTAMYYCVRHKRGLNYGMDYWGQGTSVTVSS (SEQ ID No. 9) DIVLTQSPVSLVVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQAPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPWTFGGGTKLEIK (SEQ ID No.10) F022-75 AVQFVETGGGLVQPRGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYFADSVRDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRGGERRGDYAMDYWGQGTSVTVSS (SEQ ID No. 11) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEMK (SEQ ID No. 12)

Table 2. Anti-CCR8 antibody CDRs sequence list antibody Rechain CDR Light Chain CDR F022-7 HCDR1 TYAMN (SEQ ID No. 13) LCDR1 RSSKSLLHSNGNTYLY (SEQ ID No.16) HCDR2 RVRSKSNFYATYYADSVKD (SEQ ID No.14) LCDR2 RMSNLAS (SEQ ID No.17) HCDR3 GKDAKGIYAMDF (SEQ ID No.15) LCDR3 MQHLEYPFT (SEQ ID No.18) F022-59 HCDR1 TNAMN (SEQ ID No.19) LCDR1 RSSKSLLHSNGNTYLY (SEQ ID No.16) HCDR2 RIRTKSNNYATYYADSVKD (SEQ ID No.20) LCDR2 RMSNLAS (SEQ ID No.17) HCDR3 GGYGYDVRSGMEY (SEQ ID No.21) LCDR3 MQHLEYPFT (SEQ ID No.18) F022-63 HCDR1 PYAMN (SEQ ID No. 22) LCDR1 RSSQSLVHSNGNTYLH (SEQ ID No.25) HCDR2 RIRSKSNNYATYYADSVKD (SEQ ID No.23) LCDR2 KVSNRFS (SEQ ID No. 26) HCDR3 QWVRRDYAMDY (SEQ ID No.24) LCDR3 SQSTHVPFT (SEQ ID No. 27) F022-67 HCDR1 TYAMN (SEQ ID No. 13) LCDR1 RSSKSLLHSNGNTYLY (SEQ ID No.16) HCDR2 RIRTKSNSYATYYADSVKD (SEQ ID No.28) LCDR2 RTSNLAS (SEQ ID No.30) HCDR3 GGWGYRNFVYFDV (SEQ ID No.29) LCDR3 MQHLEYPFT (SEQ ID No.18) F022-68 HCDR1 IYAMN (SEQ ID No.31) LCDR1 RASKSVSTSGYSYMH (SEQ ID No.34) HCDR2 RIRSKSNKYATFYADSVKD (SEQ ID No.32) LCDR2 LASNLES (SEQ ID No.35) HCDR3 HKRGLNYGMDY (SEQ ID No.33) LCDR3 QHSRELPWT (SEQ ID No. 36) F022-75 HCDR1 TYAMN (SEQ ID No.13) LCDR1 RSSKSLLHSNGNTYLY (SEQ ID No.16) HCDR2 RIRSKSNNYATYFADSVRD (SEQ ID No.37) LCDR2 RMSNLAS (SEQ ID No.17) HCDR3 GGERRGDYAMDY (SEQ ID No.38) LCDR3 MQHLEYPFT (SEQ ID No.18) Note: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the antibodies described in Table 2 above were identified according to the Kabat coding system.

Amino acid sequence of mouse mIgG1 heavy chain constant region (SEQ ID No. 39): AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQ TKPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

The amino acid sequence of the mouse kappa light chain constant region (SEQ ID No. 40): RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Amino acid sequence of mouse mIgG2a heavy chain constant region (SEQ ID No. 41): AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

Human hIgG1 (DLE) antibody heavy chain constant region sequence (SEQ ID No. 42): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human kappa light chain constant region sequence (SEQ ID No. 43): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Exemplarily, the full-length sequences of F022-59, F022-59-mIgG2a, and F022-59-hIgG1 (DLE) are as follows:

Amino acid sequence of F022-59 heavy chain variable region (SEQ ID No. 44): EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSRSLLYLQMNTLKTEDSAMYYCVRGGYGYDVRSGMEYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWTW NSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFT CSVLHEGLHNHHTEKSLSHSPGK

The amino acid sequence of the variable region of the light chain of F022-59 (SEQ ID No. 45): DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTVFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Amino acid sequence of F022-59-mIgG2a heavy chain variable region (SEQ ID No. 46): EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSRSLLYLQMNTLKTEDSAMYYCVRGGYGYDVRSGMEYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVK GYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT IKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNS YSCSVVHEGLHNHHTTKSFSRTPGK

The amino acid sequence of the variable region of the light chain of F022-59-mIgG2a (SEQ ID No. 47): DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTVFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELK

Amino acid sequence of F022-59-hIgG1 (DLE) heavy chain variable region (SEQ ID No. 48): EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSRSLLYLQMNTLKTEDSAMYYCVRGGYGYDVRSGMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Amino acid sequence of F022-59-hIgG1 (DLE) light chain variable region (SEQ ID No. 49): DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTVFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In addition, the amino acid sequence of the positive control antibody 4A19-mIgG2a (obtained by connecting the variable region of the 4A19 antibody in the WO2021194942A1 patent to the mIgG2a constant region) is as follows:

Amino acid sequence of 4A19-mIgG2a heavy chain (SEQ ID No. 50): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDSEMHWVRQATGQGLEWMGAIQPETGGTAYNQKFKARVTMTRDTSISTAYMELSSLRSEDTAVYYCARRRRNFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSV VHEGLHNHHTTKSFSRTPGK

Amino acid sequence of 4A19-mIgG2a light chain (SEQ ID No. 51): DIVMTQTPLSLSVTPGQPASISCRSSQSLFHSSGNTYLHWYLQKPGQPPQLLIYKVSNRFSGVPDRFSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPFTFGQGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Embodiment 4. anti- CCR8 Antibody in vitro activity analysis

Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Axis-Shield) according to the instructions. Tregs were isolated from PBMCs using EasySep Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit (Stem Cell) according to the instructions. The isolated Tregs were mixed with Dynabeads Human T-Activator CD3/CD28 (Gibco) at a ratio of 1:1 and cultured in complete medium (90% X-VIVO 15+10% FBS+500IU/ml IL-2, X-VIVO 15: Lonza, FBS: Gibco, IL-2: Tetracycline) for 5 to 8 days to activate and expand Treg cells. The binding activity of the recombinant antibodies to Raji-hCCR8, Treg (after activation), Raji, PBMC and Raji-hCCR4 (Raji cells overexpressing human CCR4) was detected by flow cytometry. The experimental method was the same as that in Example 2 above. The mIgG2a subtype antibody was detected using a PE-labeled anti-mouse Fc secondary antibody (Biolegend), and the hIgG1 (DLE) subtype antibody was detected using an Alexa Fluor 647-labeled anti-human Fab secondary antibody (Jackson). The experimental results are shown in Figures 2-6. The recombinant antibodies all showed strong binding activity to Raji-hCCR8 (Figure 2) and Treg (Figure 3). Meanwhile, the recombinant antibodies did not bind to Raji ( FIG. 4 ), PBMC ( FIG. 5 ) and Raji-hCCR4 ( FIG. 6 ), which indicates that the anti-CCR8 antibody disclosed in the present invention has good specificity.

In addition, antibody-mediated ADCC function was detected by expressing hCD16 molecules (NK-92-hCD16) in NK-92 cells. Jurkat-hCCR8 cells (Jurkat, human peripheral blood leukemia T cell line, ATCC) were stained with CFSE. Jurkat-hCCR8 cells were added to a 96-well plate at a density of 2E4/well. NK-92-hCD16 cells were added to a 96-well plate at a density of 1E5/well, followed by the addition of anti-CCR8 antibody with hIgG1 Fc (DLE) and incubation at 37 degrees for 6 hours. The 96-well plate was taken out and PI was added for staining. CSFE and PI fluorescence were detected by flow cytometry. The ratio of PI and CFSE double-positive cells in the CFSE-positive cell population was calculated, which was the cell killing rate. The experimental results are shown in Figure 7. All recombinant antibodies showed excellent ADCC activity.

Embodiment 5. anti- CCR8 Antibody in vivo activity analysis

To verify the in vivo efficacy of anti-CCR8 antibodies, MC38 cells (3E5/mouse) were subcutaneously injected into hCCR8 knock-in mice (B-hCCR8, Biocytogen) to establish a tumor model. On the 7th day after tumor inoculation, F022-7-mIgG2a, F022-59-mIgG2a, and F022-75-mIgG2a antibodies were intravenously injected, and isotype-independent target antibodies were used as negative controls, and 4A19-mIgG2a was used as a positive control. The dosage was 10 mg/kg, and the drug was administered twice a week for a total of 6 times. The tumor volume of mice was measured twice a week and calculated according to tumor volume = tumor long diameter × tumor short diameter × tumor short diameter/2. 34 days after tumor inoculation, the tumor inhibition rate of mice was calculated according to the formula: [1-(tumor volume of the treatment group after drug administration - tumor volume of the treatment group before drug administration) / (tumor volume of the control group after drug administration - tumor volume of the control group before drug administration)] × 100%. The experimental results are shown in Table 3 and Figure 8. The recombinant antibodies have significant anti-tumor effects in vivo.

Table 3. Results of the anti-CCR8 antibody inhibition of tumor growth experiment Group Average tumor volume (mm ³ ) Tumor growth inhibition (TGI, %) Isotype-mIgG2a 3353.71 / F022-7-mIgG2a 1610.05 53.32 F022-59-mIgG2a 1242.16 64.6 F022-75-mIgG2a 1803.5 47.42 4A19-mIgG2a 2534.92 25.07

The above is only a preferred embodiment of the disclosure and is not intended to limit the disclosure. For those skilled in the art, the disclosure may be subject to various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the disclosure shall be included in the protection scope of the disclosure.

without

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the figures required for use in the embodiments. It should be understood that the following figures only show certain embodiments of the present invention and should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant figures can be obtained based on these figures without creative work. Figure 1 is a diagram showing the binding test results of hybridoma purified antibodies and Raji-hCCR8. Figure 2 is a diagram showing the binding test results of recombinant antibodies and Raji-hCCR8. Figure 3 is a diagram showing the binding test results of recombinant antibodies and Treg. Figure 4 is a diagram showing the binding test results of recombinant antibodies and Raji. Figure 5 is a diagram showing the binding test results of recombinant antibodies and PBMC. Figure 6 is a graph showing the binding test results of the recombinant antibody and Raji-hCCR4. Figure 7 is a graph showing the ADCC activity test results of the recombinant antibody. Figure 8 is a graph showing the results of the anti-CCR8 antibody inhibition of tumor growth test results.

TW202423987A_112147381_SEQL.xml

Claims (16)

An anti-human CCR8 antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.3, 1, 5, 7, 9 or 11, and/or the light chain variable region of the anti-human CCR8 antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.4, 2, 6, 8, 10 or 12. An anti-human CCR8 antibody as described in claim 1, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system; Optionally, A. the heavy chain variable region of the anti-human CCR8 antibody includes the HCDR1, HCDR2 and HCDR3 in the SEQ ID No.3, and the light chain variable region of the anti-human CCR8 antibody includes the LCDR1, LCDR2 and LCDR3 in the SEQ ID No.4; B. the heavy chain variable region of the anti-human CCR8 antibody includes the SEQ ID No.1, and the light chain variable region of the anti-human CCR8 antibody includes the LCDR1, LCDR2 and LCDR3 in SEQ ID No.2; C. The heavy chain variable region of the anti-human CCR8 antibody includes the HCDR1, HCDR2 and HCDR3 in SEQ ID No.5, and the light chain variable region of the anti-human CCR8 antibody includes the LCDR1, LCDR2 and LCDR3 in SEQ ID No.6; D. The heavy chain variable region of the anti-human CCR8 antibody includes the HCDR1, HCDR2 and HCDR3 in SEQ ID No.7, and the light chain variable region of the anti-human CCR8 antibody includes the LCDR1, LCDR2 and LCDR3 in SEQ ID No.8; E. The heavy chain variable region of the anti-human CCR8 antibody includes the SEQ ID No.9, and the light chain variable region of the anti-human CCR8 antibody includes the LCDR1, LCDR2 and LCDR3 in SEQ ID No.10; or F. The heavy chain variable region of the anti-human CCR8 antibody includes the HCDR1, HCDR2 and HCDR3 in SEQ ID No.11, and the light chain variable region of the anti-human CCR8 antibody includes the LCDR1, LCDR2 and LCDR3 in SEQ ID No.12; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system; Optionally, the anti-human CCR8 antibody, wherein, a. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.19, and the HCDR2 comprises the amino acid sequence of SEQ ID NO.20, and the HCDR3 comprises the amino acid sequence of SEQ ID NO.21; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, the LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and the LCDR3 comprises the amino acid sequence of SEQ ID NO.18; b. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, the HCDR2 comprises the amino acid sequence of SEQ ID NO.14, and the HCDR3 comprises the amino acid sequence of SEQ ID NO.15; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, the LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and the LCDR3 comprises the amino acid sequence of SEQ ID NO.18; c. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.22, the HCDR2 comprises the amino acid sequence of SEQ ID NO. NO.23, and the HCDR3 comprises the amino acid sequence of SEQ ID NO.24; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.25, the LCDR2 comprises the amino acid sequence of SEQ ID NO.26, and the LCDR3 comprises the amino acid sequence of SEQ ID NO.27; d. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, the HCDR2 comprises the amino acid sequence of SEQ ID NO.28, and the HCDR3 comprises the amino acid sequence of SEQ ID NO.29; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, the LCDR2 comprises the amino acid sequence of SEQ ID NO.30, and the LCDR3 comprises the amino acid sequence of SEQ ID NO.18; e. The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31, the HCDR2 comprises the amino acid sequence of SEQ ID NO. NO.32, and the HCDR3 comprises the amino acid sequence of SEQ ID NO.33; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34, the LCDR2 comprises the amino acid sequence of SEQ ID NO.35, and the LCDR3 comprises the amino acid sequence of SEQ ID NO.36; or f. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, the HCDR2 comprises the amino acid sequence of SEQ ID NO.37, and the HCDR3 comprises the amino acid sequence of SEQ ID NO.38; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, the LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and the LCDR3 comprises the amino acid sequence of SEQ ID NO.18; Optionally, A. the anti-human CCR8 antibody comprises SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; B. The anti-human CCR8 antibody comprises the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or C. The anti-human CCR8 antibody comprises the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO. The LCDR1, LCDR2 and LCDR3 shown in NO.27. The anti-human CCR8 antibody as described in any one of claims 1-2, wherein the anti-human CCR8 antibody is a murine antibody, a chimeric antibody or a humanized antibody. An anti-human CCR8 antibody, wherein the heavy chain variable region of the anti-human CCR8 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID No.3, 1, 5, 7, 9 or 11, and/or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID No.4, 2, 6, 8, 10 or 12; Optionally, (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.4; (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.2; (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.6; (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.8; (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.10; or (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.12. The anti-human CCR8 antibody as described in any one of claims 1-4, wherein the anti-human CCR8 antibody further comprises a constant region; Optionally, the heavy chain constant region of the anti-human CCR8 antibody is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is selected from the κ or λ chain constant region; Optionally, the species of the constant region is mouse or human; Optionally, the heavy chain constant region is a mouse IgG2a constant region, a mouse IgG1 constant region or a human IgG1 constant region, and/or the light chain constant region is a mouse κ constant region or a human κ constant region; Optionally, the heavy chain constant region is a human IgG1 (DE) or a human IgG1 (DLE) constant region; Optionally, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO.39, 41 or 42, and the light chain constant region comprises the amino acid sequence of SEQ ID NO. NO.40 or 43; Optionally, the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.46, and the anti-human CCR8 antibody light chain comprises the amino acid sequence of SEQ ID NO.47; or The anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.44, and the anti-human CCR8 antibody light chain comprises the amino acid sequence of SEQ ID NO.45; or The anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.48, and the anti-human CCR8 antibody light chain comprises the amino acid sequence of SEQ ID NO.49. The anti-human CCR8 antibody as described in any one of claims 1 to 4, wherein the anti-human CCR8 antibody is a full-length antibody or any antigen-binding fragment selected from F(ab’)2, Fab’-SH, Fab’, Fab, scFab, dsFv, (dsFv)2, Fv and scFv. An antibody that competitively binds to human CCR8 as the anti-human CCR8 antibody as described in any one of claims 1 to 6, or an antibody that binds to the same epitope as the anti-human CCR8 antibody as described in any one of claims 1 to 6. An anti-human CCR8 antibody as described in any one of claims 1-7, wherein the anti-human CCR8 antibody has at least one of the following properties: A. It can specifically bind to human CCR8; optionally, it can bind to Raji cells expressing human CCR8 with an EC50 value of ≤5nM, wherein the EC50 value is determined by flow cytometry; B. It has the function of inhibiting tumor growth; C. It has ADCC (Antibody-dependent cellular cytotoxicity) activity. A multispecific antibody, comprising the anti-human CCR8 antibody as described in any one of claims 1-8. An antibody conjugate, comprising the anti-human CCR8 antibody as described in any one of claims 1 to 8. A chimeric antigen receptor (CAR), comprising an antigen binding domain, wherein the antigen binding domain comprises the anti-human CCR8 antibody as described in any one of claims 1-8. A CAR-immune cell, wherein the CAR-immune cell expresses the chimeric antigen receptor as described in claim 11. An isolated nucleic acid encoding the anti-human CCR8 antibody as described in any one of claims 1-8. A cell comprising the nucleic acid of claim 13. A drug composition comprising an anti-human CCR8 antibody as described in any one of claims 1-8, a multispecific antibody as described in claim 9, an antibody conjugate as described in claim 10, a CAR-immune cell as described in claim 12, or a nucleic acid as described in claim 13; optionally, it also comprises one or more pharmaceutically acceptable carriers, diluents or excipients. Use of the anti-human CCR8 antibody as described in any one of claims 1-8, the multispecific antibody as described in claim 9, the antibody conjugate as described in claim 10, the CAR-immune cell as described in claim 12, the nucleic acid as described in claim 13 or the drug composition as described in claim 15 in the preparation of a product having at least one of the following uses: diagnosing a disease related to abnormal expression of human CCR8, treating a disease related to abnormal expression of human CCR8 or detecting the expression of human CCR8; optionally, the disease related to abnormal expression of human CCR8 is a tumor; optionally, the tumor is breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.