Anti-CCR8 antibodies and uses thereof

Abstract

This application relates to the field of biopharmaceuticals, and in particular to an antibody or antigen-binding fragment thereof that specifically binds to CCR8, as well as pharmaceutical compositions and kits containing the same. The antibody or antigen-binding fragment thereof has ADCC activity and / or ADCP activity and simultaneously exhibits excellent tumor-inhibiting activity. Therefore, the present invention further relates to the use of the antibody or antigen-binding fragment thereof of the present invention and / or for the treatment of tumors.

Description

[Technical Field]

[0001] This application relates to the field of biopharmaceuticals. In particular, this application relates to an antibody or antigen-binding fragment thereof capable of specifically binding to CCR8, and pharmaceutical compositions and kits containing the antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof has ADCC activity and / or ADCP activity and exhibits good tumor-inhibiting effects. Therefore, the present invention also relates to the use of the antibody or antigen-binding fragment thereof in the prevention and / or treatment of tumors. [Background technology]

[0002] In recent years, significant progress has been made in tumor immunotherapy, typified by anti-PD-1 / PD-L1 monoclonal antibodies. However, the overall response rate of this therapy in solid tumors is only 10% to 30% (Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science, 2018, 359(6382): 1350-1355). Therefore, the development of new immunotherapies to improve response rates and survival in tumor patients remains an unmet clinical need.

[0003] Several studies have demonstrated that large numbers of regulatory T cells (Tregs) infiltrate the tumor microenvironment and suppress immune responses through mechanisms such as competitive binding of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), which is highly expressed on the cell surface, to activation signals from CD80 and CD86, competitive binding of the high-affinity IL-2 receptor CD25 to IL-2, and release of immunosuppressive factors such as TGF-β and IL-10, thereby promoting tumor initiation and progression (Setoguchi R, Hori S, Takahashi T, et al. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med 2005, 201(5): 723-735). Therefore, many pharmaceutical companies and research institutes are focusing on developing therapies that deplete Tregs in the tumor microenvironment, with the hope of reducing immunosuppression and enhancing patients' immune responses to tumors. Among these, several monoclonal antibodies that separately target CD25, CCR4, and CTLA-4, which are highly expressed on Tregs, and exhibit antibody-dependent cell-mediated cytotoxicity (ADCC) have entered clinical studies. However, these antibodies have limited response rates in the treatment of solid tumors and face potential side effects caused by the depletion of peripheral Tregs.

[0004] Unlike CTLA-4, CD25, CCR4, and other immune checkpoint molecules that are highly expressed on T cells or peripheral Treg cells, chemokine (C-C motif) receptor 8 (CCR8) is specifically and highly expressed on tumor-infiltrating Treg cells (Plitas G, Konopacki C, Wu K, et al. Regulatory T cells exhibit distinct features in human breast cancer. Immunity, 2016, 45(5): 1122-1134). Monoclonal antibodies targeting CCR8 have the potential to specifically eliminate tumor-infiltrating Tregs while avoiding the killing of peripheral Tregs. Therefore, there is a need in the art to develop novel CCR8-targeting antibodies that can effectively and specifically eliminate tumor-infiltrating Tregs while avoiding the adverse side effects of peripheral Treg depletion, thereby making them effective in the treatment of diseases, particularly cancer. Summary of the Invention

[0005] After extensive research, the inventors of the present application screened and obtained monoclonal antibodies against CCR8, and then further engineered a series of modifications to the heavy chain constant region of the antibodies. The obtained antibodies possess ADCC and / or ADCP activity, and these antibodies have high binding affinity and good specificity for CCR8. Some of these antibodies have enhanced ADCC or ADCP activity, and some of these antibodies simultaneously enhance ADCC and ADCP activity. In animal models, administration of the antibodies of the present invention can significantly inhibit tumor growth. Based on this, the present application also provides compositions containing the antibodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies or antigen-binding fragments thereof, and host cells containing the same, as well as related uses.

[0006] Thus, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CCR8, wherein the antibody or antigen-binding fragment thereof is: (a) a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 1 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 2 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 3 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 5 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 6 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 7 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0007] In some embodiments, the substitutions set forth in any one of (i) through (vi) are conservative substitutions.

[0008] In some embodiments, the CDRs listed in any one of (i) through (vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

[0009] In some embodiments, the CDRs listed in any one of (i) through (vi) are defined by the IMGT numbering system.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: a VH CDR1 set forth in SEQ ID NO: 1, a VH CDR2 set forth in SEQ ID NO: 2, and a VH CDR3 set forth in SEQ ID NO: 3; and / or the following three light chain CDRs: a VL CDR1 set forth in SEQ ID NO: 5, a VL CDR2 set forth in SEQ ID NO: 6, and a VL CDR3 set forth in SEQ ID NO: 7.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof is (a) (i) the sequence set forth in SEQ ID NO: 4; (ii) a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions) compared to the sequence set forth in SEQ ID NO: 4; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:4; a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: and / or (b) (iv) the sequence set forth in SEQ ID NO:8; (v) a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions) compared to the sequence set forth in SEQ ID NO: 8; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:8; A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of Includes:

[0012] In some embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:4 and a VL having the sequence set forth in SEQ ID NO:8.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises a constant region derived from a mammalian immunoglobulin or variant thereof.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a heavy chain constant region (CH) of a mammalian immunoglobulin or variant thereof, wherein the variant has one or several amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or insertions, or any combination thereof; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions, or any combination thereof); and / or (b) a light chain constant region (CL) of a mammalian immunoglobulin or variant thereof, wherein the variant has one or several amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or insertions, or any combination thereof; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions, or any combination thereof). Includes:

[0016] In one embodiment, the mammal is selected from the group consisting of a mouse and a human.

[0017] In some embodiments, the heavy chain constant region is selected from the group consisting of IgG, IgM, IgE, IgD, and IgA.

[0018] In some embodiments, the heavy chain constant region is a murine or human IgG heavy chain constant region; for example, a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region; for example, a murine IgG1, IgG2a, IgG2b, IgG2c, or IgG3 heavy chain constant region.

[0019] In one embodiment, the heavy chain constant region is selected from the group consisting of a human IgG1 heavy chain constant region and a murine IgG2a heavy chain constant region.

[0020] In one embodiment, the variant is a variant of the human IgG1 heavy chain constant region.

[0021] In one embodiment, the variant is mutated to alanine at a position corresponding to position 119 of the human IgG1 heavy chain constant region.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof is hypofucosylated or afucosylated.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 9, 10, or 13.

[0024] In some embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0025] In one embodiment, the light chain constant region is a murine or human kappa light chain constant region.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO: 11 or 14.

[0027] In some embodiments, the antigen-binding fragment is a Fab, a Fab', a (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).

[0028] In some embodiments, the antibody or antigen-binding fragment thereof of the invention is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments (1) Has ADCC activity, such as inducing the killing of CCR8-expressing cells (e.g., tumor cells) via ADCC; in some embodiments, a hypofucosylated or defucosylated antibody or antigen-binding fragment thereof has stronger ADCC activity; in such embodiments, the antibody prepared by the engineered host cell (e.g., a fucose knockout CHO cell) is a hypofucosylated or defucosylated antibody; (2) has ADCP activity, such as inducing the killing of CCR8-expressing cells (e.g., tumor cells) via ADCP; in some embodiments, an antibody or antigen-binding fragment thereof comprising a mutated heavy chain constant region (e.g., as set forth in SEQ ID NO: 10) has stronger ADCP activity; and / or (3) inhibiting tumor growth; in some embodiments, the combination of the antibody or antigen-binding fragment thereof and an additional pharmaceutically active agent (e.g., an anti-PD-L1 / TGF-βRII fusion protein) has greater tumor growth inhibition activity. The composition further has a feature selected from the group consisting of:

[0030] In another aspect, the invention provides an isolated nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof.

[0031] In another aspect, the present invention provides a vector comprising the above-described nucleic acid molecule.

[0032] In some embodiments, the vector of the invention is selected from the group consisting of a plasmid, a cosmid, a phage, and a lentivirus. In some embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (e.g., a mammal, such as a human). In some embodiments, the vector is a cloning vector or an expression vector.

[0033] In another aspect, the present invention provides a host cell comprising the above-described nucleic acid molecule or the above-described vector.

[0034] In one embodiment, the host cell is a mammalian cell.

[0035] In some embodiments, the host cell is a fucose knockout or non-knockout cell.

[0036] In such embodiments, the antibody prepared by the engineered host cell (eg, a fucose knockout CHO cell) is a hypofucosylated or defucosylated antibody.

[0037] The host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., E. coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of the invention is a mammalian cell such as a CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0038] In another aspect, the present invention provides a method for producing the above-described antibody or antigen-binding fragment thereof, the method comprising culturing the above-described host cell under conditions capable of expressing the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell.

[0039] In another aspect, the present invention provides a multispecific molecule comprising the above-described antibody or antigen-binding fragment thereof.

[0040] In one embodiment, the multispecific molecule specifically binds to CCR8 and also specifically binds to one or more other targets.

[0041] In some embodiments, the multispecific molecule is a bispecific molecule.

[0042] In some embodiments, the bispecific molecule further comprises a molecule with a second binding specificity for a second target (eg, a second antibody).

[0043] In another aspect, the present invention provides an immunoconjugate comprising the above-described antibody, or antigen-binding fragment thereof, or the above-described multispecific molecule, and a therapeutic agent conjugated to said antibody, or antigen-binding fragment thereof, or said multispecific molecule.

[0044] In one embodiment, the therapeutic agent is selected from a cytotoxic agent.

[0045] In some embodiments, the therapeutic agent is selected from the group consisting of alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof.

[0046] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC).

[0047] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned multispecific molecule, or the above-mentioned immunoconjugate, and a pharmaceutically acceptable carrier and / or excipient.

[0048] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0049] In some embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an angiogenesis inhibitor, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof, multispecific molecule or immunoconjugate and the additional pharmaceutically active agent are provided as separate components or as components in the same composition.

[0051] In some embodiments, the additional pharmaceutically active agent is an anti-PD-L1 / TGF-βRII fusion protein, hi some embodiments, the anti-PD-L1 / TGF-βRII fusion protein has a heavy chain amino acid sequence set forth in SEQ ID NO:15 and a light chain amino acid sequence set forth in SEQ ID NO:16.

[0052] In another aspect, the present invention provides a kit comprising the above-described antibody or antigen-binding fragment thereof.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substrate (e.g., a chemiluminescent substrate), or biotin.

[0054] In some embodiments, the kit further comprises a secondary antibody capable of specifically recognizing the above-mentioned antibody or antigen-binding fragment thereof.

[0055] In some embodiments, the secondary antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substrate (e.g., a chemiluminescent substrate), or biotin.

[0056] In another aspect, the present invention provides a chimeric antigen receptor comprising the antigen-binding domain of the above-described antibody or antigen-binding fragment thereof.

[0057] In some embodiments, the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of the above-mentioned antibody or antigen-binding fragment thereof.

[0058] In some embodiments, the antigen binding domain is an scFv.

[0059] In some embodiments, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

[0060] In another aspect, the present invention provides a method for inhibiting the growth of and / or killing tumor cells that express CCR8, comprising contacting said tumor cells with an effective amount of the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned multispecific molecule, or the above-mentioned immunoconjugate, or the above-mentioned pharmaceutical composition, or the above-mentioned chimeric antigen receptor.

[0061] In another aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or an immunoconjugate as described above, or a pharmaceutical composition as described above, or a chimeric antigen receptor as described above, in the manufacture of a medicament for preventing and / or treating a tumor in a subject (e.g., a human).

[0062] In some embodiments, the medicament further comprises an additional pharmaceutically active agent.

[0063] In some embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an angiogenesis inhibitor, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0064] In some embodiments, the additional pharmaceutically active agent is an anti-PD-L1 / TGF-βRII fusion protein, which in some embodiments has a heavy chain amino acid sequence set forth in SEQ ID NO:15 and a light chain amino acid sequence set forth in SEQ ID NO:16.

[0065] In one embodiment, the tumor expresses CCR8.

[0066] In some embodiments, the tumor comprises tumor cells that express CCR8. In some embodiments, the CCR8 is expressed on the surface of the tumor cells.

[0067] In some embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, and T-cell / histiocyte-rich B-cell lymphoma. lymphoma), EBV-positive and -negative PTLD, other hematopoietic malignancies such as EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, and central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumor, and brainstem glioma.

[0068] In one embodiment, the subject is a mammal, such as a human.

[0069] In another aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described above in the manufacture of a kit for determining whether a tumor is treatable with a CCR8-targeted anti-tumor therapy, comprising: (1) contacting a sample containing cells of the tumor with the antibody or antigen-binding fragment thereof; (2) detecting the structure of a complex comprising the antibody or antigen-binding fragment thereof and CCR8; Provide use.

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label.

[0071] In some embodiments, the CCR8 is mammalian (eg, human, mouse) CCR8.

[0072] In some embodiments, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, and T-cell / histiocyte-rich B-cell lymphoma. lymphoma), EBV-positive and -negative PTLD, other hematopoietic malignancies such as EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, and central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumor, and brainstem glioma.

[0073] Definition of Terms

[0074] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the means of molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genetics and recombinant DNA manipulation used herein are conventional means widely used in the corresponding fields.At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0075] As used herein, the term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in binding the antibody to antigens but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can be further subdivided into highly variable regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the respective antigen-binding sites. The distribution of amino acids in each region or domain can be determined according to the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0076] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined by various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a particular antibody, one of skill in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0077] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined by various numbering systems known in the art. In some embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined by the Kabat, Chothia, or IMGT numbering system.

[0078] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0079] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0080] As used herein, the terms "monoclonal antibody," "McAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or antibody fragment derived from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have a high degree of specificity for a single epitope on an antigen. Polyclonal antibodies, in contrast to monoclonal antibodies, generally comprise at least two or more different antibodies, which generally recognize different epitopes on the antigen. Furthermore, the modification "monoclonal" is understood only to mean that the antibody is characterized by being obtained from a population of highly homologous antibodies, and is not intended to require that the antibody be prepared by any particular method.

[0081] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, including hybridoma technology (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, e.g., U.S. Patent Application No. 4,816,567), or phage antibody library technology (see, e.g., Clackson et al. Nature 352: 624-628, 1991 or Marks et al. J. Mol. Biol. 222: 581-597, 1991).

[0082] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, also referred to as an "antigen-binding portion," that retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen. See generally Chapter 7 of Fundamental Immunology (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabodies, single-domain antibodies, chimeric antibodies, linear antibodies, nanobodies (Domantis technology), probodies, and polypeptides comprising the minimal portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0083] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains." Here, a "full-length heavy chain" refers to a polypeptide chain consisting, from N- to C-terminal, of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; if the full-length antibody is an IgE isotype, it optionally also contains a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting, from N- to C-terminal, of a VH, CH1, HR, CH2, and CH3. A "full-length light chain" is a polypeptide chain consisting, from N- to C-terminal, of a light chain variable region (VL) and a light chain constant region (CL). Two pairs of full-length antibody chains are linked together by disulfide bonds between the CL and CH1 domains and between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention may be derived from a single species, such as human, or may be chimeric or humanized antibodies. The full-length antibody of the present invention comprises two antigen-binding sites, each formed by a pair of VH and VL, that specifically recognize / bind to the same antigen.

[0084] As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; and "F(ab') 2 The term "Fab' fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" refers to an F(ab') fragment consisting of an intact light chain and an Fd fragment of the heavy chain (consisting of the VH and CH1 domains). 2 It refers to a fragment obtained after reduction of the disulfide bond linking the two heavy chain fragments in the fragment.

[0085] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single antibody arm. An Fv fragment is generally considered the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to an antigen, although with lower affinity than the complete binding site.

[0086] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonding of the second and third constant regions of a first antibody heavy chain to the second and third constant regions of a second antibody heavy chain. The Fc fragment of an antibody has diverse functions but is not involved in antigen binding.

[0087] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL domain and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating amino acid sequence of GGGGS or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, although variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may exist between the VH and VL of the scFv. In certain embodiments of the present invention, scFv may form a dis-scFv, which refers to an antibody formed by linking two or more single scFvs in tandem.In certain embodiments of the invention, scFv may form (scFv)2, which refers to an antibody formed by linking two or more single scFvs in parallel.

[0088] As used herein, the term "single domain antibody (sdAb)" has the meaning generally understood by those skilled in the art and refers to an antibody fragment that is composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as a full-length antibody. Single domain antibodies are also called nanobodies.

[0089] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen.

[0090] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a particular antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques, or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as intact antibodies.

[0091] As used herein, unless the context clearly indicates otherwise, when the term "antibody" is used it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0092] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light and / or heavy chain is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass) and another portion of its light and / or heavy chain is derived from another antibody (which may be from the same or another species or belong to the same or another antibody class or subclass), but in any case still retains the ability to bind to a target antigen (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In some embodiments, the term "chimeric antibody" can include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody and the heavy and light chain constant regions of the antibody are derived from a second antibody.

[0093] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be inserted in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared between the sequences (i.e., percent identity = number of overlapping identical positions / total number of positions × 100%). In some embodiments, the two sequences are the same length.

[0094] The determination of percent identity between two sequences can also be carried out using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0095] As used herein, the term "variant" also refers to a polypeptide or peptide containing an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions, or insertions within the context of a polypeptide (including a polypeptide). In some cases, the term "variant" also refers to a modified polypeptide or peptide (i.e., by the covalent attachment of any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide can be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, a variant can have similar, identical, or improved function as the polypeptide or peptide from which it is derived.

[0096] As used herein, the term "specific binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which the antibody is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0097] As used herein, a detectable label of the present invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa750)), luminescent substrates (e.g., chemiluminescent substrates such as acridinium ester compounds, luminol, and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., Dynabeads (登録商標) ), colloidal gold, or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin that binds to avidin (e.g., streptavidin) modified with the above labels.

[0098] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. To express the genetic material elements contained in the vector in the host cell, the vector can be introduced into the host cell by transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication.

[0099] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK293 cell, or human cell.

[0100] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing that amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute a corresponding amino acid residue with another amino acid residue from the same family with the same side chain. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0101] The 20 conventional amino acids referenced herein are written according to conventional usage. See, for example, Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

[0102] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption retarders, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween 80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Osmotic agents include, but are not limited to, sugars, NaCl, and the like. Absorption delaying agents include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, and sorbic acid. Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or their degradation products (e.g., lactalbumin hydrolysate).In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable fluid (e.g., an aqueous or non-aqueous suspension or solution), which in certain exemplary embodiments is selected from the group consisting of water for injection (WFI), sterile water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0103] As used herein, the term "prevention" refers to a method performed to prevent or delay the onset of a disease or disorder or condition in a subject. As used herein, the term "treatment" refers to a method performed to obtain beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include (but are not limited to) alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., no further worsening), delaying or slowing the progression of disease, improvement or alleviation of the disease state, and alleviation of symptoms (partial or complete), whether detectable or undetectable. Additionally, "treatment" can refer to prolonging survival as compared to expected survival if not receiving treatment.

[0104] As used herein, the term "subject" refers to a mammal such as a human, a cynomolgus monkey, or a mouse.

[0105] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for the prevention of a disease refers to an amount sufficient to prevent, suppress, or delay the onset of the disease; an effective amount for the treatment of a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is within the capabilities of one skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the method of administration, and other concurrent treatments.

[0106] Beneficial effects of the invention

[0107] The present invention provides an antibody or antigen-binding fragment thereof that targets CCR8 and has high binding affinity and good specificity to CCR8. Furthermore, the heavy chain constant region has been modified to obtain a fully human antibody. It can specifically eliminate CCR8-positive cells by enhancing ADCC and ADCP activity while avoiding killing of CCR8-negative cells. Therefore, the antibody of the present invention can be used for various purposes, including, but not limited to, enhancing immune responses, inhibiting tumor growth, anti-infection, and detecting CCR8 protein. Furthermore, the fully human antibody of the present invention can be safely administered to human subjects without inducing an immune response. Therefore, the antibody of the present invention has great clinical value.

[0108] The embodiments of the present invention will be described in detail below in combination with drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to explain the present invention and do not limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art through the detailed description of the drawings and preferred embodiments below. [Brief explanation of the drawings]

[0109] [Figure 1]FIG. 1 shows the binding activity of the anti-CCR8 antibodies of the present application to human CCR8-overexpressing CHO cells (FIG. 1, panel A) and CHO cells (FIG. 1, panel B). [Figure 2] FIG. 2 shows the ADCC activity of the anti-CCR8 antibody of the present application based on CHO-huCCR8 / Jurkat-CD16a-NFAT luciferase reporter gene cells (FIG. 2, panel A) and CHO / Jurkat-CD16a-NFAT luciferase reporter gene cells (FIG. 2, panel B). [Figure 3] FIG. 3 shows the ADCP activity of the anti-CCR8 antibodies of the present invention based on CHO-huCCR8 / Jurkat-CD32a-NFAT luciferase reporter gene cells (FIG. 3, panel A) and CHO / Jurkat-CD32a-NFAT luciferase reporter gene cells (FIG. 3, panel B). [Figure 4] FIG. 4 shows the cell killing activity of the anti-CCR8 antibodies of the present invention based on CHO-huCCR8 / PBMC. [Figure 5] FIG. 5 shows the anti-tumor effect of the anti-CCR8 antibody of the present invention in a huCCR8 transgenic mouse CT-26 tumor model. [Figure 6] Figure 6 shows the synergistic anti-tumor effect of an anti-mouse CCR8 antibody of the invention in combination with an anti-PD-L1 / TGF-βRII fusion protein in a wild-type mouse CT-26 tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0110] Sequence information

[0111] Some sequence information relevant to the present invention is provided in Table 1 below.

[0112] Table 1: Sequence description [Table 1-1] [Table 1-2]

[0113] Specific Models for Implementing the Invention

[0114] The invention will now be described with reference to the following examples which are intended to illustrate, but not limit, the invention.

[0115] Unless otherwise specified, the experiments and methods described in the Examples were essentially carried out according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genetics, and recombinant DNA used in the present invention can be found in Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD. APPROACH (M.J. MacPherson, B.D. Hames, and G.R. Taylor, eds. (1995)); and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

[0116] Furthermore, if no particular conditions are specified in the examples, they were carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or equipment used without indicating the manufacturer are conventional products available commercially. Those skilled in the art will appreciate that the examples are illustrative and are not intended to limit the scope of the protection sought by the present invention. All known examples and other documents referred to herein are incorporated herein by reference in their entirety. [Example]

[0117] Example 1 Preparation of antibodies

[0118] 1.1 Screening for fully human anti-CCR8 antibodies using yeast display technology

[0119] Based on a yeast antibody display library (Adimab, see WO 2009036379, WO 2010105256, and WO 2012009568), multiple methods, including binding and enrichment of human CCR8-overexpressing cells, were used to screen and enrich for yeast cells capable of specifically binding to human CCR8. The yeast cells obtained by screening were induced with shaking at 30°C for 48 hours to secrete and express the target anti-CCR8 antibody (full-length IgG). After induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 minutes, and the supernatant was collected. The anti-CCR8 antibody in the supernatant was purified using protein A and eluted with an acetic acid solution at pH 2.0, and the anti-CCR8 antibody was collected.

[0120] 1.2 Antibody affinity maturation

[0121] To obtain anti-human CCR8 antibodies with higher affinity, the antibodies screened above were optimized by the following methods, including but not limited to: CDRH1 / CDRH2 mutation screening; VHmut mutation screening; CDRL1 / CDRL2 / CDRL3 mutation screening, and the like. The specific CDR1 to CDR3 sequences (encoded by the IMGT coding system) and VH and VL sequences of the screened antibodies are shown in Table 1.

[0122] 1.3 Antibody expression and purification

[0123] The heavy chain variable region of the ADI-68741 clone candidate (having the amino acid sequence set forth in SEQ ID NO: 4) was fused to the human IgG1 wild-type heavy chain constant region (having the amino acid sequence set forth in SEQ ID NO: 9), the human IgG1-GA modified heavy chain constant region (G1-GA) (having the amino acid sequence set forth in SEQ ID NO: 10), and the mouse IgG2a heavy chain constant region (having the amino acid sequence set forth in SEQ ID NO: 13), respectively, and cloned into the pcDNA3.1 vector. The light chain variable region (having the amino acid sequence set forth in SEQ ID NO: 8) was linked to the human immunoglobulin kappa light chain constant region (having the amino acid sequence set forth in SEQ ID NO: 11) and the mouse immunoglobulin kappa light chain constant region (having the amino acid sequence set forth in SEQ ID NO: 14), respectively, and cloned into the pcDNA3.1 vector.

[0124] The transient expression and purification procedures using CHO cells or fucose knockout CHO cells are as follows: The pcDNA3.1 vector carrying the antibody heavy and light chains was introduced into CHO cells or fucose knockout CHO cells using chemical transfection, and the cells were then cultured at 37°C and 8% CO for 7 days. The cell suspension was collected and centrifuged at 13,000 rpm for 20 minutes. The supernatant was collected and purified with protein A. The purity of the antibody was detected by SEC, and the endotoxin content was simultaneously controlled. Finally, five antibodies were obtained and named ADI-68741-G1 (the variable regions of the clone candidate were constructed with the human IgG1 wild-type heavy chain and the constant region of the human immunoglobulin kappa light chain), ADI-68741-GA (the variable regions of the clone candidate were constructed with the human IgG1-GA modified heavy chain and the constant region of the human immunoglobulin kappa light chain), ADI-68741-G1 (defucosylated) (the construction method was the same as ADI-68741-G1, except that a fucose knockout CHO cell expression system was used), ADI-68741-GA (defucosylated) (the construction method was the same as ADI-68741-GA, except that a fucose knockout CHO cell expression system was used), and ADI-68741-mG2a (the variable regions of the clone candidate were constructed with the mouse IgG2a heavy chain and the constant region of the mouse immunoglobulin kappa light chain).

[0125] Example 2 Binding activity of anti-CCR8 antibodies to human CCR8-overexpressing CHO cells or empty CHO cells

[0126] Specifically, human CCR8-overexpressing CHO cells (designated CHO-huCCR8 cells) were generated through pressure screening by transfection of the pCHO1.0 vector (purchased from Invitrogen) containing human CCR8 cDNA (having the amino acid sequence set forth in SEQ ID NO: 12) into the MCS. The expanded CHO-huCCR8 cells or empty CHO cells were adjusted to the appropriate cell density and added to a 96-well flow plate. After centrifugation, serially diluted test samples were added and incubated at 4°C for 30 minutes, followed by washing twice with PBS. The corresponding fluorescent secondary antibody diluted to the appropriate concentration was added, incubated at 4°C for 30 minutes, and washed twice with PBS. The cells were resuspended in PBS and detected using a CytoFlex flow cytometer. The corresponding MFI was calculated. Graphs were generated using Graphpad software, and the results are shown in Figure 1. The anti-CCR8 antibodies of the present invention, ADI-68741-G1, ADI-68741-GA, ADI-68741-G1 (defucosylated), and ADI-68741-GA (defucosylated), can all specifically bind to CCR8 expressed on CHO cells, and their EC 50 The binding activity values ​​for β-lactamase were 1.65 nM, 1.21 nM, 2.41 nM, and 2.30 nM, respectively, whereas there was no binding activity to empty CHO cells.

[0127] Example 3 Detection of in vitro ADCC activity of anti-CCR8 antibodies

[0128] Based on the luciferase reporter gene system, the in vitro ADCC activity of the anti-CCR8 antibody of the present invention was detected.

[0129] Specifically, Jurkat-CD16a-NFAT-Luciferase-ADCC effector cells (purchased from Promega) were expanded to 4 × 10 cells / well in 1640 medium containing 10% low-IgG FBS. 6CHO-huCCR8 cells and empty CHO cells were resuspended in 1640 medium containing 10% low IgG FBS to a concentration of 0.8 × 10 6 The cells were diluted to 1000 cells / mL. The two cell suspensions were mixed thoroughly with Jurkat-CD16a-NFAT-Luciferase-ADCC effector cells at a 1:1 ratio, and 50 μL of each was added to a sterile 96-well white-bottom plate. Serially diluted antibody samples to be tested in 1640 medium containing 10% low IgG FBS were then added and incubated at 37°C and 5% CO2 for 6 hours. After incubation, the cells were removed and equilibrated at room temperature for 5 minutes. Bio-Glo™ reagent was added at 100 μL / well, and luminescence signals were read using a multifunctional microplate reader. The results are shown in Figure 2. The results indicated that the anti-CCR8 antibody of the present invention exhibited ADCC activity only in CHO-huCCR8 cells, and not in the defucosylated antibody ADI-68741-G1 (defucosylated) (EC 50 <0.01 nM) and ADI-68741-GA (defucosylated) (EC 50 =0.04nM) is ADI-68741-G1 (EC 50 =0.57nM) and ADI-68741-GA (EC 50 = 0.85 nM).

[0130] Example 4 Detection of in vitro ADCP activity of anti-CCR8 antibodies

[0131] Based on the luciferase reporter gene system, the in vitro ADCP activity of the anti-CCR8 antibody of the present invention was detected.

[0132] Specifically, Jurkat-CD32a-NFAT-Luciferase-ADCP effector cells (purchased from Rhino Bio) were expanded to 4 × 10 cells / mL in 1640 medium. 6 CHO-huCCR8 cells and empty CHO cells were resuspended in 1640 medium to 3.2 × 10 cells / mL. 6The cells were diluted to 1000 cells / mL. The two cell suspensions were mixed thoroughly with Jurkat-CD32a-NFAT-Luciferase-ADCP effector cells at a 1:1 ratio, and 50 μL of each was added to a sterile 96-well white-bottom plate. The antibody samples to be tested, serially diluted with 1640 medium, were then added and incubated at 37°C and 5% CO2 for 6 hours. After incubation, the cells were removed and equilibrated at room temperature for 5 minutes. Bio-Glo™ reagent was added at 100 μL / well, and the luminescence signal was read using a multifunctional microplate reader. The results are shown in Figure 3. The results indicated that the anti-CCR8 antibody of the present invention exhibited ADCP activity only in CHO-huCCR8 cells, and that the Fc-terminal GA-engineered antibody, ADI-68741-GA (EC 50 =0.56 nM) and ADI-68741-GA (defucosylated) (EC 50 =2.24nM) is ADI-68741-G1 (EC 50 = 4.90 nM), demonstrating stronger ADCP activity than ADI-68741-G1 (defucosylated) (not fitted).

[0133] Example 5 Detection of anti-CCR8 antibodies in PBMC cellular in vitro killing activity

[0134] Based on the PBMC killing system, the in vitro killing activity of the anti-CCR8 antibodies of the present invention was detected.

[0135] Specifically, human PBMC cells were recovered and cultured overnight to remove monocytes and allow them to adhere to the wall. PBMC cells were harvested on day 2 and plated in 1 × 10 wells in a black, clear-bottom 96-well cell culture plate. 5 CHOS-huCCR8 target cells were stained using the CellTraceViolet kit and added at a cell density of 1 × 10 4The antibody was adjusted to 1000 cells / well and seeded into the above-mentioned black, clear-bottomed 96-well cell culture plate. Serially diluted antibodies to be tested were then added to the cell wells and incubated for 48 hours. After 48 hours, DAPI fluorescent signals were collected using Cytation 5, and the corresponding killing activity was calculated. The results are shown in Figure 4. The results show that the anti-CCR8 antibody of the present invention exhibited in vitro PBMC cellular ADCC killing activity, and the defucosylated antibody ADI-68741-G1 (defucosylated) (EC 50 =0.0021 nM) and ADI-68741-GA (defucosylated) (EC 50 =0.0027nM) is ADI-68741-G1 (EC 50 =0.0213nM) and ADI-68741-GA (EC 50 = 0.0433 nM) showed stronger PBMC killing activity than IgG4.

[0136] Example 6 In vivo Pharmacodynamic Study of Anti-CCR8 Antibodies in huCCR8 KI Mice

[0137] In this experiment, to determine the anti-tumor effect of the anti-CCR8 antibody of the present invention, huCCR8 KI mice (purchased from Shanghai Model Organisms Center, Inc.) were inoculated in vivo with CT-26 colon cancer cells (purchased from Jiangsu GemPharmatech Co., Ltd.).

[0138] Specifically, we first established a mouse model bearing CT-26 cell tumors by subcutaneous inoculation. The average tumor volume was 80 mm 3 When the tumor size reached 100 mg / kg, the mice were divided into groups. The ADI-68741-mG2a antibody of the present invention was injected intraperitoneally (10 mg / kg) for treatment. The changes in tumor volume and body weight of each group of mice were monitored. The monitoring frequency was once every 2 to 3 days, and the monitoring continued for 2 to 3 weeks. The results are shown in Figure 5. The results showed that the anti-CCR8 antibody ADI-68741-mG2a of the present invention could significantly inhibit the growth of mouse tumors.

[0139] Example 7 Synergistic Pharmacodynamic Study of Anti-CCR8 Antibody and Anti-PD-L1 / TGF-βRII Fusion Protein in Wild-Type Balb / c Mice

[0140] To explore the possibility of combining anti-CCR8 antibodies with CCR8 antibodies for future clinical applications, the present invention determined the synergistic anti-tumor effect of anti-CCR8 antibodies and anti-PD-L1 / TGF-βRII fusion protein (having a heavy chain amino acid sequence set forth in SEQ ID NO: 15 and a light chain amino acid sequence set forth in SEQ ID NO: 16) using CT-26 tumor cells inoculated into wild-type Balb / c mice.

[0141] Specifically, we first established a mouse model bearing CT-26 cell tumors by subcutaneous inoculation. The average tumor volume was 150 mm 3 When tumor size reached 100 mg / kg / day, the mice were divided into groups and treated with anti-CCR8 antibody and / or anti-PD-L1 / TGF-βRII fusion protein via intraperitoneal injection. The changes in tumor volume and body weight of mice in each group were monitored. The monitoring frequency was once every 2 to 3 days and continued for 2 to 3 weeks. The dose and route of administration are shown in Figure 2. The results are shown in Figure 6. The results showed that the tumor-inhibiting activity of the group treated with anti-CCR8 antibody in combination with anti-PD-L1 / TGF-βRII fusion protein was significantly better than that of the corresponding two single-drug treatment groups. This suggests that the combination of CCR8 antibody and anti-PD-L1 / TGF-βRII fusion protein has potential for future clinical development.

[0142] Table 2: Dosage regimens for anti-CCR8 antibodies and anti-PD-L1 / TGF-βRII fusion proteins [Table 2]

[0143] Although the specific use of the present invention has been described in detail, it is understood that those skilled in the art can make various modifications and changes based on all known teachings, and these modifications are within the scope of protection of the present invention. All aspects of the present invention are provided by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CCR8, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 1 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 2 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 3 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; and / or (b) a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 5 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 6 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 7 or a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., one, two, or three amino acid substitutions, deletions, or insertions) compared thereto; Including, Preferably, the substitutions set forth in any one of (i) to (vi) are conservative substitutions; Preferably, the CDRs set forth in any one of (i) to (vi) are defined according to the Kabat, IMGT, or Chothia numbering system; Preferably, the CDRs set forth in any one of (i) to (vi) are defined by the IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: VH CDR1 set forth in SEQ ID NO: 1, VH CDR2 set forth in SEQ ID NO: 2, VH CDR3 set forth in SEQ ID NO: 3, and / or the following three light chain CDRs: VL CDR1 set forth in SEQ ID NO: 5, VL CDR2 set forth in SEQ ID NO: 6, VL CDR3 set forth in SEQ ID NO: 7, An antibody or antigen-binding fragment thereof.

2. the antibody or antigen-binding fragment thereof (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 4; (ii) a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions) compared to the sequence set forth in SEQ ID NO: 4; and (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:4; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence set forth in SEQ ID NO: 8; (v) a sequence having one or several amino acid substitutions, deletions, or insertions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions) compared to the sequence set forth in SEQ ID NO: 8; and (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:8; 2. The antibody or antigen-binding fragment thereof of claim 1, comprising: Preferably, the substitutions described in (ii) or (v) are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:4 and a VL having the sequence set forth in SEQ ID NO:

8. The antibody or antigen-binding fragment thereof of claim 1.

3. 3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a constant region derived from a mammalian immunoglobulin or variant thereof, Preferably, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain constant region (CH) of a mammalian immunoglobulin or variant thereof, wherein the variant has one or several amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or insertions, or any combination thereof; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions, or any combination thereof); and / or (b) a light chain constant region (CL) of a mammalian immunoglobulin or variant thereof, wherein the variant has one or several amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or insertions, or any combination thereof; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions, or any combination thereof). Including, Preferably, the mammal is selected from a mouse or a human. An antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. 4. The antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain constant region is selected from IgG, IgM, IgE, IgD, or IgA; Preferably, the heavy chain constant region is a human or mouse IgG heavy chain constant region, e.g., a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region; e.g., a mouse IgG1, IgG2a, IgG2b, IgG2c, or IgG3 heavy chain constant region; Preferably, the heavy chain constant region is selected from the group consisting of a human IgG1 heavy chain constant region and a mouse IgG2a heavy chain constant region; Preferably, the variant is a variant of a human IgG1 heavy chain constant region; Preferably, the variant is mutated to alanine at a position corresponding to position 119 of the human IgG1 heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof is hypofucosylated or defucosylated; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 9, 10, or 13; Preferably, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region; Preferably, the light chain constant region is a mouse or human kappa light chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO: 11 or 14. The antibody or antigen-binding fragment thereof according to claim 3.

5. The antigen-binding fragment may be Fab, Fab', (Fab') ２ 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody is selected from the group consisting of a Fv, a disulfide-linked Fv, a scFv, a diabody, and a single domain antibody (sdAb); and / or the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody.

6. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule of claim 6; preferably said vector is a cloning vector or an expression vector.

8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7, Preferably, the host cell is a mammalian cell; Preferably, the host cell is a fucose knockout or non-knockout cell.

9. A method for producing an antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, comprising culturing a host cell described in claim 8 under conditions capable of expressing the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from a culture of the cultured host cell.

10. 10. An immunoconjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a therapeutic agent conjugated to said antibody or antigen-binding fragment thereof or multispecific molecule; Preferably, the therapeutic agent is selected from a cytotoxic agent; Preferably, the therapeutic agent is selected from the group consisting of alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof; Preferably, said immunoconjugate is an antibody-drug conjugate (ADC).

11. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or an immunoconjugate according to claim 10 and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active substance is a drug with antitumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an angiogenesis inhibitor, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus; Preferably, said antibody or antigen-binding fragment thereof or said immunoconjugate and said additional pharmaceutically active agent are provided as separate components or as components in the same composition; Preferably, the pharmaceutical composition wherein said additional pharmaceutically active agent is an anti-PD-L1 / TGF-βRII fusion protein.

12. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5; Preferably, the antibody or antigen-binding fragment thereof comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substrate (e.g., a chemiluminescent substrate), or biotin; Preferably, the kit further comprises a secondary antibody capable of specifically recognizing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Preferably, the kit wherein the secondary antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substrate (e.g., a chemiluminescent substrate), or biotin.

13. A chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Preferably, the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Preferably, the antigen-binding domain is an scFv; Preferably, said chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

14. 14. A method for inhibiting the growth of and / or killing tumor cells that express CCR8, comprising contacting the tumor cells with an effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, or an immunoconjugate described in claim 10, or a pharmaceutical composition described in claim 11, or a chimeric antigen receptor described in claim 13.

15. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or an immunoconjugate according to claim 10, or a pharmaceutical composition according to claim 11, or a chimeric antigen receptor according to claim 13 in the manufacture of a medicament for preventing and / or treating a tumor in a subject (e.g., a human); Preferably, the medicament further comprises an additional pharmaceutically active substance; Preferably, the additional pharmaceutically active substance is a drug with antitumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an angiogenesis inhibitor, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor, or an oncolytic virus; Preferably, the additional pharmaceutically active agent is an anti-PD-L1 / TGF-βRII fusion protein; Preferably, the tumor expresses CCR8; Preferably, the tumor comprises tumor cells that express CCR8; preferably, the CCR8 is expressed on the surface of the tumor cells; Preferably, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma (T-cell / histiocyte-rich B-cell lymphoma). lymphoma), EBV-positive and -negative PTLD, other hematopoietic malignancies such as EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, and central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumor, and brainstem glioma; Preferably, the subject is a mammal such as a human.

16. 10. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the manufacture of a kit for determining whether a tumor is treatable with a CCR8-targeted anti-tumor therapy, comprising: (1) contacting a sample containing cells of the tumor with the antibody or antigen-binding fragment thereof of any one of claims 1 to 5; (2) detecting the structure of a complex comprising the antibody or antigen-binding fragment thereof and CCR8; Preferably, the antibody or antigen-binding fragment thereof comprises a detectable label; Preferably, the CCR8 is mammalian (e.g., human, mouse) CCR8; Preferably, the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma (T-cell / histiocyte-rich B-cell lymphoma). lymphoma), EBV-positive and -negative PTLD, other hematopoietic malignancies such as EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, and central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumor, and brainstem glioma.

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