Anti-CCR8 antibodies and uses thereof

Abstract

The invention relates to the field of biological medicine, in particular to an antibody specifically binding to CCR8 or an antigen binding fragment of the antibody, and a pharmaceutical composition and a kit containing the antibody or the antigen binding fragment. The antibody or the antigen binding fragment thereof has ADCC activity and / or ADCP activity, and also has a good tumor inhibition effect. Therefore, the invention further relates to the use of the antibody or the antigen binding fragment thereof in the prevention and / or treatment of tumors.

Description

Anti-CCR8 antibodies and uses thereof Technical Field

[0001] This application relates to the field of biomedicine. Specifically, it relates to antibodies or antigen-binding fragments thereof that specifically bind to CCR8, as well as pharmaceutical compositions and kits comprising such antibodies or antigen-binding fragments. Such antibodies or antigen-binding fragments exhibit ADCC and / or ADCP activity and exhibit potent tumor-suppressing effects. Therefore, the present invention further relates to the use of such antibodies or antigen-binding fragments in the prevention and / or treatment of tumors. Background Art

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

[0003] Several studies have shown that there are a large number of regulatory T cells (Treg) infiltrating in the tumor microenvironment. They inhibit the immune response through mechanisms such as competitive binding of CD80 and CD86 activation signals by cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) highly expressed on the cell surface, competitive binding of IL-2 by the high-affinity IL-2 receptor CD25, and release of immunosuppressive factors such as TGF-β and IL-10, thereby promoting tumor occurrence and development (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 institutions are committed to developing therapies to eliminate Treg in the tumor microenvironment, hoping to relieve immunosuppression and increase the patient's immune response to tumors. Among them, several monoclonal antibodies targeting CD25, CCR4, and CTLA-4, which are highly expressed by Tregs, and exhibiting antibody-dependent cell-mediated cytotoxicity (ADCC) have entered clinical research. However, these antibodies have limited response rates in the treatment of solid tumors and face potential side effects from depleting peripheral Tregs.

[0004] Unlike immune checkpoint molecules such as CTLA-4, CD25, and CCR4, which are highly expressed on T cells or peripheral Treg cells, chemokine receptor 8 (CC motif) receptor 8 (CCR8) is specifically 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 and avoid killing peripheral Tregs. Therefore, there is a need in the art to develop new antibodies targeting CCR8 that can efficiently and specifically eliminate tumor-infiltrating Tregs while avoiding the toxic side effects of eliminating peripheral Tregs for disease treatment, especially cancer treatment.

[0005] Summary of the Invention

[0006] After extensive research, the inventors of this application screened and obtained a monoclonal antibody against CCR8. Furthermore, a series of engineering modifications were performed on the heavy chain constant region of the antibody. The obtained antibodies have ADCC and / or ADCP activity, and these antibodies have high binding affinity and good specificity to CCR8. Some antibodies have enhanced ADCC or ADCP activity, and some antibodies have the activity of simultaneously enhancing ADCC and ADCP. In animal models, administration of the antibodies of the present invention can significantly inhibit tumor growth. Based on this, the application also provides a composition containing the antibody or its antigen-binding fragment, a nucleic acid encoding the antibody or its antigen-binding fragment, a host cell containing the same, and related uses.

[0007] Therefore, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CCR8, wherein the antibody or antigen-binding fragment thereof comprises:

[0008] (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):

[0009] (i) a VH CDR1 consisting of SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

[0010] (ii) a VH CDR2 consisting of SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, and

[0011] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0012] and / or,

[0013] (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):

[0014] (iv) a VL CDR1 consisting of SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

[0015] (v) VL CDR2 consisting of SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and (vi) VL CDR3 consisting of SEQ ID NO: 7, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

[0016] In certain embodiments, the substitution of any one of (i)-(vi) is a conservative substitution.

[0017] In certain embodiments, the CDRs described in any of (i)-(vi) are defined according to the Kabat, IMGT, or Chothia numbering systems.

[0018] In certain embodiments, the CDRs of any of (i)-(vi) are defined according to the IMGT numbering system.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 1, VH CDR2 as shown in SEQ ID NO: 2, VH CDR3 as shown in SEQ ID NO: 3; and / or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 5, VL CDR2 as shown in SEQ ID NO: 6, VL CDR3 as shown in SEQ ID NO: 7.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0021] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0022] (i) the sequence shown in SEQ ID NO: 4;

[0023] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 4; or

[0024] (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;

[0025] and / or

[0026] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0027] (iv) the sequence shown in SEQ ID NO: 8;

[0028] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 8; or

[0029] (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.

[0030] In certain embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH having the sequence shown in SEQ ID NO:4 and a VL having the sequence shown in SEQ ID NO:8.

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a constant region derived from a mammalian immunoglobulin or a variant thereof.

[0033] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0034] (a) a heavy chain constant region (CH) of a mammalian immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions, 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 additions, or any combination thereof; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions, or any combination thereof); and / or

[0035] (b) a light chain constant region (CL) of a mammalian immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions, 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 additions, or any combination thereof; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions, or any combination thereof).

[0036] In certain embodiments, the mammal is selected from a mouse or a human.

[0037] In certain embodiments, the heavy chain constant region is selected from IgG, IgM, IgE, IgD, or IgA.

[0038] In certain embodiments, the heavy chain constant region is a mouse or human 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.

[0039] In certain embodiments, the heavy chain constant region is selected from a human IgG1 heavy chain constant region and a mouse IgG2a heavy chain constant region.

[0040] In certain embodiments, the variant is a variant of the human IgG1 heavy chain constant region.

[0041] In certain embodiments, the variant is mutated to alanine at position corresponding to position 119 of the human IgG1 heavy chain constant region.

[0042] In certain embodiments, the antibody or antigen-binding fragment thereof is hypofucosylated or afucosylated.

[0043] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO: 9, 10 or 13.

[0044] In certain embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0045] In certain embodiments, the light chain constant region is a mouse or human kappa light chain constant region.

[0046] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) shown in SEQ ID NO: 11 or 14.

[0047] In certain embodiments, the antigen binding fragment is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb).

[0048] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are murine antibodies, chimeric antibodies, humanized antibodies, or multispecific antibodies.

[0049] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments further has a feature selected from the following:

[0050] (1) having ADCC activity, for example, inducing killing of cells expressing CCR8 (e.g., tumor cells) through ADCC; in certain embodiments, the antibody or antigen-binding fragment thereof that is hypofucosylated or afucosylated has stronger ADCC activity; in such embodiments, the antibody produced by a modified host cell (e.g., a fucose-knockout CHO cell) is a hypofucosylated or afucosylated antibody;

[0051] (2) having ADCP activity, for example, inducing killing of cells expressing CCR8 (e.g., tumor cells) through ADCP; in certain embodiments, the antibody or antigen-binding fragment thereof comprising a mutated heavy chain constant region (e.g., as shown in SEQ ID NO: 10) has stronger ADCP activity; and / or,

[0052] (3) Inhibiting tumor growth. In certain embodiments, the antibody or antigen-binding fragment thereof is used in combination with other pharmaceutically active agents (e.g., anti-PD-L1 / TGF-βRⅡ fusion protein) to have a stronger ability to inhibit tumor growth.

[0053] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described above.

[0054] In another aspect, the present invention provides a vector comprising the nucleic acid molecule as described above.

[0055] In certain embodiments, the vector of the present invention is selected from a plasmid, a cosmid, a phage, or a lentivirus. In certain embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., a mammal, such as a human). In certain embodiments, the vector is a cloning vector or an expression vector.

[0056] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned vector.

[0057] In certain embodiments, the host cell is a mammalian cell.

[0058] In certain embodiments, the host cell is a fucose knockout or non-knockout cell.

[0059] In such embodiments, the antibody produced by the engineered host cells (eg, fucose knockout CHO cells) is a hypofucosylated or afucosylated antibody.

[0060] 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., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NSO 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 certain embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0061] On the other hand, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof as described above, which comprises culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0062] In another aspect, the present invention provides a multispecific molecule comprising the above-described antibody or antigen-binding fragment thereof.

[0063] In certain embodiments, the multispecific molecule specifically binds CCR8 and additionally specifically binds one or more other targets.

[0064] In certain embodiments, the multispecific molecule is a bispecific molecule.

[0065] In certain embodiments, the bispecific molecule further comprises a molecule with a second binding specificity for a second target (eg, a second antibody).

[0066] In another aspect, the present invention provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof or the multispecific molecule as described above, and a therapeutic agent linked to the antibody or antigen-binding fragment thereof or the multispecific molecule.

[0067] In certain embodiments, the therapeutic agent is selected from a cytotoxic agent.

[0068] In certain embodiments, the therapeutic agent is selected from the group consisting of an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, and any combination thereof.

[0069] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0070] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described above, or the multispecific molecule as described above, or the immunoconjugate as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0071] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0072] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0073] In certain 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 of the same composition.

[0074] In certain embodiments, the additional pharmaceutically active agent is an anti-PD-L1 / TGF-βRII fusion protein. In certain embodiments, the anti-PD-L1 / TGF-βRII fusion protein has a heavy chain amino acid sequence as shown in SEQ ID NO: 15 and a light chain amino acid sequence as shown in SEQ ID NO: 16.

[0075] In another aspect, the present invention provides a kit comprising the above-mentioned antibody or antigen-binding fragment thereof.

[0076] In certain embodiments, the antibody or antigen-binding fragment thereof is detectably labeled, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.

[0077] In certain embodiments, the kit further comprises a second antibody that specifically recognizes the aforementioned antibody or antigen-binding fragment thereof.

[0078] In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.

[0079] In another aspect, the present invention provides a chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof as described above.

[0080] In certain embodiments, the antigen binding domain comprises the heavy chain variable region and the light chain variable region of the antibody or antigen binding fragment thereof as described above.

[0081] In certain embodiments, the antigen binding domain is a scFv.

[0082] In certain embodiments, the chimeric antigen receptor is expressed by an immune effector cell (eg, a T cell).

[0083] On the other hand, the present invention provides a method for inhibiting the growth of tumor cells expressing CCR8 and / or killing the tumor cells, which comprises contacting the tumor cells with an effective amount of the antibody or antigen-binding fragment thereof as described above, or the multispecific molecule as described above, or the immunoconjugate as described above, or the pharmaceutical composition as described above, or the chimeric antigen receptor as described above.

[0084] In another aspect, the present invention provides a use of the antibody or antigen-binding fragment thereof as described above, or the multispecific molecule as described above, or the immunoconjugate as described above, or the pharmaceutical composition as described above, or the chimeric antigen receptor as described above, in the preparation of a medicament for preventing and / or treating tumors in a subject (e.g., a human).

[0085] In certain embodiments, the medicament further comprises an additional pharmaceutically active agent.

[0086] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0087] In certain embodiments, the additional pharmaceutically active agent is an anti-PD-L1 / TGF-βRII fusion protein. In certain embodiments, the anti-PD-L1 / TGF-βRII fusion protein has a heavy chain amino acid sequence as shown in SEQ ID NO: 15 and a light chain amino acid sequence as shown in SEQ ID NO: 16.

[0088] In certain embodiments, the tumor expresses CCR8.

[0089] In certain embodiments, the tumor comprises tumor cells expressing CCR8. In certain embodiments, the CCR8 is expressed on the surface of the tumor cells.

[0090] In certain embodiments, the tumor is selected from 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 ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

[0091] In certain embodiments, the subject is a mammal, such as a human.

[0092] In another aspect, the present invention provides a use of the aforementioned antibody or antigen-binding fragment thereof in preparing a kit for detecting whether a tumor can be treated by an anti-tumor therapy targeting CCR8;

[0093] (1) contacting a sample containing the tumor cells with the antibody or antigen-binding fragment thereof as described above;

[0094] (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and CCR8.

[0095] In certain embodiments, the antibody or antigen-binding fragment thereof is detectably labeled.

[0096] In certain embodiments, the CCR8 is mammalian (eg, human, mouse) CCR8.

[0097] In certain embodiments, the tumor is selected from 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 ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

[0098] Definition of terms

[0099] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0100] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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 domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of 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 (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (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 in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site. The distribution of amino acids among regions or domains can follow 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.

[0101] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of each heavy chain and light chain contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to 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 given antibody, a person skilled 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 (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0102] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.

[0103] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0104] 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. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0105] As used herein, the terms "monoclonal antibody," "single antibody," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or an antibody fragment from a population of highly homologous antibody molecules, that is, a population of identical antibody molecules except for possible spontaneous natural mutations. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies, and these different antibodies typically recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates the characteristic of the antibody as being obtained from a population of highly homologous antibodies, and is not to be understood as requiring the antibody to be prepared by any particular method.

[0106] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, e.g., U.S. patent application 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).

[0107] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is 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 (technology from Domantis), probodies, and polypeptides that comprise at least a 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.

[0108] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that, in the direction from N-terminus to C-terminus, consists 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; and, when the full-length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the direction from N-terminus to C-terminus. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be derived from a single species, such as humans; they can also be chimeric antibodies or humanized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0109] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "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 antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; 2 The fragment obtained after the disulfide bond between the two heavy chain fragments in the fragment consists of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains).

[0110] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form 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 CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0111] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0112] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and 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 repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can 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 by 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 also be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0113] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment 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 the full-length antibody. Single-domain antibodies are also called nanobodies.

[0114] 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 the antigen.

[0115] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0116] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0117] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may 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.

[0118] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order 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., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid 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 the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0119] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in 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.

[0120] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, 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. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0121] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) of the 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.

[0122] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optical or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), 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, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0123] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0124] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0125] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the 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 of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds 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 replace the corresponding amino acid residue with another amino acid residue from the same side chain family. 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).

[0126] The twenty conventional amino acids referred to herein are written in accordance with 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 single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0127] 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 the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, 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. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0128] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0129] As used herein, the term "subject" refers to a mammal, such as a human, a cynomolgus monkey, or a mouse.

[0130] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactic amount is an amount sufficient to prevent, arrest, or delay the onset of the disease; a therapeutic amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered treatments.

[0131] Advantageous Effects of the Invention

[0132] The present invention provides antibodies or antigen-binding fragments thereof targeting CCR8, which have high binding affinity and good specificity to CCR8. Furthermore, the heavy chain constant region is modified to obtain a fully human antibody, which can specifically eliminate CCR8-positive cells through enhanced ADCC and ADCP activity while avoiding killing CCR8-negative cells. Therefore, the antibodies of the present invention can be used for a variety of purposes, including but not limited to enhancing immune response, inhibiting tumor growth, anti-infection and detecting CCR8 protein. In addition, the fully human antibodies of the present invention can be safely administered to human subjects without triggering an immunogenic response. Therefore, the antibodies of the present invention have significant clinical value.

[0133] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] FIG1 shows the binding activity of the anti-CCR8 antibody of the present application to CHO cells overexpressing human CCR8 ( FIG1A ) and CHO cells ( FIG1B ).

[0135] FIG2 shows the ADCC activity of the anti-CCR8 antibody of the present application based on CHO-huCCR8 / Jurkat-CD16a-NFAT luciferase reporter gene cells ( FIG2A ) and CHO / Jurkat-CD16a-NFAT luciferase reporter gene cells ( FIG2B ).

[0136] FIG3 shows the ADCP activity of the anti-CCR8 antibody of the present invention based on CHO-huCCR8 / Jurkat-CD32a-NFAT luciferase reporter gene cells ( FIG3A ) and CHO / Jurkat-CD32a-NFAT luciferase reporter gene cells ( FIG3B ).

[0137] FIG4 shows the cell-killing activity of the anti-CCR8 antibody of the present invention based on CHO-huCCR8 / PBMC.

[0138] FIG5 shows the anti-tumor effect of the anti-CCR8 antibody of the present invention in the huCCR8 transgenic mouse CT-26 tumor model.

[0139] Figure 6 shows the study of the synergistic anti-tumor effect of the anti-mouse CCR8 antibody of the present invention and the anti-PD-L1 / TGF-βRⅡ fusion protein in the wild-type mouse CT-26 tumor model.

[0140] Sequence information

[0141] Information on the partial sequences involved in the present invention is provided in Table 1 below.

[0142] Table 1: Description of sequences DETAILED DESCRIPTION

[0143] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0144] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially 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, genomics, and recombinant DNA used in the present invention can be found in Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE. CULTURE) (RI Freshney, ed. (1987)).

[0145] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0146] Example 1. Preparation of antibodies

[0147] 1.1 Screening of fully human anti-CCR8 antibodies using yeast display technology

[0148] Based on the yeast antibody display library (Adimab, see WO2009036379, WO2010105256 and WO2012009568), multiple methods such as cell binding enrichment with overexpressed human CCR8 were used to screen and enrich yeast cells that specifically bind to human CCR8. The yeast cells obtained by screening were induced by 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 harvested. The anti-CCR8 antibody in the supernatant was purified using Protein A and eluted with pH 2.0 acetic acid solution to harvest the anti-CCR8 antibody.

[0149] 1.2 Antibody affinity maturation

[0150] In order to obtain anti-human CCR8 antibodies with higher affinity, the above-screened antibodies were optimized by, but not limited to, the following methods: CDRH1 / CDRH2 mutation screening; VHmut mutation screening; CDRL1 / CDRL2 / CDRL3 mutation screening, etc. The specific CDR1-3 sequences (encoded by the IMGT coding system) and VH and VL sequences of the screened antibodies are shown in Table 1.

[0151] 1.3 Antibody Expression and Purification

[0152] The heavy chain variable region of candidate clone ADI-68741 (amino acid sequence shown in SEQ ID NO:4) was linked to the human IgG1 wild-type heavy chain constant region (amino acid sequence shown in SEQ ID NO:9), the human IgG1-GA modified heavy chain constant region (G1-GA) (amino acid sequence shown in SEQ ID NO:10), and the mouse IgG2a heavy chain constant region (amino acid sequence shown in SEQ ID NO:13), and cloned into the pcDNA3.1 vector; its light chain variable region (amino acid sequence shown in SEQ ID NO:8) was linked to the human immunoglobulin kappa light chain constant region (amino acid sequence shown in SEQ ID NO:11) and the mouse immunoglobulin kappa light chain constant region (amino acid sequence shown in SEQ ID NO:14), and cloned into the pcDNA3.1 vector.

[0153] Transient expression and purification were performed using a CHO cell or fucose-depleted CHO cell expression system. The specific procedures were as follows: pcDNA3.1 vectors carrying the antibody heavy and light chains were chemically transfected into CHO cells or fucose-depleted CHO cells. The cells were cultured at 37°C, 8% CO₂ for 7 days. The cell suspension was harvested and centrifuged at 13,000 rpm for 20 minutes. The supernatant was purified using Protein A. The antibody purity was assessed by SEC and the endotoxin content was controlled. Finally, five antibodies were obtained, named ADI-68741-G1 (the variable regions of the candidate clones were constructed to the wild-type heavy chain constant region of human IgG1 and the human immunoglobulin kappa light chain constant region, respectively), ADI-68741-GA (the variable regions of the candidate clones were constructed to the modified heavy chain constant region of human IgG1-GA and the human immunoglobulin kappa light chain constant region, respectively), ADI-68741-G1 (afucosylated) (the construction method is the same as ADI-68741-G1, the difference is that it uses a fucose-knockout CHO cell expression system), ADI-68741-GA (afucosylated) (the construction method is the same as ADI-68741-GA, the difference is that it uses a fucose-knockout CHO cell expression system), and ADI-68741-mG2a (the variable regions of the candidate clones were constructed to the heavy chain constant region of mouse IgG2a and the mouse immunoglobulin kappa light chain constant region, respectively).

[0154] Example 2. Binding activity of anti-CCR8 antibodies to CHO cells overexpressing human CCR8 or null CHO cells

[0155] Specifically, CHO cells overexpressing human CCR8 (named CHO-huCCR8 cells) were generated by pressure screening of the pCHO1.0 vector (purchased from Invitrogen) containing human CCR8 (amino acid sequence as shown in SEQ ID NO: 12) cDNA cloned into MCS. The expanded cultured CHO-huCCR8 cells or empty CHO cells were adjusted to an appropriate cell density and added to a 96-well flow plate. After centrifugation, gradient dilutions of the test samples were added and incubated at 4°C for 30 minutes. Wash twice with PBS, add the corresponding fluorescent secondary antibody diluted to the appropriate concentration, incubate at 4°C for 30 minutes, and wash twice with PBS. Resuspend the cells in PBS, detect on a CytoFlex flow cytometer, and calculate the corresponding MFI. Graphpad software was used to plot the results, as shown in Figure 1. The anti-CCR8 antibodies ADI-68741-G1, ADI-68741-GA, ADI-68741-G1 (afucosylated), and ADI-68741-GA (afucosylated) of the present invention can all specifically bind to CCR8 expressed on CHO cells, with EC50s for binding to CHO-huCCR8 cells of 1.65 nM, 1.21 nM, 2.41 nM, and 2.30 nM, respectively, while having no binding activity to empty CHO cells.

[0156] Example 3. Detection of ADCC activity of anti-CCR8 antibodies in vitro

[0157] The in vitro ADCC activity of the anti-CCR8 antibody of the present invention was detected based on the luciferase reporter gene system.

[0158] Specifically, Jurkat-CD16a-NFAT-Luciferase-ADCC effector cells (purchased from Promega) were expanded and resuspended to 4×10 cells in 1640 medium containing 10% low IgG FBS. 6 Resuspend CHO-huCCR8 cells and empty CHO cells in 1640 medium containing 10% low IgG FBS and dilute to 0.8×10 cells / mL. 6cells / mL. Mix the two cell suspensions described above with Jurkat-CD16a-NFAT-Luciferase-ADCC effector cells at a 1:1 ratio, and add 50 μL / well to a sterile 96-well white-bottom plate. Add serial dilutions of the antibody sample to be tested in 1640 medium supplemented with 10% low-IgG FBS. Incubate at 37°C, 5% CO2 for 6 hours. After incubation, remove the cells, equilibrate at room temperature for 5 minutes, and add 100 μL / well of Bio-Glo™ reagent. Read the fluorescence signal using a multi-function microplate reader. The results are shown in Figure 2 , which indicate that the anti-CCR8 antibodies of the present invention exhibited ADCC activity only in the CHO-huCCR8 cell system, and that the defucosyl-modified antibodies ADI-68741-G1 (afucosylated) (EC50 < 0.01 nM) and ADI-68741-GA (afucosylated) (EC50 = 0.04 nM) exhibited stronger ADCC activity than ADI-68741-G1 (EC50 = 0.57 nM) and ADI-68741-GA (EC50 = 0.85 nM).

[0159] Example 4. In vitro ADCP activity detection of anti-CCR8 antibodies

[0160] The in vitro ADCP activity of the anti-CCR8 antibody of the present invention was detected based on the luciferase reporter gene system.

[0161] Specifically, Jurkat-CD32a-NFAT-Luciferase-ADCP effector cells (purchased from Rhino Bio) were expanded and resuspended in 1640 medium to 4 × 10 6 Resuspend CHO-huCCR8 cells and empty CHO cells in 1640 medium and dilute to 3.2×10 cells / mL. 6cells / mL. Mix the two cell suspensions described above with Jurkat-CD32a-NFAT-Luciferase-ADCP effector cells at a 1:1 ratio, and add 50 μL / well to a sterile 96-well white-bottom plate. Add the antibody sample to be tested, serially diluted in 1640 medium. Incubate at 37°C, 5% CO2 for 6 hours. After incubation, remove the cells, equilibrate at room temperature for 5 minutes, and add 100 μL / well of Bio-Glo™ reagent. Read the fluorescence signal using a multi-function microplate reader. The results are shown in Figure 3 , which indicate that the anti-CCR8 antibodies of the present invention exhibited ADCP activity only in the CHO-huCCR8 cell system, and that the Fc-terminal GA-engineered antibodies ADI-68741-GA (EC50 = 0.56 nM) and ADI-68741-GA (afucosylated) (EC50 = 2.24 nM) exhibited stronger ADCP activity than ADI-68741-G1 (EC50 = 4.90 nM) and ADI-68741-G1 (afucosylated) (Not fit).

[0162] Example 5. Detection of PBMC cell killing activity of anti-CCR8 antibodies in vitro

[0163] The in vitro killing activity of the anti-CCR8 antibody of the present invention was detected based on the PBMC killing system.

[0164] Specifically, human PBMC cells were revived and cultured overnight to remove monocytes. The next day, PBMC cells were collected and plated at 1×10 cells per well. 5 The target cells CHOS-huCCR8 were stained using the CellTraceViolet kit and the cell density was adjusted to 1×10 per well. 4 Each well was inoculated into the above-mentioned 96-well clear-bottom, black-edged cell culture plate. Subsequently, the test antibody after gradient dilution was added to the cell wells and incubated for 48 hours. After 48 hours, the DAPI fluorescence signal was collected using Cytation 5 and the corresponding killing intensity was calculated. The results are shown in Figure 4. The results show that the anti-CCR8 antibodies of the present invention exhibit in vitro ADCC killing activity against PBMC cells, and the defucosylated antibodies ADI-68741-G1 (afucosylated) (EC50 = 0.0021nM) and ADI-68741-GA (afucosylated) (EC50 = 0.0027nM) exhibited stronger PBMC killing activity than ADI-68741-G1 (EC50 = 0.0213nM) and ADI-68741-GA (EC50 = 0.0433nM).

[0165] Example 6. Pharmacodynamics study of anti-CCR8 antibodies in huCCR8KI mice

[0166] In this experiment, huCCR8KI mice (purchased from Shanghai South Model Organisms Co., Ltd.) were inoculated with CT-26 colon cancer cells (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to determine the anti-tumor effect of the anti-CCR8 antibody of the present invention.

[0167] Specifically, a CT-26 cell tumor-bearing mouse model was first established by subcutaneous inoculation. 3 The mice were divided into groups and treated with the ADI-68741-mG2a antibody of the present invention (10 mg / kg) by intraperitoneal injection. Tumor volume and body weight changes in each group were monitored every two to three days for two to three weeks. The results, shown in Figure 5, demonstrate that the anti-CCR8 antibody ADI-68741-mG2a of the present invention significantly inhibited tumor growth in mice.

[0168] Example 7. Synergistic Pharmacodynamic Study of Anti-CCR8 Antibody and Anti-PD-L1 / TGF-βRⅡ Fusion Protein in Wild-Type Balb / c Mice

[0169] To explore potential combinations for future clinical applications of CCR8 antibodies, the present invention employed the method of inoculating CT-26 tumor cells in wild-type Balb / c mice to determine the synergistic anti-tumor effects of anti-CCR8 antibodies and anti-PD-L1 / TGF-βRⅡ fusion protein (heavy chain amino acid sequence as shown in SEQ ID NO: 15, light chain amino acid sequence as shown in SEQ ID NO: 16).

[0170] Specifically, a CT-26 cell tumor-bearing mouse model was first established by subcutaneous inoculation. 3 Mice were divided into groups and treated with anti-CCR8 antibodies and / or anti-PD-L1 / TGF-βRⅡ fusion proteins via intraperitoneal injection. Tumor volume and body weight changes in each group were monitored every 2-3 days for 2 to 3 weeks. The dosage and route of administration are shown in Table 2. The results, shown in Figure 6, demonstrate that the combination of anti-CCR8 antibodies and anti-PD-L1 / TGF-βRⅡ fusion proteins significantly outperformed the corresponding monotherapy groups in tumor suppression activity. This suggests that CCR8 antibodies have the potential for combined development with anti-PD-L1 / TGF-βRⅡ fusion proteins in future clinical studies.

[0171] Table 2: Dosing regimens for anti-CCR8 antibodies and anti-PD-L1 / TGF-βRⅡ fusion proteins

[0172] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that can specifically bind 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 SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, (ii) a VH CDR2 consisting of SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) 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 SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, (v) a VL CDR2 consisting of the sequence of SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, and (vi) a VL CDR3 consisting of the sequence of SEQ ID NO: 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; Preferably, the substitution in any one of (i) to (vi) is a conservative substitution; Preferably, the CDRs described in any one of (i) to (vi) are defined according to the Kabat, IMGT or Chothia numbering systems; Preferably, the CDRs of any one of (i) to (vi) are defined according to the IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises: the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 1, VH CDR2 as shown in SEQ ID NO: 2, VH CDR3 as shown in SEQ ID NO: 3; and / or, the following three light chain CDRs: VL CDR1 as shown in SEQ ID NO: 5, VL CDR2 as shown in SEQ ID NO: 6, VL CDR3 as shown in SEQ ID NO:

7.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 4; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 4; or (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 shown in SEQ ID NO: 8; (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 8; or (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; 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 shown in SEQ ID NO: 4 and a VL having the sequence shown in SEQ ID NO:

8.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises a constant region derived from a mammalian immunoglobulin or a variant thereof; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain constant region (CH) of a mammalian immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions, 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 additions, or any combination thereof; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions, or any combination thereof); and / or (b) a light chain constant region (CL) of a mammalian immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions, 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 additions, or any combination thereof; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions, or any combination thereof); Preferably, the mammal is selected from a mouse or a human.

4. The antibody or antigen-binding fragment thereof according to 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, for example, a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; for example, a mouse IgG1, IgG2a, IgG2b, IgG2c or IgG3 heavy chain constant region; Preferably, the heavy chain constant region is selected from the human IgG1 heavy chain constant region or the mouse IgG2a heavy chain constant region; Preferably, the variant is a variant of the human IgG1 heavy chain constant region; Preferably, the variant is mutated to alanine at position 119 corresponding to the human IgG1 heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof is hypofucosylated or afucosylated; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown 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) shown in SEQ ID NO: 11 or 14.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The antigen binding fragment is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody and 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 according to any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule according to claim 6; preferably, the 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 preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising culturing the host cell according to claim 8 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

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

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

12. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Preferably, the antibody or antigen-binding fragment thereof is detectably labeled, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin; Preferably, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Preferably, the second antibody further comprises a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), 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 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. The heavy chain variable region and light chain variable region of the Preferably, the antigen binding domain is a scFv; Preferably, the chimeric antigen receptor is expressed by immune effector cells (eg, T cells).

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

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the immunoconjugate according to claim 10, or the pharmaceutical composition according to claim 11, or the chimeric antigen receptor according to claim 13, in the preparation 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 agent; Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, 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 involves tumor cells expressing CCR8; preferably, the CCR8 is expressed on the surface of the tumor cells; Preferably, the tumor is selected from 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 ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas; Preferably, the subject is a mammal, such as a human.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a kit for detecting whether a tumor can be treated by an anti-tumor therapy targeting CCR8; (1) contacting a sample containing the tumor cells with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and CCR8; Preferably, the antibody or antigen-binding fragment thereof is detectably labeled; Preferably, the CCR8 is mammalian (eg, human, mouse) CCR8; Preferably, the tumor is selected from 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 ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

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