Anti-crr8 antibodies and uses thereof

Abstract

This disclosure provides antibodies that specifically bind to CCR8, antibody fragments, their antigen-binding portions, and their related applications. The anti-CCR8 antibody disclosed herein possesses the differentiated advantage of targeting CCR8 and has promising application prospects, enabling the provision of more drug options and treatment regimens for cancer patients.

Description

Technical Field

[0001] This disclosure relates to the fields of biomedical or biopharmaceutical technology, and more specifically to anti-CCR8 antibodies and their applications. Background Technology

[0002] Regulatory T cells (Tregs) are a subset of T cells that control the body's autoimmune response and are closely related to the occurrence of autoimmune diseases. They can also promote immune escape of tumor cells 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 a structure that spans seven membranes and consists of 355 amino acids, including a short extracellular N-terminus (1-35 amino acids) and intracellular C-terminal serine / threonine residues that can serve as phosphorylation sites in the receptor regulation process, thereby mediating cellular responses.

[0004] CCR8 is a chemokine receptor highly expressed in tumor-infiltrating Tregs, but its expression is lower in Treg cells from the thymus, spleen, and peripheral blood, and also lower in Th2 cells. CCR8's ligand is the chemokine CCL1, which is upregulated at sites of inflammation. CCL1 can recruit FOXP3. + CCR8 + Treg cells infiltrate tumor tissue and exert immunosuppressive functions. Simultaneously, CCL1 can also induce FOXP3. + Upregulation of CCR8 expression on the surface of Treg cells induces Ca2+ expression. 2+ The 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 for Tregs in the tumor microenvironment (TME) without inducing systemic autoimmunity. Therefore, antibodies targeting CCR8 can improve the immune function of the TME by clearing Tregs cells in the tumor microenvironment, demonstrating anti-tumor potential. Currently, several pharmaceutical companies, including Bristol-Myers Squibb (BMS), have CCR8-targeted antibody drugs in clinical trials, but all are in Phase I. Therefore, developing differentiated CCR8-targeted antibody drugs has broad application prospects and can provide cancer patients with more drug options and treatment plans. Summary of the Invention

[0006] This disclosure develops novel anti-CCR8 antibodies or antibody fragments thereof that are capable of binding to CCR8 derived from primates (humans and / or monkeys), and also provides anti-CCR8 antibodies, antibody fragments thereof, or antigen-binding portions thereof that can be used as cancer therapeutic agents.

[0007] To achieve the above objectives, this disclosure provides the following technical solution:

[0008] This disclosure provides an antibody comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, selected from any combination of the following amino acid sequences:

[0009] (a) SEQ ID NO: 3, 4, 5;

[0010] (b)SEQ ID NO: 6, 7, 5;

[0011] (c)SEQ ID NO: 8, 9, 5;

[0012] (d) SEQ ID NO: 10, 11, 12;

[0013] (e) SEQ ID NO: 20, 21, 22;

[0014] (f) SEQ ID NO: 23, 24, 22;

[0015] (g) SEQ ID NO: 25, 26, 22;

[0016] (h) SEQ ID NO: 27, 28, 29;

[0017] (i) SEQ ID NO: 37, 38, 39;

[0018] (j) SEQ ID NO: 40, 41, 39;

[0019] (k) SEQ ID NO: 42, 43, 39;

[0020] (l) SEQ ID NO: 44, 45, 46;

[0021] And includes a light chain variable region comprising LCDR1, LCDR2, and LCDR3, selected from any combination of the following amino acid sequences:

[0022] (a) SEQ ID NO: 13, 14, 15;

[0023] (b) SEQ ID NO: 16, 17, 15;

[0024] (c) SEQ ID NO: 30, 31, 32;

[0025] (d) SEQ ID NO: 33, 34, 32;

[0026] (e) SEQ ID NO: 47, 48, 49;

[0027] (f) SEQ ID NO: 50, 51, 49;

[0028] The aforementioned antibody specifically binds to one or more amino acids within the extracellular domain of CCR8, the amino acid sequence further comprising a derived sequence by adding, deleting, modifying and / or substituting one or more amino acids, and is capable of maintaining its ability to bind to CCR8.

[0029] Preferably, the antibody comprises HCDR1, HCDR2, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, selected from any combination of the following amino acid sequences:

[0030] (a) SEQ ID NO: 37, 38, 39, 47, 48, 49;

[0031] (b) SEQ ID NO: 40, 41, 39, 47, 48, 49;

[0032] (c) SEQ ID NO: 42, 43, 49, 47, 48, 49;

[0033] (d) SEQ ID NO: 44, 45, 46, 50, 51, 49;

[0034] (e) SEQ ID NO: 3, 4, 5, 13, 14, 15;

[0035] (f) SEQ ID NO: 6, 7, 5, 13, 14, 15;

[0036] (g) SEQ ID NO: 8, 9, 5, 13, 14, 15;

[0037] (h) SEQ ID NO: 10, 11, 12, 16, 17, 15;

[0038] (i) SEQ ID NO: 20, 21, 22, 30, 31, 32;

[0039] (j) SEQ ID NO: 23, 24, 22, 30, 31, 32;

[0040] (k) SEQ ID NO: 25, 26, 22, 30, 31, 32;

[0041] (l) SEQ ID NO: 27, 28, 29, 33, 34, 32;

[0042] The aforementioned antibody specifically binds to one or more amino acids within the extracellular domain of CCR8, the amino acid sequence further comprising a derived sequence by adding, deleting, modifying, and / or substituting one or more amino acids, and is capable of maintaining its ability to bind to CCR8.

[0043] Preferably, the heavy chain further includes a heavy chain constant region and / or the light chain further includes a light chain constant region.

[0044] Preferably, the variable region of the heavy chain contains the amino acid sequence shown in any one of SEQ ID NO: 1, 18, and 35.

[0045] Preferably, the variable region of the light chain contains the amino acid sequence shown in any one of SEQ ID NO: 2, 19, 36.

[0046] Preferably, the antibody described above comprises a heavy chain variable region and a light chain variable region selected from any combination of the following amino acid sequences:

[0047] (a) SEQ ID NO: 1, 2;

[0048] (b)SEQ ID NO: 18, 19;

[0049] (c)SEQ ID NO: 35, 36.

[0050] More preferably, the antibody comprises a light chain variable region and a heavy chain variable region; wherein the aforementioned heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 1, 18, or 35, or has more than 95% identity with the amino acid sequence of SEQ ID NO: 1, 18, or 35; the aforementioned light chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 2, 19, or 36, or has more than 95% identity with the amino acid sequence of SEQ ID NO: 2, 19, or 36.

[0051] More preferably, the antibody can bind to CCR8 derived from primates, preferably humans.

[0052] More preferably, the variable region of the heavy chain of the antibody further includes a human, human-derived, or human-derived frame region, and / or the variable region of the light chain of the antibody further includes a human, human-derived, or human-derived frame region.

[0053] More preferably, the antibodies mentioned above include monoclonal antibodies, chimeric antibodies, recombinant antibodies, fully human antibodies, humanized antibodies, bispecific antibodies, multispecific antibodies, antibody fragments thereof, or antigen-binding portions thereof.

[0054] More preferably, the antibodies are selected from the following combinations: (i) single-chain antibodies or single-chain variable fragments, or monovalent antibodies lacking a hinge region; (ii) Fab, Fab', F(ab')2; (iii) full-length antibodies; (iv) antibodies containing the human IgG Fc domain.

[0055] Preferably, the antibody has one or more biological activities selected from the following:

[0056] (1) It binds to both cynomolgus monkey and human CCR8;

[0057] (2) It has ADCC activity;

[0058] (3) Does not bind to human CCR4;

[0059] (4) It has the activity of blocking CCL1-mediated cell migration.

[0060] In one embodiment, this disclosure also provides a recombinant protein comprising the antibody described above, an antibody fragment thereof, or an antigen-binding fragment thereof.

[0061] Preferably, the recombinant protein further comprises a tag sequence to aid in expression and / or purification.

[0062] In another embodiment, this disclosure also provides a nucleic acid molecule that encodes the aforementioned antibody, its antibody fragment or antigen-binding fragment, or the aforementioned recombinant protein.

[0063] In another embodiment, this disclosure also provides a carrier comprising the above-described nucleic acid molecules.

[0064] Preferably, the vectors described above include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, such as adenoviruses, lentiviruses, or retroviruses.

[0065] In another embodiment, this disclosure also provides an engineered host cell comprising the above-described nucleic acid molecules or the above-described vector.

[0066] In another embodiment, this disclosure provides an antibody-drug conjugate comprising (i) a cytotoxic agent, a detection tag, a drug, a cytokine, a radionuclide, an enzyme, or a combination thereof; and (ii) the antibody, recombinant protein, or a combination thereof described above.

[0067] In a preferred embodiment, this disclosure also provides a pharmaceutical composition comprising (i) the antibody described above, an antibody fragment thereof or an antigen-binding fragment thereof, the recombinant protein described above, the nucleic acid molecule described above, the carrier described above, the engineered host cell described above, the antibody-drug conjugate described above, or / and combinations thereof; and (ii) a pharmaceutically acceptable carrier.

[0068] Preferably, the aforementioned pharmaceutical composition is used to block the binding of CCR8 to any of the CCR8 ligands, or as a neutralizing antibody.

[0069] Preferably, the aforementioned pharmaceutical composition is used to remove tumor-infiltrating Treg cells or to treat cancer.

[0070] In another embodiment, this disclosure provides a kit or drug delivery device comprising the antibody described above, an antibody fragment thereof or an antigen-binding fragment thereof, the recombinant protein described above, the nucleic acid molecule described above, the vector described above, the engineered host cell described above, the antibody-drug conjugate described above, and the pharmaceutical composition described above.

[0071] Preferably, the kit described above further includes a second antibody against the first antibody.

[0072] This disclosure provides a method for preparing recombinant polypeptides, the method comprising:

[0073] (i) Culturing engineered host cells under suitable expression conditions; and

[0074] (ii) Isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody, its antigen-binding fragment, or the recombinant protein described above.

[0075] Compared with existing technologies, the beneficial effects of the disclosed anti-CCR8 antibody are as follows:

[0076] (1) It has a strong binding ability with primates, especially cynomolgus monkeys and human CCR8.

[0077] (2) It has high antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP) activity.

[0078] (3) It does not bind nonspecifically to human CCR4.

[0079] (4) It can block CCL1-mediated cell migration and has a high inhibition rate.

[0080] (5) It has advantages as a method for detecting human CCR8, or as a drug or for the prevention and treatment of human CCR8-related diseases.

[0081] In one embodiment, this disclosure provides Attached Figure Description

[0082] The accompanying drawings, which form part of this application, are used to provide a further understanding of this disclosure. The illustrative embodiments of this disclosure and their descriptions are used to explain this disclosure and do not constitute an undue limitation of this disclosure. In the drawings:

[0083] Figure 1A The results show the cell affinity assay between the anti-human CCR8 humanized antibody and HEK293T-h.CCR8.

[0084] Figure 1B The results show the cell affinity assay between the anti-human CCR8 humanized antibody and HEK293T-h.CCR8.

[0085] Figure 2 The results show the cell affinity assay between the anti-human CCR8 humanized antibody and HEK293T-c.CCR8.

[0086] Figure 3A The results show the binding of the anti-human CCR8 humanized antibody to HEK293T-h.CCR4. Count: cell number; Control: control group.

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

[0088] Figure 4A The results show the cell affinity assay between the anti-human CCR8 humanized antibody and HEK293T-h.CCR8.

[0089] Figure 4B The results are for ADCC activity assay of humanized anti-human CCR8 antibody.

[0090] Figure 4C The results are for ADCC activity assay of humanized anti-human CCR8 antibody.

[0091] Figure 5 The results are for the detection of ADCP activity of the humanized anti-human CCR8 antibody.

[0092] Figure 6 The results of the assay for the blockade of CCL1-mediated cell migration activity by the humanized anti-human CCR8 antibody.

[0093] Figure 7A The results show the cell affinity of 13F3H6-hz with HEK293T-h.CCR8 after incubation in human plasma. Control: control group.

[0094] Figure 7BThe results show the cell affinity of 13F3H6-eADCC with HEK293T-h.CCR8 after incubation in human plasma. Control: control group.

[0095] Figure 8 The results show the blood drug concentrations of 13F3H6-hz and 13F3H6-eADCC in rats.

[0096] Figure 9 This is the in vivo efficacy test result of the anti-human CCR8 humanized antibody in the human CCR8 transgenic mouse MC38 model. Control: control group.

[0097] Figure 10 The results show the changes in body weight in the human CCR8 transgenic mouse MC38 model using anti-human CCR8 humanized antibody. Control: control group. Detailed Implementation

[0098] The present disclosure will be further described below with reference to the accompanying drawings and specific embodiments. The scope of protection of this disclosure is not limited to the following embodiments. It should also be understood that the terminology used in the embodiments of this disclosure is for describing specific implementations and is not intended to limit the scope of protection of this disclosure, nor is it a unique limitation. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this disclosure, and the scope of protection of this disclosure is defined by the appended claims and any equivalents.

[0099] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In other instances, the meaning of certain terms used herein will be clarified in the specification. Experimental methods in the following embodiments, without specific conditions specified, are common knowledge and general knowledge to those skilled in the art. The embodiments and features described in this application can be combined with each other.

[0100] The features and advantages of this disclosure can be further understood through the following detailed description in conjunction with the accompanying drawings. The provided embodiments are merely illustrative of the methods of this disclosure and do not limit the rest of the content disclosed in this disclosure in any way.

[0101] The antibodies disclosed herein refer to proteins that specifically bind to CCR8 in primates (preferably humans), including monoclonal antibodies, chimeric antibodies, recombinant antibodies, fully human antibodies, humanized antibodies, bispecific antibodies, multispecific antibodies, antibody fragments thereof, or their antigen-binding portions. 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', and F(ab')2; full-length antibodies; and antibodies containing the human IgG Fc domain, etc.

[0102] This disclosure also includes recombinant proteins, wherein the recombinant proteins are antibodies of this disclosure, antibody fragments thereof, or antigen-binding fragments thereof, and tag sequences for aiding expression and / or purification.

[0103] This disclosure includes nucleic acid molecules or polynucleotides (light chain variable regions or heavy chain variable regions) encoding antibodies of this disclosure. This disclosure also includes expression vectors containing such 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).

[0104] This disclosure also includes cells for expressing the antibodies of this disclosure, the cells containing the above-described nucleic acid molecules or the above-described vectors.

[0105] The antibodies disclosed herein can be used as drug conjugates, and may also include cytotoxic agents, detection tags, drugs, cytokines, radionuclides, enzymes or combinations thereof; as well as the antibodies, recombinant proteins or combinations thereof disclosed herein.

[0106] The antibody disclosed herein can be used as a pharmaceutical composition, particularly for recognizing human CCR8. The composition comprises the antibody of this disclosure, an antibody fragment thereof, or an antigen-binding fragment thereof; a recombinant protein; a nucleic acid molecule; a vector; an engineered host cell; an antibody-drug conjugate; or / and combinations thereof. In addition, it includes a pharmaceutically acceptable vector, such as an AAV vector. Therefore, the pharmaceutical composition of this disclosure can inhibit the binding of CCR8 to any of its ligands, and can be used for the treatment and / or prevention of cancer, the removal of tumor-infiltrating Treg cells, or as a neutralizing antibody.

[0107] abbreviations

[0108] CCR8: Chemokine (CC motif) receptor 8.

[0109] CCL1: Chemokinetic (CC motif) ligand 1.

[0110] Th2: Helper T cells 2.

[0111] CDR: Complementarity Determinant Region.

[0112] FR: Amino acid residues in the antibody variable region other than CDR residues.

[0113] V H Antibody heavy chain variable region.

[0114] V L : Antibody light chain variable region.

[0115] IgG: Immunoglobulin G.

[0116] Kabat: An immunoglobulin matching and numbering system proposed by Elvin A. Kabat.

[0117] Chothia: An immunoglobulin numbering system proposed by Chothia et al.

[0118] IMGT: A numbering system based on the International Immunogenetic Information System initiated by Lefranc et al.

[0119] EC 50 Half-maximal effect concentration, which is the concentration that can cause 50% of the maximum effect.

[0120] ELISA: Enzyme-linked immunosorbent assay.

[0121] FACS: Flow Cytometry.

[0122] PCR: Polymerase chain reaction.

[0123] HRP: Horseradish peroxidase.

[0124] ADCC: Antibody-dependent cell-mediated cytotoxicity.

[0125] ADCP: Antibody-dependent cell-mediated phagocytosis.

[0126] DMEM: Dulbecco's Modified Eagle Medium.

[0127] RPMI 1640: Roswell Park Memorial Institute 1640 medium.

[0128] FBS: Fetal bovine serum.

[0129] Example 1: Construction of CCR8 overexpression cell line and expression of antigen protein

[0130] The amino acid sequence of human CCR8 was referenced from P51685 in the UniProt protein database. The amino acid sequence of human CCR4 (C-Cchemokine receptor type 4) was referenced from P51679 in the UniProt protein database. The amino acid sequence of cynomolgus monkey CCR8 was referenced from XP_015300839.1 in the NCBI protein database. These amino acid sequences were codon-optimized by Anhui General Technology Co., Ltd., and synthesized on the lentiviral vector pLVX or pCDNA3.4.

[0131] Viruses were prepared using a lentiviral packaging system and then used to infect HEK293T and BaF3 cells. Stable cell lines were obtained through selection with puromycin (Invivogen, catalog number: ant-pr-1): HEK293T-h.CCR8 (human), HEK293T-h.CCR4 (human), HEK293T-c.CCR8 (cynomolgus monkey), and BaF3-h.CCR8 (human). After successful construction, the positive rate of target expression was determined by flow cytometry (FACS) for subsequent experiments.

[0132] The N-terminal extracellular domain of human and cynomolgus monkey CCR8 proteins contains 35 amino acids (1-35 AA). The N-terminal extracellular domain of human and cynomolgus monkey CCR8 proteins was synthesized into 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 (1-35 AA) and cynomolgus monkey c.CCR8 (1-35 AA) antigen proteins were purified using ProA packing material (GE, Mabselect XL).

[0133] Example 2: Preparation of anti-human CCR8 murine monoclonal antibody

[0134] Wild-type mice aged 5-6 weeks from three strains—Balb / C, Kunming (KM), and CD1—were immunized in two batches. The first batch underwent intraperitoneal and dorsal immunization using the human h.CCR8 (1-35AA) protein antigen, while the second batch underwent tail vein immunization using 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).

[0135] The experimental procedure is 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-chilled PBS, and the cells were resuspended in PBS (containing 1% BSA) at 3 × 10⁻⁶ cells per well. 5Cells were seeded into 96-well conical plates, and serum diluted 100, 300, and 900 times was added. Cells were incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 1 μL of anti-mouse fluorescent secondary antibody was added to each well, and the cells were incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed using a flow cytometer (Beckman, CytoFLEX). Hybridoma fusion was performed on mice with high titers of HEK293T-h.CCR8 and HEK293T-c.CCR8 but low titers of HEK293T-h.CCR4.

[0136] Mouse spleen and lymph nodes were used to prepare cell suspensions, which were then mixed with SP2 / 0 mouse myeloma cells at a 1:1 ratio. The mixtures were resuspended in cell electrofusion buffer and electrofusion were performed using a BTX-ECM2001 cell fusion reactor. After electrofusion, the cells were resuspended in complete fusion medium (RPMI 1640 + 20% FBS + OPI + 1×HAT) and aliquoted at 20,000-25,000 cells per well into 96-well cell culture plates. The cells were cultured at 37°C and 5% CO2. Following culture, HEK293T-h.CCR8, HEK293T-h.CCR4, and HEK293T-c.CCR8 cells were screened using FACS to obtain parent clones with high affinity for human and cynomolgus monkey CCR8 cells on the cell membrane but without binding to human CCR4. These clones were then used for single-clone screening. Monoclonal hybridoma cells were cultured in serum-free medium (Zhuhai Kairui Biotechnology, K04114). The collected monoclonal supernatant was purified using ProA (GE, Mabselect XL) packing material to obtain mouse antibodies.

[0137] Example 3: Evaluation of anti-human CCR8 murine monoclonal antibody

[0138] The affinity of the anti-human CCR8 murine monoclonal antibody was evaluated using FACS. HEK293T-h.CCR8 and HEK293T-c.CCR8 cells were collected by digestion and centrifugation, then washed three times with pre-chilled PBS, and resuspended in PBS (containing 1% BSA) at 3 × 10⁻⁶ cells per well. 5 Cells were seeded into 96-well conical plates, serially diluted mouse antibody was added, and incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 1 μL of anti-mouse fluorescent secondary antibody was added to each well, and the cells were incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed using a flow cytometer (Beckman, CytoFLEX). The data were imported into GraphPad Prism for plotting and EC50 was calculated. 50 value.

[0139] The specificity of the anti-human CCR8 murine monoclonal antibody was evaluated using FACS. The experimental procedure was as follows: HEK293T-h.CCR4 cells were digested, centrifuged to collect the cells, and washed three times with pre-chilled PBS; the cells were resuspended in 1% BSA (in PBS), and 3 × 10⁻⁶ BSA was added to each well of a 96-well conical plate. 5 Cells were collected in 50 μL wells. The antibody was diluted to 20 μg / mL with 1% BSA (in PBS), and 50 μL was added to each well to achieve a final antibody concentration of 10 μg / mL. The mixture was incubated at 4°C for 1 hour. Cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 50 μL of 1% BSA (in PBS). 1 μL of fluorescent secondary antibody was added to each well, and the mixture was incubated at 4°C for 0.5 hours. Cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 200 μL of PBS for sequencing. The results are shown in Table 1. Clones with high affinity for HEK293T-h.CCR8 and no non-specific binding to HEK293T-h.CCR4 were selected for sequencing, i.e., HEK293T-h.CCR8 EC was selected. 50 Clones with low values ​​and low HEK293T-h.CCR4 positivity rates. Among all murine antibodies, only 13F3H6 showed a strong binding to HEK293T-c.CCR8, and its EC50 value was low. 50 The concentration was 287.8 ng / mL; all other murine antibodies either did not bind to cynomolgus monkey CCR8 or bound very weakly. The murine antibody clones that were sequenced are: 76H1F11, 96F1C4, and 13F3H6.

[0140] Table 1. Summary of Evaluation Results of Murine Antibodies

[0141]

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

[0143] Approximately 1 × 10⁶ of the aforementioned 13F3H6, 96F1C4, and 76H1F11 candidate hybridoma cells were collected. 5 RNA was extracted from samples using Trizol (Invitrogen, 15596026), and cDNA was obtained by reverse transcription using PrimeScript RT reagent (TAKARA, RR047A) via PolyA. Upstream primers were designed for the heavy and light chains, and downstream primers were designed for the CH1 region of the heavy chain and the CL region of the light chain. The products were amplified by PCR (Gold Medal MIX, Qingke Biotechnology, TSE101), and fragments were recovered using an agarose gel extraction kit. The samples were then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained sequences are shown in Table 2.

[0144] Table 2. Mouse antibody sequence listing

[0145]

[0146]

[0147] Example 5: Humanization of anti-human CCR8 murine monoclonal antibody

[0148] Using the CDR grafting method, the human germline sequence with the highest homology to the original mouse sequence was first identified using the conventional BLAST method and used as a template. The CDR of the mouse antibody was then grafted onto the human template to construct a chimera. Based on structural analysis, the FR amino acids in the mouse antibody that could retain their original conformation were identified, and the corresponding amino acids in the chimera were reverted to mouse amino acids to maintain the original affinity. The constructed humanized antibody was then subjected to calculation and immunogenicity analysis to identify high immunogenic fragments and replace low immunogenic fragments. The humanized sequence and CDR definition are shown in Table 3.

[0149] Table 3. Humanized Antibody Sequence Listing

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] Example 6: Preparation of humanized monoclonal antibody against human CCR8

[0157] The amino acid sequences of the variable regions of the heavy chain and light chain of the humanized monoclonal antibodies 96F1C4-hz, 13F3H6-hz, and 76H1F11-hz against human CCR8, as well as the variable region of the control antibody (BM sequence derived from patent: CN 113260381A), were submitted to Universal Biotech 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 ADCC (antibody-dependent cell-mediated cytotoxicity) enhancement of the 13F3H6-hz antibody. It is obtained by mutating five amino acids (F243L, R292P, Y300L, L235V, P396L) at the Fc terminus of the wild-type IgG1 of 13F3H6-hz (SEQ ID NO:53) to obtain a candidate antibody with stronger ADCC activity. After plasmid synthesis, it was extracted by shaking and transfected into HEK293E cells via PEImax. Expression was carried out for approximately 7 days, and the supernatant was collected by centrifugation. The expression supernatant was purified using MabSelectSure affinity chromatography-mass spectrometry. All purified antibodies were ultrafiltered into PBS buffer, their concentration was determined, and they were aliquoted and stored at -20°C.

[0158] Table 4. Correspondence between CDR, variable region and constant region of humanized antibody

[0159]

[0160] Example 7: Affinity evaluation of humanized monoclonal antibody against human CCR8

[0161] The affinity of the humanized monoclonal antibody against human CCR8 was detected by FACS. The experimental procedure was as follows: HEK293T-h.CCR8 cells were digested, centrifuged to collect the cells, and washed three times with pre-chilled PBS; the cells were resuspended in 1% BSA (in PBS), and 3 × 10⁶ cells were added to each well of a 96-well conical plate. 5 50 μL of cells were collected. The antibody was diluted with 1% BSA (in PBS) to a starting concentration of 40 μg / mL. The antibody was then 3-fold diluted to 10 concentration points. 50 μL of each antibody was added to the cells to bring the final antibody concentration to 20 μg / mL. The mixture was incubated at 4°C for 1 hour. Cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 50 μL of 1% BSA (in PBS). 1 μL of fluorescent secondary antibody (Biolegend, catalog number: 410708) was added to each well, and the mixture was incubated at 4°C for 0.5 hours. Cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 200 μL of PBS for analysis.

[0162] Experimental results are as follows Figure 1A , Figure 1B As shown, 96F1C4-hz, 13F3H6-hz, and 76H1F11-hz all have high affinity for human CCR8, and their EC50... 50 The concentrations were 79.64 ng / mL, 104.3 ng / mL, and 321.8 ng / mL, respectively. The affinity of the control antibody BM for human CCR8 was 109.43 ± 9.98 ng / mL (n = 2), indicating that the affinities of 96F1C4-hz and 13F3H6-hz were comparable to those of the control antibody BM, while the affinity of 76H1F11-hz was slightly weaker.

[0163] Example 8: Evaluation of species affinity of humanized monoclonal antibody against human CCR8

[0164] The affinity of the humanized monoclonal antibody against human CCR8 to cynomolgus monkey CCR8 cells was determined by FACS. The experimental procedure was as follows: HEK293T-c.CCR8 cells were collected by digestion and centrifugation, washed three times with pre-chilled PBS, and resuspended in 1% BSA (in PBS) at 3 × 10⁻⁶ cells per well. 5 Cells were seeded into 96-well conical plates, serially diluted antibodies were added and incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 1 μL of fluorescent secondary antibody (Biolegend, catalog number: 410708) was added to each well and incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry (Beckman, model: CytoFLEX).

[0165] Experimental results are as follows Figure 2 As shown, 13F3H6-hz has a strong binding affinity to CCR8 in cynomolgus monkeys, and its EC50... 50 The value was 220.7 ng / mL. The control antibody BM bound weakly to cynomolgus monkey CCR8, while 96F1C4-hz and 76H1F11-hz did not bind to cynomolgus monkey CCR8.

[0166] Example 9: Specificity evaluation of humanized monoclonal antibody against human CCR8

[0167] The specificity of the humanized monoclonal antibody against human CCR8 was determined by FACS. The experimental procedure was as follows: HEK293T-h.CCR4 cells were collected by digestion and centrifugation, washed three times with pre-chilled PBS, and resuspended in 1% BSA (in PBS) at 3 × 10⁻⁶ cells per well. 5 Cells were seeded into 96-well conical plates, and the antibody to be tested was added to a final concentration of 20 μg / mL. The cells were incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 1 μL of fluorescent secondary antibody (Biolegend, catalog number: 410708) was added to each well and incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry (Beckman, model: CytoFLEX).

[0168] Experimental results are as follows Figure 3A , 3B As shown, 96F1C4-hz, 13F3H6-hz, and 76H1F11-hz do not specifically bind to human CCR4.

[0169] Example 10: Evaluation of the endocytic activity of humanized monoclonal antibody against human CCR8

[0170] The endocytic activity of the humanized monoclonal antibody against human CCR8 was detected by FACS. The experimental procedure was as follows: HEK293T-h.CCR8 cells were collected by digestion and centrifugation, incubated with the antibody at a concentration of 10 μg / mL at 4°C for 1 hour, washed three times with PBS, resuspended in DMEM + 10% FBS, divided into two aliquots, and incubated at 37°C for 0 hours and 5 hours respectively. After washing three times with PBS, 1 μL of fluorescent secondary antibody was added, and the cells were incubated at 4°C for 0.5 hours. After washing three times with PBS, the cells were resuspended and loaded onto the analyzer. The endocytosis rate was calculated using the following formula:

[0171] Internalization rate (%) = [1 - (average fluorescence value of 5-hour test sample - average fluorescence value of 5-hour negative control sample) / (average fluorescence value of 0-hour test sample - average fluorescence value of 0-hour negative control sample)] × 100.

[0172] Experimental results showed that both 96F1C4-hz and 13F3H6-hz could mediate CCR8 endocytosis, with endocytosis rates of 49.57% and 51.22% at 5 hours, respectively, compared to 54.41% mediated by the control antibody BM.

[0173] Example 11: Evaluation of antibody-dependent cell-mediated cytotoxicity (ADCC) activity of humanized monoclonal antibody against human CCR8

[0174] The affinity of 13F3H6-eADCC for 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, catalog number: 109-605-088). The detection results are as follows: Figure 4A As shown, 13F3H6-eADCC has a high affinity for human CCR8, and its EC50... 50 The affinity was 24.37 ng / mL. In the same experimental system, the affinity of 13F3H6-hz, control antibody BM, and human CCR8 was 20.26 ng / mL and 49.19 ng / mL, respectively, indicating that the affinity of 13F3H6-hz, 13F3H6-eADCC, and control antibody BM was basically equivalent.

[0175] The ADCC activity of the humanized monoclonal antibody against human CCR8 was detected using a luciferase reporter system. The experimental steps were as follows: HEK293T-h.CCR8 cells were collected by digestion and centrifugation, resuspended in DMEM + 1% FBS, and seeded at 50,000 cells per well; Jurkat-NFAT-CD16a cells were collected by centrifugation, resuspended in RPMI 1640 + 1% FBS, and seeded at 100,000 cells per well; serially diluted antibody was 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 readings were taken using a microplate reader (MD, i3x).

[0176] Experimental results are as follows Figure 4B , 4C As shown, 96F1C4-hz, 13F3H6-hz, 76H1F11-hz, and 13F3H6-eADCC all exhibit strong ADCC activity, and their EC50 values ​​are high. 50 The concentrations were 5.2 ng / mL, 9.77 ng / mL, 14.78 ng / mL, and 1.17 ng / mL, respectively. The EC50 of the control antibody BM... 50 The value was 17.19 ± 6.4 ng / mL (n = 2). These results indicate that the ADCC activities of 96F1C4-hz, 13F3H6-hz, and 13F3H6-eADCC were all superior to the control antibody BM.

[0177] Example 12: Evaluation of antibody-dependent cell-mediated phagocytosis (ADCP) activity of humanized monoclonal antibody against human CCR8

[0178] The ADCP activity of the humanized monoclonal antibody against human CCR8 was detected using a luciferase reporter system. The experimental steps were as follows: HEK293T-h.CCR8 cells were collected by digestion and centrifugation, resuspended in DMEM + 1% FBS, and seeded at 50,000 cells per well; Jurkat-FcγRIIa-H131 (Novizan, DD1304) cells were collected by centrifugation, resuspended in RPMI 1640 + 1% FBS, and seeded at 100,000 cells per well; serially diluted antibody was 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 readings were taken using a microplate reader (MD, i3x).

[0179] Experimental results are as follows Figure 5 As shown, 96F1C4-hz, 13F3H6-hz, and 13F3H6-eADCC all exhibit strong ADCP activity, and their EC50 values ​​are high. 50The concentrations were 107.3 ng / mL, 112.1 ng / mL, and 64.68 ng / mL, respectively. The EC50 of the control antibody BM... 50 The value was 75.08 ng / mL. These results indicate that the ADCP activities of 96F1C4-hz, 13F3H6-hz, and 13F3H6-eADCC are essentially equivalent to those of the control antibody BM.

[0180] Example 13: Assay of blocking CCL1-mediated cell migration with humanized monoclonal antibody against human CCR8

[0181] In this embodiment, the blocking activity of the anti-human CCR8 humanized monoclonal antibody against CCL1-mediated cell migration was detected by the Transwell assay.

[0182] The experimental steps are as follows: BaF3-h.CCR8 cells were collected by digestion and centrifugation, resuspended in culture medium (RPMI 1640 + 10% FBS + 1% P / S), and the density was adjusted to 800,000 / mL. 125 μL was added to the upper chamber of a Transwell (Corning, catalog number: 3421), and 125 μL of antibody diluted with culture medium was added. CCL1 was diluted to 5 ng / mL with culture medium, and 500 μL was added to the lower chamber. The cells were incubated at 37℃ and 5% CO2 for 5 hours. After incubation, the upper chamber was discarded, the liquid in the lower chamber was mixed, and 200 μL was aspirated and 20 μL of CellTiter (Suzhou Ruian Biotechnology, catalog number: RA-GL11-A) was added. After shaking for 3 minutes, the reading was taken using a microplate reader (MD, i3x), and the inhibition rate was calculated.

[0183] Experimental results are as follows Figure 6 As shown, 96F1C4-hz, 13F3H6-hz, and 13F3H6-eADCC can all block CCL1-mediated cell migration, with inhibition rates of 60.07%, 67.98%, and 73.52% at 50 μg / mL, respectively, while the inhibition rate of the control antibody BM was 83.07%.

[0184] The summarized data from the above embodiments are shown in Table 5. Compared with the control antibody BM, 13F3H6-hz, 13F3H6-hz-eADCC, and 96F1C4-hz showed stronger affinity for human CC8; however, only 13F3H6-hz exhibited strong affinity for cynomolgus monkey CCR8. The ADCP 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; among them, 13F3H6-hz-eADCC showed higher ADCP activity than the control antibody BM. In addition, the inhibitory effects of the 96F1C4-hz, 76H1F11-hz, 13F3H6-hz, and 13F3H6-hz-eADCCs disclosed herein on CCL1-mediated cell inhibition are comparable to those of the control antibody BM.

[0185] Table 5. Comparison of data between the antibody and the control antibody disclosed herein.

[0186]

[0187]

[0188] The above description is merely a preferred embodiment of this disclosure and is not intended to limit the scope of this disclosure. All documents mentioned in this disclosure are incorporated herein by reference in their entirety. Furthermore, it should be understood that after reading the foregoing teachings of this disclosure, those skilled in the art can make various alterations or modifications to this disclosure within the spirit and principles of this disclosure, and such equivalent modifications also fall within the scope defined by the claims of this application.

[0189] Example 14: Accelerated stability assay of anti-human CCR8 humanized antibody

[0190] The accelerated stability of the anti-human CCR8 humanized antibody was determined using the following method. The experimental procedure was as follows: The anti-human CCR8 humanized antibody was ultrafiltered into PBS (pH=7.4) at a concentration of 5 mg / mL. After filtration and sterilization, the sample was incubated at 37℃ for 0, 7, and 14 days, and subjected to 4 and 8 freeze-thaw cycles at -80℃. The purity of the sample was then determined 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*300 mm; Mobile phase: 61 mmol / L Na2HPO4, 39 mmol / L NaH2PO4, 200 mmol / L NaCl, 5% IPA; Instrument parameters: Sample chamber temperature: 8℃; Column temperature: 30℃; Flow rate: 0.5 mL / min; Injection volume: 20 μg; Detection wavelength: 280 nm; Isocratic run: 30 min.

[0191] The CE-SDS detection method is as follows: Dilute the sample to 4 mg / ml with ultrapure water. Take 25 μl into a centrifuge tube, add 75 μl of premixed solution (SDS Sample buffer + iodoacetamide), mix well, and centrifuge at 10000g for 1 minute. Incubate at 70℃ for 5 minutes. After removal, cool to room temperature, centrifuge at 10000g for 1 minute, mix well, and transfer to a sample vial.

[0192] The analysis was performed using a SCIEX PA800 plus capillary electrophoresis system. The instrument parameters were set as follows: capillary: 30.2cm*50μm (bare tube), effective length: 20cm, capillary column temperature: 25℃, sample tray temperature: 15℃, detection wavelength setting: 220nm; acquisition frequency: 4Hz.

[0193] The experimental results are shown in Table 6. 13F3H6-hz and 13F3H6-eADCC have good stability under accelerated and repeated freeze-thaw conditions. The purity of SEC is above 99%, the non-reduced purity of CE-SDS is around 90%, and the reduced purity of CE-SDS is above 97%, indicating that 13F3H6-hz and 13F3H6-eADCC have good accelerated stability.

[0194] Table 6. Accelerated stability study of anti-CCR8 humanized antibodies

[0195]

[0196] *Note: ND indicates not detected.

[0197] Example 15: Plasma stability assay of anti-human CCR8 humanized antibody

[0198] Humanized candidate antibodies 13F3H6-hz and 13F3H6-eADCC were diluted to 20 μg / mL using human plasma and sterilized by filtration through a 0.22 μm filter. Two time points were set for each candidate antibody: 7 days and 14 days. Each tube contained 500 μL, was sealed with sealing film, and incubated at 37°C. Samples were collected at 7 days and 14 days and stored at -80°C. The affinity of the plasma-incubated samples was detected by FACS, as described in the examples above.

[0199] Experimental results are as follows Figure 7A , 7B As shown, the affinity of 13F3H6-hz and 13F3H6-eADCC did not change significantly after 7 and 14 days of incubation in plasma, indicating that 13F3H6-hz and 13F3H6-eADCC can remain stable in human plasma.

[0200] Example 16: Detection of low pH incubation stability of anti-human CCR8 humanized antibody

[0201] The 13F3H6-hz and 13F3H6-eADCC ultrafiltration was replaced in acetate-sodium acetate buffer (pH=3.5) and incubated at room temperature for 4 hours. After incubation, the purity of the sample was detected by SEC-HPLC and CE-SDS, and the detection methods were as described in the above examples.

[0202] The experimental results are shown in Table 7. After incubation at low pH, the purity of 13F3H6-hz and 13F3H6-eADCC did not change significantly, indicating that both can remain stable under low pH conditions.

[0203] Table 7. Stability of anti-CCR8 humanized antibody under low pH incubation

[0204]

[0205]

[0206] Example 17: Pharmacokinetic Detection of Anti-Human CCR8 Humanized Antibody in Rats

[0207] To verify the metabolism of humanized anti-human CCR8 antibodies in rats, male rats (Chengdu Dashuo) were injected with the test samples 13F3H6-hz and 13F3H6-eADCC via tail vein injection, with 3 rats per group and a dosage of 10 mg / kg. Serum samples were collected at 1 h, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, and 336 h after administration.

[0208] Blood drug concentration was detected by ELISA, and the detection method is as follows:

[0209] (1) Coating: Dilute Human IgG-heavy and light chain monkey-adsorbed Antibody (BETHYL, A80-319A) to a final concentration of 1 μg / mL with coating buffer, add 100 μL / well to the microplate, cover with a membrane, and incubate overnight (15-18 h) at 2-8℃. (2) Washing: Discard the coating buffer, wash 3 times with 0.025% PBST, 300 μL / well, and blot dry. (3) Blocking: Add blocking buffer (2% BSA), 300 μL / well, cover with a membrane, and incubate at 37℃ for 2 h ± 20 min. (4) Washing: Wash 3 times with 0.025% PBST, 300 μL / well, and blot dry. (5) Add samples: 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 microplate in duplicate, 100 μL / well, cover with membrane, and incubate at 37℃ for 1 h ± 5 min. (6) Wash plate: Wash 3 times with 0.025% PBST, 300 μL / well, and pat dry. (7) Add secondary antibody: Dilute Goat Anti-Human IgG, Monkey ads-HRP (Southern Biotech, 2049-05) 8000 times with diluent (2% BSA), 100 μL / well, cover with membrane, and incubate at 37℃ for 1 h ± 5 min. (8) Wash plate: Wash 3 times with 0.025% PBST, 300 μL / well, and pat dry. (9) Color development: Add 100 μL of TMB substrate color development solution to each well, cover with a membrane, and incubate at 15–25°C in the dark for 2–15 min. (10) Termination: Add 50 μL of stop solution (1M H2SO4) to each well to terminate the reaction. (11) Detection: Use a microplate reader to read the OD value of each well at a detection wavelength of 450 nm (reference wavelength 620 nm). (12) Data processing and analysis: Use GraphPad Prism8 software to fit the OD values ​​of the standards obtained by the microplate reader to a standard curve equation using the 4-parameter method, and calculate the concentration results of each sample.

[0210] The experimental results are shown in Table 8 and Figure 8 As shown, the half-life of 13F3H6-hz and 13F3H6-eADCC in rats is approximately 12 days.

[0211] Table 8. In vivo metabolic study of anti-CCR8 humanized antibody in rats

[0212] Example 18: In vivo efficacy detection of humanized anti-human CCR8 antibody

[0213] 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 Southern Model Biotechnology Co., Ltd.) were purchased. MC38 cells (Sichuan Xiapaisen) grown to the logarithmic growth phase were digested with trypsin and resuspended in PBS. Each mouse was subcutaneously inoculated with 5*10 cells. 6 Cells, waiting for the tumor to grow to 100-200m 3 The mice were administered the drug intravenously twice a week at a dose of 10 mg / kg for a total of six weeks. Tumor volume and mouse weight were measured during each administration. The main observation indicators for this experiment were: 1) TGI (%), calculated using the formula: TGI (%) = (1 - T / C) × 100% (where T and C represent the relative tumor volume of the treatment group, Control group, and control antibody BM at a specific time point, respectively); 2) Endpoint tumor volume and size photographs, and tumor weight.

[0214] Experimental results are as follows Figure 9 As shown, both 13F3H6-hz and 13F3H6-eADCC inhibited MC38 tumor growth in mice, with TGIs of 29.5% and 47.0%, respectively, while the TGI of the control antibody was 18.2%. Mouse weight changes are shown in the figure. Figure 10 As shown, the anti-CCR8 humanized antibody had no effect on mouse body weight throughout the entire administration process.

Claims

1. An antibody that specifically binds to CCR8, characterized in that, It includes heavy chain variable regions containing HCDR1, HCDR2 and HCDR3, and light chain variable regions containing LCDR1, LCDR2 and LCDR3. According to the Chothia numbering system, the amino acid sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 37, 38, 39, 47, 48 and 49, respectively. According to the Abm numbering system, the amino acid sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 40, 41, 39, 47, 48 and 49, respectively. According to the Kabat numbering system, the amino acid sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 42, 43, 39, 47, 48 and 49, respectively. According to the IMGT numbering system, the amino acid sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 44, 45, 46, 50, 51 and 49, respectively.

2. The antibody according to claim 1, characterized in that, The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:

35.

3. The antibody according to claim 1, characterized in that, The variable region of the light chain contains the amino acid sequence shown in SEQ ID NO:

36.

4. The antibody according to claim 1, characterized in that, The heavy chain variable region and light chain variable region sequences are selected from the following amino acid sequence combinations: The heavy chain variable region shown in SEQ ID NO: 35 and the light chain variable region shown in SEQ ID NO:

36.

5. The antibody according to claim 1, characterized in that, The heavy chain variable region further includes human, human-derived, or non-human-derived frame regions, and / or the light chain variable region further includes human, human-derived, or non-human-derived frame regions.

6. The antibody according to claim 1, characterized in that, The antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, an antibody fragment thereof, or its antigen-binding portion.

7. The antibody according to claim 6, characterized in that, The antibody is a fully human antibody, a humanized antibody, or a multispecific antibody.

8. The antibody according to claim 7, characterized in that, The antibody is a bispecific antibody.

9. The antibody according to claim 6, characterized in that, The antibody is selected from the following combinations: (i) single-chain antibody or single-chain variable fragment, or monovalent antibody lacking a hinge region; (ii) Fab, Fab', F(ab')2; (iii) full-length antibody; (iv) antibody containing the human IgG Fc domain.

10. The antibody according to any one of claims 1-9, characterized in that, The antibody has one or more of the following biological activities: (1) It binds to both cynomolgus monkey and human CCR8; (2) It has ADCC activity; (3) It possesses ADCP activity; (4) Does not bind to human CCR4; (5) It has the activity of blocking CCL1-mediated cell migration.

11. A recombinant protein, characterized in that, Includes the antibody as described in any one of claims 1-10.

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

13. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 12.

14. The carrier according to claim 13, characterized in that, The vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, or mammalian cell viruses.

15. An engineered host cell, characterized in that, It comprises the nucleic acid molecule of claim 12 or the vector of claim 13.

16. A pharmaceutical composition, characterized in that, Include: (i) the antibody according to any one of claims 1-10, the recombinant protein according to claim 11, the nucleic acid molecule according to claim 12, the vector according to claim 13, the engineered host cell according to claim 15, and / or combinations thereof; and (ii) Pharmaceutically acceptable carriers.

17. The pharmaceutical composition according to claim 16, characterized in that, The pharmaceutical composition is used to block the binding of CCR8 to any of the CCR8 ligands, or as a neutralizing antibody.

18. The pharmaceutical composition according to claim 17, characterized in that, Its function is to remove tumor-infiltrating Treg cells, or to treat cancer.

19. A reagent kit or drug delivery device, characterized in that, The pharmaceutical composition comprises the antibody according to any one of claims 1-10, the recombinant protein according to claim 11, the nucleic acid molecule according to claim 12, the vector according to claim 13, the engineered host cell according to claim 15, and the pharmaceutical composition according to claim 16.

20. A method for preparing recombinant polypeptides, characterized in that, The method includes: (i) Culturing the engineered host cells of claim 15 under suitable expression conditions; and (ii) Isolating a recombinant polypeptide from a culture, wherein the recombinant polypeptide is an antibody as described in any one of claims 1-10.

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