WO2024094003A1 - 抗ccr8的抗体及其用途 - Google Patents

抗ccr8的抗体及其用途 Download PDF

Info

Publication number
WO2024094003A1
WO2024094003A1 PCT/CN2023/128490 CN2023128490W WO2024094003A1 WO 2024094003 A1 WO2024094003 A1 WO 2024094003A1 CN 2023128490 W CN2023128490 W CN 2023128490W WO 2024094003 A1 WO2024094003 A1 WO 2024094003A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
binding fragment
amino acid
sequence
Prior art date
Application number
PCT/CN2023/128490
Other languages
English (en)
French (fr)
Inventor
戴爽
翟天航
黄威峰
曾竣玮
Original Assignee
普米斯生物技术(珠海)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 普米斯生物技术(珠海)有限公司 filed Critical 普米斯生物技术(珠海)有限公司
Publication of WO2024094003A1 publication Critical patent/WO2024094003A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

  • the present application relates to the field of biomedicine, and in particular, to an antibody or an antigen-binding fragment thereof that specifically binds to CCR8, and a pharmaceutical composition and a kit comprising the antibody or the antigen-binding fragment thereof.
  • the antibody or the antigen-binding fragment thereof has ADCC activity and/or ADCP activity, and has a good tumor-suppressing effect. Therefore, the present invention further relates to the use of the antibody or the antigen-binding fragment thereof in preventing and/or treating tumors.
  • Treg regulatory T cells
  • CTL-4 cytotoxic T lymphocyte-associated antigen-4
  • chemokine receptor 8 (CC motif) receptor 8, CCR8
  • CC motif CC motif receptor 8
  • 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.
  • the inventors of the present 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 with CCR8 and good specificity. Some antibodies have enhanced ADCC or ADCP activity, and some antibodies have the activity of enhancing ADCC and ADCP at the same time. In animal models, the administration of the antibodies of the present invention can significantly inhibit tumor growth.
  • the present 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 it, and related uses.
  • the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CCR8, wherein the antibody or the antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH CDR1 which consists of the following sequence: 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,
  • VH CDR2 which consists of the following sequence: 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) compared thereto, and
  • VH CDR3 which consists 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;
  • VL light chain variable region
  • CDRs complementarity determining regions
  • VL CDR1 which consists of the following sequence: 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,
  • VL CDR2 which consists of the following sequence: 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
  • VL CDR3 which consists of the following sequence: 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.
  • substitution of any of (i)-(vi) is a conservative substitution.
  • the CDRs described in any of (i)-(vi) are numbered according to Kabat, IMGT, or Chothia. Number system definition.
  • the CDRs of any of (i)-(vi) are defined according to the IMGT numbering system.
  • the antibody or its antigen-binding fragment 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.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitution described in (ii) or (v) is a conservative substitution.
  • the antibody or its antigen-binding fragment comprises: a VH having a sequence as shown in SEQ ID NO:4 and a VL having a sequence as shown in SEQ ID NO:8.
  • the antibody or antigen-binding fragment thereof comprises a constant region derived from a mammalian immunoglobulin or a variant thereof.
  • the antibody or antigen-binding fragment thereof comprises:
  • the sequence has one or more amino acid substitutions, deletions or additions or any combination thereof (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
  • CL light chain constant region
  • the mammal is selected from a mouse or a human.
  • the heavy chain constant region is selected from IgG, IgM, IgE, IgD or IgA.
  • 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.
  • the heavy chain constant region is selected from a human IgG1 heavy chain constant region and a mouse IgG2a heavy chain constant region.
  • the variant is a variant of the human IgG1 heavy chain constant region.
  • the variant is mutated to alanine at position corresponding to position 119 of the human IgG1 heavy chain constant region.
  • the antibody or antigen-binding fragment thereof is hypofucosylated or afucosylated.
  • the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) shown in SEQ ID NO:9, 10 or 13.
  • the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.
  • the light chain constant region is a mouse or human kappa light chain constant region.
  • the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) shown in SEQ ID NO:11 or 14.
  • the antigen binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).
  • the antibodies or antigen-binding fragments thereof of the invention are murine antibodies, chimeric antibodies, humanized antibodies, or multispecific antibodies.
  • the antibody or antigen-binding fragment thereof described in any of the above embodiments further has a feature selected from the following:
  • the low-fucosylated or non-fucosylated antibody or its antigen-binding fragment has stronger ADCC activity; in such embodiments, the antibody prepared by the modified host cell (for example, a fucose knockout CHO cell) is a low-fucosylated or non-fucosylated antibody;
  • 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,
  • the antibody or its antigen-binding fragment is used in combination with other pharmaceutically active agents (e.g., anti-PD-L1/TGF- ⁇ RII fusion protein) to have a stronger ability to inhibit tumor growth.
  • other pharmaceutically active agents e.g., anti-PD-L1/TGF- ⁇ RII fusion protein
  • the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described above.
  • the present invention provides a vector comprising the nucleic acid molecule as described above.
  • the vector of the present invention is selected from plasmid, cosmid, phage, lentivirus.
  • 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).
  • the vector is a cloning vector or an expression vector.
  • the present invention provides a host cell comprising the nucleic acid molecule or the vector as described above.
  • the host cell is a mammalian cell.
  • the host cell is a fucose knockout or non-knockout cell.
  • the antibody produced by the engineered host cells is a hypofucosylated or afucosylated antibody.
  • 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.
  • 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).
  • 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.
  • the present invention provides a multispecific molecule comprising the antibody or antigen-binding fragment thereof as described above.
  • the multispecific molecule specifically binds CCR8 and additionally specifically binds one or more other targets.
  • the multispecific molecule is a bispecific molecule.
  • the bispecific molecule further comprises a molecule with a second binding specificity for a second target (eg, a second antibody).
  • a second target eg, a second antibody
  • 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.
  • the therapeutic agent is selected from a cytotoxic agent.
  • the therapeutic agent is selected from 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.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present invention provides a pharmaceutical composition
  • 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.
  • the pharmaceutical composition further comprises an additional pharmaceutically active agent.
  • 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.
  • 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.
  • the antibody or antigen-binding fragment thereof, multispecific molecule or immunoconjugate is The additional pharmaceutically active agent may be provided as a separate component or as a component of the same composition.
  • the additional pharmaceutically active agent is an anti-PD-L1/TGF- ⁇ RII fusion protein.
  • the anti-PD-L1/TGF- ⁇ RII 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.
  • the present invention provides a kit comprising the above-mentioned antibody or antigen-binding fragment thereof.
  • 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.
  • an enzyme eg, horseradish peroxidase
  • a radionuclide e.g., a radionuclide
  • a fluorescent dye eg, a fluorescent dye
  • a luminescent substance eg, a chemiluminescent substance
  • biotin biotin
  • the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof as described above.
  • 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.
  • a detectable label such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • the present invention provides a chimeric antigen receptor, which comprises the antigen binding domain of the antibody or antigen binding fragment thereof as described above.
  • 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.
  • the antigen binding domain is a scFv.
  • the chimeric antigen receptor is expressed by an immune effector cell (eg, a T cell).
  • an immune effector cell eg, a T cell
  • 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.
  • the present invention provides a 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 preparation of a medicament for preventing and/or treating tumors in a subject (e.g., a human).
  • a subject e.g., a human
  • the medicament further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, antiangiogenic agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • a drug with anti-tumor activity such as an alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, antiangiogenic agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • the additional pharmaceutically active agent is an anti-PD-L1/TGF- ⁇ RII fusion protein.
  • the anti-PD-L1/TGF- ⁇ RII 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.
  • the tumor expresses CCR8.
  • the tumor comprises a tumor cell that expresses CCR8.
  • the CCR8 is expressed on the surface of the tumor cell.
  • 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 fungoids, Merkel cell carcinoma and other malignant blood diseases, such as classical Hodgkin's 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
  • CHL
  • the subject is a mammal, such as a human.
  • the present invention provides a use of the above-mentioned 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;
  • the antibody or antigen-binding fragment thereof is detectably labeled.
  • the CCR8 is mammalian (eg, human, mouse) CCR8.
  • 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 fungoids, Merkel cell carcinoma and other hematological 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
  • CHL
  • antibody refers to an immunoglobulin molecule that is usually 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 ⁇ , ⁇ , ⁇ , ⁇ or ⁇ , and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively.
  • the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains 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 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.
  • VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form antigen binding sites, respectively.
  • the distribution of amino acids in various 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.
  • CDR complementarity determining region
  • the variable region of the heavy chain and light chain each contains three CDRs, named CDR1, CDR2, and CDR3.
  • 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.
  • 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.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • 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 an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.
  • the terms “monoclonal antibody”, “single antibody”, and “mAb” have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
  • Monoclonal antibodies have high specificity for a single epitope on an antigen.
  • Polyclonal antibodies, relative to monoclonal antibodies generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen.
  • the modifier "monoclonal” only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.
  • 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).
  • 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".
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementary determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.
  • full-length antibody means an antibody consisting of two “full-length heavy chains” and two “full-length light chains”.
  • full-length heavy chain refers to a polypeptide chain that 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 in the direction from N to C; and, when the full-length antibody is an IgE isotype, optionally also includes a heavy chain constant region CH4 domain.
  • a "full-length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N to C.
  • 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 to C.
  • VL light chain variable region
  • CL light chain constant region
  • 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 antibody of the present invention may be from a single species, such as a human; it may also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention comprises 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.
  • the term “Fd” means an antibody fragment consisting of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989))
  • the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • the term “F(ab') 2 fragment” means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • the term “Fab'fragment” means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.
  • the term "Fv" means 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 variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.
  • Fc means an antibody fragment formed by 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 of an antibody bound via a disulfide bond.
  • the Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.
  • 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 a 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.
  • GGGGS linker having the amino acid sequence
  • Other linkers useful 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.
  • scFv can form di-scFv, which refers to two or more single scFvs in series to form an antibody.
  • scFv can form (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.
  • single-domain antibody has the meaning generally understood by those skilled in the art, which 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.
  • 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.
  • Antibody antigen-binding fragments 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 antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • 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 the 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 (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • the term “chimeric antibody” may include an antibody in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody, and the heavy chain and light chain constant regions of the antibody are derived from a second antibody.
  • the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • the sequences are aligned for optimal comparison purposes (for example, 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 determination of the 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. U.S.A. 87: 2264-2268, as improved in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 5873-5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.
  • variant in the context of a polypeptide (including a polypeptide), also refers to a polypeptide or peptide comprising an amino acid sequence that has been changed by introducing amino acid residue substitutions, deletions or additions. 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).
  • a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection/blocking groups, proteolytic cleavage, connection to a cell 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.
  • a variant has a function similar, identical or improved to the polypeptide or peptide from which it is derived.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed.
  • the strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction.
  • 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.
  • the detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means.
  • labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -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
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • plasmids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • 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, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses 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.
  • 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.
  • 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
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • 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.
  • 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 with residues physically or functionally similar to the 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.
  • 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
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives.
  • pH regulators 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, and preservatives.
  • Flushing liquid such as buffered saline
  • alcohol and polyol such as glycerol
  • Preservatives include but are not limited to various antibacterial agents 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, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (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.
  • the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • such sterile injectable liquid is 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 buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • 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 buffer solution e.g., phosphate buffer solution
  • Ringer's solution e.g., Ringer's solution
  • prevention refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject.
  • treatment refers to a method implemented in order to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results 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 partially or completely), whether detectable or undetectable.
  • treatment can also refer to prolonging survival compared to the expected survival (if not receiving treatment).
  • the term "subject" refers to a mammal, such as a human, a cynomolgus monkey, or a mouse.
  • the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • a disease prevention effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of the disease
  • a disease treatment effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have 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 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 mode of administration of the drug, and other treatments administered simultaneously, etc.
  • the present invention provides an antibody or antigen-binding fragment thereof targeting CCR8, which has high binding affinity and good specificity to CCR8. Further, the heavy chain constant region is modified to obtain a fully human antibody, which can be enhanced by ADCC and ADCP activities specifically eliminate CCR8-positive cells while avoiding killing of 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 humanized antibodies of the present invention can be safely administered to human subjects without inducing immunogenic reactions. Therefore, the antibodies of the present invention have great clinical value.
  • FIG. 1 shows the binding activity of the anti-CCR8 antibody of the present application to CHO cells overexpressing human CCR8 (A in FIG. 1 ) and CHO cells (B in FIG. 1 ).
  • 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 (A in FIG2 ) and CHO/Jurkat-CD16a-NFAT luciferase reporter gene cells (B in FIG2 ).
  • 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 (A in FIG3 ) and CHO/Jurkat-CD32a-NFAT luciferase reporter gene cells (B in FIG3 ).
  • FIG. 4 shows the cell killing activity of the anti-CCR8 antibody of the present invention based on CHO-huCCR8/PBMC.
  • FIG5 shows the anti-tumor effect of the anti-CCR8 antibody of the present invention in the huCCR8 transgenic mouse CT-26 tumor model.
  • 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- ⁇ RII fusion protein in the wild-type mouse CT-26 tumor model.
  • yeast antibody display library (Adimab, see WO2009036379, WO2010105256 and WO2012009568)
  • multiple methods such as overexpression of human CCR8 cell binding enrichment were used to screen and enrich yeast cells that specifically bind to human CCR8.
  • the yeast cells obtained by screening were shaken and induced at 30°C for 48 hours to secrete and express the target anti-CCR8 antibody (full-length IgG). After the induction, the yeast cells were removed by centrifugation at 1300rpm for 10 minutes and the supernatant was harvested.
  • the anti-CCR8 antibody in the supernatant was purified using Protein A, eluted with pH2.0 acetic acid solution, and the anti-CCR8 antibody was harvested.
  • 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.
  • the heavy chain variable region of candidate clone ADI-68741 (amino acid sequence as shown in SEQ ID NO: 4) was connected to the human IgG1 wild-type heavy chain constant region (amino acid sequence as shown in SEQ ID NO: 9), and the human IgG1-GA modified heavy chain constant region was connected to the human IgG1 wild-type heavy chain constant region (amino acid sequence as shown in SEQ ID NO: 9).
  • the heavy chain constant region (G1-GA) (amino acid sequence as shown in SEQ ID NO: 10) and the heavy chain constant region of mouse IgG2a (amino acid sequence as shown in SEQ ID NO: 13) were cloned into the pcDNA3.1 vector; the light chain variable region (amino acid sequence as shown in SEQ ID NO: 8) was connected to the human immunoglobulin kappa light chain constant region (amino acid sequence as shown in SEQ ID NO: 11) and the mouse immunoglobulin kappa light chain constant region (amino acid sequence as shown in SEQ ID NO: 14) and cloned into the pcDNA3.1 vector.
  • Transient expression and purification via CHO cell or fucose knockout CHO cell expression system the specific operation is as follows: Use chemical transfection method to transfer pcDNA3.1 vector with antibody heavy chain and light chain into CHO cells or fucose knockout CHO cells, culture for 7 days at 37°C, 8% CO 2. Collect cell fluid and centrifuge at 13000rpm for 20 minutes. Take the supernatant, purify the supernatant with Protein A, detect the purity of the antibody by SEC, and control the endotoxin content at the same time.
  • 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-knocked-out 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-knocked-out CHO cell expression system
  • ADI-68741-mG2a the variable regions of the candidate clones were constructed to the heavy chain constant region of mouse
  • Example 2 Binding activity of anti-CCR8 antibodies to CHO cells overexpressing human CCR8 or empty CHO cells
  • CHO cells (named CHO-huCCR8 cells) overexpressing human CCR8 were produced by pressure screening by transfecting the pCHO1.0 vector (purchased from Invitrogen) cloned into the MCS human CCR8 (amino acid sequence as shown in SEQ ID NO: 12).
  • 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, the gradient diluted sample to be tested was added and incubated at 4°C for 30 minutes. Wash twice with PBS, add the corresponding fluorescent secondary antibody diluted to an appropriate concentration, incubate at 4°C for 30 minutes, and wash twice with PBS.
  • the anti-CCR8 antibodies ADI-68741-G1, ADI-68741-GA, ADI-68741-G1 (afucosylated), and ADI-68741-GA (afucosylated) of the present invention can specifically bind to CCR8 expressed on CHO cells, and the EC50 for binding to CHO-huCCR8 cells are 1.65nM, 1.21nM, 2.41nM, and 2.30nM, respectively, while there is no binding activity to empty CHO cells.
  • the in vitro ADCC activity of the anti-CCR8 antibody of the present invention was detected based on the luciferase reporter gene system.
  • Jurkat-CD16a-NFAT-Luciferase-ADCC effector cells purchased from Promega
  • 1640 medium containing 10% low IgG FBS a medium containing 10% low IgG FBS
  • CHO-huCCR8 cells and empty CHO cells were resuspended in 1640 medium containing 10% low IgG FBS and diluted to 0.8 ⁇ 10 6 cells/mL.
  • the above two cell suspensions were mixed with Jurkat-CD16a-NFAT-Luciferase-ADCC effector cells 1:1, 50 ⁇ L/well was added to a sterile 96-well white bottom plate, and the antibody sample to be tested was diluted in a gradient of 1640 medium containing 10% low IgG FBS.
  • the in vitro ADCP activity of the anti-CCR8 antibody of the present invention was detected based on the luciferase reporter gene system.
  • Jurkat-CD32a-NFAT-Luciferase-ADCP effector cells purchased from Rhino Bio
  • CHO-huCCR8 cells and empty CHO cells were resuspended in 1640 medium and diluted to 3.2 ⁇ 10 6 cells/mL.
  • the above two cell suspensions were mixed evenly with Jurkat-CD32a-NFAT-Luciferase-ADCP effector cells in a 1:1 ratio, 50 ⁇ L/well was added to a sterile 96-well white bottom plate, and the antibody sample to be tested was diluted in a gradient of 1640 medium. Incubate for 6 hours at 37°C and 5% CO 2.
  • the in vitro killing activity of the anti-CCR8 antibody of the present invention was detected.
  • human PBMC cells were revived, cultured overnight to remove monocytes, and PBMC cells were collected the next day and added to a 96-well transparent bottom black-edged cell culture plate at 1 ⁇ 10 5 cells per well.
  • the target cells CHOS-huCCR8 were stained with the CellTraceViolet kit, and after adjusting the cell density, 1 ⁇ 10 4 cells per well were inoculated into the above-mentioned 96-well transparent bottom black-edged cell culture plate.
  • the gradiently diluted antibodies to be tested were 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.
  • huCCR8KI mice purchased from Shanghai South Model Biotechnology Co., Ltd.
  • CT-26 colon cancer cells purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.
  • a CT-26 cell tumor-bearing mouse model was first established by subcutaneous inoculation.
  • the mice were divided into groups and treated with the ADI-68741-mG2a antibody of the present invention (10 mg/Kg) by intraperitoneal injection.
  • the tumor volume and body weight changes of each group of mice were monitored.
  • the monitoring frequency was 2-3 days/time, and the monitoring was continued for 2 to 3 weeks.
  • Figure 5 show that the anti-CCR8 antibody ADI-68741-mG2a of the present invention can significantly inhibit the growth of mouse tumors.
  • the present invention used CT-26 tumor cells inoculated in wild-type Balb/c mice to determine the synergistic anti-tumor effect of anti-CCR8 antibodies and anti-PD-L1/TGF- ⁇ RII 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).
  • a CT-26 cell tumor-bearing mouse model was first established by subcutaneous inoculation. When the average tumor volume grew to 150 mm 3 , the mice were divided into groups and treated with anti-CCR8 antibodies and/or anti-PD-L1/TGF- ⁇ RII fusion proteins by intraperitoneal injection. The changes in tumor volume and body weight of mice in each group were monitored. The monitoring frequency was 2-3 days/time, and the monitoring was continued for 2 to 3 weeks. The dosage and method of administration are shown in Table 2. The results are shown in Figure 6. The results show that anti-CCR8 antibodies and anti-PD-L1/TGF- ⁇ RII fusion proteins have a significant effect on the growth of CT-26 cells.
  • the tumor suppression activity of the ⁇ RII fusion protein combination group was significantly better than that of the corresponding two single-drug treatment groups, suggesting that CCR8 antibodies have the potential to be developed in combination with anti-PD-L1/TGF- ⁇ RII fusion proteins in future clinical research stages.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本申请涉及生物医药领域，具体而言，本申请涉及特异性结合CCR8的抗体或其抗原结合片段以及包含其的药物组合物和试剂盒。所述抗体或其抗原结合片段具有ADCC活性和/或ADCP活性，同时具有很好的抑制肿瘤的效果。因此，本发明进一步涉及所述抗体或其抗原结合片段在预防和/治疗肿瘤中的用途。

Description

抗CCR8的抗体及其用途 技术领域
本申请涉及生物医药领域，具体而言，本申请涉及特异性结合CCR8的抗体或其抗原结合片段以及包含所述抗体或其抗原结合片段的药物组合物和试剂盒。所述抗体或其抗原结合片段具有ADCC活性和/或ADCP活性，同时具有较好的抑制肿瘤的效果。因此，本发明进一步涉及所述抗体或其抗原结合片段在预防和/治疗肿瘤中的用途。
背景技术
近年来，以抗PD-1/PD-L1单克隆抗体为代表的肿瘤免疫治疗取得了突破性进展。但是该疗法在实体肿瘤中整体响应率仅为10％～30％(Ribas A,Wolchok JD.Cancer immunotherapy using checkpoint blockade.Science,2018,359(6382):1350-1355)。因此，开发新的免疫疗法提高肿瘤患者的响应率和生存期仍是未被满足的临床需求。
多项研究表明，在肿瘤微环境中存在大量调节性T细胞(regulatory T cells,Treg)浸润，通过细胞表面高表达的细胞毒T淋巴细胞相关抗原-4(cytotoxic T lymphocyte-associated antigen-4,CTLA-4)竞争结合CD80、CD86激活信号，高亲和力IL-2受体CD25竞争结合IL-2，释放免疫抑制因子TGF-β、IL-10等机制抑制免疫应答，从而促进肿瘤发生发展(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)。因此，多家药企和研究机构致力于开发清除肿瘤微环境Treg的疗法，期望解除免疫抑制，增加患者对肿瘤的免疫应答。其中，多款分别靶向Treg高表达的CD25、CCR4、CTLA-4，具有抗体依赖性的细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity，ADCC)的单克隆抗体已进入临床研究阶段。但这些抗体在实体瘤治疗上响应率有限，同时面临清除外周Treg带来的潜在副作用。
不同于CTLA-4、CD25、CCR4等免疫检查点分子在T细胞或外周Treg细胞上高表达等特性，趋化因子受体8(chemokine(C-C motif)receptor 8，CCR8)在肿瘤浸润的Treg细胞上特异性高表达(Plitas G,Konopacki C,Wu K,et al.Regulatory T cells exhibit distinct features in human breast cancer.Immunity,2016,45(5):1122-1134)。靶向CCR8的单克隆抗体具有特异性清除肿瘤浸润的Treg，避免对外周Treg造成杀伤的潜力。因 此，本领域存在开发高效特异性清除肿瘤浸润的Treg，同时避免清除外周Treg带来毒副作用的靶向CCR8新的抗体需求，用于疾病治疗，尤其是癌症治疗。
发明内容
本申请的发明人经过大量的研究，筛选获得了一株抗CCR8的单克隆抗体，进一步的，对抗体的重链恒定区进行一系列工程化改造。获得的抗体具有ADCC和/或ADCP活性，并且，这些抗体与CCR8的结合亲和力高，特异性好，部分抗体具有增强的ADCC或ADCP活性，部分抗体具有同时增强ADCC和ADCP的活性。在动物模型中，施用本发明的抗体可以显著抑制肿瘤生长。基于此，本申请还提供了含有所述抗体或其抗原结合片段的组合物，编码所述抗体或其抗原结合片段的核酸及包含其的宿主细胞，以及相关用途。
因此，在一个方面，本发明提供了能够特异性结合CCR8的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
和/或，
(b)包含下述3个互补决定区(CDRs)的轻链可变区(VL)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:5，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:6，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和(vi)VL CDR3，其由下述序列组成：SEQ ID NO:7，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，(i)-(vi)中任一项所述的置换为保守置换。
在某些实施方案中，(i)-(vi)任一项中所述的CDR根据Kabat、IMGT或Chothia编 号系统定义。
在某些实施方案中，(i)-(vi)中任一项所述的CDR根据IMGT编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：如下3个重链CDRs：如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:2所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:5所示的VL CDR1、如SEQ ID NO:6所示的VL CDR2、如SEQ ID NO:7所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:4所示的序列；
(ii)与SEQ ID NO:4所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:8所示的序列；
(v)与SEQ ID NO:8所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:8所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:4所示的序列的VH和具有如SEQ ID NO:8所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于哺乳动物免疫球蛋白的恒定区或其变体。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)哺乳动物免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序 列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；和/或
(b)哺乳动物免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)。
在某些实施方案中，所述哺乳动物选自小鼠或人。
在某些实施方案中，所述重链恒定区选自IgG、IgM、IgE、IgD或IgA。
在某些实施方案中，所述重链恒定区是小鼠或人的IgG重链恒定区；例如，人IgG1、IgG2、IgG3或IgG4重链恒定区；例如，小鼠IgG1、IgG2a、IgG2b、IgG2c或IgG3重链恒定区。
在某些实施方案中，所述重链恒定区选自人IgG1重链恒定区和小鼠IgG2a重链恒定区。
在某些实施方案中，所述变体是人IgG1重链恒定区的变体。
在某些实施方案中，所述变体在对应于人IgG1重链恒定区的第119位位置上突变为丙氨酸。
在某些实施方案中，所述抗体或其抗原结合片段是低岩藻糖基化或无岩藻糖基化的。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:9、10或13所示的重链恒定区(CH)。
在某些实施方案中，所述轻链恒定区是κ轻链恒定区或λ轻链恒定区。
在某些实施方案中，所述轻链恒定区是小鼠或人的κ轻链恒定区。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:11或14所示的轻链恒定区(CL)。
在某些实施方案中，所述抗原结合片段选自Fab、Fab’、(Fab’)2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)。
在某些实施方案中，本发明的抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体、或多特异性抗体。
在某些实施方案中，上述任一项实施方案中所述的抗体或其抗原结合片段进一步具有选自下列的特征：
(1)具有ADCC活性，例如通过ADCC来诱导杀伤表达CCR8的细胞(例如肿瘤细胞)；在某些实施方案中，低岩藻糖基化或无岩藻糖基化的所述抗体或其抗原结合片段具有更强的ADCC活性；在此类实施方案中，通过改造的宿主细胞(例如，岩藻糖敲除的CHO细胞)制备获得的抗体是低岩藻糖基化或无岩藻糖基化的抗体；
(2)具有ADCP活性，例如通过ADCP来诱导杀伤表达CCR8的细胞(例如肿瘤细胞)；在某些实施方案中，包含突变的重链恒定区(例如，如SEQ ID NO:10所示)的所述抗体或其抗原结合片段具有更强的ADCP活性；和/或，
(3)抑制肿瘤生长，在某些实施方案中，所述抗体或其抗原结合片段与其它的药学活性剂(例如，抗PD-L1/TGF-βRⅡ融合蛋白)联用具有更强的抑制肿瘤生长的能力。
在另一个方面，本发明提供了一种分离的核酸分子，其编码如前所述的抗体或其抗原结合片段。
在另一方面，本发明提供了一种载体，其包含如前所述的核酸分子。
在某些实施方案中，本发明的载体选自质粒、粘粒、噬菌体、慢病毒。在某些实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的抗体或其抗原结合片段。在某些实施方案中，所述载体为克隆载体或表达载体。
在另一个方面，本发明提供了一种宿主细胞，其包含如前所述的核酸分子或如前所述的载体。
在某些实施方案中，所述宿主细胞是哺乳动物细胞。
在某些实施方案中，所述宿主细胞是岩藻糖敲除或未敲除的细胞。
在此类实施方案中，通过改造的宿主细胞(例如，岩藻糖敲除的CHO细胞)制备获得的抗体是低岩藻糖基化或无岩藻糖基化的抗体。
宿主细胞可以是真核细胞(例如哺乳动物细胞、昆虫细胞、酵母细胞)或原核细胞(例如大肠杆菌)。合适的真核细胞包括但不限于NS0细胞、Vero细胞、Hela细胞、COS细胞、CHO细胞、ExpiCHO细胞、HEK293细胞、Expi293细胞、BHK细胞和MDCKII细胞。适宜的昆虫细胞包括但不限于Sf9细胞。在某些实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DXB11、 ExpiCHO、CHO DG44)。
本发明另一方面，提供了制备如前所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养如前所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
本发明另一方面，提供了一种多特异性分子，其包含如前所述的抗体或其抗原结合片段。
在某些实施方案中，所述多特异性分子特异性结合CCR8，并且额外地特异性结合一个或多个其他靶标。
在某些实施方案中，所述多特异性分子是双特异性分子。
在某些实施方案中，所述双特异性分子还包含一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
本发明另一方面，提供了一种免疫缀合物，其包含如前所述的抗体或其抗原结合片段或如前所述的多特异性分子，以及连接于所述抗体或其抗原结合片段或多特异性分子的治疗剂。
在某些实施方案中，所述治疗剂选自细胞毒剂。
在某些实施方案中，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合。
在某些实施方案中，所述免疫缀合物是抗体-药物偶联物(ADC)。
本发明另一方面，提供了一种药物组合物，其包含如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子或者如前所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，药物组合物还包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，所述抗体或其抗原结合片段、多特异性分子或免疫缀合物与所 述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。
在某些实施方案中，另外的药学活性剂是抗PD-L1/TGF-βRⅡ融合蛋白。在某些实施方案中，抗PD-L1/TGF-βRⅡ融合蛋白具有如SEQ ID NO:15所示的重链氨基酸序列，和如SEQ ID NO:16所示的轻链氨基酸序列。
本发明另一方面，提供了一种试剂盒，其含有如前所述的抗体或其抗原结合片段。
在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
在某些实施方案中，所述试剂盒还包括第二抗体，其特异性识别如前所述的抗体或其抗原结合片段。
在某些实施方案中，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
本发明另一方面，提供了一种嵌合抗原受体，其包含如前所述的抗体或其抗原结合片段的抗原结合结构域。
在某些实施方案中，所述抗原结合结构域包含如前所述的抗体或其抗原结合片段的重链可变区和轻链可变区。
在某些实施方案中，所述抗原结合结构域是scFv。
在某些实施方案中，所述嵌合抗原受体由免疫效应细胞(例如T细胞)所表达。
本发明另一方面，提供了一种抑制表达CCR8的肿瘤细胞生长和/或杀伤所述肿瘤细胞的方法，其包括将所述肿瘤细胞与有效量的如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，或如前所述的免疫缀合物，或如前所述的药物组合物，或如前所述的嵌合抗原受体接触。
本发明另一方面，提供了一种如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，或如前所述的免疫缀合物，或如前所述的药物组合物，或如前所述的嵌合抗原受体，在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/治疗肿瘤。
在某些实施方案中，药物还包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化 剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，另外的药学活性剂是抗PD-L1/TGF-βRⅡ融合蛋白。在某些实施方案中，抗PD-L1/TGF-βRⅡ融合蛋白具有如SEQ ID NO:15所示的重链氨基酸序列，和如SEQ ID NO:16所示的轻链氨基酸序列。
在某些实施方案中，所述肿瘤表达CCR8。
在某些实施方案中，所述肿瘤涉及CCR8的肿瘤细胞。在某些实施方案中，所述CCR8在所述肿瘤细胞表面上表达。
在某些实施方案中，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
在某些实施方案中，所述受试者为哺乳动物，例如人。
本发明另一方面，提供了一种如前所述的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测肿瘤是否能够通过靶向CCR8的抗肿瘤疗法来治疗；
(1)将含有所述肿瘤细胞的样品与如前所述的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与CCR8之间复合物的形成。
在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记。
在某些实施方案中，所述CCR8是哺乳动物(例如，人，小鼠)的CCR8。
在某些实施方案中，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞 淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2 和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单克隆抗体”、“单抗”、“mAb”具有相同的含义且可互换使用可互换，其是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。此外，修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得，不能理解为需要通过任何特定方法来制备所述抗体。
本发明的单克隆抗体可以通过多种技术进行制备，例如杂交瘤技术(参见，例如Kohler等人，Nature,256:495，1975)，重组DNA技术(参见，例如美国专利申请4,816,567)，或噬菌体抗体库技术(参见，例如Clackson等.Nature352：624-628,1991，或Marks等.J.Mol.Biol.222：581-597,1991)。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental  Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’)2、Fd、Fv、互补决定区(CDR)片段、scFv、双抗体(diabody)、单域抗体(single domain antibody)、嵌合抗体、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)、、probody和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv)2，其指的是两个或两个以上单个scFv并联而形成抗体。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力。单域抗体也称为纳米抗体(nanobody)。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。在某些实施方案中，术语“嵌合抗体”可包括这样的抗体，其中抗体的重链和轻链可变区来自第一抗体，而抗体的重链和轻链恒定区来自第二抗体。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。为了测定两个氨基酸序列或两个核酸序列的百分比同一性，为了最佳比较目的将序列进行比对(例如，可在第一氨基酸序列或核酸序列中引入缺口以与第二氨基酸或核酸序列最佳比对)。然后比较对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置被与第二序列中的对应位置相同的氨基酸残基或核苷酸占据时，则分子在该位置上是同一的。两个序列之间的百分比同一性是由序列所共享的同一性位置的数目的函数(即，百分比同一性＝同一重叠位置的数目/位置的总数×100％)。在某些实施方案中，两个序列长度相同。
两个序列之间的百分比同一性的测定还可使用数学算法来实现。用于两个序列的比较的数学算法的一个非限制性实例是Karlin和Altschul的算法，1990,Proc.Natl.Acad.Sci.U.S.A.87:2264-2268，如同Karlin和Altschul,1993,Proc.Natl.Acad.Sci.U.S.A.90:5873-5877中改进的。将这样的算法整合至Altschul等人,1990,J.Mol.Biol.215:403的NBLAST和XBLAST程序中。
如本文中所使用的，术语“变体”，在多肽的情境中(包括多肽)也指包含已通过引入氨基酸残基置换、缺失或添加改变的氨基酸序列的多肽或肽。在某些情况下，术语“变体”还指已被修饰(即，通过将任何类型的分子共价连接至多肽或肽)的多肽或肽。例如，但非限制性地，多肽可以被修饰，例如通过糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知保护/封闭基团进行的衍生化、蛋白水解切割、连接至细胞配体或其它蛋白质等。衍生多肽或肽可使用本领域技术人员已知的技术通过化学修饰来产生，所述技术包括但不限于特异性化学切割、乙酰化、甲酰化、衣霉素的代谢合成等。此外，变体具有与其所源自的多肽或肽相似、相同或改善的功能。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
如本文中所使用的，本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物、鲁米诺及其衍生物、钌衍生物如三联吡啶钌)、磁珠(例如，)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓 冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中所使用的，术语“受试者”是指哺乳动物，例如人、食蟹猴、小鼠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病有效量是指，足以预防，阻止，或延迟所述疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明提供了靶向CCR8的抗体或其抗原结合片段，其与CCR8的结合亲和力高，特异性好。进一步的，对重链恒定区进行改造并获得了全人源抗体，其可以通过增强的 ADCC和ADCP活性特异性清除CCR8阳性的细胞，同时避免对CCR8阴性细胞的杀伤。因此，本发明的抗体可以用于多种用途，包括但不限于增强免疫应答，抑制肿瘤生长，抗感染和检测CCR8蛋白。此外，本发明的全人源抗体可安全地施用给人受试者，而不引发免疫原性反应。因此，本发明的抗体具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了本申请抗CCR8抗体与过表达人CCR8的CHO细胞(图1中的A)和CHO细胞(图1中的B)的结合活性。
图2显示了本申请抗CCR8抗体基于CHO-huCCR8/Jurkat-CD16a-NFAT荧光素酶报告基因细胞(图2中的A)和CHO/Jurkat-CD16a-NFAT荧光素酶报告基因细胞(图2中的B)的ADCC活性。
图3显示了本发明抗CCR8抗体基于CHO-huCCR8/Jurkat-CD32a-NFAT荧光素酶报告基因细胞(图3中的A)和CHO/Jurkat-CD32a-NFAT荧光素酶报告基因细胞(图3中的B)的ADCP活性。
图4显示了本发明抗CCR8抗体基于CHO-huCCR8/PBMC的细胞杀伤活性。
图5显示了本发明抗CCR8抗体在huCCR8转基因小鼠CT-26肿瘤模型中的抗肿瘤效果。
图6显示了本发明抗小鼠CCR8抗体在野生型小鼠CT-26肿瘤模型中与抗PD-L1/TGF-βRⅡ融合蛋白的协同抗肿瘤效果研究。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述


具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物 化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.抗体的制备
1.1酵母展示技术筛选抗CCR8全人源抗体
基于酵母抗体展示文库(Adimab，参见WO2009036379、WO2010105256和WO2012009568)，采用过表达人CCR8细胞结合富集等多重方法，筛选富集获得特异性结合人CCR8的酵母细胞。将通过筛选获得的酵母细胞在30℃下震荡诱导48小时以分泌表达目标抗CCR8抗体(全长IgG)。诱导结束之后，1300rpm离心10分钟去除酵母细胞，收获上清液。使用Protein A对上清液中的抗CCR8抗体进行纯化，pH2.0醋酸溶液洗脱，收获抗CCR8抗体。
1.2抗体亲和力成熟
为了获得更高亲和力的抗人CCR8抗体，通过但不限于以下方法对上述经筛选获得的抗体进行优化，CDRH1/CDRH2突变筛选；VHmut突变筛选；CDRL1/CDRL2/CDRL3突变筛选等，筛选出的抗体的具体CDR1-3序列(IMGT编码系统编码)以及VH和VL序列如表1所示。
1.3抗体的表达与纯化
将候选克隆ADI-68741的重链可变区(氨基酸序列如SEQ ID NO:4所示)分别连接人IgG1野生型重链恒定区(氨基酸序列如SEQ ID NO:9所示)，人IgG1-GA改造的重 链恒定区(G1-GA)(氨基酸序列如SEQ ID NO:10所示)，以及小鼠IgG2a的重链恒定区(氨基酸序列如SEQ ID NO:13所示)，并克隆到pcDNA3.1载体中；将其轻链可变区(氨基酸序列如SEQ ID NO:8所示)分别连接人免疫球蛋白kappa轻链恒定区(氨基酸序列如SEQ ID NO:11所示)和小鼠免疫球蛋白kappa轻链恒定区(氨基酸序列如SEQ ID NO:14所示)，并克隆到pcDNA3.1载体中。
经由CHO细胞或岩藻糖敲除的CHO细胞表达系统瞬时表达纯化，具体操作如下：使用化学转染的方法将带有抗体重链和轻链的pcDNA3.1载体转入CHO细胞或岩藻糖敲除的CHO细胞中，在37℃，8％CO2条件下，培养7天。收集细胞液，13000rpm离心20分钟。取上清液，Protein A纯化上清液，SEC检测抗体纯度，同时控制内毒素含量。最终获得抗体5种抗体，分别命名为ADI-68741-G1(候选克隆可变区分别构建至人IgG1野生型重链恒定区和人免疫球蛋白kappa轻链恒定区)、ADI-68741-GA(候选克隆可变区分别构建至人IgG1-GA改造的重链恒定区和人免疫球蛋白kappa轻链恒定区)、ADI-68741-G1(afucosylated)(构建方式同ADI-68741-G1，区别在于其使用岩藻糖敲除的CHO细胞表达系统)、ADI-68741-GA(afucosylated)(构建方式同ADI-68741-GA，区别在于其使用岩藻糖敲除的CHO细胞表达系统)、ADI-68741-mG2a(候选克隆可变区分别构建至小鼠IgG2a的重链恒定区和小鼠免疫球蛋白kappa轻链恒定区)。
实施例2.抗CCR8抗体与过表达人CCR8的CHO细胞或空CHO细胞的结合活性
具体地，通过转染克隆到MCS的人CCR8(氨基酸序列如SEQ ID NO:12所示)cDNA的pCHO1.0载体(购自Invitrogen)加压筛选产生过表达人CCR8的CHO细胞(命名为CHO-huCCR8细胞)。将扩大培养的CHO-huCCR8细胞或空CHO细胞，调整至合适细胞密度加入96孔流式板，离心后加入梯度稀释的待测样品，4℃孵育30分钟。PBS清洗两次，加入对应稀释至合适浓度的荧光二抗，4℃孵育30分钟，PBS清洗两次。加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。采用Graphpad软件作图，结果如图1所示，本发明的抗CCR8抗体ADI-68741-G1、ADI-68741-GA、ADI-68741-G1(afucosylated)、ADI-68741-GA(afucosylated)均可特异性结合CHO细胞上表达的CCR8，与CHO-huCCR8细胞结合的EC50分别为1.65nM、1.21nM、2.41nM、2.30nM，而与空CHO细胞没有结合活性。
实施例3.抗CCR8抗体体外ADCC活性检测
基于荧光素酶报告基因系统，检测本发明抗CCR8抗体体外ADCC活性。
具体地，扩大培养Jurkat-CD16a-NFAT-Luciferase-ADCC效应细胞(购自Promega)，用含10％low IgG FBS的1640培养基重悬细胞至4×106细胞/mL。用含10％low IgG FBS的1640培养基重悬CHO-huCCR8细胞，空CHO细胞，稀释至0.8×106细胞/mL。将上述2种细胞悬液分别与Jurkat-CD16a-NFAT-Luciferase-ADCC效应细胞1:1混合均匀，50μL/孔加入无菌96孔白底板内，加入含10％low IgG FBS的1640培养基梯度稀释的待检测抗体样品。37℃，5％CO2共孵育6小时。培养完成，取出细胞，室温平衡5分钟，100μL/孔加入Bio-Glo TM reagent，使用多功能酶标仪读取荧光信号值。结果如图2所示，结果表明本发明抗CCR8抗体仅在CHO-huCCR8细胞体系内体现出ADCC活性，且去岩藻糖修饰的抗体ADI-68741-G1(afucosylated)(EC50<0.01nM)和ADI-68741-GA(afucosylated)(EC50＝0.04nM)相较于ADI-68741-G1(EC50＝0.57nM)和ADI-68741-GA(EC50＝0.85nM)呈现更强的ADCC活性。
实施例4.抗CCR8抗体体外ADCP活性检测
基于荧光素酶报告基因系统，检测本发明抗CCR8抗体体外ADCP活性。
具体地，扩大培养Jurkat-CD32a-NFAT-Luciferase-ADCP效应细胞(购自Rhino Bio)，用1640培养基重悬细胞至4×106细胞/mL。用1640培养基重悬CHO-huCCR8细胞，空CHO细胞，稀释至3.2×106细胞/mL。将上述2种细胞悬液分别与Jurkat-CD32a-NFAT-Luciferase-ADCP效应细胞1:1混合均匀，50μL/孔加入无菌96孔白底板内，加入1640培养基梯度稀释的待检测抗体样品。37℃，5％CO2共孵育6小时。培养完成，取出细胞，室温平衡5分钟，100μL/孔加入Bio-Glo TM reagent，使用多功能酶标仪读取荧光信号值。结果如图3所示，结果表明本发明抗CCR8抗体仅在CHO-huCCR8细胞体系内体现出ADCP活性，且Fc端GA工程改造的抗体ADI-68741-GA(EC50＝0.56nM)和ADI-68741-GA(afucosylated)(EC50＝2.24nM)相较于ADI-68741-G1(EC50＝4.90nM)和ADI-68741-G1(afucosylated)(Not fit)呈现更强的ADCP活性。
实施例5.抗CCR8抗体体外PBMC细胞杀伤活性检测
基于PBMC杀伤系统，检测本发明抗CCR8抗体体外杀伤活性。
具体地，复苏人PBMC细胞，培养过夜贴壁去除单核细胞，第二天收集PBMC细胞并按照每孔1×105个/孔加入96孔透明底黑边细胞培养板中。将靶细胞CHOS-huCCR8通过CellTraceViolet试剂盒进行细胞染色，调整细胞密度后按照每孔1×104个/孔接种至上述96孔透明底黑边细胞培养板中。随后将梯度稀释后的待测抗体加入到细胞孔中共孵育48小时。48小时后使用Cytation 5收集DAPI荧光信号并计算对应的杀伤强度。结果如图4所示，结果表明本发明抗CCR8抗体呈现PBMC细胞的体外ADCC杀伤活性，且去岩藻糖修饰的抗体ADI-68741-G1(afucosylated)(EC50＝0.0021nM)和ADI-68741-GA(afucosylated)(EC50＝0.0027nM)相较于ADI-68741-G1(EC50＝0.0213nM)和ADI-68741-GA(EC50＝0.0433nM)呈现更强的PBMC杀伤活性。
实施例6.抗CCR8抗体在huCCR8KI小鼠体内药效学研究
本实验采用在huCCR8KI小鼠(购自上海南方模式生物科技有限公司)体内接种CT-26结肠癌细胞(购自江苏集萃药康生物科技股份有限公司)测定本发明抗CCR8抗体的抗肿瘤作用。
具体地，首先采用皮下接种的方式建立CT-26细胞荷瘤小鼠模型，待平均肿瘤体积长至80mm3时进行分组，腹腔注射给予本发明ADI-68741-mG2a抗体进行治疗(10mg/Kg)，监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周。结果如图5所示，结果表明本发明抗CCR8抗体ADI-68741-mG2a可以显著抑制小鼠肿瘤的生长。
实施例7.抗CCR8抗体与抗PD-L1/TGF-βRⅡ融合蛋白在野生型Balb/c小鼠体 内协同药效学研究
为了探索CCR8抗体未来临床应用的潜在组合，本发明采用在野生型Balb/c小鼠体内接种CT-26肿瘤细胞测定抗CCR8抗体与抗PD-L1/TGF-βRⅡ融合蛋白(重链氨基酸序列如SEQ ID NO:15所示，轻链氨基酸序列如SEQ ID NO:16所示)的协同抗肿瘤作用。
具体地，首先采用皮下接种的方式建立CT-26细胞荷瘤小鼠模型，待平均肿瘤体积长至150mm3时进行分组，腹腔注射给予抗CCR8抗体和/或抗PD-L1/TGF-βRⅡ融合蛋白治疗，监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周，给药剂量和方式如表2。结果如图6所示，结果表明抗CCR8抗体与抗PD-L1/TGF- βRⅡ融合蛋白联合用药组，肿瘤抑制活性显著优于对应的两单药治疗组。提示在未来临床研究阶段，CCR8抗体具有和抗PD-L1/TGF-βRⅡ融合蛋白联合开发的潜力。
表2：抗CCR8抗体与抗PD-L1/TGF-βRⅡ融合蛋白给药方案
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (16)

  1. 能够特异性结合CCR8的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    和/或，
    (b)包含下述3个互补决定区(CDRs)的轻链可变区(VL)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:5，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:6，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和(vi)VL CDR3，其由下述序列组成：SEQ ID NO:7，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)中任一项所述的置换为保守置换；
    优选地，(i)-(vi)任一项中所述的CDR根据Kabat、IMGT或Chothia编号系统定义；
    优选地，(i)-(vi)中任一项所述的CDR根据IMGT编号系统定义；
    优选地，所述抗体或其抗原结合片段包含：如下3个重链CDRs：如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:2所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:5所示的VL CDR1、如SEQ ID NO:6所示的VL CDR2、如SEQ ID NO:7所示的VL CDR3。
  2. 权利要求1的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:4所示的序列；
    (ii)与SEQ ID NO:4所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:8所示的序列；
    (v)与SEQ ID NO:8所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:8所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:4所示的序列的VH和具有如SEQ ID NO:8所示的序列的VL。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含来源于哺乳动物免疫球蛋白的恒定区或其变体；
    优选地，所述抗体或其抗原结合片段包含：
    (a)哺乳动物免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；和/或
    (b)哺乳动物免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；
    优选地，所述哺乳动物选自小鼠或人。
  4. 权利要求3所述的抗体或其抗原结合片段，其中，所述重链恒定区选自IgG、IgM、IgE、IgD或IgA；
    优选地，所述重链恒定区是人或小鼠的IgG重链恒定区，例如，人IgG1、IgG2、IgG3或IgG4重链恒定区；例如，小鼠IgG1、IgG2a、IgG2b、IgG2c或IgG3重链恒定区；
    优选地，所述重链恒定区选自人IgG1重链恒定区或小鼠IgG2a重链恒定区；
    优选地，所述变体是人IgG1重链恒定区的变体；
    优选地，所述变体在对应于人IgG1重链恒定区的第119位位置上突变为丙氨酸；
    优选地，所述抗体或其抗原结合片段是低岩藻糖基化或无岩藻糖基化的；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:9、10或13所示的重链恒定区(CH)；
    优选地，所述轻链恒定区是κ轻链恒定区或λ轻链恒定区；
    优选地，所述轻链恒定区是小鼠或人的κ轻链恒定区；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:11或14所示的轻链恒定区(CL)。
  5. 权利要求1-4任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’)2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)；和/或，所述抗体为鼠源抗体、嵌合抗体、人源化抗体或多特异性抗体。
  6. 分离的核酸分子，其编码权利要求1-5任一项所述的抗体或其抗原结合片段。
  7. 载体，其包含权利要求6所述的核酸分子；优选地，所述载体为克隆载体或表达载体。
  8. 宿主细胞，其包含权利要求6的核酸分子或权利要求7的载体；
    优选地，所述宿主细胞是哺乳动物细胞；
    优选地，所述宿主细胞是岩藻糖敲除或未敲除的细胞。
  9. 制备权利要求1-5任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求8所述的宿主细胞，和从培养 的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  10. 免疫缀合物，其包含权利要求1-5任一项所述的抗体或其抗原结合片段，以及连接于所述抗体或其抗原结合片段或多特异性分子的治疗剂；
    优选地，所述治疗剂选自细胞毒剂；
    优选地，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合；
    优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
  11. 药物组合物，其包含权利要求1-5任一项所述的抗体或其抗原结合片段，或者权利要求10所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述抗体或其抗原结合片段或免疫缀合物与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供；
    优选地，另外的药学活性剂是抗PD-L1/TGF-βRⅡ融合蛋白。
  12. 试剂盒，其含有权利要求1-5任一项所述的抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
    优选地，所述试剂盒还包括第二抗体，其特异性识别权利要求1-5任一项所述的抗体或其抗原结合片段；
    优选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  13. 嵌合抗原受体，其包含权利要求1-5任一项所述的抗体或其抗原结合片段的抗原结合结构域；
    优选地，所述抗原结合结构域包含权利要求1-5任一项所述的抗体或其抗原结合片 段的重链可变区和轻链可变区；
    优选地，所述抗原结合结构域是scFv；
    优选地，所述嵌合抗原受体由免疫效应细胞(例如T细胞)所表达。
  14. 一种抑制表达CCR8的肿瘤细胞生长和/或杀伤所述肿瘤细胞的方法，其包括将所述肿瘤细胞与有效量的权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求10所述的免疫缀合物，或权利要求11所述的药物组合物，或权利要求13所述的嵌合抗原受体接触。
  15. 权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求10所述的免疫缀合物，或权利要求11所述的药物组合物，或权利要求13所述的嵌合抗原受体，在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/治疗肿瘤；
    优选地，药物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，另外的药学活性剂是抗PD-L1/TGF-βRⅡ融合蛋白；
    优选地，所述肿瘤表达CCR8；
    优选地，所述肿瘤涉及CCR8的肿瘤细胞；优选地，所述CCR8在所述肿瘤细胞表面上表达；
    优选地，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤；
    优选地，所述受试者为哺乳动物，例如人。
  16. 权利要求1-5任一项所述的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测肿瘤是否能够通过靶向CCR8的抗肿瘤疗法来治疗；
    (1)将含有所述肿瘤细胞的样品与权利要求1-5任一项所述的抗体或其抗原结合片段接触；
    (2)检测所述抗体或其抗原结合片段与CCR8之间复合物的形成；
    优选地，所述抗体或其抗原结合片段带有可检测的标记；
    优选地，所述CCR8是哺乳动物(例如，人，小鼠)的CCR8；
    优选地，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
PCT/CN2023/128490 2022-11-04 2023-10-31 抗ccr8的抗体及其用途 WO2024094003A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211377345 2022-11-04
CN202211377345.X 2022-11-04

Publications (1)

Publication Number Publication Date
WO2024094003A1 true WO2024094003A1 (zh) 2024-05-10

Family

ID=90929805

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/128490 WO2024094003A1 (zh) 2022-11-04 2023-10-31 抗ccr8的抗体及其用途

Country Status (1)

Country Link
WO (1) WO2024094003A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110835371A (zh) * 2018-08-19 2020-02-25 普米斯生物技术(苏州)有限公司 抗ccr8单克隆抗体及其应用
WO2022042690A1 (zh) * 2020-08-28 2022-03-03 和铂医药(上海)有限责任公司 Ccr8抗体及其应用
WO2022081718A1 (en) * 2020-10-14 2022-04-21 Five Prime Therapeutics, Inc. Anti-c-c chemokine receptor 8 (ccr8) antibodies and methods of use thereof
CN114929278A (zh) * 2020-01-06 2022-08-19 瓦西尼斯公司 抗ccr8抗体及其用途
CN115052892A (zh) * 2020-10-16 2022-09-13 礼新医药科技(上海)有限公司 抗ccr8单克隆抗体及其用途
CN115087488A (zh) * 2020-02-14 2022-09-20 震动疗法股份有限公司 与ccr8结合的抗体和融合蛋白及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110835371A (zh) * 2018-08-19 2020-02-25 普米斯生物技术(苏州)有限公司 抗ccr8单克隆抗体及其应用
CN114929278A (zh) * 2020-01-06 2022-08-19 瓦西尼斯公司 抗ccr8抗体及其用途
CN115087488A (zh) * 2020-02-14 2022-09-20 震动疗法股份有限公司 与ccr8结合的抗体和融合蛋白及其用途
WO2022042690A1 (zh) * 2020-08-28 2022-03-03 和铂医药(上海)有限责任公司 Ccr8抗体及其应用
WO2022081718A1 (en) * 2020-10-14 2022-04-21 Five Prime Therapeutics, Inc. Anti-c-c chemokine receptor 8 (ccr8) antibodies and methods of use thereof
CN115052892A (zh) * 2020-10-16 2022-09-13 礼新医药科技(上海)有限公司 抗ccr8单克隆抗体及其用途

Similar Documents

Publication Publication Date Title
JP6783886B2 (ja) 抗ｃｔｌａ４モノクローナル抗体またはその抗原結合断片、医薬組成物および使用
WO2020135201A1 (zh) 一种抗体及其用途
JP2022130393A (ja) 抗ｃｔｌａ４－抗ｐｄ－１二機能性抗体、その医薬組成物および使用
JP7401538B2 (ja) 抗ｃｌｄｎ１８．２抗体およびその使用
JP2018502050A (ja) ＣＤ３εおよびＲＯＲ１に対する二特異性抗体
US20200347130A1 (en) CD96 Antibody, Antigen-Binding Fragment and Pharmaceutical use Thereof
US11655295B2 (en) Anti-LAG-3 antibody and use thereof
CN110734493B (zh) 抗pd-1抗体及其用途
JP2023525827A (ja) 抗ｃｄ７３抗体およびその使用
CN110799546A (zh) 重组双特异性抗体
EP4039704A1 (en) Anti-pd-1 antibody and use thereof
JP7352007B2 (ja) ヒト化抗ｖｅｇｆモノクローナル抗体
US20230094083A1 (en) Human anti-grp94 antibodies and uses
WO2022267936A1 (zh) 特异性结合糖基化ceacam5的抗体
JP2022546002A (ja) ４価の対称型二重特異性抗体
WO2024094003A1 (zh) 抗ccr8的抗体及其用途
WO2023186078A1 (zh) 针对c-Met的抗体及其用途
WO2024061224A1 (zh) 抗her2抗体及其用途
WO2023236951A1 (zh) 抗cd40抗体及其用途
CN114206931B (zh) 抗pd-1抗体及其用途
WO2023016450A1 (zh) 抗tigit抗体及其用途
WO2023216218A1 (zh) Cd22特异性结合抗体及其制备方法和用途
WO2024114525A1 (zh) B7-h3结合蛋白及其用途
WO2024007671A1 (zh) 特异性结合cd24的抗体及其用途
WO2024000259A1 (zh) Cd22特异性结合抗体及其制备方法和其在双特异cart上的用途