Antibodies that bind to human CCR8 and their use

Abstract

This invention relates to a monoclonal antibody that specifically binds to CCR8 or its antigen-binding moiety. It also relates to a nucleic acid molecule encoding the antibody or its antigen-binding moiety, an expression vector for expressing the antibody or its antigen-binding moiety, a host cell and a method for doing so, and a therapeutic method using the antibody or its antigen-binding moiety, the nucleic acid molecule, the expression vector and / or the host cell.

Description

Cross-reference of related applications

[0001] This application claims priority to the Chinese patent application No. 202310693601.4, filed on 12 June 2023, all of which are incorporated herein by reference. [Technical Field]

[0002] This application relates to an antibody or antigen-binding moiety that specifically binds to CCR8, and to the use of said antibody or antigen-binding moiety in the treatment of diseases such as cancer. [Background technology]

[0003] Chemokines can bind to chemokine receptors expressed on cells and induce cell migration through their own concentration distribution. The binding of chemokines to chemokine receptors triggers a signaling pathway, and the receptors are internalized and re-expressed on the migration surface of migrating cells. Chemokines may be produced under steady state and be responsible for routine immune cell migration, or they may be produced under inflammatory stimuli and recruit immune cells to the site of infection or chronic inflammation (Moser B et al., (2004). Chemokines: multiple levels of leukocyte migration control. Trends Immunol. 25(2):75-84; Zlotnik A et al., (2012) The chemokine superfamily revisited. Immunity. 36(5):705-716).

[0004] T cells, depending on the combination of chemokine receptors on their cell surface, include regulatory T cells (Treg) that regulate or suppress the immune system and maintain self-tolerance and immune self-stability, and cytotoxic CD8 cells that recognize and kill target cells. +CCR8 may be classified into various subgroups, such as T cells. CCR8 is a chemokine receptor mainly expressed on Treg cells, and its ligands are the chemokines CCL1 and CCL18. The primary ligand CCL1 is mainly produced by activated monocytes, macrophages, and T lymphocytes, and may also be secreted by tumor cells, while the secondary ligand CCL18 may be secreted by tumor-associated macrophages (Moser B et al., (2022) Chemokine Receptor-Targeted Therapies: Special Case for CR8. Cancers 14(3):511). Through the interaction between chemokines and chemokine receptors, Treg cells migrate to the site of inflammation, trigger the CCR8 signaling pathway, and balance immunity and tolerance.

[0005] The tumor microenvironment (TME) is rich in various chemokines, and massive Treg infiltration is observed in the TME of various tumor tissues, which promotes tumor growth by suppressing the anti-tumor immune response. CCR8 expression is mainly restricted to such tumor-infiltrating Tregs and hardly occurs in peripheral blood monocyte Tregs. It has been reported that CCR8 is upregulated only in tumor Tregs compared to Tregs present in normal tissues. In preclinical studies, tumor Tregs were found to highly express CCR8 in mouse models of colorectal cancer, melanoma, breast cancer, and urothelial carcinoma. Antibody therapies targeting CCR8, for example, through antibody-dependent cell-mediated cytotoxicity (ADCC), can stimulate CCR8 expression. + It can selectively reduce tumor Treg cells, induce an anti-tumor immune response, and significantly suppress tumor growth. Furthermore, CCR8 is also expressed in cutaneous memory T cells, and such cells are thought to be the origin of mycosis fungoides (MF) tumor cells. +Removing CCR8 cells can suppress the progression of T-cell lymphoma (Giustiniani J et al., (2022) CCR8 is a new therapeutic target in cutaneous T-cell lymphomas. Blood Adv. 6(11):3507-3512). Therefore, CCR8 present in tumors + Treg cells and CCR8 present in the skin + T cells may be a suitable target for cancer immunotherapy, and targeting CCR8 could enable cancer treatment.

[0006] However, as a seven-transmembrane protein, CCR8 is difficult to maintain its native conformation after purification. Various strategies exist in this field for producing antigens for animal immunization, including the synthesis of a portion of the extracellular domain or immunization using DNA molecules. However, when peptides that cannot exhibit their natural conformation are used as antigens, the produced antibodies may not recognize the native target, and DNA immunization may be insufficient to induce animal immunization. Therefore, there are many challenges in immunizing against this target, and new methods are needed to enhance animal immunization and produce superior antibodies.

[0007] Any reference to any document in this application does not constitute an endorsement that such documents are available as prior art to this application. [Overview of the Initiative]

[0008] The inventors of this application screened for novel CCR8 antibodies in animal immunology by using a combination of a DNA plasmid containing a CCR8 coding sequence, mRNA encoding CCR8, and / or cells overexpressing CCR8.

[0009] The CCR8 antibody of this application exhibits the following advantages over prior art antibodies such as BMS-986340 and GS-1811: i) Equivalent or superior CCR8 binding ability and binding specificity, ii) Equivalent or superior CCR8-CCL1 inhibitory ability, and iii) Equivalent or superior CCR8+ It possesses the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) in cells, iv) equivalent or greater internalization activity, and / or v) equivalent or greater in vivo antitumor effect.

[0010] Therefore, in the first aspect, the present application relates to an isolated monoclonal antibody (e.g., mouse-derived, chimeric, or humanized antibody) or its antigen-binding moiety, wherein the isolated monoclonal antibody or its antigen-binding moiety can specifically bind to CCR8 (e.g., human, monkey CCR8), and i) a heavy chain variable region that may include VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 may each include the amino acid sequences shown in (1) SEQ ID NOs: 1, 2, and 3, (2) SEQ ID NOs: 7, 8, and 9, (3) SEQ ID NOs: 13, 2, and 14, or (4) SEQ ID NOs: 18, 19, and 20, and / or ii) a light chain variable region that may include VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 may include a light chain variable region that may contain the amino acid sequences shown in (1) SEQ ID NOs: 4, 5, and 6, (2) SEQ ID NOs: 10, 11, and 12, (3) SEQ ID NOs: 15, 16, and 17, or (4) SEQ ID NOs: 21, 16, and 17, respectively. Further variants of the antibody or antigen-binding moiety are provided, in which each CDR contains up to approximately three amino acid substitutions (e.g., one, two, or three amino acid residue substitutions) compared to the antibody or its antigen-binding moiety. In some embodiments, the isolated monoclonal antibody of this application or its antigen-binding moiety can specifically bind to CCR8 and include a heavy chain variable region comprising: i) a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, wherein the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 are shown in (1) SEQ ID NOs: 1, 2 and 3, (2) SEQ ID NOs: 7, 8 and 9, (3) SEQ ID NOs: 13, 2 and 14, or (4) SEQ ID NOs: 18, 19 and 20, and / or ii) a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein the amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 are shown in (1) SEQ ID NOs: 4, 5 and 6, (2) SEQ ID NOs: 10, 11 and 12, (3) SEQ ID NOs: 15, 16 and 17, or (4) SEQ ID NOs: 21, 16 and 17, respectively.

[0011] The isolated monoclonal antibody or its antigen-binding moiety of this application may include a heavy chain variable region and a light chain variable region, and VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may each contain the amino acid sequences shown in (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, (2) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, (3) SEQ ID NOs: 13, 2, 14, 15, 16, and 17, or (4) SEQ ID NOs: 18, 19, 20, 21, 16, and 17. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may each contain the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may each contain the amino acid sequences shown in SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively. Further variants of the antibody or antigen-binding moiety are provided, each containing up to approximately three amino acid residue substitutions (e.g., one, two, or three amino acid residue substitutions) compared to the antibody or its antigen-binding moiety. In some embodiments, the isolated monoclonal antibody of this application or its antigen-binding moiety may include a heavy chain variable region and a light chain variable region, and the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may be shown as (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, (2) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, (3) SEQ ID NOs: 13, 2, 14, 15, 16, and 17, or (4) SEQ ID NOs: 18, 19, 20, 21, 16, and 17, respectively.

[0012] The heavy chain variable region of the antibody or its antigen-binding portion of the present application may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 22, 24, 26, 27, 29, 31, 33, or 35.

[0013] The light chain variable region of the antibody or its antigen-binding portion of the present application may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 23, 25, 28, 30, 32, 34, or 36.

[0014] The antibody or antigen-binding portion of the present application may include a heavy chain variable region and a light chain variable region, the heavy chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 22, 24, 26, 27, 29, 31, 33, or 35, and / or the light chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 23, 25, 28, 30, 32, 34, or 36. In some embodiments, the heavy chain variable region may include the amino acid sequence shown in SEQ ID NOs: 22, 24, 26, 27, 29, 31, 33, or 35, and the light chain variable region may include the amino acid sequence shown in SEQ ID NOs: 23, 25, 28, 30, 32, 34, or 36. In some other embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NOs: 22, 24, 26, 27, 29, 31, 33, or 35, and the amino acid sequence of the light chain variable region is shown in SEQ ID NOs: 23, 25, 28, 30, 32, 34, or 36.

[0015] The isolated monoclonal antibody or its antigen-binding moiety of this application may include a heavy chain variable region and a light chain variable region, each containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (1) SEQ ID NOs. 22 and 23, (2) SEQ ID NOs. 24 and 25, (3) SEQ ID NOs. 26 and 25, (4) SEQ ID NOs. 27 and 28, (5) SEQ ID NOs. 29 and 30, (6) SEQ ID NOs. 31 and 32, (7) SEQ ID NOs. 33 and 34, or (8) SEQ ID NOs. 35 and 36. In some embodiments, the heavy-chain variable region and the light-chain variable region may each include the amino acid sequences shown in (1) SEQ ID NOs: 22 and 23, (2) SEQ ID NOs: 24 and 25, (3) SEQ ID NOs: 26 and 25, (4) SEQ ID NOs: 27 and 28, (5) SEQ ID NOs: 29 and 30, (6) SEQ ID NOs: 31 and 32, (7) SEQ ID NOs: 33 and 34, or (8) SEQ ID NOs: 35 and 36. In some other embodiments, the amino acid sequences of the heavy-chain variable region and the light-chain variable region may each include the amino acid sequences shown in (1) SEQ ID NOs: 22 and 23, (2) SEQ ID NOs: 24 and 25, (3) SEQ ID NOs: 26 and 25, (4) SEQ ID NOs: 27 and 28, (5) SEQ ID NOs: 29 and 30, (6) SEQ ID NOs: 31 and 32, (7) SEQ ID NOs: 33 and 34, or (8) SEQ ID NOs: 35 and 36.

[0016] In some embodiments, the isolated monoclonal antibody of this application or its antigen-binding moiety may include a heavy chain variable region and a light chain variable region, and VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may include the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. The heavy chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 22, 24, or 26. The light chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 23 or 25. In some embodiments, the heavy chain variable region and the light chain variable region may each contain amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (1) SEQ ID NOs. 22 and 23, (2) SEQ ID NOs. 24 and 25, or (3) SEQ ID NOs. 26 and 25.

[0017] In some embodiments, the isolated monoclonal antibody of this application or its antigen-binding moiety may include a heavy chain variable region and a light chain variable region, and VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may include the amino acid sequences shown in SEQ ID NOs. 7, 8, 9, 10, 11, and 12, respectively. The heavy chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 28 or 30 may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, the heavy chain variable region and the light chain variable region may each contain amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (1) SEQ ID NOs. 27 and 28, or (2) SEQ ID NOs. 29 and 30.

[0018] In some embodiments, the isolated monoclonal antibody of this application or its antigen-binding moiety may include a heavy chain variable region and a light chain variable region, and VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may include the amino acid sequences shown in SEQ ID NOs. 13, 2, 14, 15, 16, and 17, respectively. The heavy chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 31 or 33. The light chain variable region may include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs.32 or 34.In some embodiments, the heavy chain variable region and the light chain variable region may each include an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (1) SEQ ID NOs.31 and 32, or (2) SEQ ID NOs.33 and 34.

[0019] This application further provides an isolated monoclonal antibody or its antigen-binding moiety that can specifically bind to CCR8 and may include i) a heavy chain variable region that may include VH CDR1, VH CDR2 and VH CDR3, and ii) a light chain variable region that may include VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 are included in the selected heavy chain variable region and light chain variable region. CDR3 may be included, and the selected heavy chain variable region and light chain variable region may each contain the amino acid sequences shown in (1) SEQ ID NOs: 22 and 23, (2) SEQ ID NOs: 24 and 25, (3) SEQ ID NOs: 26 and 25, (4) SEQ ID NOs: 27 and 28, (5) SEQ ID NOs: 29 and 30, (6) SEQ ID NOs: 31 and 32, (7) SEQ ID NOs: 33 and 34, or (8) SEQ ID NOs: 35 and 36.

[0020] The isolated monoclonal antibody or its antigen-binding moiety of this application may include a heavy chain constant region and / or a light chain constant region, wherein the N-terminus of the heavy chain constant region is ligated to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is ligated to the C-terminus of the light chain variable region. The heavy chain constant region may be an IgG, IgD, IgA, IgM, or IgE heavy chain constant region, preferably a heavy chain constant region or a functional fragment thereof that has FcR and / or complement protein binding ability, either naturally or after modification, such as a fragment including the hinge region, CH2, and CH3 of the heavy chain constant region. In one embodiment, the heavy chain constant region is an IgG1 heavy chain constant region, for example, a human IgG1 heavy chain constant region, and includes, for example, the amino acid sequence shown in SEQ ID NO: 37. The light chain constant region may be a κ or λ light chain constant region. In some embodiments, the light chain constant region may be a human κ light chain constant region, and includes, for example, the amino acid sequence shown in SEQ ID NO: 38.

[0021] In some embodiments, the antibody of this application comprises two heavy chains and two light chains, or is composed of two heavy chains and two light chains, each heavy chain comprising the heavy chain constant region sequence, the heavy chain variable region sequence and / or CDR sequence, and each light chain comprising the light chain constant region sequence, the light chain variable region sequence and / or CDR sequence. In some embodiments, the antibody of this application or its antigen-binding moiety may be Fab, F(ab')2 fragment, Fv, scFv or (scFv)2, etc.

[0022] The antibody or its antigen-binding moiety in this application may be, for example, derived from mouse, chimeric, or humanized.

[0023] The antibody or its antigen-binding moiety in this application may be of the antagonistic type.

[0024] The antibody or antigen-binding moiety of this application i) binds to human CCR8, ii) binds to monkey CCR8, iii) inhibits CCR8-CCL1 binding / interaction, and iv) CCR8 + Internalized by cells, v) CCR8 +It can induce ADCC in cells (if the heavy chain constant region has FcR binding ability), and / or vi) have an endogenous antitumor effect.

[0025] This application further provides an immune complex comprising the antibody or its antigen-binding moiety, wherein the antibody or its antigen-binding moiety is linked to a therapeutic agent, such as a cytotoxic molecule or an anticancer agent. This application further provides a bispecific molecule comprising the antibody or its antigen-binding moiety, wherein the antibody or its antigen-binding moiety is linked to a second functional group having a different binding specificity than the antibody or its antigen-binding moiety, such as a second antibody. In another embodiment, the antibody or its antigen-binding moiety may be part of a chimeric antigen receptor (CAR) or a genetically modified T cell receptor (TCR). This application further provides immune cells comprising the CAR and / or TCR, including T cells and NK cells. The antibody or its antigen-binding moiety may be encoded by an oncolytic virus and may be carried by an oncolytic virus.

[0026] This application further comprises the antibody or its antigen-binding portion, immune complex, bispecificity molecule, and / or nucleic acid molecule encoding CAR / TCR. This application may further provide expression vectors and host cells. The expression vector may comprise the nucleic acid molecule of this application. The host cell may comprise the expression vector of this application or incorporate the nucleic acid molecule of this application into its genome.

[0027] This application further provides a method for producing the antibody or its antigen-binding portion, immune complex, bispecific molecule, or CAR / TCR using the host cells of this application, the method comprising (i) expressing the antibody or its antigen-binding portion, immune complex, bispecific molecule, or CAR / TCR in host cells, and (ii) isolating the antibody or its antigen-binding portion, immune complex, bispecific molecule, or CAR / TCR from host cells or a culture thereof.

[0028] This application further provides compositions comprising the antibody or its antigen-binding moiety, immune complex, bispecific molecule, immune cell, oncolytic virus, nucleic acid molecule, expression vector, or host cell. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a PD-1 antibody.

[0029] In another embodiment, the present application provides a method for treating or alleviating a CCR8-related disease in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present application to the subject. The CCR8-related disease may be tumors, including solid tumors and hematological malignancies. Solid tumors include, but are not limited to, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), gastric cancer, microsatellite-stable colorectal cancer, and cervical cancer. Hematological malignancies include, but are not limited to, cutaneous T-cell lymphomas, and may be T-cell lymphomas. In some embodiments, the pharmaceutical composition of the present application may be administered together with at least one other antibody, such as a PD-1 antibody. In another embodiment, the pharmaceutical composition of the present application may be administered together with a cytokine (e.g., IL-2 and / or IL-21) or a costimulatory antibody (e.g., CD137 antibody and / or GITR antibody). In another embodiment, the antibody of the present application or its antigen-binding moiety may be administered together with a chemotherapeutic agent, which may be a cytotoxic agent.

[0030] This application further provides a method for relieving or mitigating immunosuppression in a subject, the method comprising administering an effective amount of the pharmaceutical composition of this application to the subject. In some embodiments, the method is used to relieve or mitigate immunosuppression in the tumor microenvironment, and comprises administering an effective amount of the pharmaceutical composition of this application to the tumor site.

[0031] This application further provides a method for enhancing the immune response in a subject, the method comprising administering an effective amount of the pharmaceutical composition of this application to the subject.

[0032] This application also relates to the use of the above-mentioned compositions, particularly pharmaceutical compositions, in the manufacture of drugs for treating or alleviating CCR8-associated tumors, relieving or alleviating immunosuppression, or enhancing the immune response.

[0033] The antibody or its antigen-binding portion of this application may also be used, for example, for in vitro antigen detection.

[0034] In yet another aspect, the application provides a method for producing a CCR8 antibody, the method comprising: i) administering to an animal a DNA molecule containing a CCR8 coding sequence, an RNA molecule encoding CCR8, and / or cells capable of overexpressing CCR8; ii) collecting lymph node cells and / or spleen cells from the animal; and iii) fusing the lymph node cells and / or spleen cells with myeloma cells to produce hybridomas.

[0035] The DNA molecule containing the CCR8 coding sequence may be a DNA molecule that contains the CCR8 coding sequence and is capable of expressing the CCR8 protein in an animal body. The DNA molecule may be a DNA vector, such as a lentiviral vector or a plasmid. In some embodiments, the DNA molecule may be a vector containing a suitable promoter and the CCR8 coding sequence. The promoter may be a constitutive promoter or an inductive promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the DNA molecule may be injected into an animal, for example, subcutaneously or into the peritoneal cavity, by means of a gene gun.

[0036] The RNA molecule encoding CCR8 may be an RNA molecule containing a CCR8 coding sequence and capable of expressing (translating) the CCR8 protein in an animal body. In some embodiments, the RNA molecule can express the human CCR8 protein in an animal body. The RNA molecule may be an mRNA molecule. In some embodiments, the RNA molecule may be a circular RNA molecule. In some embodiments, the RNA molecule may contain a suitable promoter and a CCR8 coding sequence. The promoter may be a constitutive promoter or an inductive promoter. In some embodiments, the promoter may be a constitutive promoter. The RNA molecule may be injected directly into the animal, for example, the peritoneal cavity. The RNA molecule may be injected into the animal with the aid of lipid molecules (e.g., micelles, liposomes, lipid nanoparticles (LNPs)), virus-like particles (VLPs), polymer molecules (e.g., polylactic acid-glycolic acid copolymers (PLGA), polyethyleneimine (PEI), polylysine (PLL), poly(β-aminoester) (PBAE), polymer nanoparticles), exosomes, etc. In one embodiment, the RNA molecule is encapsulated by LNPs.

[0037] Cells capable of overexpressing CCR8 may be cells that express the CCR8 protein in the animal body. These cells may be eukaryotic cells. In some embodiments, these cells may be injected, for example, into the peritoneal cavity of the animal.

[0038] In some embodiments, the method of the present application comprises i) administering an RNA molecule capable of expressing the CCR8 protein to an animal; ii) collecting lymph node cells and / or spleen cells from the animal; and iii) fusing the lymph node cells and / or spleen cells with myeloma cells to produce a hybridoma. The RNA molecule can express the CCR8 protein in the animal. In some embodiments, the RNA molecule can express the human CCR8 protein in the animal.

[0039] All documents referenced or mentioned in this application (including, but not limited to, all documents, patents, and disclosed patent applications referenced herein) ("References of this Application"), all documents referenced or mentioned in the References of this Application, and manufacturer manuals, instructions, product specifications, and product sheets for any products mentioned in this application or any References of this Application are incorporated by reference into this application and may be used in carrying out the present invention. More specifically, all references are incorporated by reference into this application so that each document is incorporated by reference into this application. Any Genbank sequences mentioned herein are incorporated by reference into this application.

[0040] In this application, particularly in the claims, terms such as "include" and "contain" may have the meanings given by the Patent Law of the People's Republic of China, and the term "substantially composed of..." may have the meanings given by the Patent Law of the People's Republic of China, allowing for the presence of elements not explicitly stated, but excluding elements present in the prior art or elements that affect the fundamental or novel properties of the present invention. [Brief explanation of the drawing]

[0041] The following provides a specific explanation in an illustrative manner, but it is not intended to limit the present invention to the above-described specific embodiments, and can be better understood by referring to the drawings. [Figure 1] Figure 1A shows the binding ability of the chimeric antibodies 117G9F9, 125C1E6, and 154G7C3 of this application to HEK293 cells expressing human CCR8. Figure 1B shows the binding ability of the chimeric antibody 224E9A3 of this application to HEK293 cells expressing human CCR8. [Figure 2] This application demonstrates the binding ability of the chimeric antibody to HEK293 cells expressing monkey CCR8. [Figure 3] This application demonstrates the inhibitory ability of the chimeric antibody to CCR8-CCL1 binding / interaction. [Figure 4]This application demonstrates the ADCC-inducing activity of the chimeric antibody against CCR8+ cells. [Figure 5] Figure 5A shows the binding ability of the humanized antibody of this application to HEK293 cells expressing human CCR8. Figure 5B shows the binding ability of the humanized antibody of this application to HEK293 cells expressing monkey CCR8. Figure 5C shows the binding ability of the humanized antibody of this application to parental HEK293 cells. [Figure 6] Figure 6A shows the ADCC-inducing activity of the humanized 125C1E6 antibody of this application against CCR8+ cells. Figure 6B shows the ADCC-inducing activity of the humanized 154G7C3 antibody and 117G9F9 antibody of this application against CCR8+ cells. [Figure 7] Figure 7A shows the inhibitory ability of the humanized 125C1E6 antibody of this application to CCR8-CCL1 binding / interaction. Figure 7B shows the inhibitory abilities of the humanized 125C1E6 antibody, 154G7C3 antibody, and 117G9F9 antibody of this application to CCR8-CCL1 binding / interaction. [Figure 8] This application demonstrates the intracellular relocation activity of the humanized antibody. [Figure 9] Figure 9A shows the epitope competitive binding status between the humanized 125C1E6 antibody of this application and the positive control. In the experiment, the 125C1E6 antibody is added first, followed by BMS-986340, GS-1811-mIgG2a, or 125C1E6. Figure 9B shows the epitope competitive binding status between the humanized 125C1E6 antibody of this application and the positive control. In the experiment, BMS-986340 is added first, followed by 125C1E6-VH3-VL1, or BMS-986340. Figure 9C shows the epitope competitive binding status between the humanized 125C1E6 antibody of this application and the positive control. In the experiment, GS-1811 is added first, followed by 125C1E6. [Modes for carrying out the invention]

[0042] To better understand this application, some terms are defined first. Other definitions are enumerated through the "Modes for Carrying Out the Invention."

[0043] The terms "one" or "one type" refer to one item / one type of item, or multiple items / multiple types of items. For example, "one antibody" refers to one antibody or multiple antibodies.

[0044] The term "CCR8" refers to CC chemokine receptor 8, a member of the G protein coupling receptor (GPCR) family. The term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific to human CCR8 may, in some cases, cross-react with the CCR8 protein of another species, such as monkeys. In other embodiments, an antibody specific to human CCR8 protein may be completely specific to human CCR8 protein without cross-reacting with other species or other types of proteins, and may cross-react with CCR8 proteins of some other species but not all other species.

[0045] The term "human CCR8" refers to CCR8 proteins with human amino acid sequences, for example, CCR8 proteins with the amino acid sequence of GenBank index number AAI07160.1 (Strausberg, R. Let al., (2002) Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences Proc. Natl. Acad. Sci. USA 99(26):16899-16903). The term "monkey CCR8" refers to CCR8 proteins with monkey amino acid sequences, for example, CCR8 proteins with the amino acid sequence of GenBank index number AFR51945.1 (Wang, L. et al., (2012), Submitted (26-JUL-2012) Department of Cellular Immunology and Pharmacology, Millennium Pharmaceuticals Inc.).

[0046] As used herein, the term "antibody" is intended to include IgG, IgA, IgD, IgE, and IgM full-length antibodies, as well as any antigen-binding fragments (i.e., antigen-binding portions) thereof. A full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain consists of a heavy-chain variable region (V H or VH for short) and a heavy-chain constant region. The heavy-chain constant region consists of three domains, namely C H1 C H2 and C H3 Each light chain consists of a light-chain variable region (V L or VL for short) and a light-chain constant region. The light-chain constant region consists of one domain C L The V H region and the V L region may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are distinguished by relatively conserved framework regions (FRs). Each V H and V L consists of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding regions that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including binding to various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system. The "functional fragment" of an antibody constant region refers to a fragment having a necessary function within the constant region, for example, a fragment retaining the FcR / complement system component binding activity within the heavy-chain constant region, such as the Fc fragment.

[0047] As used herein, the term "antigen-binding portion" (or antibody portion for short) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the CCR8 protein). It has been demonstrated that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody are (i) V L V H CL and C H1 (ii) a monovalent fragment called a Fab fragment, (ii) a bivalent fragment called an F(ab')2 fragment, which contains two Fab fragments linked by a disulfide bridge in a hinge region, and (iii) V H and C H1 Fd fragments consisting of (iv) antibody single arm V L and V H The Fv fragment, (v)V, is composed of the following: H dAb fragment composed of (Ward et al., (1989) Nature 341:544-546), (vi) an isolated complementarity-determining region (CDR), and (vii) a fragment containing a single variable domain and a heavy chain constant domain, dAb-V L It includes the two domains of the Fv fragment, V. L and V H These are encoded by separate genes, but they can be linked together using a synthetic linker via recombination, which allows them to form a single protein chain, and in the single protein chain, V L and V H These pairs form a monovalent molecule. Such single-chain antibodies are intended to be included in the meaning of the term. These antibody fragments are obtained using common techniques known to those skilled in the art, and the fragments are screened for function in the same manner as complete antibodies.

[0048] As used herein, the term “isolated antibody” refers to an antibody that substantially does not contain other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to the CCR8 protein substantially does not contain antibodies that specifically bind to antigens other than the CCR8 protein. However, an isolated antibody that specifically binds to the human CCR8 protein may cross-bind to other antigens, such as CCR8 proteins of other species. Furthermore, an isolated antibody substantially does not contain other cellular material and / or chemical substances.

[0049] The terms "monoclonal antibody," "monoclonal antibody," or "monoclonal antibody composition" refer to antibody molecular products consisting of a single molecule. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a specific epitope.

[0050] The term "mouse-derived antibody" refers to an antibody in which the variable region framework and CDR region are derived from a mouse germline immunoglobulin sequence. Furthermore, if the antibody includes a constant region, the constant region is also derived from a mouse germline immunoglobulin sequence. The mouse-derived antibodies of this application may include amino acid residues not encoded by the mouse germline immunoglobulin sequence, such as mutations introduced by in vitro random mutations or point mutations, or by in vivo somatic mutations. However, the term "mouse-derived antibody" does not include antibodies in which a CDR sequence derived from another mammalian species is inserted into the mouse framework sequence.

[0051] The term "chimeric antibody" refers to an antibody obtained by combining genetic material from one species with genetic material from another species. In particular, in this application, a chimeric antibody refers to an antibody obtained by combining non-human genetic material with human genetic material.

[0052] The term "humanized antibody" refers to an antibody that originates from a non-human species but has had its protein sequence modified to increase its similarity to antibodies naturally produced in humans.

[0053] The terms "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are used interchangeably in this specification with the term "antibody that specifically binds to an antigen."

[0054] In this specification, the terms "specifically recognizes" a target, e.g., human CCR8, or "specifically binds" to a target, e.g., human CCR8, mean that the antibody or antigen-binding fragment can distinguish such target biomolecule from one or more reference molecules, and that its binding affinity or activity to the target biomolecule is, for example, 1, 5, or 10 times higher than that of other reference molecules. Specific measurement methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA testing, and peptide scanning.

[0055] The term "substantially unbound" to proteins or cells means that the substance does not bind to proteins or cells, or does not bind with high affinity. In other words, K, which binds to proteins or cells. D is 1.0 × 10 -6 M or more, preferably 1.0 × 10 -5 M or higher, comfort level 1.0 × 10 -4 M or more, 1.0×10 -3 M or more, more preferably 1.0 × 10 -2 It is M or higher.

[0056] "EC 50 The term "half-maximal effect concentration" also refers to the antibody concentration that produces 50% of the maximum effect.

[0057] "I C 50 The term "half-inhibitory concentration" refers to the concentration of a drug or inhibitor needed to suppress a specified biological process by half.

[0058] The terms “antibody-dependent cell-mediated cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to cell-mediated immune defense, in which immune system effector cells actively lyse target cells to which cell membrane surface antigens and antibodies, such as the CCR8 antibody of this application, are bound.

[0059] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, but mammals, e.g., non-human primates, sheep, dogs, cats, cattle, and horses are preferred.

[0060] The term "therapeutic dose" refers to the amount of the antibody of this application sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). The therapeutic dose is relevant to the disease being treated, and those skilled in the art can easily determine the actual effective dose.

[0061] In this specification, “sequence identity” refers to the proportion of nucleotides / amino acids in a given sequence that are identical to those in a reference sequence after sequence alignment, and gaps are introduced in the sequence alignment as needed to maximize the percentage of sequence identity between the two sequences. Those skilled in the art can determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences by aligning two or more sequences using various methods, such as computer software, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0062] The difficulty in manufacturing CCR8 antibodies lies in the fact that, as a seven-transmembrane protein, it is difficult to maintain its native configuration after purification. When peptides that cannot exhibit their natural configuration are used as antigens, the produced antibodies may not be able to recognize the natural target, and DNA immunization may be insufficient to induce animal immunization.

[0063] The inventors of this application screened for novel CCR8 antibodies with superior properties compared to prior art by using a combination of a DNA plasmid containing a CCR8 coding sequence and cells overexpressing CCR8, or by administering mRNA encoding CCR8. The inventors found that, in the case of multi-pass transmembrane proteins, mRNA induces higher antibody levels than DNA molecules.

[0064] Accordingly, in one embodiment, the present application provides a novel animal immunization method or a method for producing antibodies by animal immunization, the method comprising immunizing an animal using an RNA molecule (e.g., an mRNA molecule) encoding the CCR8 protein.

[0065] Specifically, this application provides a method for producing a CCR8 antibody, the method comprising: i) administering to an animal a DNA molecule containing a CCR8 coding sequence and capable of expressing the CCR8 protein in the animal body, an RNA molecule containing a CCR8 coding sequence and capable of expressing (translating) the CCR8 protein in the animal body, and / or cells capable of overexpressing the CCR8 protein in the animal body; ii) collecting lymph node cells and / or spleen cells from the animal; and iii) fusing the lymph node cells and / or spleen cells with myeloma cells to produce a hybridoma.

[0066] In some embodiments, the method of the present application comprises: i) administering to an animal a DNA molecule containing a CCR8 coding sequence and capable of expressing the CCR8 protein in the animal body, and cells capable of overexpressing the CCR8 protein in the animal body; ii) collecting lymph node cells and / or spleen cells from the animal; and iii) fusing the lymph node cells and / or spleen cells with myeloma cells to produce hybridomas.

[0067] In some embodiments, the method of this application comprises i) administering an RNA molecule capable of expressing the CCR8 protein in the animal body to an animal, ii) collecting lymph node cells and / or spleen cells from the animal, and iii) fusing the lymph node cells and / or spleen cells with myeloma cells to produce a hybridoma. In some embodiments, the RNA molecule can express the human CCR8 protein in the animal body. The RNA molecule may be an mRNA molecule, for example, a circular RNA molecule. In some embodiments, the RNA molecule may be a vector containing a suitable promoter and a CCR8 coding sequence. The promoter may be a constitutive promoter or an inductive promoter. In some embodiments, the promoter is a constitutive promoter. The RNA molecule may be injected directly into the animal, for example, the peritoneal cavity. RNA molecules may be injected into animals with the aid of lipid molecules (e.g., micelles, liposomes, lipid nanoparticles (LNPs)), virus-like particles (VLPs), polymer molecules (e.g., polylactic acid-glycolic acid copolymers (PLGA), polyethyleneimine (PEI), polylysine (PLL), poly(β-aminoester) (PBAE), polymer nanoparticles), exosomes, etc. In one embodiment, the RNA molecule is encapsulated by LNPs.

[0068] The CCR8 antibody of this application exhibits, compared to antibodies disclosed in the prior art, such as BMS-986340 and GS-1811, i) equivalent or better (human or monkey) CCR8 binding ability and binding specificity, ii) equivalent or better CCR8-CCL1 inhibitory ability, and iii) equivalent or better CCR8 + It possesses the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) in cells, iv) equivalent or greater internalization activity, and / or v) equivalent or greater in vivo antitumor effect.

[0069] Preferably, the antibody of this application is a monoclonal antibody. The antibody may also be, for example, a mouse-derived, chimeric, or humanized monoclonal antibody.

[0070] The exemplary antibodies or antigen-binding moieties of this application have their structure and chemical properties described below.

[0071] The sequences of the heavy chain variable region and light chain variable region of the antibody or its antigen-binding moiety in this application are shown in Tables 1 and 2. The CDRs of the heavy chain variable region and the light chain variable region were determined by the Kabat numbering system, and the CDRs thus determined are shown in Table 1. The CDRs of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding moiety in this application may also be determined by the Chothia, IMGT, AbM, or Contact numbering system.

[0072] The antibody or antigen-binding moiety of this application may include a heavy chain constant region, for example, a natural or modified region having FcR binding ability, particularly high FcR binding ability. In some embodiments, the heavy chain constant region may be an IgG1 constant region, for example, a human IgG1 constant region containing the amino acid sequence shown in SEQ ID NO: 37. The light chain constant region may be a κ constant region, for example, a human κ constant region that may contain the amino acid sequence shown in SEQ ID NO: 38.

[0073] V of other CCR8 antibodies that bind to human CCR8 H and / or V L The sequence (or CDR sequence) is the V of the antibody of this application. H and / or V L It can be "mixed and paired" with the sequence (or CDR sequence). Preferably, V H and V L If (or the CDRs within it) are mixed and paired, a specific V H / V L V in the pair H The sequence is structurally similar to V H It may be replaced with an array. Similarly, preferably a specific V H / V L V in the pair L The sequence is structurally similar to V L It will be replaced with an array.

[0074] Therefore, in one embodiment, the antibody or antigen-binding portion of the present application is (a) Heavy chain variable region containing the amino acid sequence shown in Table 1 or Table 2, and (b) A light chain variable region containing the amino acid sequence shown in Table 1 or Table 2, or another CCR8 antibody that specifically binds to human CCR8. L Includes.

[0075] In another embodiment, the antibody or antigen-binding portion of the present application is (a) CDR1, CDR2 and CDR3 of the heavy chain variable region shown in Table 1 or Table 2, and (b) The light chain variable regions CDR1, CDR2, and CDR3 shown in Table 1 or Table 2, or the CDR of another CCR8 antibody that specifically binds to human CCR8.

[0076] In another embodiment, the antibody of this application or its antigen-binding moiety includes the heavy chain variable region CDR2 of a CCR8 antibody, and the CDRs of another antibody that binds to human CCR8, for example, the heavy chain variable region CDR1 and / or CDR3, and / or the light chain variable regions CDR1, CDR2 and / or CDR3 of another CCR8 antibody.

[0077] Furthermore, it is known in this field that the CDR3 domain can independently determine the binding specificity of antibodies to the same antigen, regardless of CDR1 and / or CDR2. Therefore, it is predicted that multiple antibodies with the same binding specificity can be generated based on the CDR3 sequence. For example, Klimka et al.,British J.of Cancer 83(2):252-260(2000);Beiboer et al,J.Mol.Biol.296:833-849(2000);Rader et al.,Proc.Natl.Acad.Sci USA95:8910-8915(1998);Barbas et al. See al.

[0078] In another embodiment, the antibody or antigen-binding moiety of the present application comprises the CDR2 of the heavy chain variable region of a CCR8 antibody, and at least the CDR3 of the heavy chain and / or light chain variable region of a CCR8 antibody, or the CDR3 of the heavy chain and / or light chain variable region of another CCR8 antibody, wherein the antibody or antigen-binding moiety can specifically bind to human CCR8. Preferably, these antibodies or antigen-binding moieties (a) competitively bind to CCR8, (b) retain functional properties, (c) bind to the same epitope, and / or (d) have a binding affinity similar to that of the CCR8 antibody or antigen-binding moiety of the present application. In another embodiment, the antibody or antigen-binding moiety may further comprise the CDR2 of the light chain variable region of the CCR8 antibody or antigen-binding moiety of the present application, or the CDR2 of the light chain variable region of another CCR8 antibody, wherein the antibody or antigen-binding moiety specifically binds to human CCR8. In another embodiment, the antibody of the present application may include the CCR8 antibody of the present application or the heavy / light chain variable region CDR1 of its antigen-binding moiety, or the heavy and / or light chain variable region CDR1 of another CCR8 antibody, wherein the antibody or its antigen-binding moiety specifically binds to human CCR8.

[0079] In another embodiment, the antibody of this application or its antigen-binding moiety comprises a heavy and / or light chain variable region sequence or CDR1, CDR2, and CDR3 sequences having one or more conservative modifications to the CCR8 antibody or its antigen-binding moiety of this application. In the art, it is known that some conservative sequence modifications do not cause loss of antigen-binding ability. See, for example, Brummell et al., (1993) Biochem 32:1180-8.

[0080] Therefore, in one embodiment, the antibody or its antigen-binding portion includes a heavy chain variable region and / or a light chain variable region, and the heavy chain variable region and the light chain variable region each include CDR1, CDR2, and CDR3, (a) The CDR1 of the heavy chain variable region includes the sequences shown in Table 1, and / or their conservative modifications, and / or (b) The CDR2 of the heavy chain variable region includes the sequences shown in Table 1 and / or their conservative modifications, and / or (c) The CDR3 of the heavy chain variable region includes the sequences shown in Table 1, and / or their conservative modifications, and / or (d) The CDR1 and / or CDR2 and / or CDR3 of the light chain variable region include the sequences and / or their conservative modifications shown in Table 1. (e) The antibody or its antigen-binding portion specifically binds to human CCR8.

[0081] The antibody of this application possesses one or more of the following functional characteristics, such as high affinity and high specific binding to human CCR8, and correspondingly enhanced ADCC-inducing ability in CCR8-positive cells, such as CCR8-positive tumor cells.

[0082] In some embodiments, the antibody or its antigen-binding moiety may be, for example, derived from mouse, chimeric, or humanized.

[0083] As used herein, the term “conservative sequence modification” refers to amino acid modifications that do not significantly affect or alter the antibody binding properties. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or its antigen-binding moiety of this application by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitution involves substituting an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These amino acid residue families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibody or its antigen-binding moiety can be substituted with other amino acid residues from the same side-chain family, and the resulting antibody can be tested for retention function (i.e., the functions described herein) using the functional tests described herein.

[0084] The antibody or its antigen-binding portion of this application is one or more V of the CCR8 antibody or its antigen-binding portion of this application. H / V L Genetically modified antibodies can be produced using antibodies containing a sequence as a starting material. To improve binding affinity and / or increase similarity to antibodies naturally produced in some species, one or two variable regions (i.e., V) can be modified. H and / or V LAn antibody can be genetically modified by modifying one or more residues within its (e.g., one or more CDR regions and / or one or more framework regions). Alternatively, an antibody can be genetically modified, for example, by modifying residues within its constant region to alter its effector function.

[0085] In some embodiments, transplantation of the CDR region can be used to genetically modify the variable region of an antibody. The antibody or its antigen-binding moiety interacts with the target antigen primarily through amino acid residues located in the six heavy-chain and light-chain complementarity-determining regions (CDRs). For this reason, the amino acid residues within the CDR are more diverse among individual antibodies than the sequences outside the CDR. Because the CDR sequence is responsible for the main antibody-antigen interaction, recombinant antibodies that mimic the characteristics of a particular native antibody can be expressed by constructing expression vectors in which the CDR sequence of a specific native antibody is transplanted into the framework sequence of a different antibody with different properties.

[0086] Therefore, another embodiment of the present application relates to an isolated monoclonal antibody or its antigen-binding moiety, wherein the monoclonal antibody or its antigen-binding moiety comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3 having the sequences of the present application, and the light chain variable region comprising CDR1, CDR2 and CDR3 having the sequences of the present application. These antibodies are V of the monoclonal antibody of the present application. H and V L It includes the CDR sequence, but may also include sequences from different frameworks.

[0087] Such framework sequences can be obtained from publicly available DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences used for human heavy chain and light chain variable region genes can be obtained from the Vbase human germline sequence database. In another embodiment, germline DNA sequences used for human heavy chain and light chain variable region genes can be obtained from the Genbank database.

[0088] We compare antibody protein sequences with protein sequence databases using one of the sequence similarity search methods known in this field, called gap BLAST (Altschul et. al., (1997)).

[0089] The framework sequence used in the antibody or its antigen-binding portion of this application is preferably structurally similar to the framework sequence used in the antibody or its antigen-binding portion of this application. H The CDR1, CDR2, and CDR3 sequences may be transplanted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence was obtained, or the CDR sequence may be transplanted into a framework region having one or more mutations compared to the germline sequence. For example, in some cases, it is beneficial to mutate residues within the framework region to maintain or enhance the antigen-binding ability of the antibody.

[0090] Another type of variable region modification is used to improve one or more binding properties (e.g., affinity) of the target antibody. H and / or V L This involves mutating amino acid residues within the CDR1, CDR2, and / or CDR3 regions. Mutations can be introduced by point mutations or PCR-mediated mutations, and their effects on antibody binding or other functional properties can be evaluated in in vitro or in vivo tests known in the art. Preferably, conservative modifications known in the art are introduced. Mutations may be amino acid substitutions, additions, or deletions, but substitutions are preferred. It is also common to change one, two, three, four, or five or fewer residues within the CDR region.

[0091] In another embodiment, the present application provides an isolated CCR8 monoclonal antibody or its antigen-binding moiety, the CCR8 monoclonal antibody or its antigen-binding moiety comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising (a) the sequence of the present application, or an amino acid sequence in which 1, 2, 3, 4, or 5 amino acids are substituted, deleted, or added. H (b) the CDR1 region of (b) the sequence of this application, or an amino acid sequence in which 1, 2, 3, 4 or 5 amino acids are substituted, deleted or added. H (c) the CDR2 region of (c) the sequence of this application, or an amino acid sequence in which 1, 2, 3, 4 or 5 amino acids are substituted, deleted or added. H (d) the CDR3 region of (d) the sequence of this application, or an amino acid sequence in which 1, 2, 3, 4 or 5 amino acids are substituted, deleted or added. L (e) the CDR1 region of the present application, or an amino acid sequence in which 1, 2, 3, 4, or 5 amino acids are substituted, deleted, or added. L (f) the CDR2 region of and (f) the sequence of this application, or an amino acid sequence in which 1, 2, 3, 4 or 5 amino acids are substituted, deleted or added. L This includes the CDR3 area.

[0092] The gene-modified antibody of this application is, for example, used to alter the properties of the antibody. H and / or V L This includes genetically modified framework residues. Framework modification involves mutating one or more residues in the framework region, or by extension, one or more CDR regions, in order to remove a T cell epitope and reduce the potential immunogenicity of the antibody. This method is also called “deimmunization” and is described in detail in U.S. Patent Application 20030153043.

[0093] Furthermore, as an alternative to modifications within the framework or CDR region, one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity, can typically be altered by genetically modifying the antibody of this application to include genetic modifications in the Fc region. Alternatively, one or more functional properties of the antibody can be altered by chemically modifying the antibody (for example, by adding one or more chemical functional groups to the antibody) or by modifying its glycosylation.

[0094] In one embodiment, C H1 By modifying or altering the hinge region, for example, by increasing or decreasing the number of cysteine ​​residues in the hinge region, the method is further described in U.S. Patent No. 5,677,425. H1 By modifying the cysteine ​​residues in the hinge region, for example, the assembly of the heavy and light chains or the stability of the antibody can be increased / decreased.

[0095] In another embodiment, the biological half-life of an antibody is increased or decreased by mutating the Fc hinge region of the antibody. More specifically, one or more amino acid mutations are made in the C of the Fc hinge fragment. H2 -C H3 By introducing it into the linking region, the antibody has weakened SpA binding ability compared to the SpA binding of the natural Fc-hinge domain. This method is described in detail in U.S. Patent No. 6,165,745.

[0096] In another embodiment, the glycosylation of an antibody is modified. For example, a deglycosylated antibody (i.e., an antibody lacking glycosylation) can be produced. By modifying glycosylation, for example, the affinity of the antibody to an antigen can be increased. Such glycosylation modification can be achieved, for example, by changing one or more glycosylation sites in the antibody sequence. For example, glycosylation of a site can be removed by making one or more amino acid substitutions to remove a glycosylation site in one or more variable region frameworks. Such deglycosylation can increase the affinity of the antibody to an antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0097] Furthermore, antibodies with altered glycosylation types can be produced, for example, low-fucosylated antibodies with a reduced number of fucose residues, or antibodies having an increased bisected GlcNac structure. It has been demonstrated that altered glycosylation patterns can increase the ADCC activity of antibodies. Such glycosylation modifications may be carried out, for example, by expressing antibodies in host cells with altered glycosylation systems. Cells with altered glycosylation systems are known in the art and include, but are not limited to, Slc35c1 gene knockout cell lines, FUT8 knockout cell lines, mutant CHO cell line Lec13, rat fusion tumor cell line YB2 / 0, cell lines containing small interfering RNAs that specifically act on the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. These can be used as host cells to express the recombinant antibodies of this application to produce antibodies with altered glycosylation.

[0098] Another modification of antibodies as used herein is polyethylene glycolation (PEGylation). For example, antibodies can be PEGylated to increase their biological (e.g., serum) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that one or more PEG groups adhere to the antibody or antibody fragment. Preferably, PEGylation is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similarly reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to any form of PEG for inducing other proteins, e.g., mono(C1-C1) 10 ) comprising alkoxy- or aryloxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody requiring PEGylation is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this application. See, for example, EPO154316 and EP0401384.

[0099] The antibodies or antigen-binding moieties of this application can be characterized by their various physical properties in order to detect and / or distinguish their classes.

[0100] For example, an antibody or its antigen-binding moiety may contain one or more glycosylation sites in the light chain or heavy chain variable region. These glycosylation sites can increase the immunogenicity of the antibody or alter the pK value of the antibody due to changes in antigen binding. Glycosylation is known to occur in motifs containing NXS / T sequences. In some cases, preferably, the CCR8 antibody or its antigen-binding moiety does not contain glycosylation in the variable region. This can be achieved by selecting an antibody that does not contain glycosylation motifs in the variable region, or by mutating residues in the glycosylation region.

[0101] In preferred embodiments, the antibody or its antigen-binding moiety does not contain an asparagine isomerization site. Deamidation of asparagine occurs in the NG or DG sequence and can lead to the construction of an isoaspartic acid residue that introduces a kink into the polypeptide chain, reducing its stability (isoaspartic acid effect).

[0102] Each antibody or its antigen-binding moiety has a unique isoelectric point (pI) that is substantially within the pH range of 6 to 9.5. The pI of IgG1 antibodies is typically within the pH range of 7 to 9.5, and the pI of IgG4 antibodies is substantially within the pH range of 6 to 8. Antibodies with pIs outside the normal range are presumed to have some unfolding structures under in vivo conditions and may be unstable. Therefore, preferably, the pI value of CCR8 antibodies is within the normal range. This can be achieved by selecting antibodies with pIs within the normal range or by mutating uncharged surface residues.

[0103] The monoclonal antibodies of this application may be produced using the somatic cell hybridization (hybridoma) technique described in Kohler and Milstein (1975) Nature 256:495. Other methods for producing monoclonal antibodies include single B cell antibody production techniques and phage display techniques. Methods for producing chimeric or humanized antibodies are also known in the art.

[0104] The antibodies or their antigen-binding moieties of this application can also be generated in a host cell transfectoma using, for example, a combination of recombinant DNA technology and a gene transfection method (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, the gene is operably ligated to transcriptional and translational regulatory sequences by inserting partial or full-length light and heavy chain encoding DNA obtained by standard molecular biology techniques into one or more expression vectors. In this case, the term “operably ligated” means that the antibody gene is ligated to the vector such that the transcriptional and translational regulatory sequences in the vector perform a predetermined function of regulating the transcription and translation of the antibody gene.

[0105] The term "regulatory sequence" includes promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Regulatory sequences used for expression in mammalian host cells preferably include viral elements that induce high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus-derived promoters and / or enhancers, such as the adenovirus major late promoter (AdMLP) and polyomaviruses. Alternatively, non-viral regulatory sequences such as ubiquitin promoters or β-globin promoters may be used. Furthermore, the regulatory elements consist of sequences from different sources; for example, the SRα promoter subsystem includes sequences derived from the SV40 early promoter and long-terminal repeat sequences of human T-cell leukemia virus type I. The expression vector and expression regulatory sequences are selected to be compatible with the expression host cells used.

[0106] The antibody light chain gene and the antibody heavy chain gene may be inserted into the same or different expression vectors. In a preferred embodiment, V H C in vector H It is operably connected to V L C in vector L A full-length antibody gene can be constructed by inserting a variable region into an expression vector encoding the heavy chain constant region and light chain constant region of a desired isotype, so that they can be operably linked. Alternatively, a recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from host cells. The antibody chain gene can be cloned into the vector so that the signal peptide is linked to the amino terminus of the antibody chain gene within the reading frame. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0107] In addition to antibody chain genes and regulatory sequences, the recombinant expression vector of this application may also contain other sequences, such as sequences that regulate the replication of the vector within host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes may be used to select host cells into which the vector has been introduced. For example, selectable marker genes can typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Selectable marker genes preferably include dihydrofolate reductase (DHFR) genes (for methotrexate selection / amplification in dhfr host cells) and neo genes (for G418 selection).

[0108] Regarding the expression of light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" include various techniques for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. While it is theoretically possible to express the antibodies or their antigen-binding moieties in prokaryotic or eukaryotic host cells, the antibodies are preferably expressed in eukaryotic cells, and most preferably in mammalian host cells, because eukaryotic cells, especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete antibodies that are properly folded and possess immunological activity.

[0109] Mammalian host cells for expressing the recombinant antibody of this application preferably include Slc35C1 gene knockout cell lines, FUT8 knockout cell lines, mutant CHO cell line Lec13, rat fusion tumor cell line YB2 / 0, cell lines containing small interfering RNA that specifically acts on the FUT8 gene, cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II, Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells administered with a DHFR-selectable marker), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a time sufficient to express the antibody in the host cells, preferably for a time sufficient to secrete the antibody in a culture medium in which the host cells proliferate. The antibody or its antigen-binding portion may be recovered from the culture medium using a protein purification method.

[0110] In another embodiment, the application provides nucleic acid molecules encoding the heavy / light chain variable region or CDR of the antibody or its antigen-binding moiety. The nucleic acid may be present in whole cells, in cell lysates, or in a partially purified or substantially pure form. The nucleic acid is “isolated” or “substantially pure” after being purified by standard techniques from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins. The nucleic acid of the application may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0111] The nucleic acids of the present application may be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas produced from transgenic mice carrying human immunoglobulin genes), the cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas may be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), the nucleic acids encoding such antibodies may be collected from the gene library.

[0112] The nucleic acid molecules of the present application preferably include those encoding the V H and V L sequences or CDRs of a CCR8 monoclonal antibody. When DNA fragments encoding V H and V L are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, converting the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, the DNA fragments encoding V H or V L are operably linked to another DNA fragment encoding another protein such as an antibody constant region or a flexible linker. The term "operably linked" refers to the linking of two DNA fragments such that the amino acid sequences encoded by both DNA fragments are in the reading frame.

[0113] The isolated DNA encoding the V H region is the DNA encoding V H operably linked to the heavy chain constant region (C H1 C H2 and C H3It may be converted into a full-length heavy-chain gene by operably linking it with another DNA molecule encoding H The DNA encoding the V region may be operably linked to another DNA molecule encoding only the constant region of the heavy-chain C H1 .

[0114] The isolated DNA encoding the V L region may be converted into a full-length light-chain gene by operably linking it with the DNA encoding V L and another DNA molecule encoding the light-chain constant region C L . The sequences of human light-chain constant region genes are known in the art, and DNA fragments containing these regions may be obtained by standard PCR amplification. In a preferred embodiment, the light-chain constant region may be the κ and λ constant regions.

[0115] To construct the scFv gene, the DNA fragments encoding V H and V L are operably linked to another fragment encoding a flexible linker, so that the V H and V L sequences can be expressed as a continuous single-chain protein, and the V H and V L regions are linked via the flexible linker.

[0116] The antibody or its antigen-binding moiety of this application can be combined with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxic molecules, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibody and therapeutic agent may be crosslinked by a linker, which is cleavable and is, for example, a peptide linker, a disulfide linker, or a hydrazone linker. More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs may be manufactured as described, for example, in U.S. Patents 7,087,600, 6,989,452 and 7,129,261, PCT Publications WO02 / 096910, WO07 / 038,658, WO07 / 051,081, WO07 / 059,404, WO08 / 083,312 and WO08 / 103,693, U.S. Patent Publications 20060024317, 20060004081 and 20060247295. In this application, the CCR8 antibody is CCR8 + Cells, especially CCR8 + Because it can specifically bind to and internalize Treg cells, it can be compounded with cytotoxic molecules, thereby allowing the cytotoxic molecules to interact with CCR8. + Treg cells are specifically damaged, and CCR8 + This can achieve the objective of killing Treg cells. In particular, cytotoxic molecules can be internalized by antibodies to CCR8 + It can enter Treg cells. The cytotoxic molecule may be any small molecule compound or protein molecule that damages the target cell, such as a tubulin polymerization inhibitor or a DNA damage agent.

[0117] In another embodiment, the application relates to a bispecific molecule comprising an antibody or its antigen-binding moiety, which is linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), in order to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The term “bispecific molecule” includes molecules having three or more specificities.

[0118] Bispecific molecules may appear in a variety of forms and sizes. At one end of the size spectrum, bispecific molecules retain the conventional antibody form except that they have two binding arms, each with a different specificity, instead of having two binding arms with the same specificity. At the other end is a bispecific molecule called a Bs(scFv)2 construct, which consists of two single-chain antibody fragments (scFv) linked by a peptide chain. Intermediate-sized bispecific molecules contain two different F(ab) fragments linked by a peptide linker. These and other forms of bispecific molecules may be produced by genetic modification, somatic cell hybridization, or chemical methods.

[0119] This application further provides a chimeric antigen receptor comprising a CCR8 single-chain antibody scFv, wherein the scFv comprises the heavy-chain and light-chain CDR or heavy-chain and light-chain variable regions described in this application.

[0120] The CCR8 chimeric antigen receptor may include (a) an extracellular antigen-binding domain containing CCR8 scFv, (b) a transmembrane domain, and (c) an intracellular signaling domain.

[0121] This application further provides immune cells, such as T cells or NK cells, which include the chimeric antigen receptor of this application.

[0122] Oncolytic viruses preferentially infect and kill cancer cells. The antibody or its antigen-binding portion of this application may be used together with an oncolytic virus. Furthermore, the antibody or the oncolytic virus encoding its antigen-binding portion may be introduced into the body of a test subject.

[0123] In another embodiment, the present application provides a composition comprising the antibody or its antigen-binding moiety, a nucleic acid molecule, an expression vector, a host cell, an immune complex, a chimeric antigen receptor, an immune cell, a bispecific antibody, and / or an oncolytic virus. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. The composition may optionally contain one or more other pharmaceutically active ingredients, e.g., another antitumor antibody, an immunoenhancing antibody, a non-antibody antitumor agent, or an immunoenhancing agent. The compositions of the present application may be used in combination, for example, with another anticancer agent or another immunoenhancing agent.

[0124] A pharmaceutical composition may contain any number of excipients. Available excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of appropriate excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0125] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or bolus infusion). Depending on the route of administration, the active ingredient may be coated with a material to protect it from acids and other natural conditions that may inactivate it. "Pareral administration," unlike enteral and topical administration, is usually performed by injection and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intramembrane, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and bolus infusions. Alternatively, the antibodies of this application may be administered via parenteral routes, e.g., topical, epidermal, or mucosal routes, e.g., intranasal, oral, vaginal, rectal, sublingual, or topically.

[0126] The pharmaceutical composition may be in the form of a sterile aqueous solution or dispersion. They may also be prepared as microemulsions, liposomes, or other ordered structures suitable for high-concentration drugs.

[0127] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the target of treatment and the specific method of administration, and is the amount of the composition that substantially produces a therapeutic effect. In percentage terms, this amount is about 0.01 to about 99% of the active ingredient that can be bound to a pharmaceutically acceptable carrier.

[0128] The administration regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, it may be administered as a single bolus, in divided doses over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosing unit form to facilitate administration and ensure dose uniformity. A dosing unit form refers to a physically distinct unit appropriate as a unit dose for the target of treatment, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in relation to the pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained-release formulation, in which case the required frequency of administration is reduced.

[0129] When administering antibodies, the dose may be approximately 0.001 to 100 mg per kg of host body weight. An exemplary treatment regimen involves weekly administration.

[0130] The therapeutically effective dose of the pharmaceutical composition of this application results in a reduction in the severity of disease symptoms and an increase in the frequency and duration of asymptomatic periods. For example, in the treatment of a subject with a tumor, the therapeutically effective dose preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to an untreated subject. The therapeutically effective dose of the therapeutic antibody can reduce the size of the tumor or alleviate the symptoms of the subject, and the subject may be a human or another mammal.

[0131] The pharmaceutical composition may also be a sustained-release reagent comprising an implant and a microencapsulation delivery system. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid, can be used. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0132] The pharmaceutical composition may be administered by medical devices, for example, (1) needleless subcutaneous injection devices (e.g., U.S. Patents No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824 and 4,596,556), (2) microinjection pumps (U.S. Patent No. 4,487,603), (3) transdermal administration devices (U.S. Patent No. 4,486,194), (4) bolus injection devices (U.S. Patents No. 4,447,233 and 4,447,224), and (5) infiltration devices (U.S. Patents No. 4,439,196 and 4,475,196).

[0133] In some embodiments, components in the compositions of this application may be prepared to ensure appropriate biological distribution. For example, to ensure that the therapeutic antibody or its antigen-binding portion of this application crosses the blood-brain barrier, the antibody may be prepared in liposomes and may further contain target functional groups to enhance selective delivery to specific cells or organs. See, for example, U.S. Patents 4,522,811, 5,374,548, 5,416,016, and 5,399,331.

[0134] This application also relates to in vivo gene therapy, in which nucleic acid molecules encoding the antibody or its antigen-binding portion, immune complex, or bispecific molecule of this application are directly introduced into a subject. For example, the nucleic acid sequence encoding the antibody or antigen-binding portion of this application is introduced into target cells by local injection via a suitable delivery vector, such as a nucleic acid construct having or not having an adeno-associated virus vector. Other selectable viral vectors include, but are not limited to, retrovirus, adenovirus, herpes simplex virus, and papillomavirus vectors. Physical transfer of the viral vector into the body can be achieved by local injection of the desired nucleic acid construct or other suitable delivery vector containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or particle bombardment (gene gun).

[0135] The pharmaceutical compositions of this application have various in vitro and extracorporeal uses, for example, relating to the treatment of cancer, or more generally to immune enhancement in patients with diseases such as cancer. The pharmaceutical compositions may be administered to human subjects, for example, to suppress tumor growth in the body.

[0136] Considering the ability of the pharmaceutical compositions of this application to inhibit the proliferation and survival of tumor cells, this application provides a method for inhibiting the growth of tumor cells in a subject, the method comprising administering the pharmaceutical compositions of this application to the subject in order to inhibit tumor growth in the subject. Non-limiting examples of tumors that can be treated with the compositions of this application include, but are not limited to, solid tumors and hematological malignancies. Solid tumors include, but are not limited to, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), gastric cancer, microsatellite-stable colorectal cancer, and cervical cancer (regardless of whether it is primary or metastatic). Hematological malignancies include, but are not limited to, cutaneous T-cell lymphomas. Furthermore, the pharmaceutical compositions of this application may be used to treat refractory or recurrent malignancies.

[0137] This application provides a combination therapy in which the pharmaceutical composition of this application is administered together with one or more other antibodies or non-antibody therapeutic agents, the combination therapy can effectively suppress tumor growth in a subject. In one embodiment, this application provides a method for suppressing tumor growth in a subject, the method comprising administering the pharmaceutical composition of this application and one or more other antibodies, such as PD-1 antibodies, to the subject. In some embodiments, the subject is human. In another embodiment, this application provides a method for treating cancer, in which the pharmaceutical composition of this application is administered together with a chemotherapeutic agent, the chemotherapeutic agent may be a cytotoxic agent. Other therapies that can be used in combination with the pharmaceutical composition of this application include, but are not limited to, immunosuppressant administration, interleukin-2 (IL-2) administration, radiotherapy, surgery, or hormone deprivation.

[0138] The compositions of this application may also be used to relieve or alleviate immunosuppression in a subject. In particular, this application further provides a method for relieving or alleviating immunosuppression in a subject, the method comprising administering an effective amount of the pharmaceutical composition of this application to the subject. In some embodiments, the method is used to relieve or alleviate immunosuppression in the tumor microenvironment, and comprises administering an effective amount of the pharmaceutical composition of this application to the tumor site.

[0139] The compositions of this application may also be used to enhance the immune response. Specifically, this application provides a method for enhancing the immune response in a subject, the method comprising administering an effective amount of the pharmaceutical composition of this application to the subject.

[0140] The therapeutic agent combinations discussed herein may be administered simultaneously as a single composition on a pharmaceutically acceptable carrier, or each agent may be administered simultaneously as separate compositions on a pharmaceutically acceptable carrier. In another embodiment, the therapeutic agent combination may be administered sequentially.

[0141] Furthermore, when combination therapy is administered multiple times and the drugs are administered sequentially, the order of sequential administration at each time point can be reversed or kept the same, and sequential administration may be combined with simultaneous administration or any combination thereof.

[0142] Various aspects and embodiments of this application will be discussed with reference to the accompanying drawings and the following embodiments. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference in their entirety. Although this application has been described with reference to exemplary embodiments, many equivalent modifications and changes will be apparent to those skilled in the art in providing this application. Thus, the exemplary embodiments of this application are illustrative and not limiting. Various modifications can be made to the above embodiments without departing from the spirit and scope of this application. Examples

[0143] The following examples are presented for illustrative purposes only and do not limit the present invention in any way. Example 1: Production of anti-human CCR8 monoclonal antibody (mAb)

[0144] To produce antibodies against human CCR8, wild-type female mice, including BALB / C, A / J, or SJL strains, were immunized with various antigens, including DNA / mRNA encoding human CCR8 and cell lines overexpressing human CCR8. Specifically, these antigens were used individually or in combination for immunization. For example, a DNA plasmid encoding CCR8 (GenScript) and a CHO-K1 cell line overexpressing human CCR8 (CHO-K1 / hu-CCR8, Cat#:RD00915, GenScript) were used together for immunization. Every two weeks, gold particles coated with DNA plasmid (6 μg) were injected subcutaneously or intraperitoneally into the mice three times using the Helios gene gun system (Bio-Rad), followed by intraperitoneal injection of CHO-K1 / hu-CCR8 cells (5 x 10⁶ cells per mouse). 6 (Each cell was injected.) Alternatively, mice were injected with 20 / 40 μg of mRNA LNP (TheraRNA) every two weeks for three doses.

[0145] Furthermore, in order to evaluate the serum titer of immunized animals and screen / identify CCR8-specific antibodies, human and monkey CCR8 coding genes were synthesized, inserted into pLVX-Puro vectors, and lentiviral packaged. These were then used to infect HEK293 cells, which were subsequently screened with puromycin to produce stable cell lines based on the HEK293 cell line, namely HEK293 / hu-CCR8 cells (Cat#:RD00953, GenScript) and HEK293 / cyno CCR8 cells (Cat#:RD01063, GenScript), respectively.

[0146] After evaluating the serum titers of immunized mice, mice with the highest human CCR8 antibody titer were selected. Their lymph nodes and / or spleens were excised, and lymph node cells and / or spleen cells were isolated and fused with myeloma cells to produce hybridomas. Specifically, isolated spleen cells and / or lymphocytes were mixed with mouse myeloma cell lineage SP2 / 0 in a 3:1 ratio, and then fused cells were formed by electrofusion. Next, the fused cells were placed in a 96-well plate and incubated for 2 weeks in a selective medium containing hypoxanthine-aminopterin-thymidine (HAT) to produce hybridomas. Using HEK293 / hu-CCR8 cells, anti-human CCR8 antibodies were screened for from the supernatant of each hybridoma well by flow cytometry. Subsequently, positive hybridomas were subcloned by restriction dilution to obtain single clones.

[0147] The ability of antibodies expressed in monoclonal supernatants to bind to HEK293 cells overexpressing human / monkey CCR8 was analyzed by flow cytometry. First, HEK293 cells overexpressing human / monkey CCR8, i.e., HEK293 / hu-CCR8 and HEK293 / cyno CCR8, were cultured and harvested, and incubated with anti-CCR8 antibody monoclonal supernatant. Subsequently, samples were analyzed using a flow cytometer (BD Canto II), and two antibodies, BMS-986340 (manufactured according to the information disclosed in WO2021194942A1) and GS-1811 (manufactured according to the information disclosed in WO2021163064A2), were used as positive control antibodies, while unmodified parental HEK293 cells were used as negative control cells for the FACS binding assay.

[0148] Initial screening results showed that antibodies produced by the monoclonal 154G7C3 and 117G9F9 in this application bound only to cells expressing human CCR8, while antibodies produced by 125C1E6 and 224E9A3 bound to HEK293 cells expressing both human and monkey CCR8. Several clones of 125C1E6, 154G7C3, 117G9F9, and 224E9A3 were sequenced, and unique clones were selected for use in subsequent research and development. Example 2: Production of Chimeric Antibodies

[0149] The coding sequences of the mouse-derived heavy chain and light chain variable regions, which were screened and sequenced, were fused to the N-terminuses of the coding sequences of the human IgG1 heavy chain constant region and the human κ light chain constant region, respectively. These were then constructed downstream of the signal peptide in the expression plasmid pTT5, and secretory expression was performed. The amino acid sequences of each heavy chain and light chain variable region are shown in Table 1, and the amino acid sequences of the human IgG1 heavy chain constant region and the human κ light chain constant region are shown in SEQ ID NOs. 37 and 38, respectively.

[0150] [Table 1-1] [Table 1-2] [ka]

[0151] Using PEImax 40,000 (Cat#:24765-1, Polysciences, Inc.), plasmids containing the heavy and light chain coding sequences of each antibody were combined and transfected into CHO-3E7 cells for transient expression of anti-CCR8 antibodies. After 24 hours, expression / secretion was enhanced by adding Tryptone N-1 supplement. After 6 days of shaking culture at 37°C and 5% CO2, the supernatant was collected and the antibodies were purified using a chromatography column containing a protein A agarose gel. The purified antibodies were then stored in PBS solution and used for subsequent analytical validation. Example 3: Identification of in vitro activity of chimeric antibodies Detection of antibody binding to cells overexpressing human CCR8 using flow cytometry.

[0152] To further study the binding ability and binding specificity of the antibody of this application to human CCR8, 1 × 10⁶ in 100 μl of culture medium 5 Each HEK293 / hu-CCR8 cell was incubated with 50 μl of the anti-CCR8 chimeric antibody of this application in an ELISA plate at 4°C for 1 hour. Each antibody was then diluted in a 3-fold gradient in the cell / antibody mixture to a starting concentration of 300 nM. Antibodies BMS-986340 and GS-1811 were used as positive controls, and hIgG1 was used as a negative control. After washing the ELISA plate twice with PB, goat anti-human IgG (H+L) (Jackson, 109-605-088) labeled with a fluorophore (iFluor 647) was added to each well, and incubated at 4°C for 0.5 hours. Subsequently, the samples were analyzed by flow cytometry, and antibody-antigen binding curves were generated with mean fluorescence intensity (geometric mean fluorescence intensity) on the y-axis and antibody concentration on the x-axis. Raw data were plotted using GraphPad Prism v6.02 software, and EC 50 We made that decision.

[0153] Of these anti-CCR8 chimeric antibodies, as shown in Figures 1A and 1B, the monoclonal antibodies of this application include 125C1E6, 154G7C3, 117G9F9, and 224E9A3, all of which bind to cells overexpressing human CCR8 and exhibit binding ability equivalent to that of the positive controls BMS-986340 and GS-1811. Detection of antibody binding to cells overexpressing monkey CCR8 using flow cytometry.

[0154] The cross-reactivity of anti-CCR8 chimeric antibodies with monkey CCR8 was further tested using the same method steps as above, except that HEK293 / cyno-CCR8 cells were used instead of HEK293 / hu-CCR8 cells.

[0155] The results are shown in Figure 2. The 224E9A3 antibody bound to cells expressing monkey CCR8 with a high binding capacity comparable to that of BMS-986340, while GS-1811 did not show binding to cells expressing monkey CCR8 in the FACS assay. CCR8-CCL1 inhibition analysis by detecting CCR8-mediated calcium flow

[0156] The binding of ligand CCL1 to the chemokine receptor CCR8 can induce CCR8-mediated calcium influx; therefore, the inhibitory ability of anti-CCR8 antibodies on CCL1-CCR8 signaling can be determined by measuring intracellular calcium flow.

[0157] Simply put, 20 μl of 7.5 × 10 5CHO-K1 / hu-CCR8 cells were inoculated into a 384-well assay plate at 37°C for 16 hours. 10 μl of antibody was added to the assay plate, and each antibody was diluted in a 5-fold gradient in the cell / antibody mixture to a starting concentration of 1 μM, creating a total of 8 concentration points. For 117G9F9, it was diluted in a 5-fold gradient in the cell / antibody mixture to a starting concentration of 0.4 μM, creating a total of 8 concentration points. The working dye solution (Cat#: R7446, Molecular Devices) from the FLIPR® Calcium 4 assay kit was added, and the mixture was incubated in the dark at 37°C for 1 hour. BMS-986340 and GS-1811 were used as positive controls. 35 μl of 10 nM recombinant human CCL1 (GenScript, Z02911) was added, followed by FLIPR TM The calcium flux fluorescence signal is measured using the TETRA system (Molecular Devices), and the data is analyzed using GraphPad Prism to obtain IC (Indicative Convolution). 50 The value was retrieved.

[0158] The results are shown in Figure 3, IC 50 According to the results, the anti-CCR8 antibody molecule of this application showed superior inhibitory activity against CCL1-CCR8 signaling compared to the positive control. Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by anti-CCR8 antibodies

[0159] The ADCC-inducing activity of anti-CCR8 chimeric antibodies was evaluated using a reporter gene-based ADCC biological activity assay.

[0160] Simply put, CHO-K1 / hu-CCR8 target cells are cultured and harvested, and 1 × 10⁶ cells are used. 4Cells were inoculated into a 96-well plate at a cell density of 1.5 × 10¹⁶ cells per well, and the cells were placed in 40 μl of culture medium. 20 μl of the antibody of this application or positive controls (BMS-986340 and GS-1811) were added to the 96-well plate, and each antibody was diluted in a 10-fold gradient in the cell / antibody mixture to a starting concentration of 10 μg / ml (approximately 60 nM), creating a total of 7 concentration points. The well plate was incubated at 37°C under 5% CO2 conditions for 30 minutes. Next, 1.5 × 10¹⁶ cells were added to the 96-well plate. 6 40 μl of GS-J2C / CD16A cells (ADCC reporter cell line, Cat#:RD00830, GenScript), which are effector cells, were added and incubated at 37°C and 5% CO2 for 6 hours. The 96-well plate was removed, and the activation of the ADCC reporter cell line was analyzed using the Bio-Glo® analysis kit. Luminescence data was detected by PheraStar (BMG) for ADCC reporter gene activation signal analysis and analyzed by GraphPad Prism 6.0.

[0161] The measurement results are shown in Figure 4. Compared to the positive control, all of the anti-CCR8 chimeric antibodies of this application exhibited equivalent or greater activity in detecting CCR8. + It can induce ADCC in cells. Example 4 Humanization of antibodies

[0162] For the humanization of 117G9F9, 125C1E6, and 154G7C3, the mouse-derived antibodies were aligned with human Ig germline sequences to obtain the sequence that best matched overall. The human germline sequence with high homology to the obtained mouse-derived antibody was used as the receptor, and the CDR of the mouse-derived antibody was transplanted into the receptor framework. Subsequently, sequence analysis was performed on the newly produced antibody to determine whether high-risk sites for potential post-translational modifications, including deamidation, isomerization, oxidation, and unpaired cysteine ​​residues that may affect antibody binding activity and stability, were present in the newly constructed sequence.

[0163] Referring to the steps of the method in Example 2, DNA sequences encoding the heavy chain and light chain variable regions of the humanized antibody were synthesized and fused to the N-terminuses of the encoding sequences of the human IgG1 heavy chain constant region and the human κ light chain constant region, respectively. These were then constructed downstream of the signal peptide in the expression plasmid pTT5, and secretory expression was performed. The amino acid sequences of each heavy chain and light chain variable region are shown in Table 2, and the amino acid sequences of the human IgG1 heavy chain constant region and the human κ light chain constant region are shown in SEQ ID NOs: 37 and 38, respectively.

[0164] [Table 2] Example 5: Identification of the activity of humanized antibodies Detection of the binding ability of humanized antibodies to cells overexpressing human or monkey CCR8 using flow cytometry.

[0165] The binding ability and binding specificity of the humanized antibody to cells overexpressing human or monkey CCR8 and HEK293 parent cells were tested according to the steps of the method in Example 3.

[0166] Figure 5A shows the antibody-antigen binding curves between humanized antibodies and cells overexpressing human CCR8. It can be seen that all of the humanized antibodies in this application exhibit high binding affinity to cells overexpressing human CCR8. In particular, the binding affinity of 125C1E6-VH1.1-VL1 and 125C1E6-VH3-VL1 is higher than that of BMS-986340 and GS-1811, specifically showing higher span values ​​and / or lower EC values. 50 Show the value.

[0167] Figure 5B shows the antibody-antigen binding curves of humanized antibodies and cells overexpressing monkey CCR8. Both 125C1E6-VH1.1-VL1 and 125C1E6-VH3-VL1 bound to cells expressing monkey CCR8 with a much higher binding capacity than BMS-986340, and no binding of GS-1811 to monkey CCR8 was detected.

[0168] Figure 5C shows the antibody-antigen binding curves between humanized antibodies and HEK293 cells. Two positive controls, BMS-986340 and GS-1811, showed weak binding to parental HEK293 cells, but 125C1E6-VH1.1-VL1 and 125C1E6-VH3-VL1 did not bind to HEK293 cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by humanized antibodies

[0169] Referring to the steps of the method in Example 3, the ADCC-inducing activity of the humanized antibody was evaluated, and both the humanized antibody and the positive control were diluted in a 10-fold gradient in the cell / antibody mixture to a starting concentration of 10 μg / ml.

[0170] The results are shown in Figures 6A and 6B, and all humanized anti-CCR8 antibodies + The ADCC-inducing activity on cells is equivalent to that of the positive control. CCR8-CCL1 inhibition analysis by detecting CCR8-mediated calcium flow

[0171] Referring to the steps of the method in Example 3, the inhibitory ability of the humanized antibody against CCL1-CCR8 signaling was determined by measuring the intracellular calcium flow rate. Each antibody was diluted in a cell / antibody mixture at a 5-fold gradient, with a starting concentration of 1 μM, resulting in a total of 8 concentration points.

[0172] As shown in Figures 7A and 7B, 125C1E6-VH1.1-VL1, 125C1E6-VH3-VL1, and 154G7C3-VH1.1-VL2 all showed in vitro CCR8-CCL1 inhibitory activity equivalent to or greater than that of the positive control GS-1811. Antibody internalization analysis using the IncuCyte live cell analysis system

[0173] The internalization status of antibodies was analyzed using the Incucyte real-time live cell system (Sartorius).

[0174] Simply put, 50 μl of 2 × 10 5CHO-K1 / hu-CCR8 cells were inoculated into 96-well assay plates at 37°C for 16 hours. Antibodies were labeled before internalization measurement. Specifically, humanized antibody and Incucyte Fabfluor pH dye were diluted in a 2-fold gradient with complete growth medium (90% F12K medium + 10% fetal bovine serum) to a starting concentration of 4 μg / ml, creating 8 concentration points. The diluted humanized antibody and Incucyte Fabfluor pH dye were then mixed in a 1:1 volume ratio and incubated in a 96-well round-bottom plate at 37°C in the dark for 15 minutes. 50 μl of labeled humanized antibody was added to the 96-well assay plate and incubated in an Incucyte S3 cell incubator at 37°C for 24 hours. The internalization rate of the antibody was detected by measuring the red fluorescence in the CHO-K1 / hu-CCR8 cells. BMS-986340 and GS-1811 were used as positive controls, and human IgG1 was used as a negative control. Raw data was derived from Incucyte 2022B and analyzed using Incucyte 2022B, Microsoft Office Excel 2016, and GraphPad Prism 6. The overall fluorescence intensity (RCU × μm) of each fluorescence image was measured. 2 ) and antibody concentration were plotted.

[0175] As shown in Figure 8, both the humanized antibodies 125C1E6-VH1.1-VL1 and 125C1E6-VH3-VL1 showed higher internalization activity than the positive control. Furthermore, the humanized antibodies 154G7C3-VH1.1-VL2 and 117G9F9-VH2.1-VL1 showed internalization activity equivalent to that of the positive control. Example 6: Antigen Epitope Competitive Binding Measurement

[0176] To determine whether humanized antibodies bind to the same or similar epitopes as the positive control, competitive binding of each antibody to HEK293 / hu-CCR8 cells was measured by FACS.

[0177] Simply put, first, a 125C1E6-VH3-VL1 antibody containing hIgG1-Fc (i.e., SEQ ID NO: 37) with a final concentration of 0.45 nM and 1 × 10⁻¹⁰ 5Each HEK293 / hu-CCR8 cell was incubated at 4°C for 0.2 hours to a solution volume of 100 μl. Then, 50 μl of a mouse Fc-containing antibody containing BMS-986340-mIgG2a (the heavy chain constant region of BMS-986340 was replaced with the heavy chain constant region of mouse IgG2a of SEQ ID NO: 39, and the light chain constant region was replaced with SEQ ID NO: 40), GS-1811-mIgG2a (the heavy chain constant region of GS-1811-mIgG2a was replaced with SEQ ID NO: 39, and the light chain constant region was replaced with SEQ ID NO: 40), and 125C1E6-mIgG2c (purified from hybridoma supernatant), or mouse IgG1 was added. Each antibody was diluted in the cell / antibody mixture using a 3-fold gradient to a starting concentration of 300 nM and incubated at 4°C for 0.8 hours. Subsequently, 125C1E6-VH3-VL1 bound to the cell surface was detected using goat anti-human IgG(H+L) labeled with the fluorophore (iFluor 647). The data was analyzed using GraphPad Prism 6.0. The results are shown in Figure 9A.

[0178] Similarly, antigen epitope competition between several antibodies was further determined by first adding BMS-986340-mIgG2a at a final concentration of 0.28 nM, followed by the addition of the competing antibody 125C1E6-VH3-VL1, BMS-986340, or human IgG1, or by first adding GS-1811-mIgG2a at a final concentration of 0.28 nM, followed by the addition of 125C1E6-VH3-VL1, or human IgG1. Each competing antibody was diluted in the cell / antibody mixture at a 3-fold gradient under the same conditions as above, with a starting concentration of 300 nM. BMS-986340-mIgG2a or GS-1811-mIgG2a bound to the cell surface was detected using goat anti-mouse IgG(H+L) labeled with fluorophore (iFluor 647). The results are shown in Figures 9B and 9C, respectively. [ka]

[0179] As shown in Figure 9A, both BMS-986340-mIgG2a and GS-1811-mIgG2a can compete with 125C1E6-VH3-VL1 for the CCR8 binding site. The results shown in Figures 9B and 9C are consistent with those in Figure 8A. It can be seen that the antigen epitope to which the 125C1E6 antibody binds may overlap with the CCR8 epitopes of the two positive controls.

[0180] Although this application has been described with reference to one or more embodiments, it should be understood that this application is not limited to these embodiments. The description in this application is intended to cover all variations and equivalents that fall within the spirit and scope of the appended claims. All documents referenced herein are incorporated herein by reference in their entirety.

Claims

1. It can specifically bind to CCR8, i) A heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 each contain (1) the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, (2) SEQ ID NOs: 7, 8, and 9, (3) SEQ ID NOs: 13, 2, and 14, or (4) SEQ ID NOs: 18, 19, and 20, or each CDR contains an amino acid sequence having 1 to 3 amino acid substitutions compared to the above amino acid sequences, and / or ii) An isolated monoclonal antibody or its antigen-binding moiety comprising a light chain variable region including VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 each contain an amino acid sequence shown in (1) SEQ ID NOs: 4, 5, and 6, (2) SEQ ID NOs: 10, 11, and 12, (3) SEQ ID NOs: 15, 16, and 17, or (4) SEQ ID NOs: 21, 16, and 17, or an amino acid sequence having 1 to 3 amino acid substitutions in each CDR compared to the above amino acid sequence.

2. An isolated monoclonal antibody or antigen-binding moiety thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences shown in (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, (2) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, (3) SEQ ID NOs: 13, 2, 14, 15, 16, and 17, or (4) SEQ ID NOs: 18, 19, 20, 21, 16, and 17.

3. The isolated monoclonal antibody or antigen-binding moiety according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 22, 24, 26, 27, 29, 29, 31, 33, or 35.

4. An isolated monoclonal antibody or its antigen-binding moiety according to any one of claims 1 to 3, wherein the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 23, 25, 28, 30, 32, 34, or 36.

5. An isolated monoclonal antibody or antigen-binding moiety thereof according to any one of claims 1 to 4, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (1) SEQ ID NOs: 22 and 23, (2) SEQ ID NOs: 24 and 25, (3) SEQ ID NOs: 26 and 25, (4) SEQ ID NOs: 27 and 28, (5) SEQ ID NOs: 29 and 30, (6) SEQ ID NOs: 31 and 32, (7) SEQ ID NOs: 33 and 34, or (8) SEQ ID NOs: 35 and 36.

6. An isolated monoclonal antibody or antigen-binding moiety according to any one of claims 1 to 5, further comprising a heavy chain constant region and / or a light chain constant region.

7. The isolated monoclonal antibody or its antigen-binding moiety according to claim 6, wherein the heavy chain constant region is the IgG1 heavy chain constant region.

8. The isolated monoclonal antibody or its antigen-binding moiety according to claim 7, wherein the heavy chain variable region is a human IgG1 heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO:

37.

9. The isolated monoclonal antibody or its antigen-binding moiety according to claim 6, wherein the light chain constant region is a κ-light chain constant region.

10. The isolated monoclonal antibody or its antigen-binding moiety according to claim 9, wherein the light chain constant region is a human κ light chain constant region comprising the amino acid sequence shown in SEQ ID NO:

38.

11. An isolated monoclonal antibody or antigen-binding moiety thereof according to any one of claims 1 to 10, which is derived from mouse, chimeric, or humanized.

12. i) It can bind to human CCR8, ii) It can inhibit CCR8-CCL1 binding / interaction, and iii) CCR8 ＋ It can be internalized by cells, and iv) CCR8 ＋ An isolated monoclonal antibody or antigen-binding moiety thereof according to any one of claims 1 to 11, which is capable of inducing antibody-dependent cell-mediated cytotoxicity against cells and / or v) has an endogenous antitumor effect.

13. An immune complex comprising i) an antibody or antigen-binding portion thereof as described in any one of claims 1 to 12, and ii) a cytotoxic molecule, wherein i) and ii) are linked via a linker or directly.

14. A bispecific molecule comprising i) an antibody or antigen-binding moiety according to any one of claims 1 to 12, and ii) a second functional group, wherein i) and ii) bind to different antigens or different epitopes of the same antigen and are linked to each other.

15. A chimeric antigen receptor comprising i) an extracellular antigen-binding domain, ii) a transmembrane domain, and iii) an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises an antibody or its antigen-binding portion as described in any one of claims 1 to 12.

16. An immune cell comprising the chimeric antigen receptor described in claim 15.

17. An oncolytic virus that expresses an antibody or its antigen-binding portion according to any one of claims 1 to 12.

18. A nucleic acid molecule encoding an antibody or antigen-binding portion thereof according to any one of claims 1 to 12, an immune complex according to claim 13, a bispecific molecule according to claim 14, or a chimeric antigen receptor according to claim 15.

19. An expression vector comprising the nucleic acid molecule described in claim 18.

20. A host cell comprising the expression vector described in claim 19, or having the nucleic acid molecule described in claim 18 incorporated into its genome.

21. A composition comprising an antibody or antigen-binding portion thereof according to any one of claims 1 to 12, an immune complex according to claim 13, a bispecific molecule according to claim 14, a chimeric antigen receptor according to claim 15, an immune cell according to claim 16, an oncolytic virus according to claim 17, a nucleic acid molecule according to claim 18, an expression vector according to claim 19, or a host cell according to claim 20.

22. The composition according to claim 21, which is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

23. The composition according to claim 21 or 22, further comprising a PD-1 antibody.

24. Use of the composition according to any one of claims 21 to 23 in the manufacture of a drug for treating or alleviating CCR8-related cancer.

25. The use according to claim 24, wherein the CCR8-related cancer is a solid tumor.

26. The use according to claim 25, wherein the solid tumor is non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), gastric cancer, microsatellite-stable colorectal cancer, or cervical cancer.

27. The use according to claim 24, wherein the CCR8-related cancer is a T-cell lymphoma.

28. A method for producing a CCR8 antibody, comprising: i) administering to an animal a DNA molecule capable of expressing the CCR8 protein, an RNA molecule capable of expressing the CCR8 protein, and / or cells capable of overexpressing the CCR8 protein; ii) collecting lymph node cells and / or spleen cells from the animal; and iii) fusing the lymph node cells and / or spleen cells with myeloma cells to produce a hybridoma.

29. The method according to claim 28, i) administering an RNA molecule capable of expressing the CCR8 protein to an animal, ii) collecting lymph node cells and / or spleen cells from the animal, and iii) fusing the lymph node cells and / or spleen cells with myeloma cells to produce a hybridoma.

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