Antibody binding to human CCR8 and the use thereof

Abstract

The present application relates to a monoclonal antibody that specifically binds to CCR8 or an antigen-binding portion thereof. The present application also relates to a nucleic acid molecule encoding the antibody or the antigen-binding portion thereof; an expression vector, host cell and method for expressing the antibody or the antigen-binding portion thereof; and a therapeutic method using the antibody or the antigen-binding portion thereof, the nucleic acid molecule, the expression vector and / or the host cell.

Description

Antibodies binding to human CCR8 and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310693601.4 filed on June 12, 2023, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] The present application relates to an antibody or an antigen-binding portion thereof that specifically binds to CCR8, and the use of the antibody or antigen-binding portion in treating diseases such as cancer. Background Art

[0004] Chemokines can bind to chemokine receptors expressed on cells and guide cell migration through their own concentration distribution. The binding of chemokine receptors to chemokines can trigger a signaling pathway, and the receptors will be internalized and expressed again on the forward side of the moving cell. Chemokines can be produced under steady state, responsible for daily immune cell migration, or they can be produced under inflammatory stimulation to recruit immune cells to sites 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).

[0005] T cells can be divided into different subgroups according to the combination of chemokine receptors on the cell surface, such as regulatory T cells (Treg) that regulate or suppress the immune system and maintain self-tolerance and immune homeostasis, and cytotoxic CD8 T cells that recognize and kill target cells. +T cells, etc. CCR8 is a chemokine receptor mainly expressed in Tregs, and its ligands are the chemokines CCL1 and CCL18. The main ligand CCL1 is mainly produced by activated monocytes, macrophages, and T lymphocytes, and can also be secreted by tumor cells, while the second ligand CCL18 can 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, Tregs migrate to the inflammatory site, triggering the CCR8 signaling pathway, and balancing immunity and tolerance.

[0006] The tumor microenvironment (TME) is rich in various chemokines, and a large number of Treg infiltrations are observed in the tumor microenvironment (TME) of various tumor tissues, which promote tumor growth by inhibiting anti-tumor immune responses. The expression of CCR8 is mainly limited to this type of tumor-infiltrating Treg, and rarely appears in Tregs of peripheral blood mononuclear cells. It has been reported that CCR8 is only upregulated in tumor Tregs compared with Tregs residing in normal tissues. In preclinical studies, it was found that in mouse models of colorectal cancer, melanoma, breast cancer, and urothelial carcinoma, tumor Tregs highly expressed CCR8. Antibody therapy targeting CCR8 can selectively reduce CCR8 through, for example, antibody-dependent cell-mediated cytotoxicity (ADCC). + Tumor Treg cells trigger anti-tumor immune responses and significantly inhibit tumor growth. In addition, CCR8 is also expressed on skin memory T cells, which are considered to be the source of tumor cells in mycosis fungoides (MF). Elimination of CCR8 + Cells may be able to inhibit the progression of T-cell lymphoma (Giustiniani J et al., (2022) CCR8 is a new therapeutic target in cutaneous T-cell lymphomas. BloodAdv. 6(11): 3507-3512). Therefore, CCR8 residing in tumors + Treg cells and CCR8 resident in the skin + T cells may be suitable targets for cancer immunotherapy, and targeting CCR8 has potential for cancer treatment.

[0007] However, as a seven-transmembrane protein, CCR8 is difficult to maintain its native conformation after purification. Various strategies have been used in the field to prepare antigens for animal immunization, including synthesizing part of the extracellular domain or immunizing with DNA molecules. However, when peptides that cannot present their natural conformation are used as antigens, the antibodies produced may not be able to recognize the natural target, and DNA immunization may not be sufficient to induce strong animal immunity. Therefore, immunization against this target is full of challenges, and new methods are needed to strengthen animal immunity and generate better antibodies.

[0008] The citation of any document in this application does not constitute an admission that these documents are prior art to this application.

[0009] Summary of the Invention

[0010] The inventors of the present application screened for novel CCR8 antibodies by using a DNA plasmid containing a CCR8 coding sequence, mRNA encoding CCR8, and / or cells overexpressing CCR8 in combination for animal immunization.

[0011] The CCR8 antibody of the present application has i) comparable or better CCR8 binding ability and binding specificity, ii) comparable or higher CCR8-CCL1 blocking ability, iii) comparable or higher CCR8-CCL1 blocking ability, and iii) comparable or higher CCR8-CCL1 blocking ability compared to prior art antibodies such as BMS-986340 and GS-1811. + The invention also provides for the ability of the cells to elicit antibody-dependent cell-mediated cytotoxicity (ADCC), vi) equivalent or higher internalization activity, and / or v) equivalent or better in vivo anti-tumor efficacy.

[0012] Thus, in a first aspect, the present application relates to an isolated monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody), or an antigen-binding portion thereof, which can specifically bind to CCR8 (e.g., human or monkey CCR8), and can comprise i) a heavy chain variable region, which can comprise VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 can comprise the amino acid sequences of (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, respectively; and / or ii) a light chain variable region, which can comprise VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 can comprise the amino acid sequences of (1) SEQ ID NOs: 1, 2, and 3, respectively; (2) SEQ ID NOs: 7, 8, and 9, respectively; (3) SEQ ID NOs: 13, 2, and 14, or (4) SEQ ID NOs: 18, 19, and 20, respectively. NO: 4, 5 and 6; (2) SEQ ID NO: 10, 11 and 12; (3) SEQ ID NO: 15, 16 and 17; or (4) SEQ ID NO: 21, 16 and 17. Also provided are variants of the above-described antibodies or antigen-binding portions, which comprise up to about 3 amino acid substitutions (e.g., 1, 2, or 3 amino acid residue substitutions) in each CDR compared to the above-described antibodies or antigen-binding portions thereof. In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application, which is capable of specifically binding to CCR8, comprises: 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 can be as 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, respectively; 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 can be as 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: 18, 19 and 20, respectively. IDNO:21, 16 and 17.

[0013] The isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise 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 may comprise the amino acid sequences set forth 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 comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 may comprise the amino acid sequences set forth in SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively. Also provided are variants of the above antibodies or antigen-binding portions, comprising up to about 3 amino acid residue substitutions (e.g., 1, 2, or 3 amino acid residue substitutions) in each CDR compared to the above antibodies or antigen-binding portions thereof. In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 can be as shown in (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; (2) SEQ ID NO: 7, 8, 9, 10, 11 and 12; (3) SEQ ID NO: 13, 2, 14, 15, 16 and 17; or (4) SEQ ID NO: 18, 19, 20, 21, 16 and 17, respectively.

[0014] The heavy chain variable region of the antibody or antigen-binding portion thereof of the present application may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22, 24, 26, 27, 29, 31, 33, or 35.

[0015] The light chain variable region of the antibody or antigen-binding portion thereof of the present application may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23, 25, 28, 30, 32, 34, or 36.

[0016] The antibodies or antigen-binding portions thereof of the present application may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22, 24, 26, 27, 29, 31, 33, or 35; and / or the light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23, 25, 28, 30, 32, 34, or 36. In some embodiments, the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 22, 24, 26, 27, 29, 31, 33, or 35; and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 23, 25, 28, 30, 32, 34, or 36. In other embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 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 NO: 23, 25, 28, 30, 32, 34, or 36.

[0017] The isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region can respectively comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (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 can respectively comprise 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 other embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region can be shown as (1) SEQ ID NO: 22 and 23; (2) SEQ ID NO: 24 and 25; (3) SEQ ID NO: 26 and 25; (4) SEQ ID NO: 27 and 28; (5) SEQ ID NO: 29 and 30; (6) SEQ ID NO: 31 and 32; (7) SEQ ID NO: 33 and 34; or (8) SEQ ID NO: 35 and 36, respectively.

[0018] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise 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 may comprise the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. The heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 22, 24, or 26. The light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 23 or 25. In some embodiments, the heavy chain variable region and the light chain variable region can respectively comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 22 and 23; (2) SEQ ID NOs: 24 and 25; or (3) SEQ ID NOs: 26 and 25.

[0019] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise 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 may comprise the amino acid sequences of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively. The heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 27 or 29. The light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 28 or 30. In some embodiments, the heavy chain variable region and the light chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 27 and 28; or (2) SEQ ID NOs: 29 and 30, respectively.

[0020] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise 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 may comprise the amino acid sequences of SEQ ID NOs: 13, 2, 14, 15, 16, and 17, respectively. The heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 31 or 33. The light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 32 or 34. In some embodiments, the heavy chain variable region and the light chain variable region can comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 31 and 32; or (2) SEQ ID NOs: 33 and 34, respectively.

[0021] The present application also provides an isolated monoclonal antibody or antigen-binding portion thereof, which can specifically bind to CCR8 and can comprise i) a heavy chain variable region, wherein the heavy chain variable region can comprise VH CDR1, VH CDR2 and VH CDR3, and ii) a light chain variable region, wherein the light chain variable region can comprise VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 can comprise the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 contained in a selected heavy chain variable region and a light chain variable region, and the selected heavy chain variable region and the light chain variable region can respectively comprise (1) SEQ ID NO: 22 and 23; (2) SEQ ID NO: 24 and 25; (3) SEQ ID NO: 26 and 25; (4) SEQ ID NO:27 and 28; (5) SEQ ID NO:29 and 30; (6) SEQ ID NO:31 and 32; (7) SEQ ID NO:33 and 34; or (8) the amino acid sequence shown in SEQ ID NO:35 and 36.

[0022] The isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain constant region and / or a light chain constant region, wherein the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected 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 that naturally or after modification has FcR and / or complement system protein binding ability, or a functional fragment thereof, such as a fragment comprising the hinge region, CH2, and CD3 of the heavy chain constant region. In one embodiment, the heavy chain constant region is an IgG1 heavy chain constant region, such as a human IgG1 heavy chain constant region, comprising, for example, the amino acid sequence shown in SEQ ID NO: 37. The light chain constant region may be a kappa or lambda light chain constant region. In some embodiments, the light chain constant region may be a human kappa light chain constant region, comprising, for example, the amino acid sequence shown in SEQ ID NO: 38.

[0023] In some embodiments, the antibodies of the present application comprise two heavy chains and two light chains, or are composed of two heavy chains and two light chains, wherein each heavy chain comprises the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, and / or CDR sequence, and each light chain comprises the aforementioned light chain constant region sequence, light chain variable region sequence, and / or CDR sequence. In some embodiments, the antibodies of the present application or their antigen-binding portions may be Fab, F(ab')2 fragments, Fv, scFv, or (scFv)2, etc.

[0024] The antibodies of the present application, or antigen-binding portions thereof, can be, for example, murine, chimeric, or humanized.

[0025] The antibodies or antigen-binding portions thereof of the present application may be antagonistic.

[0026] The antibodies or antigen-binding portions thereof of the present application can i) bind to human CCR8, ii) bind to monkey CCR8, iii) block CCR8-CCL1 binding / interaction, vi) + Cell internalization, v) can trigger the expression of CCR8 + iv) ADCC of cells (when the heavy chain constant region has FcR binding ability), and / or iv) having an anti-tumor effect in vivo.

[0027] The application also provides an immunoconjugate containing the present application antibody or its antigen-binding portion thereof, and the antibody or its antigen-binding portion thereof is connected to a therapeutic agent such as a cytotoxic molecule or an anticancer agent. The application also provides a bispecific molecule containing the present application antibody or its antigen-binding portion thereof, the antibody or its antigen-binding portion thereof is connected with a second functional group, for example, a second antibody, and the second functional group has a binding specificity different from the present application antibody or its binding portion thereof. On the other hand, the present application's antibody or its antigen-binding portion thereof can be a part of a chimeric antigen receptor (CAR) or a genetically modified T cell receptor (TCR). The application also provides immune cells containing the above-mentioned CAR and / or TCR, including T cells, NK cells, etc. The present application's antibody or its antigen-binding portion thereof can also be encoded by an oncolytic virus or carried by an oncolytic virus.

[0028] The present application also includes nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, immunoconjugates, bispecific molecules, and / or CAR / TCRs of the present application. The present application may also provide an expression vector and a host cell. The expression vector may comprise the nucleic acid molecules of the present application. The host cell may comprise the expression vector of the present application, or have the nucleic acid molecules of the present application integrated into its genome.

[0029] The present application also provides a method of using the host cells of the present application to prepare the antibody or its antigen-binding portion, immunoconjugate, bispecific molecule, or CAR / TCR of the present application, comprising: (i) expressing the antibody or its antigen-binding portion, immunoconjugate, bispecific molecule, or CAR / TCR in the host cell, and (ii) isolating the antibody or its antigen-binding portion, immunoconjugate, bispecific molecule, or CAR / TCR from the host cell or its culture.

[0030] The present application also provides a composition comprising the antibody or antigen-binding portion thereof, immunoconjugate, bispecific molecule, immune cell, oncolytic virus, nucleic acid molecule, expression vector or host cell of the present application. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a PD-1 antibody.

[0031] On the other hand, the present application provides a method for treating or alleviating a CCR8 related disease in a subject, comprising administering a therapeutically effective amount of the present application's pharmaceutical composition to the subject. CCR8 related diseases can be tumors, including solid tumors and hematologic tumors. 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. Hematologic cancers can be T-cell lymphomas, including but not limited to, cutaneous T-cell lymphomas. In some embodiments, the pharmaceutical composition of the present application can be administered together with at least one other antibody, such as PD-1 antibodies, etc. In another embodiment, the pharmaceutical composition of the present application can be administered together with cytokines (such as IL-2 and / or IL-21) or costimulatory antibodies (such as CD137 antibodies and / or GITR antibodies). In another embodiment, the antibody of the present application, or its antigen-binding portion thereof, can be administered together with a chemotherapeutic agent, which can be a cytotoxic agent.

[0032] The present application also provides a method for relieving or alleviating immunosuppression in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of the present application. In some embodiments, the method is used to relieve or alleviating immunosuppression in a tumor microenvironment, comprising administering to the tumor site an effective amount of the pharmaceutical composition of the present application.

[0033] The present application also provides a method for enhancing an immune response in a subject, comprising administering an effective amount of the pharmaceutical composition of the present application to the subject.

[0034] The present application also relates to the use of the above-mentioned composition, in particular the pharmaceutical composition, in the preparation of a drug for treating or alleviating CCR8-related tumors, relieving or alleviating immunosuppression, or enhancing immune response.

[0035] The antibodies or antigen-binding portions thereof of the present application can also be used, for example, for in vitro antigen detection.

[0036] On the other hand, the present application provides a method for preparing CCR8 antibodies, comprising i) administering a DNA molecule containing a CCR8 coding sequence, an RNA molecule encoding CCR8, and / or cells capable of overexpressing CCR8 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 hybridomas.

[0037] The DNA molecule containing the CCR8 coding sequence can be a DNA molecule containing the CCR8 coding sequence and capable of expressing the CCR8 protein in an animal. The DNA molecule can be a DNA vector, such as a lentiviral vector, a plasmid, or the like. In some embodiments, the DNA molecule can be a vector containing a suitable promoter and the CCR8 coding sequence. The promoter can be a constitutive promoter or an inducible promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the DNA molecule can be injected into an animal, for example, subcutaneously or intraperitoneally, using a gene gun.

[0038] The RNA molecule encoding CCR8 can be an RNA molecule containing a CCR coding sequence and capable of expressing (translating) a CCR8 protein in an animal. In some embodiments, the RNA molecule can express a human CCR8 protein in an animal. The RNA molecule can be an mRNA molecule. In some embodiments, the RNA molecule can be a circular RNA molecule. In some embodiments, the RNA molecule can comprise a suitable promoter and a CCR8 coding sequence. The promoter can be a constitutive promoter or an inducible promoter. In some embodiments, the promoter can be a constitutive promoter. The RNA molecule can be directly injected into the animal, for example, into the peritoneal cavity. The RNA molecule can also be injected into the animal with the assistance of lipid molecules (such as micelles, liposomes, lipid nanoparticles (LNPs)), virus-like particles (VLPs), polymer molecules (such as poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI), polylysine (PLL), poly(β-amino ester) (PBAE), polymer nanoparticles), exosomes, etc. In one embodiment, the RNA molecule is encapsulated by LNPs.

[0039] The cell capable of overexpressing CCR8 can be a cell that expresses CCR8 protein in an animal. The cell can be a eukaryotic cell. In some embodiments, the cell can be injected into the animal body, for example, into the peritoneal cavity.

[0040] In some embodiments, the methods of the present application comprise i) administering an RNA molecule capable of expressing a 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 a CCR8 protein in the animal. In some embodiments, the RNA molecule can express a human CCR8 protein in the animal.

[0041] All documents cited or mentioned in this application (including but not limited to all documents, patents, published patent applications cited herein) ("this application's references"), all documents cited or mentioned in this application's references, and manufacturer's manuals, instructions, product specifications, and product pages for any product mentioned in this application or any of this application's references are incorporated herein by reference and may be used in practicing the present invention. More specifically, all references are incorporated herein by reference, just as each file is incorporated by reference. Any Genbank sequence mentioned in this article is incorporated herein by reference.

[0042] It should be noted that in this application, especially in the claims, terms such as "comprising" and "including" may have the meanings assigned to them by the Chinese Patent Law; and terms such as "essentially consisting of..." have the meanings assigned to them by the Chinese Patent Law, such as allowing the existence of elements not explicitly stated, but excluding elements existing in the prior art or elements that affect the basic or new characteristics of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The following detailed description is given by way of example but is not intended to limit the present invention to the specific embodiments described, and can be better understood in conjunction with the accompanying drawings.

[0044] Figures 1A and 1B show the binding ability of the chimeric antibodies 117G9F9, 125C1E6, 154G7C3 (1A), and 224E9A3 (1B) of the present application to HEK293 cells expressing human CCR8.

[0045] FIG2 shows the binding ability of the chimeric antibody of the present application to HEK293 cells expressing monkey CCR8.

[0046] FIG3 shows the blocking ability of the chimeric antibody of the present application on CCR8-CCL1 binding / interaction.

[0047] FIG4 shows the chimeric antibody of the present application for CCR8 + Cells trigger ADCC activity.

[0048] Figures 5A-5C show the binding of the humanized antibodies of the present invention to HEK293 cells expressing human CCR8 (5A) and monkey CCR8 (5B), as well as parental HEK293 cells (5C).

[0049] Figures 6A and 6B show the humanized 125C1E6 antibody (6A), 154G7C3 antibody, and 117G9F9 antibody (6B) in this application for CCR8 + Cells trigger ADCC activity.

[0050] Figures 7A and 7B show the blocking ability of the humanized 125C1E6 antibody (7A, 7B), 154G7C3 antibody, and 117G9F9 antibody (7B) of the present application on CCR8-CCL1 binding / interaction.

[0051] FIG8 shows the cell internalization activity of the humanized antibody of the present invention.

[0052] Figures 9A-9C show the epitope competition binding between the humanized 125C1E6 antibody of the present invention and the positive control, wherein in the experiment, the 125C1E6 antibody was added first, followed by BMS-986340, GS-1811-mIgG2a or 125C1E6 (9A), BMS-986340 was added first, followed by 125C1E6-VH3-VL1 or BMS-986340 (9B), or GS-1811 was added first, followed by 125C1E6 (9C). DETAILED DESCRIPTION

[0053] For a better understanding of this application, some terms are first defined. Other definitions are listed throughout the detailed description.

[0054] The term "a" or "an" refers to one or more than one item. For example, "an antibody" refers to one antibody or more than one antibody.

[0055] The term "CCR8" refers to CC chemokine receptor 8, a member of the G protein-coupled receptor (GPCR) family. The term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific for human CCR8 may, in certain circumstances, cross-react with a CCR8 protein from another species, such as monkey. In other embodiments, an antibody specific for human CCR8 may be completely specific for human CCR8 protein and not cross-react with proteins from other species or other types, or may cross-react with CCR8 proteins from some other species but not all other species.

[0056] The term "human CCR8" refers to a CCR8 protein having a human amino acid sequence, for example, a CCR8 protein having the amino acid sequence of GenBank Accession No. AAI07160.1 (Strausberg, R.L. et 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 a CCR8 protein having a monkey amino acid sequence, for example, a CCR8 protein having the amino acid sequence of GenBank Accession No. AFR51945.1 (Wang, L. et al., (2012), Submitted (26-JUL-2012) Department of Cellular Immunology and Pharmacology, Millennium Pharmaceuticals Inc.).

[0057] The term "antibody" as used herein is intended to include IgG, IgA, IgD, IgE and IgM full-length antibodies and 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 connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V H The heavy chain constant region is composed 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) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The V domains can be further divided into regions of hypervariability, termed complementarity determining regions (CDRs), separated by more conserved framework regions (FRs). H and V LIt is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including binding to a variety of immune system cells (e.g., effector cells) and the first component (C1q) of the traditional complement system. A "functional fragment" of an antibody constant region refers to a fragment that retains certain desired functions in the constant region, such as a fragment that retains FcR / complement system component binding activity in the heavy chain constant region, such as an Fc fragment.

[0058] The term "antigen-binding portion" of an antibody (or simply "antibody portion") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CCR8 protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment consisting of V L 、V H 、C L and C H1 (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) V H and C H1 (iv) an Fd fragment consisting of an antibody single-arm V L and V H Fv fragment composed of V H (vi) isolated complementarity determining regions (CDRs); and (vii) dAb-V L , a fragment comprising a single variable domain and a heavy chain constant domain. In addition, although the two domains V L and V H Encoded by different genes, they can be recombinantly linked via a synthetic linker that makes the two into a single protein chain, where V L and V H The regions are paired to form monovalent molecules. These single-chain antibodies are also intended to be included in the meaning of the term. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0059] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to a CCR8 protein is substantially free of antibodies that specifically bind to antigens other than the CCR8 protein. However, an isolated antibody that specifically binds to a human CCR8 protein may have cross-binding properties to other antigens, such as CCR8 proteins from other species. Furthermore, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0060] The term "monoclonal antibody" or "mAb" or "monoclonal antibody composition" refers to an antibody molecule preparation of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0061] The term "mouse-derived antibody" refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. The mouse-derived antibodies of the present application may contain amino acid residues that are not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo. However, the term "mouse-derived antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the mouse framework sequence.

[0062] The term "chimeric antibody" refers to an antibody that combines genetic material from one species with genetic material from another species. In particular, a chimeric antibody in the present application refers to an antibody that is derived from a combination of non-human genetic material and human genetic material.

[0063] The term "humanized antibody" refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to naturally occurring antibodies in humans.

[0064] The terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0065] As used herein, the term "specifically recognizes" or "specifically binds" to a target, such as human CCR8, refers to an antibody or antigen-binding fragment that can distinguish the target biomolecule from one or more reference molecules, and has a binding affinity or activity to the target biomolecule that is, for example, 1-fold, 5-fold, 10-fold, etc., higher than the other reference molecules. Specificity determination methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA testing, and peptide scanning.

[0066] The term "substantially not binding" to a protein or cell means not binding to the protein or cell, or not binding to the protein or cell with high affinity, i.e., the K of the binding protein or cell is less than 1%. D 1.0×10 -6M or more, more preferably 1.0×10 -5 M or more, more preferably 1.0×10 -4 M or more, 1.0×10 -3 M or more, more preferably 1.0×10 -2 M or above.

[0067] The term "EC 50 ", also known as half-maximal effect concentration, refers to the antibody concentration that causes 50% of the maximum effect.

[0068] The term "IC 50 ” refers to the half-inhibitory concentration, which is the concentration of the drug or inhibitor required to inhibit a specified biological process by half.

[0069] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse target cells that have surface antigens bound to antibodies, such as the CCR8 antibodies of the present application.

[0070] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows and horses, are preferred.

[0071] The term "therapeutically effective amount" refers to an amount of the antibody of the present invention sufficient to prevent or alleviate the symptoms associated with a disease or disorder (e.g., cancer). The therapeutically effective amount is related to the disease being treated, and those skilled in the art can easily determine the actual effective amount.

[0072] "Sequence identity" herein refers to the percentage of nucleotides / amino acids in a sequence that are identical to the nucleotides / amino acid residues in a reference sequence after sequence alignment, with spaces introduced, if necessary, to achieve the maximum percentage of sequence identity between the two sequences. Those skilled in the art can perform pairwise sequence alignment or multiple sequence alignment to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences by various methods, such as using computer software such as Clustal Omega, T-coffee, Kalign, and MAFFT.

[0073] The difficulty in preparing CCR8 antibodies lies in the fact that, as a seven-transmembrane protein, it is difficult to maintain its native conformation after purification. When peptides that cannot present their native conformation are used as antigens, the antibodies produced may not be able to recognize their native targets, and DNA immunization may not be sufficient to induce strong animal immunity.

[0074] The inventors of this application have screened for novel CCR8 antibodies with superior properties compared to existing technologies by combining a DNA plasmid containing the CCR8 coding sequence with cells overexpressing CCR8, or by administering CCR8-encoding mRNA. The inventors of this application have discovered that for multi-transmembrane proteins, mRNA elicits higher antibody levels than DNA molecules.

[0075] Therefore, in one aspect, the present application provides a novel method for animal immunization, or a method for preparing antibodies by animal immunization, comprising immunizing an animal with an RNA molecule (such as an mRNA molecule) encoding a CCR8 protein.

[0076] Specifically, the present application provides a method for preparing CCR8 antibodies, comprising i) administering to an animal a DNA molecule containing a CCR8 coding sequence and capable of expressing a CCR8 protein in the animal, an RNA molecule containing a CCR8 coding sequence and capable of expressing (translating) a CCR8 protein in the animal, and / or cells capable of overexpressing a CCR8 protein in the 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 hybridomas.

[0077] 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 a CCR8 protein in the animal, and cells capable of overexpressing a CCR8 protein in the 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 hybridomas.

[0078] In some embodiments, the methods of the present application comprise i) administering to an animal an RNA molecule capable of expressing a CCR8 protein in the 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 hybridomas. In some embodiments, the RNA molecule can express human CCR8 protein in the animal. The RNA molecule can be an mRNA molecule, such as a circular RNA molecule. In some embodiments, the RNA molecule can be a vector comprising a suitable promoter and a CCR8 coding sequence. The promoter can be a constitutive promoter or an inducible promoter. In some embodiments, the promoter is a constitutive promoter. The RNA molecule can be injected directly into the animal, for example, into the peritoneal cavity. The RNA molecule can also be injected into the animal with the aid of lipid molecules (such as micelles, liposomes, lipid nanoparticles (LNPs)), virus-like particles (VLPs), polymer molecules (such as poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI), polylysine (PLL), poly(β-amino ester) (PBAE), polymer nanoparticles), exosomes, and the like. In one embodiment, the RNA molecule is encapsulated by LNP.

[0079] The CCR8 antibodies of the present application have i) comparable or better (human or monkey) CCR8 binding ability and binding specificity, ii) comparable or higher CCR8-CCL1 blocking ability, iii) comparable or higher CCR8-CCL1 blocking ability, and iv) comparable or higher CCR8-CCL1 blocking ability compared to the antibodies disclosed in the prior art, such as BMS-986340 and GS-1811. + The invention also provides for the ability of the cells to elicit antibody-dependent cell-mediated cytotoxicity (ADCC), vi) equivalent or higher internalization activity, and / or v) equivalent or better in vivo anti-tumor efficacy.

[0080] The preferred antibody of the present application is a monoclonal antibody. In addition, the antibody can be, for example, a mouse-derived, chimeric, or humanized monoclonal antibody.

[0081] Exemplary antibodies of the present application, or antigen-binding portions thereof, are those whose structural and chemical properties are described below.

[0082] The heavy chain variable region and light chain variable region sequences of the antibodies or antigen-binding portions thereof of the present application are listed in Tables 1 and 2. The heavy chain variable region CDRs and light chain variable region CDRs are identified using the Kabat numbering system, and the CDRs identified thereby are listed in Table 1. The heavy chain variable region CDRs and light chain variable region CDRs of the antibodies or antigen-binding portions thereof of the present application may also be identified using the Chothia, IMGT, AbM, or Contact numbering systems.

[0083] The antibodies or antigen-binding portions thereof of the present application may comprise a heavy chain constant region, such as a natural or engineered heavy chain constant region having FcR binding, particularly one with high FcR binding. In some embodiments, the heavy chain constant region may be an IgG1 constant region, such as a human IgG1 constant region comprising the amino acid sequence set forth in SEQ ID NO: 37. The light chain constant region may be a kappa constant region, such as a human kappa constant region, which may comprise the amino acid sequence set forth in SEQ ID NO: 38.

[0084] V of other CCR8 antibodies that bind to human CCR8 H and / or V L The sequence (or CDR sequence) can be the same as the V H and / or V L Sequences (or CDR sequences) are "mixed and paired". Preferably, when V H and V L When (or CDRs therein) are mixed and paired, a specific V H / V L V in pairing H The sequence can be approximated by the structure of V H Similarly, specific V H / V L V in pairing L The sequence is composed of structurally similar V L Sequence substitution.

[0085] Therefore, in one embodiment, the antibody or antigen-binding portion thereof of the present application comprises:

[0086] (a) a heavy chain variable region comprising the amino acid sequence listed in Table 1 or Table 2; and

[0087] (b) a light chain variable region comprising the amino acid sequence listed in Table 1 or Table 2, or a V region of another CCR8 antibody L ,in

[0088] This antibody specifically binds to human CCR8.

[0089] In another embodiment, the antibody or antigen-binding portion thereof of the present application comprises:

[0090] (a) CDR1, CDR2, and CDR3 of the heavy chain variable region listed in Table 1 or Table 2; and

[0091] (b) CDR1, CDR2 and CDR3 of the light chain variable region listed in Table 1 or Table 2, or the light chain variable region CDRs of another CCR8 antibody, wherein the antibody specifically binds to human CCR8.

[0092] In another embodiment, the antibody of the present application or its antigen-binding portion includes the heavy chain variable region CDR2 of a CCR8 antibody and the CDRs of other antibodies that bind to human CCR8, such as the heavy chain variable region CDR1 and / or CDR3, and / or the light chain variable region CDR1, CDR2 and / or CDR3 of another CCR8 antibody.

[0093] Furthermore, it is well known in the art that the CDR3 domain, independent of CDR1 and / or CDR2, can independently determine the binding specificity of an antibody to the same antigen, and it can be predicted that multiple antibodies with the same binding specificity can be generated based on the CDR3 sequence. See, eg, 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. USA 95:8910-8915 (1998); Barbas et al. al., J. Am. Chem. Soc. 116: 2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. USA 92: 2529-2533 (1995); Ditzel et al., J. Immunol. 157: 739-749 (1996).

[0094] In another embodiment, the antibody or antigen-binding portion thereof of the present application comprises the CDR2 of the heavy chain variable region of the CCR8 antibody and at least the CDR3 of the heavy chain and / or light chain variable region of the 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 portion thereof is capable of specifically binding to human CCR8. Preferably, these antibodies or antigen-binding portions thereof (a) compete for binding to CCR8; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have similar binding affinity as the CCR8 antibody or antigen-binding portion thereof of the present application. In another embodiment, the antibody or antigen-binding portion thereof may further comprise the CDR2 of the light chain variable region of the CCR8 antibody or antigen-binding portion thereof of the present application, or the CDR2 of the light chain variable region of another CCR8 antibody, wherein the antibody or antigen-binding portion thereof specifically binds to human CCR8. In another embodiment, the antibody of the present application may include the heavy chain / light chain variable region CDR1 of the CCR8 antibody of the present application or its antigen-binding portion, or the heavy chain and / or light chain variable region CDR1 of another CCR8 antibody, wherein the antibody or its antigen-binding portion specifically binds to human CCR8.

[0095] In another embodiment, the antibodies or antigen-binding portions thereof of the present application comprise heavy and / or light chain variable region sequences or CDR1, CDR2, and CDR3 sequences that have one or more conservative modifications to the CCR8 antibodies or antigen-binding portions thereof of the present application. It is known in the art that some conservative sequence modifications do not abrogate antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8.

[0096] Thus, in one embodiment, the antibody or antigen-binding portion thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region and the light chain variable region comprising CDR1, CDR2 and CDR3, respectively, wherein:

[0097] (a) the heavy chain variable region CDR1 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0098] (b) the heavy chain variable region CDR2 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0099] (c) the heavy chain variable region CDR3 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0100] (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 comprise the sequences listed in Table 1, and / or conservative modifications thereof; and

[0101] (e) The antibody or antigen-binding portion thereof specifically binds to human CCR8.

[0102] The antibodies of the present application have one or more of the following functional characteristics, such as high affinity and high specific binding to human CCR8, and corresponding enhanced ability to induce ADCC on CCR8-positive cells such as CCR8-positive tumor cells.

[0103] In various embodiments, the antibody or antigen-binding portion thereof can be, for example, mouse, chimeric, or humanized.

[0104] The term "conservative sequence modification" as used herein refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid replacements, additions, and deletions. Modifications can be introduced into the present application's antibodies or their antigen-binding portion thereof by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacements are amino acid residues that are replaced with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region of the antibodies of the present application, or antigen-binding portions thereof, can be replaced with other amino acid residues from the same side chain group, and the resulting antibodies can be tested for retention of function (i.e., the function described above) using the functional assays described herein.

[0105] The antibody or antigen-binding portion thereof of the present application can be used with one or more V H / V L The antibody sequence is used as the starting material to prepare the genetically modified antibody. The antibody can be prepared by modifying one or two variable regions (i.e., V H and / or V L ) (e.g., in one or more CDR regions and / or one or more framework regions) to improve binding affinity and / or increase similarity to antibodies naturally produced by certain species. Alternatively, antibodies can be genetically modified by modifying residues in the constant region, for example, to alter the effector function of the antibody.

[0106] In certain embodiments, CDR regions are implanted into variable regions that can be used to genetically modify antibodies. Antibodies or their antigen-binding portions interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). For this reason, the amino acid residues within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Because the CDR sequences are responsible for the primary antibody-antigen interactions, recombinant antibodies that mimic the properties of specific natural antibodies can be expressed by constructing expression vectors that contain CDR sequences of specific natural antibodies and implant them into the backbone sequences of different antibodies with different properties.

[0107] Therefore, another embodiment of the present application relates to an isolated monoclonal antibody or antigen-binding portion thereof, which comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 having the above-mentioned sequence of the present application, and the light chain variable region comprises CDR1, CDR2 and CDR3 having the above-mentioned sequence of the present application. Although these antibodies comprise the V H and V L CDR sequences, which may contain different framework sequences.

[0108] Such backbone sequences can be obtained from public DNA databases or public references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be obtained in the Vbase human germline sequence database. As another embodiment, germline DNA sequences for human heavy and light chain variable region genes can be obtained in the Genbank database.

[0109] The antibody protein sequence is compared to protein sequence databases by using one of the sequence similarity search methods known in the art as gapped BLAST (Altschul et al., (1997)).

[0110] Preferred framework sequences for use in the antibodies or antigen-binding portions thereof of the present invention are those that are structurally similar to the framework sequences used in the antibodies or antigen-binding portions thereof of the present invention. H The CDR1, CDR2, and CDR3 sequences can be implanted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences can be implanted into a framework region that contains one or more mutations compared to the germline sequence. For example, in some cases, it may be beneficial to mutate residues in the framework region to maintain or enhance the antigen binding ability of the antibody.

[0111] Another type of variable region modification is to modify V H and / or V L The amino acid residues in the CDR1, CDR2 and / or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the target antibody. Point mutations or PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The mutations can be amino acid replacements, additions, or deletions, but are preferably replacements. In addition, no more than one, two, three, four, or five residues in the CDR regions are typically changed.

[0112] In another embodiment, the present application provides an isolated CCR8 monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, comprising: (a) V H CDR1 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; (b) V H CDR2 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; (c) V H CDR3 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (d) V L CDR1 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (e) V L CDR2 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; and (f) V L The CDR3 region comprises the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added.

[0113] The genetically modified antibodies of the present application are included in V H and / or V L Genetic modifications are made to the framework residues of the antibody to, for example, alter the properties of the antibody. Framework modifications include mutating one or more residues in the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach, also known as "deimmunization," is described in more detail in U.S. Patent Application No. 20030153043.

[0114] In addition, as an alternative to modifications within the framework or CDR regions, the antibodies of the present application can be genetically engineered to include genetic modifications in the Fc region, generally to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cellular cytotoxicity. In addition, the antibodies of the present application can be chemically modified (e.g., one or more chemical functional groups can be added to the antibody), or modified to alter its glycosylation to alter one or more functional properties of the antibody.

[0115] In one embodiment, C H1 The hinge region of the hinge region is modified to change, for example, increase or decrease the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Patent No. 5,677,425. H1The cysteine ​​residues in the hinge region can be used to, for example, facilitate the assembly of the heavy and light chains or to increase / decrease the stability of the antibody.

[0116] In another embodiment, the Fc hinge region of the antibody is mutated to increase or decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2 -C H3 The linker region is then used to create an antibody that has reduced SpA binding relative to the native Fc-hinge domain SpA binding. This approach is described in more detail in US Patent No. 6,165,745.

[0117] In another embodiment, the glycosylation of the antibody is modified. For example, a deglycosylated antibody (i.e., the antibody lacks glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such glycosylation modification can be achieved by, for example, altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that position. Such deglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0118] In addition, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced amounts of fucose residues, or antibodies with increased bisecting GlcNac structures. Altered glycosylation forms have been shown to increase the ADCC activity of antibodies. Such glycosylation modifications can be performed, for example, by expressing the antibody in a host cell with an altered glycosylation system. Cells with altered glycosylation systems are known in the art, including but not limited to Slc35c1 gene knockout cell lines, FUT8 knockout cell lines, variant CHO cell lines Lec13, rat hybridoma cell lines YB2 / 0, cell lines containing small interfering RNA specifically for the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. They can be used as host cells for expressing the recombinant antibodies of the present application to prepare antibodies with altered glycosylation.

[0119] Another modification of the antibodies herein is polyethylene glycolation (PEGylation). Antibodies can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is performed by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any form of PEG used to derivatize other proteins, such as mono(C1-C 10 ) alkoxy- or aryloxypolyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present application. See, for example, EPO 154316 and EP 0401384.

[0120] The antibodies or antigen-binding portions thereof of the present application can be characterized by various physical properties to detect and / or distinguish their species.

[0121] For example, an antibody or antigen-binding portion thereof may contain one or more glycosylation sites in the light or heavy chain variable region. These glycosylation sites may result in increased antibody immunogenicity or an altered antibody pK value due to altered antigen binding. Glycosylation is known to occur in motifs containing the N-X-S / T sequence. In some cases, it is preferred that the CCR8 antibody or antigen-binding portion thereof not contain variable region glycosylation. This can be achieved by selecting an antibody that does not contain a glycosylation motif in the variable region or by mutating residues in the glycosylation region.

[0122] In a preferred embodiment, the antibody or antigen-binding portion thereof does not contain an asparagine isomerization site. Deamidation of asparagine may occur in NG or DG sequences, creating an isoaspartic acid residue that introduces a kink into the polypeptide chain and reduces its stability (isoaspartic acid effect).

[0123] Each antibody or antigen-binding portion thereof will have a unique isoelectric point (pI), generally falling within the pH range of 6-9.5. The pI of IgG1 antibodies generally falls within the pH range of 7-9.5, while the pI of IgG4 antibodies generally falls within the pH range of 6-8. It is speculated that antibodies with pIs outside the normal range may have some unfolded structures and be unstable under in vivo conditions. Therefore, it is preferred that the pI value of the CCR8 antibody falls within the normal range. This can be achieved by selecting antibodies with pIs within the normal range or by mutating uncharged surface residues.

[0124] The monoclonal antibodies of the present application can be prepared using the somatic cell hybridization (hybridoma) technology of Kohler and Milstein (1975) Nature 256:495. Other methods for preparing monoclonal antibodies include single B cell antibody preparation technology and phage display technology. Chimeric or humanized antibody preparation methods are also well known in the art.

[0125] The antibodies or antigen-binding portions thereof of the present application can also be produced in host cell transfectomas using, for example, recombinant DNA technology in combination with gene transfection methods (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biological techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this case, the term "operably linked" refers to the connection of the antibody gene to the vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating antibody gene transcription and translation.

[0126] The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses, such as the adenovirus major late promoter (AdMLP), and polyomavirus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter may be used. In addition, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1. The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

[0127] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In a preferred embodiment, the variable region is inserted into an expression vector encoding the heavy chain constant region and the light chain constant region of the desired subtype to construct a full-length antibody gene, so that V H With the C in the carrier H Operatively connected, V L With the C in the carrier L Operably connected. Alternatively, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the antibody chain gene in the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0128] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present application may carry other sequences, such as sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. Selectable marker genes can be used to select the host cell into which the vector has been introduced. For example, conventional selectable marker genes confer drug resistance, such as G418, hygromycin, or methotrexate resistance, to the host cell into which the vector has been introduced. Preferred selectable marker genes include dihydrofolate reductase (DHFR) genes (for methotrexate selection / amplification of dhfr host cells) and neo genes (for G418 selection).

[0129] For the expression of light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The term "transfection" in its various forms includes a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextrose transfection, and the like. Although it is theoretically feasible to express the present antibody or its antigen-binding portion in prokaryotic or eukaryotic host cells, it is preferred that the antibody be expressed in eukaryotic cells, most preferably in mammalian host cells, because eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete properly folded and immunologically active antibodies than prokaryotic cells.

[0130] Preferred mammalian host cells for expressing the recombinant antibodies of the present application include Slc35C1 gene knockout cell lines, FUT8 knockout cell lines, variant CHO cell lines Lec13, rat hybridoma cell lines YB2 / 0, cell lines containing small interfering RNA specifically for the FUT8 gene, cell lines that co-express β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II, Chinese hamster ovary (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 an antibody gene is introduced into a mammalian host cell, the antibody is prepared by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or preferably sufficient to allow secretion of the antibody into the culture medium in which the host cell is grown. The antibody or its antigen-binding portion can be recovered from the culture medium using a protein purification method.

[0131] On the other hand, the present application provides nucleic acid molecules encoding the heavy chain / light chain variable region or CDR of the antibody or antigen-binding portion thereof of the present application. The nucleic acid can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acid of the present application can 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.

[0132] The nucleic acid of the present application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes), cDNA encoding the light chain and heavy chain of the antibody prepared by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene repositories (e.g., using phage display technology), nucleic acids encoding such antibodies can be collected from gene banks.

[0133] The preferred nucleic acid molecules of the present application include V encoding CCR8 monoclonal antibody H and V L Once the sequences encoding V H and V L These DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. H or V L The term "operably linked" refers to a DNA fragment that is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" means that the two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments are in the reading frame.

[0134] Code V H The isolated DNA of the region can be operably linked to the V H The DNA encoding the heavy chain constant region (C H1 、C H2 and C H3 The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, but is preferably IgG1. For Fab fragment heavy chain genes, the V H The DNA of the heavy chain C region can be operably linked to theH1 The constant region of another DNA molecule is attached.

[0135] Code V L The isolated DNA of the region can be operably linked to the V L DNA encoding the light chain constant region C L The light chain constant region is converted into a full-length light chain gene by using another DNA molecule from the human light chain constant region. The sequences of human light chain constant region genes are known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0136] To create scFv genes, the V H and V L The DNA fragment can be operably linked to another fragment encoding a flexible linker, such that V H and V L The sequence can be expressed as a continuous single-chain protein, where V H and V L The regions are connected by this flexible linker.

[0137] The antibody of the present application or its antigen-binding portion thereof can be coupled with therapeutic agent to form an immunoconjugate (immunoconjugate), such as antibody-drug conjugate (ADC). Suitable therapeutic agent includes cytotoxic molecules, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In ADC, antibody and therapeutic agent can be cross-linked by a joint, and the joint can be cut, such as a peptide joint, a disulfide joint or a hydrazone joint. More preferably, the joint is a peptide joint, 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 can be prepared as described in U.S. Patents 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02 / 096910; WO 07 / 038,658; WO 07 / 051,081; WO 07 / 059,404; WO 08 / 083,312; and WO 08 / 103,693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295. In the present application, since CCR8 antibodies can bind to CCR8 + cells, especially CCR8+ Treg cells specifically bind and internalize cytotoxic molecules, which can be coupled to CCR8 + Treg cells cause damage, killing CCR8 + The purpose of Treg cells. In particular, cytotoxic molecules can be internalized by antibodies and enter CCR8 + In Treg cells, cytotoxic molecules can be any small molecule compound or protein molecule that damages target cells, such as microtubule polymerization inhibitors, DNA damaging agents, etc.

[0138] On the other hand, the present application relates to a bispecific molecule comprising an antibody of the present application or an antigen-binding portion thereof linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to generate a bispecific molecule that binds to at least two different binding sites or targeting molecules. The term "bispecific molecule" includes molecules with three or more specificities.

[0139] Bispecific molecules can appear in a variety of formats and sizes. At one end of the size spectrum, bispecific molecules maintain the traditional antibody format, 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 extreme are bispecific molecules composed of two single-chain antibody fragments (scFv) linked by a peptide chain, called Bs(scFv)2 constructs. Bispecific molecules of intermediate size include two different F(ab) fragments connected by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic modification, somatic cell hybridization, or chemical methods.

[0140] The present application also provides a chimeric antigen receptor comprising a CCR8 single-chain antibody scFv, wherein the scFv comprises the heavy chain and light chain CDRs, or the heavy chain and light chain variable regions described in the present application.

[0141] The CCR8 chimeric antigen receptor may comprise (a) an extracellular antigen binding domain comprising a CCR8 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0142] The present application also provides an immune cell, such as a T cell or a NK cell, which contains the chimeric antigen receptor of the present application.

[0143] Oncolytic viruses preferentially infect and kill cancer cells. The antibodies or antigen-binding portions thereof of the present application can be used together with oncolytic viruses. In addition, oncolytic viruses encoding the antibodies or antigen-binding portions thereof of the present application can be introduced into the subject.

[0144] On the other hand, the present application provides a composition comprising an antibody or antigen-binding portion thereof, a nucleic acid molecule, an expression vector, a host cell, an immunoconjugate, a chimeric antigen receptor, an immune cell, a bispecific antibody, and / or an oncolytic virus of the present application. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The composition may optionally comprise one or more other pharmaceutically active ingredients, such as another anti-tumor antibody, or an immune enhancing antibody, or a non-antibody anti-tumor agent, or an immunopotentiator. The composition of the present application can be used in combination with, for example, another anticancer agent or another immunopotentiator.

[0145] The pharmaceutical composition may contain any number of excipients. Useful excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0146] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or push injection). Based on the difference in route of administration, the active ingredient can be wrapped in a material to protect it from the influence of acid and other natural conditions that may inactivate it. "Parenteral administration" refers to a mode different from intestinal and topical, usually carried out by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, on the dura mater and intrasternal injection and push injection. Alternatively, the antibody of the present application can be administered by non-parenteral route, such as external use, epidermal administration or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

[0147] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0148] The amount of active ingredient prepared in a single dosage form together with the carrier material will vary depending on the subject being treated and the particular mode of administration, and is essentially the amount of the composition that produces a therapeutic effect. In percentage terms, this amount is about 0.01 to about 99% of the active ingredient combined with the pharmaceutically acceptable carrier.

[0149] The dosage regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a bolus may be administered, multiple divided doses may be administered over time, or the dose may be reduced or increased in proportion to the severity of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. A dosage unit form refers to physically discrete units suitable for single administration to a subject; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect together with a pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained-release formulation, in which case the required frequency of administration is reduced.

[0150] For administration of the antibody, the dosage may be about 0.001-100 mg / kg host body weight. An exemplary treatment regimen involves administration once per week.

[0151] A "therapeutically effective amount" of the pharmaceutical composition of the present application results in a reduction in the severity of disease symptoms and an increase in the frequency and duration of symptom-free periods. For example, for the treatment of a tumor-bearing subject, a "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and even more preferably by at least about 80%, compared to an untreated subject. A therapeutically effective amount of a therapeutic antibody can reduce tumor size or alleviate symptoms in a subject, which can be a human or another mammal.

[0152] The pharmaceutical composition can be a sustained-release agent, including implants and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, 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.

[0153] Pharmaceutical compositions can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Patent 4,487,603); (3) transdermal delivery devices (U.S. Patent 4,486,194); (4) push injection devices (U.S. Patents 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patents 4,439,196 and 4,475,196).

[0154] In certain embodiments, the components of the compositions of the present application can be formulated to ensure proper in vivo distribution. For example, to ensure that the therapeutic antibodies or antigen-binding portions thereof of the present application cross the blood-brain barrier, the antibodies can be formulated in liposomes, which can also additionally contain targeting 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.

[0155] The application also relates to in vivo gene therapy, wherein nucleic acid molecules encoding the application's antibodies or their antigen-binding portions thereof, immunoconjugates, or bispecific molecules are directly introduced into a subject. For example, the nucleic acid sequence encoding the application's antibodies or antigen-binding portions thereof is introduced into target cells via local injection of a nucleic acid construct with or without a suitable delivery vehicle, such as an adeno-associated viral vector. Other alternative viral vectors include, but are not limited to, retroviruses, adenoviruses, herpes simplex viruses, and papillomavirus vectors. The in vivo physical transfer of viral vectors can be achieved by local injection of the desired nucleic acid construct or other suitable delivery vehicles comprising the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or microparticle bombardment (gene gun).

[0156] The pharmaceutical compositions of the present application have various in vitro and in vivo applications, involving, for example, the treatment of cancer, or more generally, for immune enhancement in patients with diseases such as cancer. The pharmaceutical compositions can be administered to human subjects, for example, to inhibit tumor growth in vivo.

[0157] Taking into account the ability of the present application's pharmaceutical composition to inhibit tumor cell proliferation and survival, the present application provides a method for inhibiting tumor cell growth in a subject, comprising administering the present application's pharmaceutical composition to the subject, thereby inhibiting tumor growth in the subject. Non-limiting examples of tumors that can be treated by the present application's composition include, but are not limited to, solid tumors and hematologic tumors. 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, etc., whether primary or metastatic. Hematologic cancer can be a T-cell lymphoma, including but not limited to, cutaneous T-cell lymphoma. In addition, refractory or recurrent malignancies may also be treated with the present application's pharmaceutical composition.

[0158] The present application provides a combination therapy in which the pharmaceutical composition of the present application is administered together with one or more other antibodies or non-antibody therapeutic agents, which can effectively inhibit tumor growth in a subject. In one embodiment, the present application provides a method for inhibiting tumor growth in a subject, comprising administering the pharmaceutical composition of the present application and one or more other antibodies, such as PD-1 antibodies, to the subject. In certain embodiments, the subject is a human. In another aspect, the present application provides a method for treating cancer, wherein the pharmaceutical composition of the present application is administered together with a chemotherapeutic agent, which may be a cytotoxic agent. Other therapies that can be combined with the pharmaceutical composition of the present application include, but are not limited to, administration of an immunogenic agent, administration of interleukin 2 (IL-2), radiotherapy, surgery, or hormone removal.

[0159] The compositions of the present application can also be used to relieve or alleviate immunosuppression in a subject. In particular, the present application also provides a method for relieving or alleviating immunosuppression in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of the present application. In some embodiments, the method is used to relieve or alleviate immunosuppression in the tumor microenvironment, comprising administering to the tumor site an effective amount of the pharmaceutical composition of the present application.

[0160] The composition of the present application can also be used to enhance immune response. Specifically, the present application provides a method for enhancing immune response in a subject, comprising administering an effective amount of the pharmaceutical composition of the present application to the subject.

[0161] The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, each agent being in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0162] Furthermore, if multiple administrations of the combination therapy are performed and the agents are administered sequentially, the order of sequential administration at each time point can be reversed or remain the same, sequential administration can be combined with simultaneous administration, or any combination thereof.

[0163] Various aspects and embodiments of the present application will be discussed with reference to the accompanying drawings and the following examples. Other aspects and embodiments will be clear to those skilled in the art. All documents described herein are incorporated herein by reference in their entirety. Although the present application has been described in conjunction with the exemplary embodiments, many equivalent modifications and variations will be clear to those skilled in the art when the present application is given. Thus, the exemplary embodiments of the present application are exemplary and non-restrictive. Various changes may be made to the embodiments without departing from the purpose and scope of the present application.

[0164] Example

[0165] The following examples are given by way of illustration only and are not intended to limit the present invention in any way.

[0166] Example 1. Generation of anti-human CCR8 monoclonal antibodies (mAbs)

[0167] To produce antibodies against human CCR8, wild-type female mice including BALB / C, A / J or SJL were immunized with a variety of antigens, including DNA / mRNA encoding human CCR8 and cell lines overexpressing human CCR8. Specifically, the above antigens were used alone 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. Gold bullets coated with DNA plasmids (6 μg) were injected into the subcutaneous or abdominal cavity of mice three times every two weeks using the Helios gene gun system (Bio-Rad), and then CHO-K1 / hu-CCR8 cells were injected intraperitoneally (ip) (5×10 per mouse). 6 Alternatively, mice were injected three times every two weeks with 20 / 40 μg of mRNA LNP (TheraRNA).

[0168] At the same time, in order to evaluate the serum titer of immunized animals and screen / identify CCR8-specific antibodies, the human and monkey CCR8 encoding genes were synthesized, inserted into the pLVX-Puro vector, packaged by lentivirus, and infected with HEK293 cells. Subsequently, they were selected with puromycin to prepare human and monkey CCR8 overexpression 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.

[0169] After the serum titer of the immunized mice was evaluated, the mice with the highest human CCR8 antibody titer were selected, and their lymph nodes and / or spleens were taken to isolate lymph node cells and / or spleen cells, which were fused with myeloma cells to produce hybridomas. Specifically, the isolated spleen cells and / or lymphocytes were mixed with the mouse myeloma cell line SP2 / 0 in a ratio of 3:1 and then electrofused to form fused cells. The fused cells were then placed in a 96-well plate and incubated for two weeks in a selective medium containing hypoxanthine-aminopterin-thymidine (HAT) to produce hybridomas. Using HEK293 / hu-CCR8 cells, anti-human CCR8 antibodies were screened from the supernatant of each hybridoma well by flow cytometry. The positive hybridomas were then subcloned by limiting dilution to obtain single clones.

[0170] Flow cytometry was used to analyze the ability of the antibodies expressed in the monoclonal supernatant to bind to HEK293 cells overexpressing human / monkey CCR8. First, HEK293 cells overexpressing human / monkey CCR8, namely HEK293 / hu-CCR8 and HEK293 / cyno CCR8, were cultured and harvested and incubated with anti-CCR8 antibody monoclonal supernatant. The samples were then analyzed by flow cytometry (BD Canto II). BMS-986340 (prepared according to the disclosure of WO2021194942A1) and GS-1811 (prepared according to the disclosure of WO2021163064A2) were used as positive control antibodies, and unengineered parental HEK293 cells were used as negative control cells for FACS binding assays.

[0171] Initial screening results showed that antibodies produced by the monoclonal clones 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. Clones 125C1E6, 154G7C3, 117G9F9, and 224E9A3 were sequenced, and unique clones were selected for further development.

[0172] Example 2. Preparation of chimeric antibodies

[0173] The selected and sequenced mouse heavy and light chain variable region coding sequences were fused to the N-terminus of the human IgG1 heavy chain constant region and human kappa light chain constant region coding sequences, respectively, and inserted into the expression plasmid pTT5, downstream of the signal peptide, for secretory expression. The amino acid sequences of the heavy and light chain variable regions are listed in Table 1, and the amino acid sequences of the human IgG1 heavy chain constant region and human kappa light chain constant region are shown in SEQ ID NOs: 37 and 38, respectively.

[0174] Table 1. Heavy chain and light chain variable regions and CDR information of the monoclonal antibodies of this application

[0175] Human IgG1 heavy chain constant region:

[0176] Human kappa light chain constant region:

[0177] Plasmids encoding the heavy and light chains of each antibody were transfected into CHO-3E7 cells using PEImax 40,000 (Cat#: 24765-1, Polysciences, Inc.) 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 by chromatography on a protein A-Sepharose column. The purified antibodies were then stored in PBS for subsequent analytical verification.

[0178] Example 3. In vitro activity identification of chimeric antibodies

[0179] Flow cytometry detection of antibody binding to cells overexpressing human CCR8

[0180] To further study the binding ability and specificity of the antibody of the present application to human CCR8, 1×10 5 HEK293 / hu-CCR8 cells were incubated with 50 μl of the present anti-CCR8 chimeric antibody at 4°C in an ELISA plate for 1 hour, wherein each antibody was diluted 3-fold in the cell / antibody mixture, with a starting and ending concentration of 300 nM. Antibodies BMS-986340 and GS-1811 were used as positive controls, and hIgG1 was used as a negative control. After the ELISA plate was washed 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. The samples were then analyzed by flow cytometry, and an antibody-antigen binding curve was generated with the mean fluorescence intensity (geometric mean of fluorescence intensity) as the ordinate and the antibody concentration as the abscissa. The raw data were plotted using GraphPadPrism v6.02 software, and the EC values ​​were determined. 50 .

[0181] Among these anti-CCR8 chimeric antibodies, as shown in Figures 1A and 1B, the monoclonal antibodies of the present application, including 125C1E6, 154G7C3, 117G9F9 and 224E9A3, all bound to cells overexpressing human CCR8, with comparable binding affinity compared to the positive controls BMS-986340 and GS-1811.

[0182] Flow cytometry detection of antibody binding to cells overexpressing monkey CCR8

[0183] The cross-reactivity of the anti-CCR8 chimeric antibody with monkey CCR8 was further tested using the same protocol as above, except that HEK293 / cyno-CCR8 cells were used instead of HEK293 / hu-CCR8 cells.

[0184] As shown in FIG2 , the 224E9A3 antibody bound to monkey CCR8-expressing cells with a binding affinity comparable to that of BMS-986340, while GS-1811 did not show binding to monkey CCR8-expressing cells in FACS assay.

[0185] CCR8-CCL1 blocking assay by detecting CCR8-mediated calcium flux

[0186] The binding of the ligand CCL1 to the chemokine receptor CCR8 can induce CCR8-mediated calcium influx, so the blocking effect of anti-CCR8 antibodies on CCL1-CCR8 signaling can be determined by measuring cellular calcium flux.

[0187] Briefly, 20 μl of 7.5×10 5 CHO-K1 / hu-CCR8 cells were seeded at 400 μg / ml in a 384-well assay plate and cultured at 37°C for 16 hours. 10 μl of antibody was added to the assay plate, so that each antibody was diluted 5-fold in the cell / antibody mixture, starting and ending at a concentration of 1 μM, with a total of 8 concentration points. For 117G9F9, it was diluted 5-fold in the cell / antibody mixture, starting and ending at a concentration of 0.4 μM, with a total of 8 concentration points. The working dye solution in the Calcium 4 Detection Kit (Cat#: R7446, Molecular Devices) was incubated at 37°C in the dark for 1 hour. BMS-986340 and GS-1811 were used as positive controls. After adding 35 μl of 10 nM recombinant human CCL1 (GenScript, Z02911), the calcium flux fluorescence signal was measured using the FLIPR™ TETRA system (Molecular Devices), and the data were analyzed using GraphPad Prism to obtain the IC 50 value.

[0188] The results are shown in Figure 3. According to IC 50 The results show that compared with the positive control, the anti-CCR8 antibody molecules of the present application show better blocking activity on CCL1-CCR8 signaling.

[0189] Antibody-dependent cellular cytotoxicity (ADCC) induced by anti-CCR8 antibodies

[0190] A reporter gene-based ADCC bioassay was used to evaluate the ADCC-inducing activity of anti-CCR8 chimeric antibodies.

[0191] Briefly, CHO-K1 / hu-CCR8 target cells were cultured and harvested, and 1 × 10 4The cells were seeded into a 96-well plate at a density of 10 μg / well, where the cells were in 40 μl of culture medium. 20 μl of the present antibody or positive control (BMS-986340 and GS-1811) was added to the 96-well plate, so that each antibody was diluted 10-fold in the cell / antibody mixture, starting and ending at a concentration of 10 μg / ml (about 60 nM), with a total of 7 concentration points, and the plate was incubated at 37°C and 5% CO2 for 30 minutes. Then, 40 μl of 1.5×10 6 / ml GS-J2C / CD16A cells (ADCC reporter cell line, Cat#: RD00830, GenScript) as effector cells were incubated at 37°C, 5% CO2 for 6 hours. The activation of ADCC reporter cell lines was analyzed using an assay kit. Luminescence data were detected by PheraStar (BMG) for ADCC reporter gene activation signal analysis and analyzed using GraphPad Prism 6.0.

[0192] The results are shown in FIG4 . Compared with the positive control, the anti-CCR8 chimeric antibodies of the present application can induce CCR8 expression with comparable or higher activity. + ADCC of cells.

[0193] Example 4. Humanization of antibodies

[0194] For the humanization of 117G9F9, 125C1E6, and 154G7C3, these mouse antibodies were aligned with human Ig germline sequences to obtain the best overall match. The human germline sequences obtained with high homology to the mouse antibodies were used as acceptors, and the CDRs of the mouse antibodies were transplanted into the acceptor framework. The newly generated antibodies were then sequence analyzed to determine whether there were high-risk sites of potential post-translational modifications in the newly constructed sequences, including deamidation, isomerization, oxidation, and unpaired cysteine ​​residues that may affect the binding activity and stability of the antibodies.

[0195] Following the methods and steps described in Example 2, DNA sequences encoding the heavy and light chain variable regions of the humanized antibody were synthesized and fused to the N-termini of the coding sequences for the human IgG1 heavy chain constant region and the human kappa light chain constant region, respectively. The sequences were then inserted into the expression plasmid pTT5, downstream of the signal peptide, for secretory expression. The amino acid sequences of the heavy and light chain variable regions are listed in Table 2, and the amino acid sequences of the human IgG1 heavy chain constant region and the human kappa light chain constant region are shown in SEQ ID NOs: 37 and 38, respectively.

[0196] Table 2. Sequence information of humanized antibodies

[0197] Example 5. Activity identification of humanized antibodies

[0198] Flow cytometry was used to detect the binding of humanized antibodies to cells overexpressing human or monkey CCR8.

[0199] According to the method steps in Example 3, the binding ability and binding specificity of the humanized antibodies to cells overexpressing human or monkey CCR8 and HEK293 parental cells were tested.

[0200] Figure 5A shows the antibody-antigen binding curves of humanized antibodies to cells overexpressing human CCR8. It can be seen that the humanized antibodies of the present application all show 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, which is specifically manifested by higher span values ​​and / or smaller EC values. 50 value.

[0201] Figure 5B shows the antibody-antigen binding curves of humanized antibodies to cells overexpressing monkey CCR8. As can be seen, both 125C1E6-VH1.1-VL1 and 125C1E6-VH3-VL1 bound to cells expressing monkey CCR8 with much higher binding affinity than BMS-986340, while GS-1811 showed no detectable binding to monkey CCR8.

[0202] Figure 5C shows the antibody-antigen binding curves of the humanized antibodies to HEK293 cells. The two positive controls, BMS-986340 and GS-1811, showed weak binding to parental HEK293 cells, while 125C1E6-VH1.1-VL1 and 125C1E6-VH3-VL1 did not bind to HEK293 cells.

[0203] Antibody-dependent cellular cytotoxicity (ADCC) induced by humanized antibodies

[0204] The ADCC activity of the humanized antibody was evaluated by referring to the method steps in Example 3, wherein the humanized antibody and the positive control were diluted 10-fold in the cell / antibody mixture, with a starting and ending concentration of 10 μg / ml.

[0205] The results are shown in Figures 6A and 6B. All humanized anti-CCR8 antibodies were specific for CCR8. + The ADCC activity of the cells was comparable to that of the positive control.

[0206] CCR8-CCL1 blocking assay by detecting CCR8-mediated calcium flux

[0207] Referring to the method steps in Example 3, the blocking ability of humanized antibodies on CCL1-CCR8 signaling was determined by measuring cellular calcium flux, wherein each antibody was diluted 5-fold in the cell / antibody mixture, starting and ending at a concentration of 1 μM, for a total of 8 concentration points.

[0208] As shown in Figures 7A and 7B, 125C1E6-VH1.1-VL1, 125C1E6-VH3-VL1, and 154G7C3-VH1.1-VL2 all showed comparable or better in vitro CCR8-CCL1 blocking activity than the positive control GS-1811.

[0209] Antibody internalization analysis using the IncuCyte live cell analysis system

[0210] Antibody internalization was analyzed using the Incucyte Real-Time Live Cell System (Sartorius).

[0211] Briefly, 50 μl of 2 × 10 5 CHO-K1 / hu-CCR8 cells at 4 μg / ml were seeded in a 96-well assay plate and incubated at 37°C for 16 hours. Simultaneously, the antibodies were labeled prior to the internalization assay. Specifically, the humanized antibody and Incucyte Fabfluor pH dye were each diluted two-fold in complete growth medium (90% F12K medium + 10% fetal bovine serum) at a starting concentration of 4 μg / ml across eight concentration points. The diluted humanized antibody was then mixed with Incucyte Fabfluor pH dye in a 1:1 volume ratio and incubated in a 96-well round-bottom plate at 37°C in the dark for 15 minutes. Fifty μl of labeled humanized antibody was added to the 96-well assay plate and incubated at 37°C in an Incucyte S3 cell culture incubator for 24 hours. Antibody internalization rates were detected by measuring red fluorescence in CHO-K1 / hu-CCR8 cells. BMS-986340 and GS-1811 served as positive controls, and human IgG1 served as a negative control. Raw data were exported from Incucyte 2022B and analyzed using Incucyte 2022B, Microsoft Office Excel 2016, and GraphPad Prism 6. The overall fluorescence intensity (RCU × μm 2 ) and antibody concentrations.

[0212] As shown in Figure 8, both humanized antibodies 125C1E6-VH1.1-VL1 and 125C1E6-VH3-VL1 showed higher internalization activity than the positive control. In addition, humanized antibodies 154G7C3-VH1.1-VL2 and 117G9F9-VH2.1-VL1 showed internalization activity comparable to that of the positive control.

[0213] Example 6. Epitope competition binding assay

[0214] To determine whether the humanized antibodies bound to the same or similar epitope as the positive control, competition for binding of each antibody to HEK293 / hu-CCR8 cells was measured by FACS.

[0215] Briefly, 125C1E6-VH3-VL1 antibody containing hIgG1-Fc (i.e., SEQ ID NO: 37) at a final concentration of 0.45 nM was first mixed with 1×10 5 HEK293 / hu-CCR8 cells were incubated at 4°C for 0.2 hours in a 100 μl solution. Subsequently, 50 μl of mouse Fc-containing antibodies, including BMS-986340-mIgG2a (BMS-986340 heavy chain constant region replaced with the mouse IgG2a heavy chain constant region of SEQ ID NO: 39, and the light chain constant region replaced with SEQ ID NO: 40), GS-1811-mIgG2a (GS-1811-mIgG2a heavy chain constant region replaced with SEQ ID NO: 39, and the light chain constant region replaced with SEQ ID NO: 40), and 125C1E6-mIgG2c (purified from hybridoma supernatant), or mouse IgG1, were added. Each antibody was serially diluted 3-fold in the cell / antibody mixture, starting at a starting concentration of 300 nM, and incubated at 4°C for 0.8 hours. Then, goat anti-human IgG (H+L) labeled with fluorophore (iFluor 647) was used to detect 125C1E6-VH3-VL1 bound to the cell surface. The data were analyzed by GraphPad Prism 6.0. The results are shown in Figure 9A.

[0216] Similarly, epitope competition between the antibodies was further determined by adding BMS-986340-mIgG2a at a final concentration of 0.28 nM followed by the competing antibodies 125C1E6-VH3-VL1, BMS-986340, or human IgG1; or by adding GS-1811-mIgG2a at a final concentration of 0.28 nM followed by 125C1E6-VH3-VL1 or human IgG1. Other conditions were the same as above. Each competing antibody was serially diluted three-fold into the cell / antibody mixture, starting at a final concentration of 300 nM. Fluorophore (iFluor 647)-labeled goat anti-mouse IgG (H+L) was used to detect cell-surface-bound BMS-986340-mIgG2a or GS-1811-mIgG2a. The results are shown in Figures 9B and 9C, respectively.

[0217] mIgG2a heavy chain constant region:

[0218] Light chain constant region:

[0219] As shown in Figure 9A, both BMS-986340-mIgG2a and GS-1811-mIgG2a 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. This indicates that the epitope bound by the 125C1E6 antibody overlaps with the CCR8 epitope of the two positive controls.

[0220] Although the present application has been described in conjunction with one or more embodiments, it should be understood that the present application is not limited to these embodiments. The description in this application is intended to cover all variants and equivalents, all of which are included in the subject matter and scope of the appended claims. All documents cited in this article are incorporated herein by reference in their entirety.

Claims

1. An isolated monoclonal antibody or antigen-binding portion thereof, which is capable of specifically binding to CCR8, comprising: i) a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 respectively comprise 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, or comprise an amino acid sequence having 1 to 3 amino acid substitutions in each CDR compared to the above amino acid sequences; and / or ii) a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1, VL CDR2 and VL CDR3 respectively comprise 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, or comprise an amino acid sequence having 1-3 amino acid substitutions in each CDR compared with the above amino acid sequences.

2. The isolated monoclonal antibody or antigen-binding portion thereof of 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 respectively comprise 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 portion thereof of 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 to SEQ ID NO: 22, 24, 26, 27, 29, 31, 33, or 35.

4. The isolated monoclonal antibody or antigen binding portion thereof of any one of claims 1-3, 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 to SEQ ID NO:23, 25, 28, 30, 32, 34, or 36.

5. The isolated monoclonal antibody or antigen-binding portion thereof of 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 respectively comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (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. The isolated monoclonal antibody or antigen binding portion thereof 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 antigen binding portion thereof according to claim 6, wherein the heavy chain constant region is an IgG1 heavy chain constant region.

8. The isolated monoclonal antibody or antigen-binding portion thereof 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 antigen binding portion thereof according to claim 6, wherein the light chain constant region is a kappa light chain constant region.

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

38.

11. The isolated monoclonal antibody or antigen binding portion thereof according to any one of claims 1 to 10 which is murine, chimeric or humanized.

12. The isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 11, which i) is capable of binding to human CCR8, ii) is capable of blocking CCR8-CCL1 binding / interaction, iii) is capable of being regulated by CCR8 + Cell internalization, vi) can trigger the expression of CCR8 + The invention relates to antibody-dependent cell-mediated cytotoxicity of cells, and / or v) having an in vivo anti-tumor effect.

13. An immunoconjugate comprising i) the antibody or antigen binding portion thereof according to 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) the antibody or antigen binding portion thereof of any one of claims 1-12, and ii) a second functional group, wherein i) and ii) bind to different antigens or different epitopes of the same antigen, and i) and ii) are linked.

15. A chimeric antigen receptor comprising i) an extracellular antigen binding domain, ii) a transmembrane domain, and iii) an intracellular signal transduction domain, wherein the extracellular antigen binding domain comprises the antibody or antigen binding portion thereof of any one of claims 1-12. An immune cell comprising the chimeric antigen receptor according to claim 15 .

17. An oncolytic virus expressing the antibody or antigen-binding portion thereof according to any one of claims 1 to 12.

18. A nucleic acid molecule encoding the antibody or antigen binding portion thereof of any one of claims 1-12, the immunoconjugate of claim 13, the bispecific molecule of claim 14, or the chimeric antigen receptor of claim 15. An expression vector comprising the nucleic acid molecule of claim 18 .

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

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

22. The composition according to claim 21, which is a pharmaceutical composition, further comprising 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 preparation of a medicament 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 T-cell lymphoma.

28. A method for preparing CCR8 antibodies, comprising i) administering a DNA molecule capable of expressing CCR8 protein, an RNA molecule capable of expressing CCR8 protein, and / or a cell capable of overexpressing 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 hybridomas.

29. The method according to claim 28, wherein i) an RNA molecule capable of expressing CCR8 protein is administered to an animal, ii) lymph node cells and / or spleen cells of the animal are collected, and iii) the lymph node cells and / or spleen cells are fused with myeloma cells to produce hybridomas.

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