Anti-CCR8 antibodies and uses thereof

Abstract

The invention relates to an antibody specifically binding to CCR8, an antibody fragment, an antigen binding part thereof, and related applications thereof. The anti-CCR8 antibody has the differentiated advantage of targeting CCR8, and provides medication selection and treatment schemes for tumor patients.

Description

Anti-CCR8 antibodies and their applications Technical Field

[0001] The present disclosure relates to the field of biomedicine or biopharmaceutical technology, and more particularly to anti-CCR8 antibodies and applications thereof. Background Art

[0002] Regulatory T cells (Tregs) are a type of T cell subpopulation that controls the body's own immune reactivity and is closely related to the occurrence of autoimmune diseases. They can also induce tumor cells to escape the immune system and accelerate tumor cell proliferation. Therefore, the development of antibody drugs targeting Tregs has become an important research direction in tumor immunotherapy.

[0003] CCR8 (CC chemokine receptor type 8) is a G protein-coupled receptor with seven transmembrane spans and is composed of 355 amino acids, including a short extracellular N-terminus (1-35 amino acids). The serine / threonine residues at the intracellular C-terminus can serve as phosphorylation sites during receptor regulation, thereby mediating cellular responses.

[0004] CCR8 is a chemokine receptor that is highly expressed in tumor-infiltrating Tregs, but is expressed at lower levels in Tregs in the thymus, spleen, and peripheral blood, and also in Th2 cells. The ligand for CCR8 is the chemokine CCL1, which is upregulated at sites of inflammation. CCL1 can recruit FOXP3. + CCR8 + Treg cells infiltrate into tumor tissues and perform immunosuppressive functions. At the same time, CCL1 can also induce FOXP3 + CCR8 expression on the surface of Treg cells is upregulated, inducing Ca 2+ Flow induces STAT3-dependent upregulation of FOXP3, CD39, IL-10, and granzyme B expression, thereby enhancing the immunosuppressive activity of these tumor-infiltrating Tregs cells.

[0005] Studies have found that CCR8 is significantly upregulated in tumor-infiltrating Tregs compared to Tregs in normal tissues, indicating that CCR8 is a promising therapeutic target on Tregs in the tumor microenvironment (TME) and does not cause systemic autoimmunity. Therefore, antibodies targeting CCR8 will improve the immune function of the TME by clearing Tregs cells in the tumor microenvironment and have anti-tumor potential. Currently, several pharmaceutical companies such as Bristol-Myers Squibb (BMS) have CCR8-targeted antibody drugs in the clinical research stage, but all are in clinical phase I. Therefore, the development of CCR8-targeted antibody drugs with differentiated advantages has broad application prospects and can also provide more medication options and treatment options for cancer patients.

[0006] Summary of the Invention

[0007] The present disclosure discloses the development of novel anti-CCR8 antibodies or antibody fragments thereof that are capable of binding to CCR8 derived from primates (human and / or monkey), and also provides anti-CCR8 antibodies, antibody fragments thereof, or antigen-binding portions thereof that can be used as cancer therapeutic drugs.

[0008] To achieve the above objectives, the present disclosure provides the following technical solutions:

[0009] The present disclosure provides an antibody comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 selected from any one of the following amino acid sequence combinations:

[0010] (a) SEQ ID NO: 3, 4, 5;

[0011] (b) SEQ ID NO: 6, 7, 5;

[0012] (c) SEQ ID NO: 8, 9, 5;

[0013] (d) SEQ ID NO: 10, 11, 12;

[0014] (e) SEQ ID NO: 20, 21, 22;

[0015] (f) SEQ ID NO: 23, 24, 22;

[0016] (g) SEQ ID NO: 25, 26, 22;

[0017] (h) SEQ ID NO: 27, 28, 29;

[0018] (i) SEQ ID NO: 37, 38, 39;

[0019] (j) SEQ ID NO: 40, 41, 39;

[0020] (k) SEQ ID NO: 42, 43, 39;

[0021] (l) SEQ ID NO: 44, 45, 46;

[0022] and comprising a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, selected from any one of the following amino acid sequence combinations:

[0023] (a) SEQ ID NO: 13, 14, 15;

[0024] (b) SEQ ID NO: 16, 17, 15;

[0025] (c) SEQ ID NO: 30, 31, 32;

[0026] (d) SEQ ID NO: 33, 34, 32;

[0027] (e) SEQ ID NO: 47, 48, 49;

[0028] (f) SEQ ID NO: 50, 51, 49;

[0029] The aforementioned antibody specifically binds to one or more amino acids in the extracellular domain of CCR8. The aforementioned amino acid sequence further includes derivative sequences obtained by adding, deleting, modifying and / or replacing one or more amino acids and can maintain the ability to bind to CCR8.

[0030] Preferably, the antibody comprises HCDR1, HCDR2, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, selected from any one of the following amino acid sequence combinations:

[0031] (a) SEQ ID NO: 37, 38, 39, 47, 48, 49;

[0032] (b) SEQ ID NO: 40, 41, 39, 47, 48, 49;

[0033] (c) SEQ ID NO: 42, 43, 49, 47, 48, 49;

[0034] (d) SEQ ID NO: 44, 45, 46, 50, 51, 49;

[0035] (e) SEQ ID NO: 3, 4, 5, 13, 14, 15;

[0036] (f) SEQ ID NO: 6, 7, 5, 13, 14, 15;

[0037] (g) SEQ ID NO: 8, 9, 5, 13, 14, 15;

[0038] (h) SEQ ID NO: 10, 11, 12, 16, 17, 15;

[0039] (i) SEQ ID NO: 20, 21, 22, 30, 31, 32;

[0040] (j) SEQ ID NO: 23, 24, 22, 30, 31, 32;

[0041] (k) SEQ ID NO: 25, 26, 22, 30, 31, 32;

[0042] (l) SEQ ID NO: 27, 28, 29, 33, 34, 32;

[0043] The above-mentioned antibody specifically binds to one or more amino acids in the extracellular domain of CCR8, and the amino acid sequence further includes a derivative sequence by adding, deleting, modifying and / or replacing one or more amino acids, and can maintain the ability to bind to CCR8.

[0044] Preferably, the heavy chain further comprises a heavy chain constant region and / or the light chain further comprises a light chain constant region.

[0045] Preferably, the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 18, and 35.

[0046] Preferably, the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 19, and 36.

[0047] Preferably, the above-mentioned antibody comprises a heavy chain variable region and a light chain variable region selected from any one of the following amino acid sequence combinations:

[0048] (a) SEQ ID NO: 1, 2;

[0049] (b) SEQ ID NO: 18, 19;

[0050] (c) SEQ ID NO: 35, 36.

[0051] More preferably, the above-mentioned antibody comprises the following light chain variable region and heavy chain variable region; wherein the aforementioned heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 18, and 35, or has more than 95% identity with the amino acid sequence of SEQ ID NOs: 1, 18, and 35; the aforementioned light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 19, and 36, or has more than 95% identity with the amino acid sequence of SEQ ID NOs: 2, 19, and 36.

[0052] More preferably, the above-mentioned antibody can bind to CCR8 derived from primates, preferably humans.

[0053] More preferably, the heavy chain variable region of the above-mentioned antibody further includes a human, human or humanized framework region, and / or the light chain variable region of the antibody further includes a human, human or humanized framework region.

[0054] More preferably, the above-mentioned antibody comprises a monoclonal antibody, a chimeric antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, an antibody fragment thereof, or an antigen-binding portion thereof.

[0055] More preferably, the above-mentioned antibody is selected from the following combinations: (i) a single-chain antibody or a single-chain variable fragment, or a monovalent antibody lacking a hinge region; (ii) Fab, Fab', F(ab')2; (iii) a full-length antibody; (iv) an antibody comprising a human IgG Fc domain.

[0056] Preferably, the above-mentioned antibody has one or more biological activities selected from the following:

[0057] (1) Binds to both cynomolgus monkey and human CCR8;

[0058] (2) possess ADCC activity;

[0059] (3) does not bind to human CCR4;

[0060] (4) It has the activity of blocking CCL1-mediated cell migration.

[0061] In one embodiment, the present disclosure further provides a recombinant protein comprising the above-mentioned antibody, antibody fragment or antigen-binding fragment thereof.

[0062] Preferably, the above recombinant protein further comprises a tag sequence to facilitate expression and / or purification.

[0063] In another embodiment, the present disclosure further provides a nucleic acid molecule encoding the above-mentioned antibody, its antibody fragment or antigen-binding fragment, or the above-mentioned recombinant protein.

[0064] In another embodiment, the present disclosure also provides a vector comprising the aforementioned nucleic acid molecule.

[0065] Preferably, the above-mentioned vector comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, such as an adenovirus, a lentivirus or a retrovirus.

[0066] In another embodiment, the present disclosure also provides an engineered host cell comprising the aforementioned nucleic acid molecule or the aforementioned vector.

[0067] In another embodiment, the present disclosure provides an antibody-drug conjugate comprising (i) a cytotoxic agent, a detection label, a drug, a cytokine, a radionuclide, an enzyme, or a combination thereof; and (ii) the above-mentioned antibody, recombinant protein, or a combination thereof.

[0068] In a preferred embodiment, the present disclosure also provides a pharmaceutical composition comprising (i) the above-mentioned antibody, its antibody fragment or antigen-binding fragment thereof, the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned engineered host cell, the above-mentioned antibody-drug conjugate, or / and a combination thereof; and (ii) a pharmaceutically acceptable carrier.

[0069] Preferably, the pharmaceutical composition is used to inhibit the binding of CCR8 to any of the CCR8 ligands, or is used as a neutralizing antibody.

[0070] Preferably, the aforementioned pharmaceutical composition is used to eliminate the effects of tumor-infiltrating Treg cells, or to treat cancer.

[0071] In another embodiment, the present disclosure provides a kit or drug delivery device comprising the above-mentioned antibody, its antibody fragment or antigen-binding fragment thereof, the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned engineered host cell, the above-mentioned antibody-drug conjugate, and the above-mentioned pharmaceutical composition.

[0072] Preferably, the above kit further comprises a secondary antibody against the first antibody.

[0073] The present disclosure provides a method for preparing a recombinant polypeptide, the method comprising:

[0074] (i) culturing the engineered host cell under conditions suitable for expression; and

[0075] (ii) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the above-mentioned antibody, antigen-binding fragment thereof or recombinant protein.

[0076] Compared with the prior art, the anti-CCR8 antibody disclosed herein has the following beneficial effects:

[0077] (1) It has strong binding ability with primates, especially crab-eating macaques and human CCR8.

[0078] (2) It has high antibody-dependent cell-mediated cytotoxicity (ADCC) and cell phagocytosis (ADCP) activities.

[0079] (3) It does not non-specifically bind to human CCR4.

[0080] (4) It can block CCL1-mediated cell migration with a high inhibition rate.

[0081] (5) It has advantages as a method for detecting human CCR8, or as a drug for preventing or treating human CCR8-related diseases.

[0082] In one embodiment, the present disclosure provides BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present disclosure. The exemplary embodiments of the present disclosure and their descriptions are intended to explain the present disclosure and do not constitute an improper limitation of the present disclosure. In the accompanying drawings:

[0084] FIG1A shows the results of affinity testing of anti-human CCR8 humanized antibodies to HEK293T-h.CCR8 cells.

[0085] FIG1B shows the results of affinity testing of anti-human CCR8 humanized antibodies to HEK293T-h.CCR8 cells.

[0086] FIG2 shows the results of affinity testing of anti-human CCR8 humanized antibodies to HEK293T-c.CCR8 cells.

[0087] Figure 3A shows the binding assay results of anti-human CCR8 humanized antibody to HEK293T-h.CCR4. Count: cell number; Control: control group.

[0088] Figure 3B shows the binding assay results of anti-human CCR8 humanized antibody to HEK293T-h.CCR4. Count: cell number; Control: control group.

[0089] FIG4A shows the results of affinity testing of anti-human CCR8 humanized antibodies to HEK293T-h.CCR8 cells.

[0090] FIG4B shows the ADCC activity test results of the anti-human CCR8 humanized antibody.

[0091] FIG4C shows the ADCC activity test results of the anti-human CCR8 humanized antibody.

[0092] FIG5 shows the ADCP activity test results of the anti-human CCR8 humanized antibody.

[0093] Figure 6 shows the results of the detection of anti-human CCR8 humanized antibody blocking CCL1-mediated cell migration activity.

[0094] Figure 7A shows the results of affinity testing of 13F3H6-hz to HEK293T-h.CCR8 cells after incubation in human plasma.

[0095] Figure 7B shows the results of affinity testing of 13F3H6-eADCC cells with HEK293T-h.CCR8 cells after incubation in human plasma.

[0096] FIG8 shows the blood drug concentration test results of 13F3H6-hz and 13F3H6-eADCC in rats.

[0097] Figure 9 shows the results of in vivo efficacy testing of anti-human CCR8 humanized antibodies in the human CCR8 transgenic mouse MC38 model.

[0098] Figure 10 shows the results of body weight change detection of anti-human CCR8 humanized antibody in human CCR8 transgenic mouse MC38 model. DETAILED DESCRIPTION

[0099] The present disclosure is further described below in conjunction with the accompanying drawings and specific examples. The protection content of the present disclosure is not limited to the following examples. It should also be understood that the terms used in the examples of the present disclosure are intended to describe specific embodiments, rather than to limit the scope of protection of the present disclosure, and are not exclusive limitations. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present disclosure, and the scope of protection of the present disclosure is the attached claims and any equivalents thereof.

[0100] All technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this disclosure pertains. In other cases, certain terms used herein will be clarified in the specification. The experimental methods in the following examples, where specific conditions are not specified, are generally within the knowledge and common general knowledge of those skilled in the art. The embodiments and features in the embodiments of this application may be combined with each other.

[0101] The features and advantages of the present disclosure can be further understood through the following detailed description in conjunction with the accompanying drawings. The embodiments provided are merely illustrative of the method of the present disclosure and do not limit the rest of the disclosure in any way.

[0102] The antibodies disclosed herein refer to proteins that specifically bind to primate (preferably human) CCR8, and include monoclonal antibodies, chimeric antibodies, recombinant antibodies, fully human antibodies, humanized antibodies, bispecific antibodies, multispecific antibodies, antibody fragments thereof, or antigen-binding portions thereof. Specifically, they also include single-chain antibodies (Fv) or single-chain variable fragments, or monovalent antibodies lacking a hinge region; disulfide-stabilized Fv (dsFv); antigen-binding fragments (Fab, Fab', F(ab')2); full-length antibodies; and antibodies containing the human IgG Fc domain.

[0103] The present disclosure also encompasses recombinant proteins, wherein the recombinant protein is an antibody, an antibody fragment or an antigen-binding fragment thereof of the present disclosure, and a tag sequence to facilitate expression and / or purification.

[0104] The present disclosure includes nucleic acid molecules or polynucleotides encoding the antibodies of the present disclosure (light chain variable region or heavy chain variable region). The present disclosure also includes expression vectors containing the nucleic acid molecules, wherein the vector types include bacterial plasmids, bacteriophages, yeast plasmids; plant cell viruses, mammalian cell viruses (such as adenoviruses, lentiviruses or retroviruses).

[0105] The present disclosure also includes cells for expressing the antibodies of the present disclosure, wherein the cells comprise the aforementioned nucleic acid molecules or the aforementioned vectors.

[0106] The antibodies of the present disclosure can be used as drug conjugates, which further include a cytotoxic agent, a detection label, a drug, a cytokine, a radionuclide, an enzyme, or a combination thereof; and the antibodies, recombinant proteins, or a combination thereof of the present disclosure.

[0107] The antibodies disclosed herein can be used as pharmaceutical compositions, especially for recognizing human CCR8, and the compositions comprise the antibodies disclosed herein, their antibody fragments or antigen-binding fragments thereof; recombinant proteins; nucleic acid molecules; vectors; engineered host cells; antibody-drug conjugates; or / and combinations thereof. In addition, a pharmaceutically acceptable carrier, such as an AAV vector, is also included. Therefore, the pharmaceutical compositions disclosed herein can hinder the binding of CCR8 to any of the CCR8 ligands, and can be used to treat and / or prevent cancer, remove tumor-infiltrating Treg cells, or be used as neutralizing antibodies.

[0108] Abbreviations

[0109] CCR8: Chemokine (CC motif) receptor 8.

[0110] CCL1: Chemokine (CC motif) ligand 1.

[0111] Th2: Helper T cell 2.

[0112] CDR: complementarity determining region.

[0113] FR: amino acid residues in the antibody variable region excluding CDR residues.

[0114] V H : Antibody heavy chain variable region.

[0115] V L : Antibody light chain variable region.

[0116] IgG: Immunoglobulin G.

[0117] Kabat: Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat.

[0118] Chothia: Immunoglobulin numbering system proposed by Chothia et al.

[0119] IMGT: A numbering system based on the International Immunogenetics Information System initiated by Lefranc et al.

[0120] EC 50 : Half-maximal effect concentration, that is, the concentration that can cause 50% of the maximum effect.

[0121] ELISA: enzyme-linked immunosorbent assay.

[0122] FACS: flow cytometry.

[0123] PCR: polymerase chain reaction.

[0124] HRP: horseradish peroxidase.

[0125] ADCC: Antibody-dependent cell-mediated cytotoxicity.

[0126] ADCP: antibody-dependent cell-mediated phagocytosis.

[0127] DMEM: Dulbecco's Modified Eagle Medium.

[0128] RPMI1640: Roswell Park Memorial Institute 1640 medium.

[0129] FBS: fetal bovine serum.

[0130] Example 1: Construction of CCR8 overexpressing cell line and antigen protein expression

[0131] The amino acid sequence of human CCR8 is referenced at P51685 in the UniProt protein database. The amino acid sequence of human CCR4 (CC chemokine receptor type 4) is referenced at P51679 in the UniProt protein database. The amino acid sequence of cynomolgus macaque CCR8 is referenced at XP_015300839.1 in the NCBI protein database. The above amino acid sequences were codon-optimized by Anhui General Company and synthesized in the lentiviral vector pLVX or pCDNA3.4.

[0132] Viruses were prepared using a lentiviral packaging system and subsequently infected HEK293T and BaF3 cells. Stable HEK293T-h.CCR8 (human), HEK293T-h.CCR4 (human), HEK293T-c.CCR8 (cynomolgus macaque), and BaF3-h.CCR8 (human) cell lines were generated by selection with puromycin (Invivogen, Catalog No. ant-pr-1). After successful construction, the positive rate of target expression was determined by flow cytometry (FACS) for subsequent experiments.

[0133] The N-terminal extracellular domain of human and cynomolgus macaque CCR8 proteins consists of 35 amino acids (1-35AA). The N-terminal extracellular domain of human and cynomolgus macaque CCR8 proteins were synthesized in pTT5-hFc or pTT5-mFc vectors. The synthesized plasmids were transiently transfected into HEK293-EBNA cells using PEImax (Polysciences, 24765-1). After 7 days of expression, the human h.CCR8 (1-35AA) and cynomolgus macaque c.CCR8 (1-35AA) antigen proteins were purified using ProA filler (GE, MabselectXL).

[0134] Example 2: Preparation of anti-human CCR8 mouse monoclonal antibody

[0135] Wild-type mice, aged 5-6 weeks, from three strains (Balb / C, Kunming (KM), and CD1) were immunized in two batches. The first batch was immunized intraperitoneally and at multiple sites on the back with human h.CCR8 (1-35AA) protein antigen, while the second batch was immunized intravenously with the pCDNA3.4-h.CCR8 plasmid. After four immunizations in the first batch and five immunizations in the second batch, serum titers were measured using flow cytometry (FACS).

[0136] The experimental steps were as follows: HEK293T-h.CCR8, HEK293T-h.CCR4, and HEK293T-c.CCR8 cells were collected by digestion and centrifugation, then washed three times with pre-cooled PBS, and resuspended in PBS (containing 1% BSA) at 3 × 10 cells per well. 5Cells were plated into 96-well conical-bottom plates and incubated with 100-, 300-, and 900-fold diluted serum at 4°C for 1 hour. After three washes with pre-chilled PBS, 1 μL of anti-mouse fluorescent secondary antibody was added to each well and incubated at 4°C for 0.5 hour. After three washes with pre-chilled PBS, the cells were resuspended and analyzed using a flow cytometer (Beckman, CytoFLEX). Hybridomas were fused to mice with high titers for HEK293T-h.CCR8 and HEK293T-c.CCR8 and low titers for HEK293T-h.CCR4.

[0137] Mouse spleens and lymph nodes were harvested to prepare a cell suspension, which was then mixed with SP2 / 0 mouse myeloma cells at a 1:1 ratio. The cells were resuspended in electrofusion buffer and electrofusion was performed using BTX-ECM2001. After electrofusion, the cells were resuspended in complete fusion medium (RPMI1640 + 20% FBS + OPI + 1× HAT) and aliquoted at 20,000–25,000 cells per well into a 96-well cell culture plate. The cells were incubated at 37°C in 5% CO2. Following incubation, FACS screening was performed on HEK293T-h.CCR8, HEK293T-h.CCR4, and HEK293T-c.CCR8 cells. A primary clone with high affinity for human and cynomolgus macaque CCR8 on the cell membrane and no binding to human CCR4 was obtained for monoclonal screening. Monoclonal hybridoma cells were cultured in serum-free medium (Zhuhai Kairui Biotechnology, K04114), and the collected monoclonal supernatants were purified using ProA (GE, Mabselect XL) filler to obtain mouse antibodies.

[0138] Example 3: Evaluation of anti-human CCR8 mouse monoclonal antibodies

[0139] The affinity of the anti-human CCR8 mouse monoclonal antibody was evaluated by FACS. HEK293T-h.CCR8 and HEK293T-c.CCR8 cells were collected by digestion and centrifugation, then washed three times with pre-cooled PBS and resuspended in PBS (containing 1% BSA). 3×10 cells were added to each well. 5 Cells were plated into 96-well conical bottom plates, and serially diluted mouse antibodies were added. The cells were incubated at 4°C for 1 hour, washed three times with pre-cooled PBS, and 1 μL of anti-mouse fluorescent secondary antibody was added to each well. The cells were incubated at 4°C for 0.5 hours, washed three times with pre-cooled PBS, and resuspended. The cells were detected by flow cytometry (Beckman, model: CytoFLEX). The values ​​were imported into GraphPad Prism for plotting and calculating EC. 50 value.

[0140] The specificity of the anti-human CCR8 mouse monoclonal antibody was evaluated by FACS. The experimental steps were as follows: HEK293T-h.CCR4 cells were digested, collected by centrifugation, and washed three times with pre-chilled PBS; the cells were resuspended in 1% BSA (in PBS) and 3×10 cells were added to each well of a 96-well conical bottom plate. 5 Cells were collected in a volume of 50 μL; the antibody to be tested was diluted to 20 μg / mL with 1% BSA (in PBS), and 50 μL / well was added to the cells to make the final concentration of the antibody 10 μg / mL, mixed, and incubated at 4°C for 1 hour; the cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended with 50 μL 1% BSA (in PBS), 1 μL fluorescent secondary antibody was added to each well, mixed, and incubated at 4°C for 0.5 hours; the cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended with 200 μL PBS for detection. The experimental results are shown in Table 1. The clones with high affinity for HEK293T-h.CCR8 and no non-specific binding to HEK293T-h.CCR4 were selected for sequencing, that is, HEK293T-h.CCR8EC 50 Among all mouse antibodies, only 13F3H6 has strong binding to HEK293T-c.CCR8, and its EC 50 The binding rate of all other mouse antibodies to cynomolgus monkey CCR8 was 287.8 ng / mL. All other mouse antibodies did not bind to cynomolgus monkey CCR8 or bound very weakly. The mouse antibody clone numbers for sequencing are as follows: 76H1F11, 96F1C4, and 13F3H6.

[0141] Table 1. Summary of mouse antibody evaluation results

[0142] Example 4: Sequencing of anti-human CCR8 mouse monoclonal antibody

[0143] The above 13F3H6, 96F1C4, and 76H1F11 candidate hybridoma cells were collected at a rate of approximately 1×10 5 RNA was extracted using Trizol (Invitrogen, 15596026), and then reverse transcribed using the PrimeScript RT reagent (TAKARA, RR047A) kit to obtain cDNA via PolyA. Upstream primers were designed upstream of the heavy and light chains, and downstream primers were designed for the heavy chain CH1 region and the light chain CL region, respectively. Products were amplified by PCR (Gold MIX, Qingke Biotechnology, TSE101), and fragments were recovered using an agarose gel extraction kit. Samples were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The resulting sequences are shown in Table 2.

[0144] Table 2. Mouse Antibody Sequence List

[0145] Example 5: Humanization of anti-human CCR8 murine monoclonal antibody

[0146] Using the CDR grafting method, the human germline sequence with the highest homology to the original mouse sequence is first found through the conventional BLAST method and used as a template; the CDR of the mouse antibody is transplanted onto the human template to construct a chimera; based on structural analysis of the FR amino acids in the mouse antibody that can retain its original conformation, the corresponding amino acids in the chimera are backmutated to mouse amino acids to maintain the original affinity; the constructed humanized antibody is subjected to calculations and immunogenicity analysis to find highly immunogenic fragments and replace them with less immunogenic fragments. The humanized sequence and CDR definition method are shown in Table 3.

[0147] Table 3. Humanized antibody sequence list

[0148] Example 6: Preparation of anti-human CCR8 humanized monoclonal antibody

[0149] The variable region amino acid sequences of the heavy and light chain variable regions of the anti-human CCR8 humanized monoclonal antibodies 96F1C4-hz, 13F3H6-hz, and 76H1F11-hz, as well as the control antibody (BM sequence from patent: CN 113260381A) were submitted to Universal Biotechnology for gene synthesis. After codon optimization, they were constructed into the pTT5-h.IgG1 vector (light chain constant region SEQ NO: 52, heavy chain constant region SEQ NO: 53). 13F3H6-hz-eADCC is an enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) version of the 13F3H6-hz antibody. It involves subjecting the wild-type IgG1 Fc terminus of 13F3H6-hz (i.e., SEQ ID NO: 53) to five amino acid mutations, namely, F243L, R292P, Y300L, L235V, and P396L (i.e., SEQ ID NO: 54), to obtain a candidate antibody with enhanced ADCC activity. After plasmid synthesis, the plasmid was shaken for extraction and transfected into HEK293E cells using PEImax. Expression was performed for approximately 7 days, and the supernatant was collected by centrifugation. The expression supernatant was purified using MabSelectSure affinity filler. All purified antibodies were ultrafiltered into PBS buffer, the concentration was determined, and the antibodies were stored in aliquots at -20°C.

[0150] Table 4. Correspondence between humanized antibody CDR, variable region and constant region

[0151] Example 7: Affinity evaluation of anti-human CCR8 humanized monoclonal antibodies

[0152] The affinity of humanized monoclonal antibodies against human CCR8 was determined by FACS. The experimental steps were as follows: HEK293T-h.CCR8 cells were digested, collected by centrifugation, and washed three times with pre-chilled PBS; the cells were resuspended in 1% BSA (in PBS) and added to a 96-well conical bottom plate at 3×10 per well. 5 cells in a volume of 50 μL; dilute the test antibody with 1% BSA (in PBS) starting at 40 μg / mL, dilute 3-fold, and 10 concentration points, taking 50 μL / well and adding it to the cells to make the final antibody concentration of 20 μg / mL, mix well, and incubate at 4°C for 1 hour; collect the cells by centrifugation, wash three times with pre-cooled PBS, resuspend the cells with 50 μL 1% BSA (in PBS), add 1 μL of fluorescent secondary antibody (Biolegend, catalog number: 410708) to each well, mix well, and incubate at 4°C for 0.5 hour; collect the cells by centrifugation, wash three times with pre-cooled PBS, resuspend the cells with 200 μL PBS, and detect them on the instrument.

[0153] The experimental results are shown in Figure 1A and Figure 1B. 96F1C4-hz, 13F3H6-hz, and 76H1F11-hz all have high affinity for human CCR8. 50 The affinity of the control antibody BM to human CCR8 was 109.43±9.98 ng / mL (n=2), indicating that the affinity of 96F1C4-hz and 13F3H6-hz was comparable to that of the control antibody BM, while the affinity of 76H1F11-hz was slightly weaker.

[0154] Example 8: Species Affinity Evaluation of Anti-Human CCR8 Humanized Monoclonal Antibodies

[0155] The affinity of anti-human CCR8 humanized monoclonal antibody to cynomolgus monkey CCR8 was determined by FACS. The experimental steps were as follows: HEK293T-c.CCR8 cells were collected by digestion and centrifugation, washed three times with pre-cooled PBS, and resuspended in 1% BSA (in PBS). 3×10 cells were plated per well. 5 Cells were plated into 96-well conical-bottom plates, and gradiently diluted antibodies were added and incubated at 4°C for 1 hour. After washing three times with pre-cooled PBS, 1 μL of fluorescent secondary antibody (Biolegend, Catalog No.: 410708) was added to each well and incubated at 4°C for 0.5 hour. After washing three times with pre-cooled PBS, the cells were resuspended and detected by flow cytometer (Beckman, Model: CytoFLEX).

[0156] The experimental results are shown in Figure 2. 13F3H6-hz has a strong binding to cynomolgus monkey CCR8, and its EC 50 The control antibody BM bound weakly to cynomolgus monkey CCR8, while 96F1C4-hz and 76H1F11-hz did not bind to cynomolgus monkey CCR8.

[0157] Example 9: Specificity evaluation of anti-human CCR8 humanized monoclonal antibodies

[0158] The specificity of humanized monoclonal antibodies against human CCR8 was determined by FACS. The experimental steps were as follows: HEK293T-h.CCR4 cells were collected by digestion and centrifugation, washed three times with pre-cooled PBS, and resuspended with 1% BSA (in PBS). 3×10 cells were added to each well. 5 Cells were plated into 96-well pointed-bottom plates, and the antibody to be detected was added at a final concentration of 20 μg / mL and incubated at 4°C for 1 hour. After washing three times with pre-cooled PBS, 1 μL of fluorescent secondary antibody (Biolegend, Catalog No.: 410708) was added to each well and incubated at 4°C for 0.5 hour. After washing three times with pre-cooled PBS, the cells were resuspended and detected by flow cytometer (Beckman, Model: CytoFLEX).

[0159] The experimental results are shown in Figures 3A and 3B , and 96F1C4-hz, 13F3H6-hz, and 76H1F11-hz do not non-specifically bind to human CCR4.

[0160] Example 10: Evaluation of endocytic activity of anti-human CCR8 humanized monoclonal antibodies

[0161] The endocytic activity of a humanized anti-human CCR8 monoclonal antibody was assessed by FACS. The experimental steps were as follows: HEK293T-h.CCR8 cells were harvested by digestion and centrifugation, incubated with 10 μg / mL of the antibody to be tested for 1 hour at 4°C, washed three times with PBS, and resuspended in DMEM + 10% FBS. The cells were divided into two aliquots and incubated at 37°C for 0 and 5 hours, respectively. After washing three times with PBS, 1 μL of fluorescent secondary antibody was added and incubated at 4°C for 0.5 hour. After washing three times with PBS, the cells were resuspended and loaded onto the microarray. The endocytic rate was calculated using the following formula:

[0162] Internalization rate (%) = [1-(average fluorescence value of the test sample at 5 hours-average fluorescence value of the negative control sample at 5 hours) / (average fluorescence value of the test sample at 0 hours-average fluorescence value of the negative control sample at 0 hours)]×100.

[0163] The experimental results showed that both 96F1C4-hz and 13F3H6-hz could mediate the endocytosis of CCR8, with the endocytosis rates of the two being 49.57% and 51.22% respectively after 5 hours, while the endocytosis rate mediated by the control antibody BM was 54.41%.

[0164] Example 11: Evaluation of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activity of Anti-Human CCR8 Humanized Monoclonal Antibodies

[0165] The affinity of 13F3H6-eADCC to human CCR8 was detected using the method described in Example 7. The fluorescent secondary antibody used was anti-human Alexa Fluor 647 secondary antibody (The Jackson Laboratory, Cat. No. 109-605-088). The test results are shown in Figure 4A. 13F3H6-eADCC has a high affinity for human CCR8. Its EC 50 In this experimental system, the affinities of 13F3H6-hz and control antibody BM to human CCR8 were 20.26 ng / mL and 49.19 ng / mL, respectively, indicating that the affinities of 13F3H6-hz, 13F3H6-eADCC, and control antibody BM are basically equivalent.

[0166] ADCC activity of humanized anti-human CCR8 monoclonal antibodies was detected using a luciferase reporter assay. The experimental steps were as follows: HEK293T-h.CCR8 cells were harvested by digestion and centrifugation, resuspended in DMEM + 1% FBS, and plated at 50,000 cells per well. Jurkat-NFAT-CD16a cells were harvested by centrifugation, resuspended in RPMI1640 + 1% FBS, and plated at 100,000 cells per well. Serially diluted test antibodies were added and incubated at 37°C for 5 hours. After incubation, 50 μL of Bio-Glory One-Step Luciferase Substrate (Suzhou Ruian, RA-GO04) was added to each well, and the cells were read using a microplate reader (MD, i3x).

[0167] The experimental results are shown in Figures 4B and 4C. 96F1C4-hz, 13F3H6-hz, 76H1F11-hz and 13F3H6-eADCC all have strong ADCC activity. 50 The EC values ​​of control antibody BM were 5.2 ng / mL, 9.77 ng / mL, 14.78 ng / mL and 1.17 ng / mL respectively. 50 The value was 17.19±6.4 ng / mL (n=2). The above results indicate that the ADCC activities of 96F1C4-hz, 13F3H6-hz, and 13F3H6-eADCC are all superior to those of the control antibody BM.

[0168] Example 12: Evaluation of Antibody-Dependent Cell-Mediated Phagocytosis (ADCP) Activity of Humanized Anti-Human CCR8 Monoclonal Antibodies

[0169] The ADCP activity of a humanized anti-human CCR8 monoclonal antibody was detected using a luciferase reporter assay. The experimental steps were as follows: HEK293T-h.CCR8 cells were harvested by digestion and centrifugation, resuspended in DMEM + 1% FBS, and plated at 50,000 cells per well. Jurkat-FcγRIIa-H131 (Novagen, DD1304) cells were harvested by centrifugation, resuspended in RPMI1640 + 1% FBS, and plated at 100,000 cells per well. Serial dilutions of the test antibody were added and incubated at 37°C for 5 hours. After incubation, 50 μL of Bio-Glory One-Step Luciferase Substrate (Suzhou Ruian, RA-GO04) was added to each well, and the cells were read using a microplate reader (MD, i3x).

[0170] The experimental results are shown in Figure 5. 96F1C4-hz, 13F3H6-hz and 13F3H6-eADCC all have strong ADCP activity. 50 The EC values ​​of control antibody BM were 107.3 ng / mL, 112.1 ng / mL, and 64.68 ng / mL, respectively. 50 The above results indicate that the ADCP activity of 96F1C4-hz, 13F3H6-hz, and 13F3H6-eADCC is basically equivalent to that of the control antibody BM.

[0171] Example 13: Anti-human CCR8 humanized monoclonal antibody blocking CCL1-mediated cell migration experiment

[0172] In this example, the blocking activity of anti-human CCR8 humanized monoclonal antibody on CCL1-mediated cell migration was detected by Transwell assay.

[0173] The experimental steps are as follows: BaF3-h.CCR8 cells were collected by digestion and centrifugation, resuspended in culture medium (RPMI1640 + 10% FBS + 1% P / S) and adjusted to a density of 800,000 / mL. 125 μL was added to the upper chamber of a Transwell (Corning, Catalog No.: 3421), and 125 μL of antibody diluted in culture medium was added; CCL1 was diluted to 5 ng / mL in culture medium, and 500 μL was added to the lower chamber; the cells were placed in a 37°C, 5% CO2 incubator and incubated for 5 hours; after incubation, the upper chamber was discarded, the liquid in the lower chamber was mixed, 200 μL was aspirated, 20 μL of CellTiter (Suzhou Ruian Bio, Catalog No.: RA-GL11-A) was added, and the cells were shaken for 3 minutes. The readings were read using a microplate reader (MD, i3x), and the inhibition rate was calculated.

[0174] The experimental results are shown in Figure 6. 96F1C4-hz, 13F3H6-hz, and 13F3H6-eADCC were all able to block CCL1-mediated cell migration, with inhibition rates at 50 μg / mL being 60.07%, 67.98%, and 73.52%, respectively, while the inhibition rate of the control antibody BM was 83.07%.

[0175] To summarize the above examples, see Table 5 for a comparative table of integrated data. Compared to the control antibody BM, 13F3H6-hz, 13F3H6-hz-eADCC, and 96F1C4-hz exhibited stronger affinities for human CC8. Among these, only 13F3H6-hz exhibited stronger affinity for cynomolgus macaque CCR8. The ADCC activities of the four clones, 96F1C4-hz, 76H1F11-hz, 13F3H6-hz, and 13F3H6-hz-eADCC, were all higher than those of the control antibody BM. Furthermore, 13F3H6-hz-eADCC exhibited higher ADCP activity than the control antibody BM. In addition, the inhibitory effects of 96F1C4-hz, 76H1F11-hz, 13F3H6-hz, and 13F3H6-hz-e in blocking CCL1-mediated cell ADCC were comparable to those of the control antibody BM.

[0176] Table 5. Comparison of various data between the antibodies disclosed herein and control antibodies

[0177] The foregoing is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. All documents mentioned in this disclosure are incorporated herein by reference in their entirety. In addition, it should be understood that after reading the above teachings of the present disclosure, those skilled in the art may make various changes or modifications to the present disclosure within the spirit and principles of the present disclosure, and such modifications in equivalent form also fall within the scope defined by the claims of this application.

[0178] Example 14: Accelerated stability testing of anti-human CCR8 humanized antibodies

[0179] The accelerated stability of a humanized anti-human CCR8 antibody was tested using the following method. The antibody was ultrafiltration-purified into PBS (pH 7.4) at a concentration of 5 mg / mL, sterilized by filtration, and stored at 37°C for 0, 7, and 14 days, followed by four and eight freeze-thaw cycles at -80°C. The purity of the sample was then assessed by SEC-HPLC and CE-SDS. The SEC-HPLC method was as follows: Instrument: Waters Alliance e2695 HPLC; Column: Thermo MabPac SEC-1, 5 μm, 7.8 x 300 mm; Mobile phase: 61 mmol / L Na₂HPO₄, 39 mmol / L NaH₂PO₄, 200 mmol / L NaCl, 5% IPA; Instrument parameters: Sample compartment temperature: 8°C; Column temperature: 30°C; Flow rate: 0.5 ml / min; Injection volume: 20 μg; Detection wavelength: 280 nm; Isocratic run: 30 min.

[0180] CE-SDS detection method is as follows: Dilute the sample to 4 mg / ml with ultrapure water. Transfer 25 μl to a centrifuge tube, add 75 μl of premix (SDS sample buffer + iodoacetamide), mix thoroughly, and centrifuge at 10,000 g for 1 minute. Incubate at 70°C for 5 minutes. Remove, cool to room temperature, centrifuge at 10,000 g for 1 minute, mix thoroughly, and transfer to a sample vial.

[0181] The analysis was performed using a SCIEX PA800 plus capillary electrophoresis instrument with the following instrument parameters: capillary: 30.2 cm*50 μm (bare tube), effective length: 20 cm; capillary column temperature: 25°C; sample tray temperature: 15°C; detection wavelength: 220 nm; acquisition frequency: 4 Hz.

[0182] The experimental results are shown in Table 6. 13F3H6-hz and 13F3H6-eADCC have good stability under accelerated and repeated freeze-thaw conditions. The SEC purity is above 99%, the CE-SDS non-reduced purity is around 90%, and the CE-SDS reduced purity is above 97%, indicating that 13F3H6-hz and 13F3H6-eADCC have good accelerated stability.

[0183] Table 6. Accelerated stability study of anti-CCR8 humanized antibodies

[0184] *Note: ND stands for Not Detected

[0185] Example 15: Plasma stability test of anti-human CCR8 humanized antibody

[0186] Humanized candidate antibodies 13F3H6-hz and 13F3H6-eADCC were diluted to 20 μg / mL in human plasma and sterilized by filtration through a 0.22 μm filter. Two time points were set for each candidate: 7 days and 14 days. Each tube contained 500 μL of the diluted solution. The tubes were sealed with parafilm and incubated in a 37°C incubator. Samples were collected on days 7 and 14 and stored in a -80°C refrigerator. The affinity of the plasma samples after incubation was determined by FACS, as described in the previous examples.

[0187] The experimental results are shown in Figures 7A and 7B. The affinity of 13F3H6-hz and 13F3H6-eADCC did not change significantly after incubation in plasma for 7 days and 14 days, indicating that 13F3H6-hz and 13F3H6-eADCC can remain stable in human plasma.

[0188] Example 16: Low pH incubation stability test of anti-human CCR8 humanized antibody

[0189] 13F3H6-hz and 13F3H6-eADCC were ultrafiltered and replaced into acetic acid-sodium acetate buffer (pH = 3.5) and incubated at room temperature for 4 hours. After incubation, the purity of the samples was detected by SEC-HPLC and CE-SDS, using the same detection methods as described in the above examples.

[0190] The experimental results are shown in Table 7. After incubation under low pH conditions, the purity of 13F3H6-hz and 13F3H6-eADCC did not change significantly, indicating that both can remain stable under low pH conditions.

[0191] Table 7. Low pH incubation stability study of anti-CCR8 humanized antibodies

[0192] Example 17: Pharmacokinetics of anti-human CCR8 humanized antibody in rats

[0193] To verify the metabolism of humanized anti-human CCR8 antibodies in rats, the test samples 13F3H6-hz and 13F3H6-eADCC were injected into the tail vein of male rats (Chengdu Dashuo), with 3 rats per group at a dose of 10 mg / kg. Serum samples were collected at 1 hour, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, and 336 hours after administration.

[0194] The blood drug concentration was detected by ELISA. The detection method is as follows:

[0195] (1) Coating: Dilute human IgG-heavy and light chain monkey-adsorbed Antibody (BETHYL, A80-319A) with coating solution to a final concentration of 1 μg / mL, add 100 μL / well to the ELISA plate, cover with film, and incubate at 2-8°C for overnight (15-18 hours).

[0196] (2) Washing: discard the coating solution, wash 3 times with 0.025% PBST, 300 μL / well, and pat dry. (3) Blocking: add blocking solution (2% BSA), 300 μL / well, cover with film, and incubate at 37°C for 2h±20min. (4) Washing: wash 3 times with 0.025% PBST, 300 μL / well, and pat dry. (5) Sample addition: add standard curve samples (13F3H6-hz and 13F3H6-eADCC starting at 50 ng / mL, 2-fold dilution, 8 concentration points) and test samples to the ELISA plate in duplicate wells, 100 μL / well, cover with film, and incubate at 37°C for 1h±5min. (6) Washing: wash 3 times with 0.025% PBST, 300 μL / well, and pat dry. (7) Adding secondary antibody: Dilute Goat Anti-Human IgG, Monkey ads-HRP (Southern Biotech, 2049-05) 8000-fold with diluent (2% BSA), add 100 μL / well, cover with film, and incubate at 37°C for 1h±5min. (8) Washing: Wash three times with 0.025% PBST, add 300 μL / well, and pat dry. (9) Color development: Add TMB substrate color development solution, add 100 μL / well, cover with film, and develop at 15-25°C in the dark for 2-15min. (10) Stopping: Add stop solution (1M H2SO4), add 50 μL / well to stop the reaction. (11) Detection: Read the OD value of each well at a detection wavelength of 450nm (reference wavelength 620nm) using a microplate reader. (12) Data processing and analysis: GraphPad Prism 8 software was used to fit the standard curve equation using the four-parameter method to the OD values ​​of the standard samples obtained by the microplate reader, and the concentration results of each sample were calculated.

[0197] The experimental results are shown in Table 8 and Figure 8. The half-life of 13F3H6-hz and 13F3H6-eADCC in rats is about 12 days.

[0198] Table 8. Metabolism study of anti-CCR8 humanized antibody in rats

[0199] Example 18: In vivo efficacy testing of anti-human CCR8 humanized antibodies

[0200] To verify the in vivo efficacy of the humanized anti-human CCR8 antibody, 6-8 week-old female human CCR8 transgenic mice C57BL / 6J-Ccr8em3(hCCR8) / Smoc (Shanghai Model Organisms Science Co., Ltd.) were purchased. MC38 cells (Sichuan Xiapaison) grown to the logarithmic growth phase were digested with trypsin and resuspended in PBS. Each mouse was subcutaneously inoculated with 5 × 10 6 cells, wait until the tumor grows to 100-200m 3 The drug was administered intravenously twice weekly at 10 mg / kg for a total of six doses, and tumor volume and mouse body weight were measured. The main outcome measures for this study were: 1) TGI (%), calculated as follows: TGI (%) = (1-T / C) × 100% (T and C are the relative tumor volumes of the treatment group, control group, and control antibody BM at a specific time point); 2) End-point tumor volume photographs and tumor weight.

[0201] The experimental results, shown in Figure 9, show that both 13F3H6-hz and 13F3H6-eADCC inhibited MC38 tumor growth in mice, with TGIs of 29.5% and 47.0%, respectively, compared to 18.2% for the control antibody. Changes in mouse body weight are shown in Figure 10, demonstrating that the humanized anti-CCR8 antibody had no effect on mouse body weight throughout the administration process.

Claims

1. An antibody that specifically binds to CCR8, characterized in that The invention comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are selected from the amino acid sequences corresponding to any of the following HCDR1, HCDR2 and HCDR3 combinations: (a) SEQ ID NOs: 3, 4, and 5; (b) SEQ ID NOs: 6, 7, and 5; (c) SEQ ID NOs: 8, 9, and 5; (d) SEQ ID NOs: 10, 11 and 12; (e) SEQ ID NOs: 20, 21 and 22; (f) SEQ ID NOs: 23, 24, and 22; (g) SEQ ID NOs: 25, 26, and 22; (h) SEQ ID NOs: 27, 28 and 29; (i) SEQ ID NOs: 37, 38 and 39; (j) SEQ ID NOs: 40, 41 and 39; (k) SEQ ID NOs: 42, 43, and 39; (1) SEQ ID NOs: 44, 45 and 46; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are selected from the amino acid sequences corresponding to any of the following combinations of LCDR1, LCDR2, and LCDR3: (a) SEQ ID NOs: 13, 14, and 15; (b) SEQ ID NOs: 16, 17, and 15; (c) SEQ ID NOs: 30, 31 and 32; (d) SEQ ID NOs: 33, 34, and 32; (e) SEQ ID NOs: 47, 48 and 49; (f) SEQ ID NOs: 50, 51 and 49.

2. The antibody according to claim 1, characterized in that Comprising HCDR1, HCDR2, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, selected from the amino acid sequences corresponding to any one of the following combinations of HCDR1, HCDR2, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: (a) SEQ ID NOs: 37, 38, 39, 47, 48, and 49; (b) SEQ ID NOs: 40, 41, 39, 47, 48, and 49; (c) SEQ ID NOs: 42, 43, 49, 47, 48, and 49; (d) SEQ ID NOs: 44, 45, 46, 50, 51 and 49; (e) SEQ ID NOs: 3, 4, 5, 13, 14, and 15; (f) SEQ ID NOs: 6, 7, 5, 13, 14, and 15; (g) SEQ ID NOs: 8, 9, 5, 13, 14, and 15; (h) SEQ ID NOs: 10, 11, 12, 16, 17, and 15; (i) SEQ ID NOs: 20, 21, 22, 30, 31 and 32; (j) SEQ ID NOs: 23, 24, 22, 30, 31 and 32; (k) SEQ ID NOs: 25, 26, 22, 30, 31 and 32; (1) SEQ ID NO: 27, 28, 29, 33, 34 and 32.

3. The antibody according to claim 1, characterized in that The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, 18 or 35.

4. The antibody according to claim 1, characterized in that The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, 19 or 36.

5. The antibody according to claim 1, characterized in that The heavy chain variable region and light chain variable region sequences are selected from any one of the following amino acid sequence combinations: (a) the heavy chain variable region set forth in SEQ ID NO: 1 and the light chain variable region set forth in SEQ ID NO: 2; (b) the heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 19; (c) The heavy chain variable region represented by SEQ ID NO: 35 and the light chain variable region represented by SEQ ID NO:

36.

6. The antibody according to claim 1, wherein the heavy chain variable region of the antibody further comprises a human, human or non-human framework region, and / or the light chain variable region of the antibody further comprises a human, human or non-human framework region.

7. The antibody according to claim 1, characterized in that The antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, an antibody fragment thereof, or an antigen-binding portion thereof.

8. The antibody of claim 7, wherein the antibody is selected from the following combinations: (i) a single-chain antibody or a single-chain variable fragment, or a monovalent antibody lacking a hinge region; (ii) Fab, Fab', F(ab')2; (iii) a full-length antibody; (iv) an antibody comprising a human IgG Fc domain.

9. The antibody according to any one of claims 1 to 8, wherein the antibody has one or more biological activities selected from the group consisting of: (1) Binds to both cynomolgus monkey and human CCR8; (2) possess ADCC activity; (3) Possess ADCP activity; (4) does not bind to human CCR4; (5) It has the activity of blocking CCL1-mediated cell migration.

10. A recombinant protein comprising the antibody, antibody fragment or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody, antibody fragment or antigen-binding fragment thereof according to any one of claims 1 to 9, or the recombinant protein according to claim 10.

12. A carrier, characterized in that Comprising the nucleic acid molecule of claim 11.

13. The carrier according to claim 12, characterized in that The vector is selected from bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or mammalian cell virus.

14. An engineered host cell, characterized in that Comprising the nucleic acid molecule according to claim 11 or the vector according to claim 12.

15. An antibody-drug conjugate, characterized in that: Include: (i) a cytotoxic agent, a detection label, a drug, a cytokine, a radionuclide, an enzyme, or a combination thereof; and (ii) the antibody according to any one of claims 1 to 9, the recombinant protein according to claim 10, or a combination thereof.

16. A pharmaceutical composition, characterized in that Include: (i) the antibody, antibody fragment or antigen-binding fragment thereof according to any one of claims 1 to 9, the recombinant protein according to claim 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the engineered host cell according to claim 14, the antibody-drug conjugate according to claim 15, or / and a combination thereof; and (ii) a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, wherein The pharmaceutical composition is used to inhibit the binding of CCR8 to any of the CCR8 ligands, or to be used as a neutralizing antibody.

18. The pharmaceutical composition according to claim 17, wherein It can be used to remove tumor-infiltrating Treg cells or to treat cancer.

19. A kit or drug delivery device, characterized in that: Comprising the antibody, antibody fragment or antigen-binding fragment thereof according to any one of claims 1 to 9, the recombinant protein according to claim 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the engineered host cell according to claim 14, the antibody-drug conjugate according to claim 15, and the pharmaceutical composition according to claim 16.

20. A method for preparing a recombinant polypeptide, the method comprising: (i) culturing the engineered host cell of claim 14 under conditions suitable for expression; and (ii) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody, an antigen-binding fragment thereof or a recombinant protein according to claims 1 to 10.

Images