WO2024120418A1 - 抗ccr8抗体及其应用 - Google Patents

抗ccr8抗体及其应用 Download PDF

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WO2024120418A1
WO2024120418A1 PCT/CN2023/136644 CN2023136644W WO2024120418A1 WO 2024120418 A1 WO2024120418 A1 WO 2024120418A1 CN 2023136644 W CN2023136644 W CN 2023136644W WO 2024120418 A1 WO2024120418 A1 WO 2024120418A1
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seq
antibody
amino acid
acid sequence
variable region
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PCT/CN2023/136644
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English (en)
French (fr)
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郭建
霍永庭
芦迪
肖亮
张轶博
欧颖烨
罗颖
张喆
李永锋
耿梦圆
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广东菲鹏制药股份有限公司
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Publication of WO2024120418A1 publication Critical patent/WO2024120418A1/zh

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  • the present disclosure relates to the field of biotechnology, and in particular, to an anti-CCR8 antibody and an application thereof.
  • Chemokine receptor 8 is a seven-transmembrane G protein-coupled receptor that belongs to the CC subfamily of chemokine receptors.
  • the present disclosure develops an anti-CCR8 antibody, which in some embodiments can enhance the body's anti-tumor immune response and improve the survival rate of tumor patients.
  • the present disclosure discloses an anti-human CCR8 antibody, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 2, 4, 6, 8, 10 or 12; in some embodiments, wherein,
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.2;
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.5, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.6;
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.7, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.8;
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.9, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.10; or
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.11, and
  • the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 12;
  • the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system; in some embodiments, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system. In some embodiments, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the IMGT numbering system.
  • the anti-human CCR8 antibody as described in any of the above items, wherein
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.14
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.15
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.19
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.20
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.21
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.22
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.23
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.24
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.25
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.26
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.27;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.28
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.29
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.30
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.32
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.33
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.35
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.36;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.37
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.38
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • the anti-human CCR8 antibody as described in any of the above items comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, respectively.
  • the anti-human CCR8 antibody comprises the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively, and the LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, respectively, and the LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27, respectively.
  • the anti-human CCR8 antibody as described in any of the above items is a murine antibody, a chimeric antibody or a humanized antibody.
  • the anti-human CCR8 antibody as described in any of the above items wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the anti-human CCR8 antibody as described in any of the above items, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9 or 11, and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the present disclosure provides an anti-human CCR8 antibody, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the anti-human CCR8 antibody as described in any of the above items, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9 or 11, and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.1
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO.2
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.4;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.6;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.8;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.12;
  • the anti-human CCR8 antibody as described in any of the above items the antibody further comprises a constant region; in some embodiments, the heavy chain constant region of the antibody is selected from the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is selected from the constant region of ⁇ or ⁇ chain; in some embodiments, the species of the constant region is mouse or human; in some embodiments, the heavy chain constant region is a mouse IgG2a constant region, a mouse IgG1 constant region, a human IgG1 constant region; in some embodiments, the heavy chain constant region is a human IgG1 (DLE) or human IgG1 (DE) constant region; and/or the light chain constant region is a mouse ⁇ constant region or a human ⁇ constant region.
  • the heavy chain constant region includes the amino acid sequence of SEQ ID NO.39, 41 or 42, and the light chain constant region includes the amino acid sequence of SEQ ID NO.40 or 43.
  • the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.44, and the light chain comprises the amino acid sequence of SEQ ID NO.45; or the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.46, and the light chain comprises the amino acid sequence of SEQ ID NO.47; or the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.48, and the light chain comprises the amino acid sequence of SEQ ID NO.49.
  • the anti-human CCR8 antibody as described in any of the above items is a full-length antibody or an antigen-binding fragment selected from any one of F(ab’)2, Fab’-SH, Fab’, Fab, scFab, dsFv, (dsFv)2, Fv and scFv.
  • the invention discloses an antibody that competes with any of the above anti-human CCR8 antibodies for binding to human CCR8, or an antibody that binds to the same epitope as any of the above anti-human CCR8 antibodies.
  • the anti-human CCR8 antibody can specifically bind to human CCR8; in some embodiments, the anti-human CCR8 antibody can bind to Raji cells expressing human CCR8 with an EC50 value of ⁇ 5nM (e.g., ⁇ 4.50nM, ⁇ 3.00nM, ⁇ 2.00nM, ⁇ 1.00nM, ⁇ 0.9nM, ⁇ 0.8nM, ⁇ 0.7nM, ⁇ 0.6nM, ⁇ 0.5nM, ⁇ 0.4nM, ⁇ 0.3nM, ⁇ 0.2nM, ⁇ 0.1nM, ⁇ 0.09nM, ⁇ 0.08nM or less), wherein the EC50 value is determined by flow cytometry; in some embodiments, the EC50 value is determined by the method of Example 2 of the present application.
  • ⁇ 5nM e.g., ⁇ 4.50nM, ⁇ 3.00nM, ⁇ 2.00nM, ⁇ 1.00nM, ⁇ 0.9nM, ⁇ 0.8nM, ⁇ 0.7
  • the anti-human CCR8 antibody has the function of inhibiting tumor growth; in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is greater than or equal to 30% (e.g., greater than 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or greater); in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is determined by the method of Example 5 of the present disclosure;
  • the anti-human CCR8 antibody has ADCC activity; in some embodiments, the ADCC activity of the anti-human CCR8 antibody is determined by the method of Example 4 of the present disclosure.
  • the anti-human CCR8 antibody as described in any of the above items has the function of inhibiting tumor growth; in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is greater than or equal to 30% (e.g., greater than 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or greater); in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is determined by the method of Example 5 of the present disclosure;
  • the anti-human CCR8 antibody as described in any of the above items has ADCC activity; in some embodiments, the ADCC activity of the anti-human CCR8 antibody is determined by the method of Example 4 of the present disclosure.
  • the present disclosure also provides a multispecific antibody, wherein the multispecific antibody comprises any of the anti-human CCR8 antibodies described above.
  • the antibody is a bispecific antibody.
  • the present disclosure also provides an antibody conjugate, which includes the anti-human CCR8 antibody described in any of the above.
  • the antibody conjugate also includes a therapeutic agent or an imaging agent coupled to the antibody; in some embodiments, the antibody conjugate also includes biotin or a biotin derivative coupled to the antibody; in some embodiments, the antibody conjugate also includes a solid phase carrier coupled to the antibody; in some embodiments, the antibody conjugate also includes a marker coupled to the antibody; in some embodiments, the marker is selected from at least one of a fluorescent dye, an enzyme, a radioactive isotope, a chemiluminescent agent, and a nanoparticle marker; in some embodiments, the marker is colloidal gold.
  • the present disclosure also provides a chimeric antigen receptor (CAR), which comprises an antigen binding domain, and the antigen binding domain comprises the anti-human CCR8 antibody described in any one of the above.
  • CAR chimeric antigen receptor
  • the present disclosure also provides a CAR-immune cell, wherein the CAR-immune cell expresses the chimeric antigen receptor described above.
  • the present disclosure also provides an isolated nucleic acid encoding the anti-human CCR8 antibody described in any one of the above.
  • the present disclosure also provides a cell comprising the nucleic acid of claim 8.
  • the present disclosure also provides a pharmaceutical composition, comprising: the anti-human CCR8 antibody, multispecific antibody, antibody conjugate, cell or nucleic acid described in any of the foregoing items.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the present disclosure also provides the use of any of the above anti-human CCR8 antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids or pharmaceutical compositions in the preparation of products having at least one of the following uses: diagnosing diseases related to abnormal human CCR8 expression, treating diseases related to abnormal human CCR8 expression or detecting human CCR8 expression; in some embodiments, the disease related to abnormal human CCR8 expression is a tumor.
  • the tumor with abnormal human CCR8 expression is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.
  • the present disclosure provides a method for treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of any of the above anti-human CCR8 antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids or pharmaceutical compositions.
  • the disease is abnormal expression of human CCR8.
  • the disease is a tumor.
  • the tumor with abnormal human CCR8 expression is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.
  • the present disclosure also provides an anti-human CCR8 antibody, a multispecific antibody, an antibody conjugate, a cell, a nucleic acid or a pharmaceutical composition as described in any of the foregoing for use as a drug.
  • the drug is used to treat a disease.
  • the disease is a disease related to abnormal expression of human CCR8.
  • the disease related to abnormal expression of human CCR8 is a tumor.
  • the tumor with abnormal expression of human CCR8 is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.
  • the treatment described in any of the above further comprises administering to the subject an additional therapeutic drug.
  • the additional therapeutic drug is a chemotherapeutic agent, such as a platinum complex, taxane, pemetrexed, gemcitabine, fluorouracil, irinotecan, etoposide or doxorubicin, etc.
  • the present disclosure also provides a method for detecting or measuring human CCR8, comprising the step of using the anti-human CCR8 antibody or antibody conjugate as described in any of the preceding items to detect or measure human CCR8.
  • the present disclosure also provides a kit, comprising the anti-human CCR8 antibody or antibody conjugate as described in any of the preceding items; in some embodiments, the kit is used to detect or measure human CCR8.
  • the present disclosure also provides a method for preparing the anti-human CCR8 antibody as described in any of the preceding items, comprising the steps of culturing the cell as described in any of the preceding items, and then isolating and purifying to obtain the anti-human CCR8 antibody.
  • the anti-human CCR8 antibodies disclosed in the present disclosure can specifically bind to human CCR8; in some embodiments, the antibodies have significant anti-tumor effects both in vitro and in vivo; in some embodiments, they also have good ADCC activity.
  • FIG1 is a graph showing the binding test results of hybridoma purified antibodies and Raji-hCCR8;
  • FIG2 is a graph showing the binding test results of the recombinant antibody and Raji-hCCR8;
  • FIG3 is a diagram showing the binding detection results of the recombinant antibody and Treg
  • FIG4 is a graph showing the binding test results of the recombinant antibody and Raji;
  • FIG5 is a graph showing the binding test results of the recombinant antibody and PBMC
  • FIG6 is a graph showing the binding test results of the recombinant antibody and Raji-hCCR4;
  • FIG7 is a graph showing the results of a recombinant antibody ADCC activity experiment
  • FIG8 is a graph showing the results of an experiment in which anti-CCR8 antibodies inhibit tumor growth.
  • amino acid refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs or amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine; common natural amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S
  • Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.
  • antibody is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, chimeric antibodies, humanized antibodies, full-length antibodies, or antigen-binding fragments thereof (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Natural antibodies refer to naturally occurring immunoglobulin molecules.
  • natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains. From N to C-terminus, each heavy chain has a heavy chain variable region (VH, also known as a variable heavy domain), followed by a heavy chain constant region, and the natural IgG heavy chain constant region generally includes three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a light chain variable region (VL, also known as a variable light domain), followed by a light chain constant region (CL, also known as a light chain constant domain).
  • VH heavy chain variable region
  • CL light chain constant region
  • full length antibody or “intact antibody” refers to an antibody that comprises a structure substantially similar to a native antibody structure, or an antibody whose heavy chain comprises an Fc region.
  • variable region refers to the domain of an antibody heavy chain or light chain that is involved in antibody binding to an antigen.
  • VH antibody heavy chain variable region
  • VL light chain variable region
  • FR conserved framework regions
  • CDR Complementarity determining region
  • VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N terminus) to the carboxyl terminus (also called the C terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDRs refer to more than two CDRs in the heavy chain and light chain of the antibody.
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering convention (see Kabat et al.
  • the anti-human CCR8 antibody disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the present invention discloses an anti-human CCR8 antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 1, 3, 5, 7, 9 or 11, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the present disclosure discloses an anti-human CCR8 antibody, wherein A. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.2; B. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.3, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.4; C.
  • the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.5, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.6; D. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.7, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.8; E. the heavy chain variable region of the antibody includes SEQ ID F. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 9, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 11, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 12.
  • the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering system.
  • the anti-human CCR8 antibody disclosed herein wherein a. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, HCDR2 comprises the amino acid sequence of SEQ ID NO.14, and HCDR3 comprises the amino acid sequence of SEQ ID NO.15; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18; b.
  • the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.19
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.20
  • HCDR3 comprises The amino acid sequence of SEQ ID NO.21
  • the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.22
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.23
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.24
  • the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.25
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.26
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.27; d.
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.28
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.29
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.30
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18; e.
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.32
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.33 amino acid sequence
  • the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.35
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.36; or f.
  • the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.37
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.38
  • the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18.
  • the anti-human CCR8 antibody of the present disclosure comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.25, SEQ ID NO.
  • the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.28 and SEQ ID NO.29, respectively, and SEQ ID NO.16, SEQ ID NO.30, respectively.
  • the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.33, SEQ ID NO.34 and SEQ ID NO.35, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.36 and SEQ ID NO.37, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively.
  • antigen-binding fragment or "antigen-binding domain” refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that is capable of specifically binding to an antigen to which the intact antibody binds.
  • antigen-binding fragments include, but are not limited to, Fv (consisting of VH and VL), Fab (consisting of one light chain and one heavy chain constant region 1 (CH1) and a heavy chain variable region), Fab' (consisting of a Fab region and a hinge region), Fab'-SH (the cysteine residue in the hinge region of the Fab' fragment carries a free thiol group), (Fab')2 (dimeric Fab'), scFv (single-chain antibody molecule, in which the light chain variable region is directly connected to the heavy chain variable region or connected by a linker), scFab (single-chain Fab), dsFv (disulfide-stabilized Fv fragment), (dsst al
  • the term "Fc region” is used to define the C-terminal region of the heavy chain of an antibody, including a native Fc region and a modified Fc region.
  • the Fc region for the antibodies described herein includes the Fc region of human IgG1, IgG2 (IgG2a, IgG2b), IgG3, and IgG4.
  • the Fc region is a human IgG1 comprising a DLE mutation (i.e., the Fc of human IgG1 undergoes S239D/A330L/I332E mutations, and the sequence is shown in SEQ ID No. 42).
  • the Fc region is a human IgG1 comprising a DE mutation (i.e., the Fc of human IgG1 undergoes S239D/I332E mutations).
  • the boundaries of the Fc region can also vary, such as the deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system).
  • the numbering convention of the Fc region is the EU numbering system, also known as the EU index.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from one species, while the other portion of the heavy and/or light chain is derived from another species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, it can be achieved by retaining the non-human CDR region and replacing the rest with a human antibody counterpart (i.e., the constant region and the framework region portion of the variable region).
  • human antibody humanized antibody
  • fully human antibody fully human antibody
  • completely human antibody refers to antibodies whose variable and constant regions are human sequences.
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise indicated, as used herein, binding “affinity” refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art.
  • KD refers to a dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as a molar concentration (M).
  • M molar concentration
  • effector function refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an Fc region of amino acid sequence mutation).
  • antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
  • the term “monoclonal antibody” refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for the natural mutations that may be present in small amounts.
  • polyclonal antibody preparations are typically comprised of a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes.
  • “Monoclonal” represents the characteristics of antibodies obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any particular method.
  • the antibody provided by the present disclosure is a monoclonal antibody.
  • antigen refers to a molecule that can be bound by an antigen binding protein (eg, an antibody) selective binding agent.
  • An antigen may have one or more epitopes that can interact with different antigen binding proteins (eg, antibodies).
  • epitope refers to an area or region on an antigen that can specifically bind to an antibody.
  • An epitope can be formed by a continuous string of amino acids (linear epitope) or contain non-continuous amino acids (conformational epitope), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen by protein properties) and become spatially close.
  • the difference between a conformational epitope and a linear epitope is that the binding of the antibody to the conformational epitope is lost in the presence of a denaturing solvent.
  • a conformational epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind to a specific epitope can be performed using routine methods in the art, such as alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.
  • An antibody that binds to the same epitope as a reference antibody or an antibody that competes for binding with a reference antibody means an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, or an antibody whose binding to the antigen is blocked by the reference antibody by 50% or more.
  • test antibody binds to the same epitope
  • routine experiments e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art
  • two antibodies are considered to bind to the same or overlapping epitope if an excess (e.g., a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amount) of one antibody inhibits binding of the other antibody to the antigen by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).
  • an antibody that is capable of binding to an antigen or an epitope within the antigen with a higher affinity than other antigens or epitopes.
  • an antibody binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of about 100 nM or less (e.g., about 10 nM, 1 nM, 0.1 nM, 0.01 nM or less).
  • KD equilibrium dissociation constant
  • the KD of an antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein).
  • KD can be measured using known methods, including but not limited to Biacore assays, Octet methods, microthermophoresis, HPLC-MS methods, and flow cytometry fluorescence sorting techniques; for example, by Measured by surface plasmon resonance assay.
  • the binding of the anti-human CCR8 antibody provided by the present disclosure to human CCR8 can also be expressed by a "half-maximal effective concentration" (EC50) value.
  • EC50 half-maximal effective concentration
  • the EC50 value can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting technology, and other binding assays).
  • antibody-dependent cellular cytotoxicity is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with effector cells with lytic activity (such as natural killer cells (NK), monocytes, macrophages, and neutrophils) via Fc ⁇ receptors (Fc ⁇ Rs) expressed on effector cells.
  • lytic activity such as natural killer cells (NK), monocytes, macrophages, and neutrophils
  • Fc ⁇ Rs Fc ⁇ receptors
  • NK cells express Fc ⁇ RIIIa
  • monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIIIa.
  • the ADCC activity of the antibodies provided herein can be assessed using in vitro assays using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on markers released from lysed cells (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins).
  • ADCP antibody-dependent cellular phagocytosis
  • complement-dependent cytotoxicity refers to a mechanism of inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death.
  • Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.
  • complement receptors e.g., CR3
  • CAR is a chimeric antigen receptor, which is an artificially modified receptor, for example, it is capable of anchoring specific molecules (such as antibodies) that recognize tumor cell surface antigens or viral antigens on immune cells (such as T cells), so that immune cells recognize tumor antigens or viral antigens and kill tumor cells or viruses.
  • T cells expressing CAR are called CAR-T (or CART).
  • chimeric antigen receptors sequentially include an extracellular domain, a transmembrane region, and an intracellular signaling domain.
  • the chimeric antigen receptor of the present disclosure can be constructed using transmembrane regions and intracellular signaling domains known in the art for constructing CAR.
  • the signal is transmitted to the cell through the hinge region and the transmembrane region, and the intracellular signal domain converts the signal into an activation signal, activating effector cells, and effector cells kill tumor cells by secreting perforin or producing cytokines, and the effector cells themselves also amplify, further expanding the immune killing effect.
  • the extracellular domain may include an antigen binding domain (also known as the part of the antibody that specifically binds to the antigen), which may be a single-chain variable fragment (scFv) that specifically binds to the antigen.
  • the single-chain variable fragment may serve as the extracellular domain of CAR, which determines the specificity of CAR-immune cells.
  • the hinge region is the extracellular domain of CAR that connects the single-chain antibody unit and the transmembrane domain. It usually maintains the stability required for robust CAR expression and activity in effector cells.
  • the hinge region of most CARs is derived from the hinge of IgG or the extracellular region of CD8 ⁇ /CD28. The type and length of the hinge region have an important influence on the functional activity of CAR.
  • the transmembrane domain connects the extracellular domain of CAR to the intracellular signal transduction domain. Commonly used transmembrane domains are derived from CD4, CD8 ⁇ , CD28 and CD3 ⁇ .
  • the choice of transmembrane domain affects the degree of activation of the CAR structure in cell function.
  • the intracellular domain is composed of a co-stimulatory domain and a signal transduction domain.
  • the CAR is a chimeric antigen receptor, and its main components may also include a transmembrane domain and an intracellular domain.
  • the CAR may also include a co-stimulatory molecule.
  • "Co-stimulatory molecule” refers to a molecule that exists on the surface of an antigen presenting cell and can bind to a co-stimulatory molecule receptor on a Th cell to produce a co-stimulatory signal.
  • Isolated nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which cells typically contain the nucleic acid molecules, but the nucleic acid molecules are present outside the chromosome or at a chromosomal position different from its natural chromosomal position.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • sequence identity refers to the degree (percentage) to which the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned (introducing gaps, if necessary, to obtain the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity).
  • percentage of sequence identity alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • a person skilled in the art can determine Applicable parameters for measuring alignment include any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • antibody conjugates include conjugates formed by conjugating anti-human CCR8 antibodies with therapeutic agents or imaging agents.
  • the antibody conjugate is formed by conjugating anti-human CCR8 antibodies with therapeutic agents via a linker; in an optional embodiment, the therapeutic agent is a cytotoxic agent; in an optional embodiment, the therapeutic agent can be selected from toxins, chemotherapeutic agents, antibiotics, radioactive isotopes and nucleolytic enzymes.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction
  • toxin refers to a substance that can have a deleterious effect on the growth or proliferation of cells (e.g., maytansine, calicheamicin, taxanes, vincristine, colchicine, auristatin, etc.).
  • Cyclotherapeutic agent refers to a chemical compound that can be used to treat cancer.
  • the antibody conjugate of the present disclosure further comprises biotin or a biotin derivative conjugated to the antibody.
  • the antibody conjugate further comprises a label coupled to the antibody.
  • the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.
  • the marker is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent agent, and a nanoparticle marker.
  • a fluorescent dye an enzyme, a radioisotope, a chemiluminescent agent, and a nanoparticle marker.
  • the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc.
  • fluorescein dyes and their derivatives for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs
  • rhodamine dyes and their derivatives for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B
  • Cy series dyes and their derivatives for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc.
  • Alexa series dyes and their derivatives for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
  • protein dyes and their derivatives for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.
  • the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
  • the radioactive isotopes include but are not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.
  • the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, lophanine and its derivatives, and peroxyoxalate and its derivatives.
  • the sodium Rice particle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
  • the colloid includes but is not limited to colloidal metal, colloidal selenium, disperse dyes, dye-labeled microspheres and latex.
  • the colloidal metal includes but is not limited to colloidal gold or colloidal silver. In an optional embodiment, the colloidal metal is colloidal gold.
  • the antibody conjugate may further include a solid phase carrier coupled to the antibody.
  • the antibody is coupled to a solid phase carrier.
  • the solid phase carrier is selected from microspheres, plates and membranes.
  • the solid phase includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.
  • the solid phase carrier is a nitrocellulose membrane.
  • linker refers to a connecting unit connecting two polypeptide fragments, which usually has a certain flexibility, and the use of the linker will not cause the loss of the original function of the protein domain.
  • the linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids.
  • the linker is selected from (GxS)y linkers, wherein x is selected from an integer of 1-5 and y is selected from an integer of 0-6.
  • the linker is selected from (GxS)y linkers, wherein x is selected from an integer of 1-5 and y is selected from an integer of 1-6.
  • the disclosed embodiments also provide a method for detecting CCR8, comprising: mixing the antibody as described in any of the preceding items, or the antibody conjugate as described in any of the preceding items, or the reagent or kit as described in any of the preceding items with a sample to be detected, so that the antibody contacts the CCR8 in the sample to be detected to form an immune complex.
  • the immune complex further comprises a second antibody, and the second antibody binds to the antibody.
  • the immune complex further comprises a second antibody, and the second antibody binds to CCR8.
  • the present disclosure also provides a method for preparing the antibody as described in any of the preceding items, comprising: a step of culturing the cells as described in any of the preceding items, and a step of purifying and recovering the antibody.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked.
  • plasmid refers to a circular double-stranded DNA loop to which additional DNA segments can be attached.
  • viral vector such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be attached to the viral genome.
  • AAV adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors can be integrated into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome.
  • expression vector or "expression construct” refers to a vector that can transform a host cell and contains a nucleic acid sequence that directs and/or controls (together with the host cell) the expression of one or more heterologous coding regions to which it is operably linked.
  • Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of the coding region to which it is operably linked.
  • host cell refers to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and offspring derived therefrom, without regard to the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Mutant offspring with the same function or biological activity as screened or selected in the initial transformed cells are included herein.
  • Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells and fungal cells.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells and HEK-293 cells.
  • CHO Chinese hamster ovary
  • NSO Chinese hamster ovary
  • SP2 cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • A549 cells e.g., 3T3 cells and HEK-293 cells.
  • Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membraneaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia pastoris.
  • Pichia pastoris Pichia pastoris
  • Pichia finlandica Pichia trehalophila
  • Pichia koclamae Pichia membraneaefaciens
  • Pichia minuta Ogataea minuta, Pichia lindneri
  • Pichia puntiae Pichia thermotolerans
  • Pichia salictaria Pichia guercuum
  • Saccharomyces cerevisiae Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucnowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.
  • Pichia spp. any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucnowense, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa.
  • progeny As used in this application, “cell”, “cell line” and “cell culture” can be used interchangeably, and all such names include progeny.
  • the words “transformant” and “converted cell” include the primary subject cell and the culture derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all progeny have exactly the same DNA content. Included are mutant progeny that have the same function or biological activity as the original transformed cell from which it was screened.
  • composition refers to a mixture containing one or more active ingredients such as the antibodies described herein and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein.
  • cyno or “cynomolgus” refers to cynomolgus monkeys (Macaca fascicularis).
  • the individual or subject is a human.
  • administering refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.
  • biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory
  • Treatment refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology.
  • the desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis.
  • the antibodies of the present disclosure are used to delay the development of the disease or slow the progression of the disease.
  • Effective amount is generally enough to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or potential causes, prevent symptoms and/or their potential causes from occurring and/or improve or ameliorate the damage (e.g., lung disease) caused by or associated with the disease state.
  • the effective amount is a therapeutically effective amount or a preventive effective amount.
  • “Therapeutically effective amount” is enough to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way.
  • Preventive effective amount is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the onset (or recurrence) possibility of the disease state or related symptoms. Complete treatment or preventive effect may not occur after administering one dose, but may occur after administering a series of doses. Thus, the therapeutic or preventive effective amount can be administered in one or more administrations.
  • a “therapeutically effective amount” and a “prophylactically effective amount” may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.
  • mice Balb/c mice were used, and CHO (Chinese hamster ovary cells) cells (CHO-hCCR8) overexpressing human CCR8 and a fusion protein of amino acids 1-35 at the N-terminus of human CCR8 and mouse Fc (CCR8N-term-mFc) were used as immunogens for multiple immunizations. This was performed once every one to two weeks, for a total of 6-10 times. After the fourth immunization, blood was collected from the mice's orbits, and the serum titer of anti-human CCR8 antibodies was detected by ELISA and flow cytometry.
  • CHO Choinese hamster ovary cells
  • CCR8N-term-mFc mouse Fc
  • the mouse serum was diluted in multiples and incubated with Raji cell lines (human BURKITT'S lymphoma cells) and Raji cell lines (Raji-hCCR8) overexpressing human CCR8, and stained with PE-labeled goat anti-mouse Fc antibodies (Biolegend), and the serum titer was detected by flow cytometry.
  • CCR8N-term-hFc protein (a fusion protein of amino acids 1-35 at the N-terminus of human CCR8 and human Fc) was coated in a 96-well plate, and serum diluted in multiples was added for incubation, followed by the addition of HRP-labeled goat anti-mouse Fc antibody (Sigma), and the serum titer was detected by ELISA.
  • the mice with the highest titer were determined based on the results of flow cytometry and ELISA, and the mice were euthanized, spleen cells were collected, and fused with the SP20 cell line.
  • Monoclonal cells with strong binding activity to Raji-hCCR8 and no binding to Raji cell line were selected for expansion culture. After one week, the supernatant was collected and the antibodies in the supernatant were purified.
  • Count the Raji-hCCR8 and Raji cells adjust the density to 2E6/ml, and add 100 ⁇ L/well of cell suspension Place in a 96-well plate, centrifuge at 400 ⁇ g for 5 minutes, and discard the supernatant.
  • the binding test results of anti-CCR8 antibodies and Raji-hCCR8 are shown in Figure 1.
  • the experimental results show that the six hybridoma purified antibodies F022-7, F022-59, F022-63, F022-66, F022-68, and F022-75 exhibited strong binding activity with Raji-hCCR8.
  • the sequences of hybridoma purified antibodies of F022-7, F022-59, F022-63, F022-66, F022-68 and F022-75 were retrieved, and the sequencing results showed that the light chain constant regions of all hybridoma purified antibodies were of mouse ⁇ type (as shown in SEQ ID No.40), the heavy chain constant regions of F022-7, F022-63 and F022-75 were of mIgG2a type (as shown in SEQ ID No.41), and the heavy chain constant regions of F022-59, F022-66 and F022-68 were of mIgG1 type (as shown in SEQ ID No.39).
  • the heavy chain constant region of the hybridoma purified antibody was replaced with mouse mIgG2a, and the other sequences remained unchanged.
  • the antibody was cloned into the pCDNA3.4A expression plasmid, and the plasmid was transfected into the EXPI293 cell line. The supernatant was collected after 7 days and purified with protein A to obtain the recombinant antibody.
  • the recombinant antibodies were named F022-7-mIgG2a, F022-59-mIgG2a, F022-63-mIgG2a, F022-66-mIgG2a, F022-68-mIgG2a, and F022-75-mIgG2a, respectively.
  • the recombinant antibody "F022-59-mIgG2a” represents the antibody formed after the heavy chain constant region of the hybridoma purified antibody “F022-59” was replaced with “mIgG2a", and the others are similar.
  • the heavy chain constant region of the hybridoma purified antibody was replaced with human hIgG1Fc (DLE) (i.e., the Fc of human IgG1 was mutated with S239D/A330L/I332E, and the sequence is shown in SEQ ID No.42), and the light chain constant region was replaced with human ⁇ light chain constant region (as shown in SEQ ID No.43), while other sequences remained unchanged, and cloned into the pCDNA3.4A expression plasmid.
  • the plasmid was transfected into the EXPI293 cell line, and the supernatant was collected after 7 days.
  • the recombinant antibodies were obtained after purification with protein A.
  • the recombinant antibodies were named F02 2-7-hIgG1(DLE), F022-59-hIgG1(DLE), F022-63-hIgG1(DLE), F022-66-hIgG1(DLE), F022-68-hIgG1(DLE), F022-75-hIgG1(DLE), for example, the recombinant antibody "F022-59-hIgG1(DLE)” means the heavy chain constant region of the hybridoma purified antibody "F022-59” is replaced by human hIgG1(DLE) and the light chain constant region is replaced by human ⁇ light chain constant region, and the others are similar.
  • the variable region sequences of the antibodies are shown in Table 1, and the CDRs sequences are shown in Table 2:
  • amino acid sequence of the mouse mIgG1 heavy chain constant region (SEQ ID No.39):
  • amino acid sequence of the constant region of the mouse mIgG2a heavy chain (SEQ ID No.41):
  • F022-59, F022-59-mIgG2a, and F022-59-hIgG1(DLE) are as follows:
  • amino acid sequence of the light chain variable region of F022-59 (SEQ ID No.45):
  • amino acid sequence of the heavy chain variable region of F022-59-mIgG2a (SEQ ID No.46):
  • amino acid sequence of the light chain variable region of F022-59-mIgG2a (SEQ ID No.47):
  • amino acid sequence of the light chain variable region of F022-59-hIgG1(DLE) (SEQ ID No.49):
  • amino acid sequence of the positive control antibody 4A19-mIgG2a (obtained by connecting the 4A19 antibody variable region in the WO2021194942A1 patent to the mIgG2a constant region) is as follows:
  • Amino acid sequence of 4A19-mIgG2a heavy chain (SEQ ID No.50):
  • amino acid sequence of 4A19-mIgG2a light chain (SEQ ID No.51):
  • PBMC Peripheral blood mononuclear cells
  • Lymphoprep Axis-Shield
  • Tregs were isolated from PBMC using EasySep Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit (Stem Cell) according to the instructions.
  • the isolated Tregs were mixed with Dynabeads Human T-Activator CD3/CD28 (Gibco) in a 1:1 ratio and cultured with complete medium (90% X-VIVO 15+10% FBS+500IU/ml IL-2, X-VIVO 15: Lonza, FBS: Gibco, IL-2: Tetracycline) for 5 to 8 days to activate and expand Treg cells.
  • the binding activity of the recombinant antibody to Raji-hCCR8, Treg (after activation), Raji, PBMC and Raji-hCCR4 (Raji cells overexpressing human CCR4) was detected by flow cytometry.
  • the experimental method was the same as in Example 2 above.
  • the mIgG2a subtype antibody was detected using a PE-labeled anti-mouse Fc secondary antibody (Biolegend), and the hIgG1 (DLE) subtype antibody was detected using an Alexa Fluor 647-labeled anti-human Fab secondary antibody (Jackson).
  • the experimental results are shown in Figures 2-6.
  • the recombinant antibodies all showed strong binding activity with Raji-hCCR8 (Figure 2) and Treg ( Figure 3). At the same time, the recombinant antibodies did not bind to Raji ( Figure 4), PBMC ( Figure 5) and Raji-hCCR4 ( Figure 6), which shows that the anti-CCR8 antibodies disclosed in the present invention have good
  • hCD16 molecules were expressed in NK-92 cells to detect antibody-mediated ADCC function.
  • Jurkat-hCCR8 cells Jurkat, human peripheral blood leukemia T cell line, ATCC
  • CFSE human peripheral blood leukemia T cell line
  • NK-92-hCD16 cells were added to a 96-well plate at a density of 1E5/well, followed by the addition of anti-CCR8 antibodies with hIgG1Fc (DLE) and incubation at 37 degrees for 6 hours. The 96-well plate was taken out and PI staining was added.
  • MC38 cells (3E5/mouse) were subcutaneously injected into hCCR8 knock-in mice (B-hCCR8, Biocytogen) to establish a tumor model.
  • F022-7-mIgG2a, F022-59-mIgG2a, and F022-75-mIgG2a antibodies were intravenously injected, and isotype-independent target antibodies were used as negative controls, and 4A19-mIgG2a was used as a positive control.
  • the dosage was 10 mg/kg, and the drug was administered twice a week for a total of 6 times.
  • the experimental results are shown in Table 3 and Figure 8 , and the recombinant antibodies have significant anti-tumor effects in vivo.

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Abstract

一种抗CCR8抗体及其应用。所述的抗CCR8抗体包括重链互补决定区和轻链互补决定区，其能够特异性结合人CCR8抗原，可用于***。

Description

抗CCR8抗体及其应用
本申请请求2022年12月07日向中国国家知识产权局提交的专利申请号为202211578659.6，发明名称为“抗CCR8抗体及其应用”的中国专利申请的优先权和权益，并且通过参照将其全文并入本申请。
技术领域
本公开涉及生物技术领域，具体地，涉及一种抗CCR8抗体及其应用。
背景技术
随着人们对肿瘤免疫微环境认识的不断提高，Treg细胞对抑制机体抗肿瘤免疫应答的作用被越来越重视。
趋化因子受体8(Chemokine receptor 8，简称CCR8)为七跨膜G蛋白偶联受体,属于趋化因子受体CC亚家族。近年来的一些研究证明，在多种小鼠肿瘤模型及人肿瘤组织中仅肿瘤组织浸润的Treg细胞广泛高表达CCR8，而在外周血中的Treg细胞及其他PBMC均不表达CCR8，CCR8已被视为肿瘤浸润Treg的表面标志。靶向CCR8是一种很有前景的癌症免疫治疗方法。但CCR8为七次跨膜的GPCR受体，胞外端非常短且不连续，相较于一般单次跨膜的蛋白而言，制备抗体的难度更大。
发明内容
本公开开发一种抗CCR8抗体。在一些实施方案中，其可增强机体抗肿瘤免疫反应，提高肿瘤患者生存率。
在一些实施方案中，本公开公开一种抗人CCR8抗体，所述抗体包含重链可变区和轻链可变区，所述重链可变区包括SEQ ID No.1、3、5、7、9或11中的HCDR1、HCDR2和HCDR3，和/或所述抗体的轻链可变区包括SEQ ID No.2、4、6、8、10或12中的LCDR1、LCDR2和LCDR3；在一些实施方案中，其中，
A.所述抗体的重链可变区包括SEQ ID No.1中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.2中的LCDR1、LCDR2和LCDR3；
B.所述抗体的重链可变区包括SEQ ID No.3中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.4中的LCDR1、LCDR2和LCDR3；
C.所述抗体的重链可变区包括SEQ ID No.5中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.6中的LCDR1、LCDR2和LCDR3；
D.所述抗体的重链可变区包括SEQ ID No.7中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.8中的LCDR1、LCDR2和LCDR3；
E.所述抗体的重链可变区包括SEQ ID No.9中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.10中的LCDR1、LCDR2和LCDR3；或者
F.所述抗体的重链可变区包括SEQ ID No.11中的HCDR1、HCDR2和HCDR3，并 且所述抗体的轻链可变区包括SEQ ID No.12中的LCDR1、LCDR2和LCDR3；
在一些实施方案中，前述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号***定义，或由Kabat编号***定义，或由Chothia编号***定义，或由Contact编号***定义，或由AbM编号***定义；在一些实施方案中，前述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3根据Kabat编号***定义。在一些实施方案中，前述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3根据IMGT编号***定义。
在一些实施方案中，如上任一项所述的抗人CCR8抗体，其中，
a.重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.14的氨基酸序列，和HCDR3包含SEQ ID NO.15的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；
b.重链可变区的HCDR1包含SEQ ID NO.19的氨基酸序列，HCDR2包含SEQ ID NO.20的氨基酸序列，和HCDR3包含SEQ ID NO.21的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；
c.重链可变区的HCDR1包含SEQ ID NO.22的氨基酸序列，HCDR2包含SEQ ID NO.23的氨基酸序列，和HCDR3包含SEQ ID NO.24的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.25的氨基酸序列，LCDR2包含SEQ ID NO.26的氨基酸序列，和LCDR3包含SEQ ID NO.27的氨基酸序列；
d.重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.28的氨基酸序列，和HCDR3包含SEQ ID NO.29的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.30的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；
e.重链可变区的HCDR1包含SEQ ID NO.31的氨基酸序列，HCDR2包含SEQ ID NO.32的氨基酸序列，和HCDR3包含SEQ ID NO.33的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.34的氨基酸序列，LCDR2包含SEQ ID NO.35的氨基酸序列，和LCDR3包含SEQ ID NO.36的氨基酸序列；或
f.重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.37的氨基酸序列，和HCDR3包含SEQ ID NO.38的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；
在一些实施方案中，如上任一项所述的抗人CCR8抗体，其包含分别如SEQ ID NO.13、SEQ ID NO.14和SEQ ID NO.15所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.16、SEQ ID NO.17和SEQ ID NO.18所示的LCDR1、LCDR2和LCDR3；或者所述抗人CCR8抗体包含分别如SEQ ID NO.19、SEQ ID NO.20和SEQ ID NO.21 所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.16、SEQ ID NO.17和SEQ ID NO.18所示的LCDR1、LCDR2和LCDR3；或者所述抗人CCR8抗体包含分别如SEQ ID NO.22、SEQ ID NO.23和SEQ ID NO.24所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.25、SEQ ID NO.26和SEQ ID NO.27所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如上任一项所述的抗人CCR8抗体，所述抗体为鼠源抗体，嵌合抗体或人源化抗体。
在一些实施方案中，如上任一项所述的抗人CCR8抗体，其中，所述抗体的重链可变区包括与SEQ ID No.1、3、5、7、9或11具有至少90％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和/或轻链可变区包括与SEQ ID No.2、4、6、8、10或12具有至少90％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。在一些实施方案中，如上任一项所述的抗人CCR8抗体，其中，所述抗体的重链可变区包括与SEQ ID No.1、3、5、7、9或11具有至少90％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，并且轻链可变区包括与SEQ ID No.2、4、6、8、10或12具有至少90％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，本公开提供一种抗人CCR8抗体，其中，所述抗体的重链可变区包括与SEQ ID No.1、3、5、7、9或11具有至少90％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和/或轻链可变区包括与SEQ ID No.2、4、6、8、10或12具有至少90％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。在一些实施方案中，如上任一项所述的抗人CCR8抗体，其中，所述抗体的重链可变区包括与SEQ ID No.1、3、5、7、9或11具有至少90％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，并且轻链可变区包括与SEQ ID No.2、4、6、8、10或12具有至少90％(例如90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗人CCR8抗体，(i)所述重链可变区包含SEQ ID NO.1的氨基酸序列，和所述轻链可变区包含SEQ ID NO.2的氨基酸序列；
(ii)所述重链可变区包含SEQ ID NO.3的氨基酸序列，和所述轻链可变区包含SEQ ID NO.4的氨基酸序列；
(iii)所述重链可变区包含SEQ ID NO.5的氨基酸序列，和所述轻链可变区包含SEQ ID NO.6的氨基酸序列；
(iv)所述重链可变区包含SEQ ID NO.7的氨基酸序列，和所述轻链可变区包含SEQ ID NO.8的氨基酸序列；
(v)所述重链可变区包含SEQ ID NO.9的氨基酸序列，和所述轻链可变区包含SEQ  ID NO.10的氨基酸序列；
(vi)所述重链可变区包含SEQ ID NO.11的氨基酸序列，和所述轻链可变区包含SEQ ID NO.12的氨基酸序列；
在一些实施方案中，如上任一项所述的抗人CCR8抗体，所述抗体还包含恒定区；在一些实施方案中，所述抗体的重链恒定区选自IgG1、IgG2、IgG3和IgG4的重链恒定区，所述轻链恒定区选自κ或λ链恒定区；在一些实施方案中，所述恒定区的种属来源为鼠或人；在一些实施方案中，所述重链恒定区为鼠IgG2a恒定区、鼠IgG1恒定区、人IgG1恒定区；在一些实施方案中，重链恒定区为人IgG1(DLE)或人IgG1(DE)恒定区；和/或轻链恒定区为鼠κ恒定区或人κ恒定区。在一些实施方案中，所述重链恒定区包括SEQ ID NO.39、41或42的氨基酸序列，轻链恒定区包括SEQ ID NO.40或43的氨基酸序列。在一些实施方案中，所述抗人CCR8抗体重链包含SEQ ID NO.44的氨基酸序列，轻链包括SEQ ID NO.45的氨基酸序列；或者所述抗人CCR8抗体重链包含SEQ ID NO.46的氨基酸序列，轻链包括SEQ ID NO.47的氨基酸序列；或者所述抗人CCR8抗体重链包含SEQ ID NO.48的氨基酸序列，轻链包括SEQ ID NO.49的氨基酸序列
在一些实施方案中，如上任一项所述的抗人CCR8抗体，所述抗体为全长抗体或为选自F(ab’)2、Fab’-SH、Fab’、Fab、scFab、dsFv、(dsFv)2、Fv和scFv中的任意一种抗原结合片段。
在一些实施方案中，发明公开与前面任一项所述的抗人CCR8抗体竞争性结合人CCR8的抗体，或与前面任一项所述的抗人CCR8抗体结合相同表位的抗体。
在一些实施方案中，如上任一项所述的抗人CCR8抗体，其中所述抗人CCR8抗体具有以下特性中的至少一种：
A.所述抗人CCR8抗体能与人CCR8特异性结合；在一些实施方案中，所述抗人CCR8抗体能以≤5nM(例如，≤4.50nM、≤3.00nM、≤2.00nM、≤1.00nM、≤0.9nM、≤0.8nM、≤0.7nM、≤0.6nM、≤0.5nM、≤0.4nM、≤0.3nM、≤0.2nM、≤0.1nM、≤0.09nM、≤0.08nM或更小)的EC50值与表达人CCR8的Raji细胞结合，其中，所述EC50值通过流式细胞分析法测定；在一些实施方案中，所述EC50值通过本申请实施例2的方法测定
B.所述抗人CCR8抗体具有抑制肿瘤生长功能；在一些实施方案中，所述抗人CCR8抗体肿瘤抑制率大于或等于30％(例如，大于30％、40％、45％、50％、60％、70％、80％、90％或更大)；在一些实施方案中，所述抗人CCR8抗体肿瘤抑制率通过本公开实施例5方法测定；
C.所述抗人CCR8抗体具有ADCC活性；在一些实施方案中，所述抗人CCR8抗体ADCC活性通过本公开实施例4的方法测定。
在一些实施方案中，如上任一项所述的抗人CCR8抗体，其中所述抗人CCR8抗体能与人CCR8特异性结合；在一些实施方案中，所述抗人CCR8抗体能以≤5nM(例如，≤4.50nM、≤3.00nM、≤2.00nM、≤1.00nM、≤0.9nM、≤0.8nM、≤0.7nM、≤0.6nM、 ≤0.5nM、≤0.4nM、≤0.3nM、≤0.2nM、≤0.1nM、≤0.09nM、≤0.08nM或更小)的EC50值与表达人CCR8的Raji细胞结合，其中，所述EC50值通过流式细胞分析法测定；在一些实施方案中，所述EC50值通过本申请实施例2的方法测定
在一些实施方案中，如上任一项所述的抗人CCR8抗体，具有抑制肿瘤生长功能；在一些实施方案中，所述抗人CCR8抗体肿瘤抑制率大于或等于30％(例如，大于30％、40％、45％、50％、60％、70％、80％、90％或更大)；在一些实施方案中，所述抗人CCR8抗体肿瘤抑制率通过本公开实施例5方法测定；
在一些实施方案中，如上任一项所述的抗人CCR8抗体，具有ADCC活性；在一些实施方案中，所述抗人CCR8抗体ADCC活性通过本公开实施例4的方法测定。
本公开还提供一种多特异性抗体，所述多特异性抗体包含前面任一项所示的抗人CCR8抗体。在一些实施方案中，所述抗体为双特异性抗体。
本公开还提供一种抗体偶联物，所述抗体偶联物包括前面任一项所述的抗人CCR8抗体。在一些实施方案中，所述抗体偶联物还包括与所述抗体偶联的治疗剂或显像剂；在一些实施方案中，所述抗体偶联物还包括与所述抗体偶联的生物素或生物素衍生物；在一些实施方案中，所述抗体偶联物还包括与所述抗体偶联的固相载体；在一些实施方案中，所述抗体偶联物还包括与所述抗体偶联的标记物；在一些实施方案中，所述标记物选自荧光染料、酶、放射性同位素、化学发光试剂和纳米颗粒类标记物中的至少一种；在一些实施方案中，所述标记物为胶体金。
本公开还提供一种嵌合抗原受体(CAR)，所述嵌合抗原受体包括抗原结合结构域，所述抗原结合结构域包括前面任一项所述的抗人CCR8抗体。
本公开还提供一种CAR-免疫细胞，所述CAR-免疫细胞表达前面所述的嵌合抗原受体。
本公开还提供一种分离的核酸，其编码前面任一项所述的抗人CCR8抗体。
本公开还提供一种细胞，其含有权利要求8所述的核酸。
本公开还提供一种药物组合物，其包含：前面任一项所述的抗人CCR8抗体、多特异性抗体、抗体偶联物、细胞或核酸，在一些实施方案中，所述药物组合物还包含一种或多种药学上可接受的载体、稀释剂或赋形剂。
本公开还提供前面任一项所述的抗人CCR8抗体、多特异性抗体、抗体偶联物、细胞、核酸或药物组合物在制备具有以下至少一种用途的产品中的应用：诊断人CCR8表达异常的相关疾病、治疗人CCR8表达异常的相关疾病或检测人CCR8的表达；在一些实施方案中，所述人CCR8表达异常的相关疾病为肿瘤。所述人CCR8表达异常的肿瘤优选为乳腺癌、卵巢癌、肾癌、胰腺癌、膀胱癌、胃癌、***、结肠癌、肉瘤、肝癌或肺癌。
在一些实施方案中，本公开提供一种治疗疾病的方法，所述方法包括向有需要的受试者施用治疗有效量的前面任一项所述的抗人CCR8抗体、多特异性抗体、抗体偶联物、细胞、核酸或药物组合物的步骤。在一些实施方案中，所述疾病为人CCR8表达异常的 相关疾病。在一些实施方案中，所述疾病为肿瘤。所述人CCR8表达异常的肿瘤优选为乳腺癌、卵巢癌、肾癌、胰腺癌、膀胱癌、胃癌、***、结肠癌、肉瘤、肝癌或肺癌。
在另一个方面，本公开还提供用作药物的前述任一项所述的抗人CCR8抗体、多特异性抗体、抗体偶联物、细胞、核酸或药物组合物。在一些实施方案中，药物用于治疗疾病。在一些实施方案中，所述疾病为人CCR8表达异常的相关疾病。在一些实施方案中，所述人CCR8表达异常的相关疾病为肿瘤。所述人CCR8表达异常的肿瘤优选为乳腺癌、卵巢癌、肾癌、胰腺癌、膀胱癌、胃癌、***、结肠癌、肉瘤、肝癌或肺癌。
在一些实施方案中，前面任一项所述的治疗，进一步包括向受试者施用另外的治疗药物。在一些实施方案中，所述另外的治疗药物为化疗剂，例如铂络合物、紫杉烷、培美曲塞、吉西他滨、氟尿嘧啶、伊立替康、依托泊苷或多柔比星等。
本公开还提供一种检测或测定人CCR8的方法，所述方法包括使用如前任一项所述抗人CCR8抗体或抗体偶联物检测或测定人CCR8的步骤。
本公开还提供一种试剂盒，其包含如前任一项所述的抗人CCR8抗体或抗体偶联物；在一些实施方案中，所述试剂盒用于检测或测定人CCR8。
本公开还提供了一种制备如前任一项所述的抗人CCR8抗体的方法，其包括培养如前任一项所述的细胞，然后分离和纯化获得抗人CCR8抗体的步骤。
本公开公开的抗人CCR8抗体能够特异性结合人CCR8；在一些实施方案中，所述抗体在体外和体内均具有显著的抗肿瘤作用；在一些实施方案中，还具有很好的ADCC活性。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为杂交瘤纯化抗体与Raji-hCCR8的结合检测结果图；
图2为重组抗体与Raji-hCCR8的结合检测结果图；
图3为重组抗体与Treg的结合检测结果图；
图4为重组抗体与Raji的结合检测结果图；
图5为重组抗体与PBMC的结合检测结果图；
图6为重组抗体与Raji-hCCR4的结合检测结果图；
图7为重组抗体ADCC活性实验结果图；
图8抗CCR8抗体抑制肿瘤生长实验结果图。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面对本公开实施例中的技术方案进行清楚、完整地描述，但是描述和实施例不应被解释为限制本公开的范围。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，均为可以通过市售购买获得的常规产品。除非另外定义，本文所用的全部技术术语和科学术语具有与本公开所属领域的普通技术人员通常所理解的相同含义。
本公开中，所用的单数形式“一个”、“一种”和“所述”包括复数指代，除非上下文清楚表明并非如此。另外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。术语“多个”的含义是至少两个，例如2个、3个等，除非另有明确具体的限定。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“氨基酸”是指天然存在的氨基酸和合成的氨基酸，以及以与天然存在的氨基酸类似的方式起作用的氨基酸类似物或氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸；常见的天然氨基酸包括丙氨酸(Ala；A)、精氨酸(Arg；R)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、半胱氨酸(Cys；C)；谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)；组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、甲硫氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)、丝胺酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。氨基酸类似物是指与天然存在的氨基酸具有相同基本化学结构(即与氢、羧基、氨基和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。此类类似物具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有与氨基酸的一般化学结构不同的结构，但是以与天然存在的氨基酸类似的方式起作用的化学化合物。
在本公开中，术语“抗体”在最广义上使用，该术语涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体、三特异性抗体、四特异性抗体等)、鼠源抗体、嵌合抗体、人源化抗体、全长抗体或其抗原结合片段(或称抗原结合部分)，只要它们展示出所期望的抗原结合活性。
“天然抗体”指天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异四聚糖蛋白，由两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个重链可变区(VH，又称作可变重域)，接着是重链恒定区，天然IgG重链恒定区通常包括三个恒定域(CH1、CH2和CH3)。类似地，从N至C端，每条轻链具有一个轻链可变区(VL，又称作可变轻域)，接着是一个轻链恒定区(CL，也称轻链恒定域)。
术语“全长抗体”或“完整抗体”指包含与天然抗体结构基本类似的结构的抗体，或重链包含Fc区的抗体。
术语抗体“可变区”或“可变域”指抗体重链或轻链中涉及抗体结合抗原的域。本文中，抗体重链可变区(VH)和轻链可变区(VL)各包含四个保守的框架区(FR)和三个互 补决定区(CDR)。术语“互补决定区”或“CDR”指可变区内主要促成与抗原特异性结合的区域；“框架”或“FR”是指可变区中除CDR残基之外的可变结构域残基。VH包含3个CDR区：HCDR1、HCDR2和HCDR3；VL包含3个CDR区：LCDR1、LCDR2和LCDR3。每个VH和VL由从氨基末端(也称N末端)排到羧基末端(也称C末端)按以下顺序排列的3个CDR和4个FR构成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。在本公开具体实施方式中，CDRs是指所述抗体的重链和轻链中2个以上CDR。可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003))等；各种编号***之间的对应关系是本领域技术人员熟知的。
在一些实施方案中，本公开抗人CCR8抗体，其包含重链可变区和轻链可变区，所述重链可变区包括SEQ ID No.1、3、5、7、9或11中的HCDR1、HCDR2和HCDR3，和/或所述抗体的轻链可变区包括SEQ ID No.2、4、6、8、10或12中的LCDR1、LCDR2和LCDR3。
在一些实施方案中，本公开抗人CCR8抗体，其包含重链可变区和轻链可变区，所述重链可变区包括SEQ ID No.1、3、5、7、9或11中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.2、4、6、8、10或12中的LCDR1、LCDR2和LCDR3。
在一些实施方案中，本公开抗人CCR8抗体，其中，A.所述抗体的重链可变区包括SEQ ID No.1中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.2中的LCDR1、LCDR2和LCDR3；B.所述抗体的重链可变区包括SEQ ID No.3中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.4中的LCDR1、LCDR2和LCDR3；C.所述抗体的重链可变区包括SEQ ID No.5中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.6中的LCDR1、LCDR2和LCDR3；D.所述抗体的重链可变区包括SEQ ID No.7中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.8中的LCDR1、LCDR2和LCDR3；E.所述抗体的重链可变区包括SEQ ID No.9中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.10中的LCDR1、LCDR2和LCDR3；或者F.所述抗体的重链可变区包括SEQ ID No.11中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.12中的LCDR1、LCDR2和LCDR3。在一些实施方案中，前述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号***定义，或由Kabat编号***定义，或由Chothia编号***定义，或由Contact编号***定义，或由AbM编号***定义。
在一些实施方案中，本公开抗人CCR8抗体，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3根据Kabat编号***定义。
在一些实施方案中，本公开抗人CCR8抗体，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3根据IMGT编号***定义。
在一些实施方案中，本公开所述的抗人CCR8抗体，其中，a.重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.14的氨基酸序列，和HCDR3包含SEQ ID NO.15的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；b.重链可变区的HCDR1包含SEQ ID NO.19的氨基酸序列，HCDR2包含SEQ ID NO.20的氨基酸序列，和HCDR3包含SEQ ID NO.21的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；c.重链可变区的HCDR1包含SEQ ID NO.22的氨基酸序列，HCDR2包含SEQ ID NO.23的氨基酸序列，和HCDR3包含SEQ ID NO.24的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.25的氨基酸序列，LCDR2包含SEQ ID NO.26的氨基酸序列，和LCDR3包含SEQ ID NO.27的氨基酸序列；d.重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.28的氨基酸序列，和HCDR3包含SEQ ID NO.29的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.30的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；e.重链可变区的HCDR1包含SEQ ID NO.31的氨基酸序列，HCDR2包含SEQ ID NO.32的氨基酸序列，和HCDR3包含SEQ ID NO.33的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.34的氨基酸序列，LCDR2包含SEQ ID NO.35的氨基酸序列，和LCDR3包含SEQ ID NO.36的氨基酸序列；或f.重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.37的氨基酸序列，和HCDR3包含SEQ ID NO.38的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列。
在一些实施方案中，本公开所述的抗人CCR8抗体，其包含分别如SEQ ID NO.13、SEQ ID NO.14和SEQ ID NO.15所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.16、SEQ ID NO.17和SEQ ID NO.18所示的LCDR1、LCDR2和LCDR3；或者所述抗人CCR8抗体包含分别如SEQ ID NO.19、SEQ ID NO.20和SEQ ID NO.21所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.16、SEQ ID NO.17和SEQ ID NO.18所示的LCDR1、LCDR2和LCDR3；或者所述抗人CCR8抗体包含分别如SEQ ID NO.22、SEQ ID NO.23和SEQ ID NO.24所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.25、SEQ ID NO.26和SEQ ID NO.27所示的LCDR1、LCDR2和LCDR3；或者所述抗人CCR8抗体包含分别如SEQ ID NO.13、SEQ ID NO.28和SEQ ID NO.29所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.16、SEQ ID NO.30 和SEQ ID NO.18所示的LCDR1、LCDR2和LCDR3；或者所述抗人CCR8抗体包含分别如SEQ ID NO.30、SEQ ID NO.31和SEQ ID NO.32所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.33、SEQ ID NO.34和SEQ ID NO.35所示的LCDR1、LCDR2和LCDR3；或者所述抗人CCR8抗体包含分别如SEQ ID NO.13、SEQ ID NO.36和SEQ ID NO.37所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.16、SEQ ID NO.17和SEQ ID NO.18所示的LCDR1、LCDR2和LCDR3。
术语“抗原结合片段”或“抗原结合域”指不同于完整抗体的分子，其包含完整抗体的部分，所述部分能够与完整抗体所结合的抗原特异性结合。抗原结合片段的实例包括但不限于Fv(由VH和VL组成)、Fab(由一条轻链和一条重链的恒定区1(CH1)及重链可变区组成)、Fab'(由Fab区和铰链区组成)、Fab'-SH(Fab'片段的铰链区的半胱氨酸残基携带游离硫醇基团)、(Fab')2(二聚化Fab')、scFv(单链抗体分子，由轻链可变区与重链可变区直接相连或通过连接子连接)、scFab(单链Fab)、dsFv(二硫键稳定化的Fv片段)、(dsFv)2(二聚化dsFv)，以及由抗体片段形成的多特异性抗体。
术语“Fc区”用于定义抗体重链的C末端区域，包括天然Fc区和改造的Fc区。在一些实施方式中，用于本文所述抗体的Fc区包括人IgG1、IgG2(IgG2a、IgG2b)、IgG3和IgG4的Fc区。在一些实施方式中，Fc区为包含DLE突变的人IgG1(也即人IgG1的Fc进行S239D/A330L/I332E突变，序列如SEQ ID No.42所示)。在一些实施方式中，Fc区为包含DE突变的人IgG1(也即人IgG1的Fc进行S239D/I332E突变)。在一些实施方式中，Fc区的边界还可以变化，例如缺失Fc区的C末端赖氨酸(根据EU编号***的残基447)或缺失Fc区的C末端甘氨酸和赖氨酸(根据EU编号***的残基446和447)。除非另有说明，Fc区的编号规则为EU编号***，又称作EU索引。
术语“嵌合”抗体指抗体中的重链和/或轻链的一部分源自一种物种，而重链和/或轻链的其它部分源自另外一种物种。
术语“人源化”抗体是保留非人抗体的反应性，同时在人中具有较低免疫原性的抗体。例如，可以通过保留非人CDR区，其余部分用人源抗体对应物(即，恒定区以及可变区的框架区部分)替换来实现。
术语“人抗体”、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，意指可变区及恒定区是人序列的抗体。
术语“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，结合“亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间1:1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(KD)表示。亲和力可以通过本领域已知的常规方法测量。术语“kassoc”或“ka”指特定抗体-抗原相互作用的缔合速率，术语“kdis”或“kd”指特定抗体-抗原相互作用的解离速率。术语“KD”指解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域公知的方法测定抗体的KD值。例如，使用生物传感***例如***测量表面等离子体共振(例如Biacore)，或 通过溶液平衡滴定法(SET)测量溶液中的亲和力。
术语“效应子功能”指那些可归于抗体Fc区(天然序列Fc区或氨基酸序列突变的Fc区)的生物学活性。抗体效应子功能的例子包括但不限于：C1q结合和补体依赖性细胞毒性、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、吞噬作用、细胞表面受体(例如B细胞受体)下调；和B细胞活化。
术语“单克隆抗体”指基本上均质的抗体的群，即在该群中包含的抗体分子的氨基酸序列是相同的，除了可能少量存在的天然突变以外。相比之下，多克隆抗体制剂通常包含在其可变结构域具有不同氨基酸序列的多种不同抗体，其通常特异性针对不同表位。“单克隆”表示从基本上均质的抗体群体获得的抗体的特征，并且不应解释为要求通过任何特定方法来生产抗体。在一些实施方式中，本公开提供的抗体是单克隆抗体。
术语“抗原”是指能够由抗原结合蛋白(例如抗体)选择性结合剂结合的分子。抗原可具有一个或多个能够与不同的抗原结合蛋白(例如抗体)相互作用的表位。
术语“表位”指能够与抗体特异性结合的抗原上的区域(area或region)。表位可以由连续氨基酸串(线性表位)形成或包含非连续氨基酸(构象表位)，例如因抗原的折叠(即通过蛋白质性质的抗原的三级折叠)而变成空间接近。构象表位和线性表位的差别在于：在变性溶剂的存在下，抗体对构象表位的结合丧失。构象表位包含处于独特空间构象的至少3，至少4，至少5，至少6，至少7，或8-10个氨基酸。筛选结合特定表位的抗体(即那些结合相同表位的)可以使用本领域例行方法来进行，例如丙氨酸扫描，肽印迹，肽切割分析，表位切除，表位提取，抗原的化学修饰(见Prot.Sci.9(2000)487-496)，和交叉阻断。
与参照抗体结合相同表位的抗体或与参照抗体竞争结合的抗体意指在竞争测定法中，将参照抗体对其抗原的结合阻断50％或更多的抗体，或其对抗原的结合被参照抗体阻断50％或更多的抗体。例如，为了测定待测抗体是否与参照抗体结合相同表位，在饱和条件下容许参照抗体结合抗原，然后在去除过量的参照抗体后，评估待测抗体结合抗原的能力；如果该待测抗体能够在参照抗体的饱和结合之后结合抗原，那么可以得出结论，该待测抗体与参照抗体结合不同表位；而如果该待测抗体在参照抗体的饱和结合之后不能够结合抗原，那么该待测抗体可结合与参照抗体结合相同的表位。为了确认待测抗体是否结合相同表位，可以使用常规实验(例如，肽突变和使用ELISA、RIA、表面等离子共振、流式细胞术或本领域可获得的任何其它定量或定性抗体结合测定的结合分析)检测。在一些实施方案中，如在竞争性结合测定中所测量的(参见例如，Junghans等，Cancer Res.50(1990)1495-1502)，如果一个过量(例如1倍、5倍、10倍、20倍或100倍的量)的抗体将另一个抗体与抗原的结合抑制至少50％、至少75％、至少90％或甚至99％或更多，则认为两个抗体结合相同或重叠的表位。
术语“能够特异性结合”、“特异性结合”或“结合”是指相比其他抗原或表位，抗体能够以更高的亲和力结合至某个抗原或该抗原内的表位。通常地，抗体以约100nM或更小(例如约10nM、1nM、0.1nM、0.01nM或更小)的平衡解离常数(KD)结合抗原或 抗原内的表位。在一些实施方式中，抗体与抗原结合的KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的10％或更低(例如1％)。可使用已知的方法来测量KD，包括但不限于Biacore测定法、Octet方法、微量热泳法、HPLC‐MS方法和流式细胞荧光分选技术；例如通过表面等离子体共振测定法所测量的。
本公开提供的抗人CCR8抗体与人CCR8的结合也可以用“半最大有效浓度”(EC50)值表示，一般EC50越小亲和力越好，表示更低的浓度下即可以与抗原结合。可以通过本领域已知的结合检测法，例如直接或间接结合检测法(例如酶联免疫吸附检测法(ELISA)、流式细胞荧光分选技术和其他结合检测法)来测定EC50值。
术语“抗体依赖性细胞的细胞毒性”、“抗体依赖性细胞介导的细胞毒性”或“ADCC”是诱导细胞死亡的机制，该机制依赖于抗体包被靶细胞与具有裂解活性的效应细胞(诸如自然杀伤细胞(NK)、单核细胞、巨噬细胞和中性粒细胞)经由效应细胞上表达的Fcγ受体(FcγR)发生的相互作用。例如，NK细胞表达FcγRIIIa，而单核细胞表达FcγRI、FcγRII和FcγRIIIa。本文提供的抗体的ADCC活性可使用体外测定，使用表达抗原的细胞作为靶细胞和NK细胞作为效应细胞进行评定。根据从裂解的细胞中释放的标记物(例如放射性底物、荧光染料或天然胞内蛋白)来检测细胞裂解。
术语“抗体依赖性细胞吞噬作用”(“ADCP”)是指通过吞噬细胞(诸如巨噬细胞或树突状细胞)的内化作用消除抗体包被的靶细胞的机制。
术语“补体依赖性细胞毒性”或“CDC”是指诱导细胞死亡的机制，其中靶结合抗体的Fc效应域结合并激活补体成分C1q，C1q继而激活补体级联，从而导致靶细胞死亡。补体的激活也可导致补体成分沉积在靶细胞表面上，这些补体成分通过结合白细胞上的补体受体(例如，CR3)来促进CDC。
本文中“CAR”为嵌合抗原受体，是人工改造受体，例如其是能够将识别肿瘤细胞表面抗原或病毒抗原的特异性分子(如抗体)锚定在免疫细胞(如T细胞)上，使免疫细胞识别肿瘤抗原或病毒抗原并杀死肿瘤细胞或病毒的受体。表达CAR的T细胞称为CAR-T(或CART)。通常，嵌合抗原受体依次包含胞外结构域、跨膜区域和胞内信号结构域。可采用本领域已知的用于构建CAR的跨膜区域和胞内信号结构域来构建本公开的嵌合抗原受体。肿瘤细胞表面的抗原(受体)与嵌合抗原受体的抗体(配体)结合时，通过铰链区和跨膜区将信号传递至胞内，胞内信号域再将信号转化为活化信号，激活效应细胞，效应细胞通过分泌穿孔素或者产生细胞因子杀伤肿瘤细胞，同时效应细胞本身也发生扩增，进一步扩大免疫杀伤作用。胞外结构域可包含抗原结合结构域(也称特异性结合抗原的抗体部分)，抗原结合结构域可以是特异性结合抗原的单链可变片段(single-chain variable fragment，scFv)。单链可变片段可作为CAR的胞外结构域，该结构域决定CAR-免疫细胞的特异性。铰链区是连接单链抗体单位和跨膜结构域的CAR细胞外结构区，它通常维持效应细胞中稳健的CAR表达和活性所需的稳定性，大多数CAR的铰链区由IgG的铰链或CD8α/CD28胞外区衍变而来。铰链区的类型和长度对CAR的功能活动有重要影响。跨膜结构域将CAR的细胞外结构域与细胞内信号转导结构域连接。 常用的跨膜结构域来源于CD4，CD8α，CD28和CD3ζ。跨膜结构域的选择影响CAR结构在细胞功能上的活化程度。胞内结构域由共刺激结构域和信号转导结构域构成。在可选的实施方式中，所述CAR为嵌合抗原受体，其主要成分还可以包括跨膜结构域和胞内结构域。在可选的实施方式中，所述CAR还可以包括共刺激分子。“共刺激分子”是指存在于抗原提呈细胞表面，能与Th细胞上的共刺激分子受体结合，产生协同刺激信号的分子。T淋巴细胞的增殖不仅需要抗原的结合，还需要接受共刺激分子信号。共刺激信号传递给T细胞主要是通过表达在抗原呈递细胞表面的共刺激分子CD80、CD86与T细胞表面的CD28分子结合。B细胞接受共刺激信号可以通过一般的病原体成分例如LPS，或者通过补体成分，或者通过激活了的抗原特异性的Th细胞表面的CD40L。可采用本领域已知的用于构建CAR的共刺激分子，包括但不限于CD27、CD28、4-1BB、OX40、ICOS、B7-H3和/或它们的任何组合。
术语“CAR-免疫细胞”是指表达CAR的免疫细胞或CAR修饰的免疫细胞，其中，免疫细胞包括但不限于T细胞、自然杀伤(NK细胞)、巨噬细胞(M细胞)、Treg细胞。
术语“核酸”在本文中可与术语“多核苷酸”互换使用，并且是指呈单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。所述术语涵盖含有已知核苷酸类似物或修饰的骨架残基或连接的核酸，所述核酸是合成的、天然存在的和非天然存在的，具有与参考核酸相似的结合特性，并且以类似于参考核苷酸的方式代谢。此类类似物的实例包括但不限于硫代磷酸酯、氨基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色***置的染色***置处。编码多肽或融合蛋白的分离的核酸指编码多肽或融合蛋白的一个或更多个核酸分子，包括在单一载体或分开的载体中的这样的一个或更多个核酸分子，和存在于宿主细胞中一个或更多个位置的这样的一个或更多个核酸分子。除非另有说明，否则特定的核酸序列还隐含地涵盖其保守修饰的变体(例如，简并密码子取代)和互补序列以及明确指明的序列。具体地，如下详述，简并密码子取代可以通过产生如下序列而获得，在这些序列中，一个或多个所选的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代。
术语“多肽”和“蛋白质”在本文中可互换使用，指氨基酸残基的聚合物。该术语适用于氨基酸聚合物，其中一个或多个氨基酸残基是相应天然存在的氨基酸的人工化学模拟物，以及适用于天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。
术语序列“同一性”指，当对两条序列进行最佳比对时(必要时引入间隙，以获取最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分)，两条序列的氨基酸/核酸在等价位置相同的程度(百分比)。为测定序列同一性百分比，比对可以通过本领域技术已知的技术来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定 适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
本公开中，“抗体偶联物”包括抗人CCR8抗体与治疗剂或显像剂偶联而成的偶联物。在可选的实施方式中，所述抗体偶联物由抗人CCR8抗体与治疗剂通过连接子偶联而成；在可选的实施方式中，所述治疗剂为细胞毒剂；在可选的实施方式中，所述治疗剂可选自毒素、化疗剂、抗生素、放射性同位素和核溶酶。
本公开中，“细胞毒剂”是指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质；“毒素”指能够对细胞的生长或增殖产生有害效果的物质(例如美登素类、卡利奇霉素、紫杉烷类、长春新碱、秋水仙碱、澳瑞他汀等)。“化疗剂”指可用于治疗癌症的化学化合物。
在可选的实施方式中，本公开所述抗体偶联物还包括与所述抗体偶联的生物素或生物素衍生物。
在可选的实施方式中，所述抗体偶联物还包括与所述抗体偶联的标记物。
在可选的实施方式中，上述标记物是指具有能够被肉眼直接观察或被仪器检测或探测到的特性例如发光、显色、放射性等特性的一类物质，通过该特性可以实现对相应目标物的定性或定量检测。
在可选的实施方式中，所述标记物选自荧光染料、酶、放射性同位素、化学发光试剂和纳米颗粒类标记物中的至少一种。在实际的使用过程中，本领域技术人员可以根据检测条件或实际需要选择其它合适的标记物，无论使用何种标记物，均属于本公开的保护范围。
在可选的实施方式中，所述荧光染料包括但不限于荧光素类染料及其衍生物(例如包括但不限于异硫氰酸荧光素(FITC)羟基光素(FAM)、四氯光素(TET)等或其类似物)、罗丹明类染料及其衍生物(例如包括但不限于红色罗丹明(RBITC)、四甲基罗丹明(TAMRA)、罗丹明B(TRITC)等或其类似物)、Cy系列染料及其衍生物(例如包括但不限于Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、Cy3等或其类似物)、Alexa系列染料及其衍生物(例如包括但不限于AlexaFluor350、405、430、488、532、546、555、568、594、610、33、647、680、700、750等或其类似物)和蛋白类染料及其衍生物(例如包括但不限于藻红蛋白(PE)、藻蓝蛋白(PC)、别藻蓝蛋白(APC)、多甲藻黄素-叶绿素蛋白(preCP)等)。
在可选的实施方式中，所述酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。在可选的实施方式中，所述放射性同位素包括但不限于212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu和18F。
在可选的实施方式中，所述化学发光试剂包括但不限于鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。在可选的实施方式中，所述纳 米颗粒类标记物包括但不限于纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
在可选的实施方式中，所述胶体包括但不限于胶体金属、胶体硒、分散型染料、染料标记的微球和乳胶。在可选的实施方式中，所述胶体金属包括但不限于胶体金或胶体银。在可选的实施方式中，所述胶体金属为胶体金。
在可选的实施方式中，所述抗体偶联物还可包括与所述抗体偶联的固相载体。抗体偶联物中，所述抗体偶联至固相载体。在可选的实施方式中，所述固相载体选自微球、板和膜。在可选的实施方式中，所述固相包括但不限于磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。在可选的实施方式中，所述固相载体为硝酸纤维素膜。
术语“连接子”、“Linker”或“接头”指连接两个多肽片段的连接单元，通常具有一定的柔性，接头的使用不会使蛋白质结构域原有的功能丧失。连接子可以是肽连接子，其包含一个或多个氨基酸，典型的约1-30个、2-24个或3-15个氨基酸。在一些实施方案中，所述连接子为选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数，在一些实施方案中，所述连接子为选自(GxS)y连接子，其中，x选自1-5的整数，y选自1-6的整数。
另一方面，本公开实施例还提供了一种检测CCR8的方法，其包括：将如前任一项所述的抗体或如前任一项所述的抗体偶联物或如前任一项所述的试剂或试剂盒与待检测样品混合，使所述抗体与待测样本中的CCR8接触，形成免疫复合物。在优选的实施方式中，所述免疫复合物还包括第二抗体，所述第二抗体与所述抗体结合。在优选的实施方式中，所述免疫复合物还包括第二抗体，所述第二抗体与CCR8结合。
另一方面，本公开实施例还提供了一种制备如前任一项所述的抗体的方法，其包括：培养如前任一项所述的细胞步骤。还包括纯化回收抗体的步骤。
在本公开公开了抗体的氨基酸序列的基础上，本领域技术人员可以采用基因工程技术或其他技术(化学合成、重组表达)制备得到该抗体，例如从能够重组表达如上任一项所述的抗体的重组细胞的培养产物中分离纯化得到该抗体，这对本领域技术人员来说是容易实现的，基于此，无论采用何种技术制备本公开的抗体，其均属于本公开的保护范围。
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，例如腺相关病毒载体(AAV或AAV2)，其中另外的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。术语“表达载体”或“表达构建体”是指可对宿主细胞进行转化，且含有指导和/或控制(连同宿主细胞一起)与其可操作地连接的一个或多个异源编码区的表达的核酸序列的 载体。表达构建体可以包括但不限于影响或控制转录、翻译且在存在内含子时影响与其可操作地连接的编码区的RNA剪接的序列。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且指已经导入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及自其衍生的后代，而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，而是可以含有突变。本文中包括具有与在初始转化细胞中筛选或选择的相同功能或生物学活性的突变体后代。宿主细胞包括原核和真核宿主细胞，其中真核宿主细胞包括但不限于哺乳动物细胞、昆虫细胞系植物细胞和真菌细胞。哺乳动物宿主细胞包括人、小鼠、大鼠、犬、猴、猪、山羊、牛、马和仓鼠细胞，包括但不限于中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，Hep G2)、A549细胞、3T3细胞和HEK-293细胞。真菌细胞包括酵母和丝状真菌细胞，包括例如巴氏毕赤酵母(Pichiapastoris)、芬兰毕赤酵母(Pichia finlandica)、海藻毕赤酵母(Pichia trehalophila)、科克拉马毕赤酵母(Pichia koclamae)、膜状毕赤酵母(Pichia membranaefaciens)、小毕赤酵母(Pichia minuta)(Ogataea minuta、Pichia lindneri)、仙人掌毕赤酵母(Pichiaopuntiae)、耐热毕赤酵母(Pichia thermotolerans)、柳毕赤酵母(Pichia salictaria)、Pichia guercuum、皮杰普毕赤酵母(Pichia pijperi)、具柄毕赤酵母(Pichia stiptis)、甲醇毕赤酵母(Pichia methanolica)、毕赤酵母属、酿酒酵母(Saccharomycescerevisiae)、酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、克鲁维酵母属、乳酸克鲁维酵母(Kluyveromyces lactis)、白色念珠菌(Candida albicans)、构巢曲霉(Aspergillus nidulans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、里氏木霉(Trichoderma reesei)、勒克氏菌(Chrysosporium lucknowense)、镰刀菌属(Fusarium sp.)、禾谷镰刀菌(Fusarium gramineum)、菜镰刀菌(Fusarium venenatum)、小立碗藓(Physcomitrella patens)和粗糙脉孢菌(Neurospora crassa)。毕赤酵母属、任何酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、任何克鲁维酵母属、白色念珠菌(Candida albicans)、任何曲霉属、里氏木霉(Trichoderma reesei)、勒克霉菌(Chrysosporium lucknowense)、任何镰刀菌属、解脂耶氏酵母(Yarrowia lipolytica)和粗糙脉孢菌(Neurospora crassa)。
如在本申请中所使用的，“细胞”、“细胞系”和“细胞培养物”可以互换使用，并且所有这样的名称均包括子代。词语“转化体”和“转化的细胞”包括原代受试者细胞和来源于其的培养物，而与传代的次数无关。还应理解的是，由于有意或无意的突变，使得并非所有子代均具有完全相同的DNA内容物。包括与筛选出其的原始转化细胞具有相同功能或生物活性的突变子代。
术语“药物组合物”表示含有一种或多种本文所述的抗体等活性成分与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。
术语“药学上可接受的载体”指药学配制剂中与活性成分不同的,且对受试者无毒的成分。药学可接受载剂包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
术语“受试者”或“个体”包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如，食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。除非指出时，否则所述术语“患者”或“受试者”在本文中可互换地使用。如本文所使用的，术语“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些实施方案中，个体或受试者是人。
“施用”或“给予”，当其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。
术语“样本”是指从受试者分离的类似流体、细胞、或组织的采集物，以及存在于受试者体内的流体、细胞或组织。示例性样本为生物流体，诸如血液、血清和浆膜液、血浆、淋巴液、尿液、唾液、囊液、泪液、***物、痰、分泌组织和器官的粘膜分泌物、***分泌物、腹水、胸膜、心包、腹膜、腹腔和其它体腔的流体、由支气管灌洗液收集的流体、滑液、与受试者或生物来源接触的液体溶液，例如细胞和器官培养基(包括细胞或器官条件培养基)、灌洗液等，组织活检样本、细针穿刺、手术切除的组织、器官培养物或细胞培养物。
“治疗(treatment或treat)”指试图改变所治疗个体的天然过程的临床干预，并且可以为了预防或者在临床病理学的过程期间实施。治疗的期望效果包括但不限于预防疾病的发生或再发生，减轻症状，减轻/减少疾病的任何直接或间接病理后果，预防转移，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。在一些实施方案中，使用本公开的抗体来延迟疾病的形成或减缓疾病的进展。
“有效量”一般是足以降低症状的严重程度及/或频率、消除这些症状及/或潜在病因、预防症状及/或其潜在病因出现及/或改良或改善由疾病状态引起或与其相关的损伤(例如肺病)的量。在一些实施例中，有效量是治疗有效量或预防有效量。“治疗有效量”是足以治疗疾病状态或症状、尤其与该疾病状态相关的状态或症状，或者以其他方式预防、阻碍、延迟或逆转该疾病状态或以任何方式与该疾病相关的任何其他不理想症状的进展的量。“预防有效量”是当给予受试者时将具有预定预防效应，例如预防或延迟该疾病状态的发作(或复发)，或者降低该疾病状态或相关症状的发作(或复发)可能性的量。完全治疗或预防效未必在给予一个剂量之后便发生，可能在给予一系列剂量之后发生。因而，治疗或预防有效量可以一次或多次给予的方式给予。“治疗有效量”和“预防有效量”可取决于多种因素变化：诸如个体的疾病状态、年龄、性别和体重，以及治疗剂或治疗剂组合在个体中引发期望的应答的能力。有效治疗剂或治疗剂组合的示例性指标包括例如患者改善的健康状况。
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。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，均为可以通过市售购买获得的常规产品。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编，1984)；《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编，1987)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《哺乳动物细胞用基因转移载体(Gene Transfer Vectors for Mammalian Cells)》(J.M.Miller和M.P.Calos编，1987)；《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)；以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编，1991)，所述文献中的每个文献均通过引用明确并入本文中。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1.抗CCR8杂交瘤抗体制备
采用Balb/c小鼠，用过表达人CCR8的CHO(中国仓鼠卵巢细胞)细胞(CHO-hCCR8)及人CCR8的N端1-35位氨基酸与小鼠Fc的融合蛋白(CCR8N-term-mFc)作为免疫原，进行多次免疫。每一到两周进行一次，共6-10次。在第四次免疫后，对小鼠进行眼眶取血，通过ELISA及流式细胞术检测抗人CCR8抗体的血清效价。将小鼠血清倍比稀释后与Raji细胞系(人BURKITT'S淋巴瘤细胞)及过表达人CCR8的Raji细胞系(Raji-hCCR8)共同孵育，并通过PE标记的山羊抗小鼠Fc抗体(Biolegend)进行染色，通过流式细胞术检测血清效价。同时，将CCR8N-term-hFc蛋白(人CCR8的N端1-35位氨基酸与人Fc的融合蛋白)包被至96孔板中，加入倍比稀释的血清孵育，随后加入HRP标记的山羊抗小鼠Fc抗体(Sigma)，通过ELISA检测血清效价。根据流式细胞术及ELISA的结果确定效价最高的小鼠，对小鼠进行安乐死，收集脾细胞，与SP20细胞系进行融合。
挑选出与Raji-hCCR8结合活性强、且不与Raji细胞系结合的上清的单克隆细胞用以扩大培养。一周后收集上清，对上清中的抗体进行纯化。
实施例2.抗CCR8抗体的结合活性筛选
对Raji-hCCR8和Raji细胞进行计数，调整密度至2E6/ml，加入100μL/孔细胞悬液 至96孔板中，400×g离心5分钟，弃上清。配置浓度为200nM的杂交瘤纯化抗体，并进行3倍梯度稀释。用100μL稀释好的杂交瘤纯化抗体重悬细胞，4℃孵育30分钟。用PBS洗涤2次后，加入100μL按1：500稀释的PE标记的山羊抗小鼠Fc抗体重悬细胞，4℃孵育30分钟。用PBS洗涤1次后，加入100μL/孔PBS重悬细胞，通过流式细胞术分析PE荧光强度。
抗CCR8抗体与Raji-hCCR8的结合检测结果如图1所示，实验结果表明，F022-7、F022-59、F022-63、F022-66、F022-68、F022-75这6个杂交瘤纯化抗体展现出了与Raji-hCCR8较强的结合活性。
实施例3.重组的抗CCR8抗体的制备
调取F022-7、F022-59、F022-63、F022-66、F022-68、F022-75杂交瘤纯化抗体的序列，测序结果证明所有杂交瘤纯化抗体的轻链恒定区均为鼠κ型(如SEQ ID No.40所示)，F022-7、F022-63和F022-75的重链恒定区为mIgG2a型(如SEQ ID No.41所示)，F022-59、F022-66和F022-68的重链恒定区为mIgG1型(如SEQ ID No.39所示)。将杂交瘤纯化抗体的重链恒定区替换为小鼠mIgG2a，其它序列不变，并克隆至pCDNA3.4A表达质粒中，将质粒转染至EXPI293细胞系中，7天后收取上清，经protein A纯化后得到重组抗体，重组抗体分别命名为F022-7-mIgG2a,F022-59-mIgG2a,F022-63-mIgG2a,F022-66-mIgG2a,F022-68-mIgG2a,F022-75-mIgG2a，例如重组抗体“F022-59-mIgG2a”表示杂交瘤纯化抗体“F022-59”的重链恒定区被“mIgG2a”替换后形成的抗体，其它依此类推。将杂交瘤纯化抗体的重链恒定区替换为人hIgG1Fc(DLE)(也即人IgG1的Fc进行S239D/A330L/I332E突变，序列如SEQ ID No.42所示)，同时轻链恒定区替换为人κ轻链恒定区(如SEQ ID No.43所示)，其它序列不变，并克隆至pCDNA3.4A表达质粒中，将质粒转染至EXPI293细胞系中，7天后收取上清，经protein A纯化后得到重组抗体，重组抗体分别命名为F022-7-hIgG1(DLE),F022-59-hIgG1(DLE),F022-63-hIgG1(DLE),F022-66-hIgG1(DLE),F022-68-hIgG1(DLE),F022-75-hIgG1(DLE)，例如重组抗体“F022-59-hIgG1(DLE)”表示杂交瘤纯化抗体“F022-59”的重链恒定区被人hIgG1(DLE)且轻链恒定区被人κ轻链恒定区替换后形成的抗体，其它依此类推。抗体的可变区序列见表1，CDRs序列见表2：
表1.抗CCR8抗体可变区序列表

表2.抗CCR8抗体CDRs序列表

备注：上述表2所述抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3 是根据Kabat编码***确认。
鼠mIgG1重链恒定区的氨基酸序列(SEQ ID No.39)：
鼠κ轻链恒定区的氨基酸序列(SEQ ID No.40)：
鼠mIgG2a重链恒定区的氨基酸序列(SEQ ID No.41)：
人hIgG1(DLE)抗体重链恒定区序列(SEQ ID No.42)：
人κ轻链恒定区序列(SEQ ID No.43)：
示例性地，F022-59、F022-59-mIgG2a、F022-59-hIgG1(DLE)的全长序列如下：
F022-59重链可变区的氨基酸序列(SEQ ID No.44)：

F022-59轻链可变区的氨基酸序列(SEQ ID No.45)：
F022-59-mIgG2a重链可变区的氨基酸序列(SEQ ID No.46)：
F022-59-mIgG2a轻链可变区的氨基酸序列(SEQ ID No.47)：
F022-59-hIgG1(DLE)重链可变区的氨基酸序列(SEQ ID No.48)：
F022-59-hIgG1(DLE)轻链可变区的氨基酸序列(SEQ ID No.49)：
另外，阳性对照抗体4A19-mIgG2a(WO2021194942A1专利中的4A19抗体可变区连接mIgG2a恒定区获得)的氨基酸序列如下：
4A19-mIgG2a重链的氨基酸序列(SEQ ID No.50)：
4A19-mIgG2a轻链的氨基酸序列(SEQ ID No.51)：
4.抗CCR8抗体体外活性分析
利用Lymphoprep(Axis-Shield)按照说明书的操作步骤分离外周血单个核细胞(PBMC)。利用EasySep Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit(Stem Cell)按照说明书的操作步骤分离PBMC中的Treg。分离好的Treg与Dynabeads Human T-Activator CD3/CD28(Gibco)按照1:1的比例混合，用完全培养基(90％X-VIVO 15+10％FBS+500IU/ml IL-2，X-VIVO 15：Lonza，FBS：Gibco，IL-2：四环生物)培养5到8天，活化扩增Treg细胞。分别通过流式细胞术检测重组抗体与Raji-hCCR8、Treg(活化后)，Raji，PBMC和Raji-hCCR4(Raji细胞过表达人CCR4)的结合活性。实验方法同上述实施例2，mIgG2a亚型抗体使用PE标记的抗小鼠Fc二抗(Biolegend)检测，hIgG1(DLE)亚型抗体使用Alexa Fluor 647标记的抗人Fab二抗(Jackson)检测。实验结果如图2-6所示，重组抗体均展现出了与Raji-hCCR8(图2)和Treg(图3)的较强的结合活性。同时，重组抗体均不与Raji(图4)，PBMC(图5)及Raji-hCCR4(图6)结合，这说明本公开的抗CCR8抗体具有较好的特异性。
另外，在NK-92细胞中通过表达hCD16分子(NK-92-hCD16)用以检测抗体介导的ADCC功能。用CFSE对Jurkat-hCCR8细胞(Jurkat，人外周血白血病T细胞系，ATCC)进行染色。按2E4/孔的密度将Jurkat-hCCR8细胞加入96孔板中。按1E5/孔的密度将NK-92-hCD16细胞加入96孔板中，随后加入抗具有hIgG1Fc(DLE)的抗CCR8抗体，37度孵育6小时。取出96孔板加入PI染色，通过流式细胞术检测CSFE及PI荧光，计算PI、CFSE双阳性的细胞在CFSE阳性细胞群中的比例，即为细胞杀伤率。实验结果如图7所示，所有重组抗体均展示出优秀的ADCC活性。
5.抗CCR8抗体体内活性分析
为验证抗CCR8抗体的体内药效，在hCCR8敲入小鼠(B-hCCR8，百奥塞图)中皮下注射MC38细胞(3E5/只)，建立肿瘤模型。在肿瘤接种第7天，将F022-7-mIgG2a,F022-59-mIgG2a,F022-75-mIgG2a抗体进行静脉注射给药，同时以同型无关靶点抗体作为阴性对照，以4A19-mIgG2a为阳性对照。给药剂量为10mg/kg，一周给药2次，共给药6次。每周测量两次小鼠肿瘤体积，按照肿瘤体积＝肿瘤长径×肿瘤短径×肿瘤短径/2计算。肿瘤接种34天后，计算小鼠肿瘤抑制率，抑制率按公式计算：[1-(治疗组给药后瘤体积-治疗组给药前瘤体积)/(对照组给药后瘤体积-对照组给药前瘤体积)]×100％。实验结果见表3及附图8所示，重组抗体在体内均有显著的抗肿瘤作用。
表3.抗CCR8抗体抑制肿瘤生长实验结果表
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (18)

  1. 一种抗人CCR8抗体，所述抗体包含重链可变区和轻链可变区，所述重链可变区包括SEQ ID No.3、1、5、7、9或11中的HCDR1、HCDR2和HCDR3，和/或所述抗体的轻链可变区包括SEQ ID No.4、2、6、8、10或12中的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的抗人CCR8抗体，其中
    所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号***定义，或由Kabat编号***定义，或由Chothia编号***定义，或由Contact编号***定义，或由AbM编号***定义；
    可选地，
    A.所述抗体的重链可变区包括SEQ ID No.3中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.4中的LCDR1、LCDR2和LCDR3；
    B.所述抗体的重链可变区包括SEQ ID No.1中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.2中的LCDR1、LCDR2和LCDR3；
    C.所述抗体的重链可变区包括SEQ ID No.5中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.6中的LCDR1、LCDR2和LCDR3；
    D.所述抗体的重链可变区包括SEQ ID No.7中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.8中的LCDR1、LCDR2和LCDR3；
    E.所述抗体的重链可变区包括SEQ ID No.9中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.10中的LCDR1、LCDR2和LCDR3；或者
    F.所述抗体的重链可变区包括SEQ ID No.11中的HCDR1、HCDR2和HCDR3，并且所述抗体的轻链可变区包括SEQ ID No.12中的LCDR1、LCDR2和LCDR3；
    可选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号***定义；
    可选地，所述的抗人CCR8抗体，其中，
    a.所述重链可变区的HCDR1包含SEQ ID NO.19的氨基酸序列，HCDR2包含SEQ ID NO.20的氨基酸序列，和HCDR3包含SEQ ID NO.21的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；
    b.所述重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.14的氨基酸序列，和HCDR3包含SEQ ID NO.15的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；
    c.所述重链可变区的HCDR1包含SEQ ID NO.22的氨基酸序列，HCDR2包含SEQ ID NO.23的氨基酸序列，和HCDR3包含SEQ ID NO.24的氨基酸序列；并且所述轻链 可变区的LCDR1包含SEQ ID NO.25的氨基酸序列，LCDR2包含SEQ ID NO.26的氨基酸序列，和LCDR3包含SEQ ID NO.27的氨基酸序列；
    d.所述重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.28的氨基酸序列，和HCDR3包含SEQ ID NO.29的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.30的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；
    e.所述重链可变区的HCDR1包含SEQ ID NO.31的氨基酸序列，HCDR2包含SEQ ID NO.32的氨基酸序列，和HCDR3包含SEQ ID NO.33的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.34的氨基酸序列，LCDR2包含SEQ ID NO.35的氨基酸序列，和LCDR3包含SEQ ID NO.36的氨基酸序列；或
    f.所述重链可变区的HCDR1包含SEQ ID NO.13的氨基酸序列，HCDR2包含SEQ ID NO.37的氨基酸序列，和HCDR3包含SEQ ID NO.38的氨基酸序列；并且所述轻链可变区的LCDR1包含SEQ ID NO.16的氨基酸序列，LCDR2包含SEQ ID NO.17的氨基酸序列，和LCDR3包含SEQ ID NO.18的氨基酸序列；
    可选地，
    A.所述抗人CCR8抗体包含分别如SEQ ID NO.19、SEQ ID NO.20和SEQ ID NO.21所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.16、SEQ ID NO.17和SEQ ID NO.18所示的LCDR1、LCDR2和LCDR3；
    B.所述抗人CCR8抗体包含分别如SEQ ID NO.13、SEQ ID NO.14和SEQ ID NO.15所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.16、SEQ ID NO.17和SEQ ID NO.18所示的LCDR1、LCDR2和LCDR3；或者
    C.所述抗人CCR8抗体包含分别如SEQ ID NO.22、SEQ ID NO.23和SEQ ID NO.24所示的HCDR1、HCDR2和HCDR3，以及分别如SEQ ID NO.25、SEQ ID NO.26和SEQ ID NO.27所示的LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1或2所述的抗人CCR8抗体，所述抗体为鼠源抗体，嵌合抗体或人源化抗体。
  4. 一种抗人CCR8抗体，其中，所述抗体的重链可变区包括与SEQ ID No.3、1、5、7、9或11具有至少90％序列同一性的氨基酸序列，和/或轻链可变区包括与SEQ ID No.4、2、6、8、10或12具有至少90％序列同一性的氨基酸序列；
    可选地，
    (i)所述重链可变区包含SEQ ID NO.3的氨基酸序列，和所述轻链可变区包含SEQ ID NO.4的氨基酸序列；
    (ii)所述重链可变区包含SEQ ID NO.1的氨基酸序列，和所述轻链可变区包含SEQ ID NO.2的氨基酸序列；
    (iii)所述重链可变区包含SEQ ID NO.5的氨基酸序列，和所述轻链可变区包含SEQ ID NO.6的氨基酸序列；
    (iv)所述重链可变区包含SEQ ID NO.7的氨基酸序列，和所述轻链可变区包含SEQ ID NO.8的氨基酸序列；
    (v)所述重链可变区包含SEQ ID NO.9的氨基酸序列，和所述轻链可变区包含SEQ ID NO.10的氨基酸序列；或
    (vi)所述重链可变区包含SEQ ID NO.11的氨基酸序列，和所述轻链可变区包含SEQ ID NO.12的氨基酸序列。
  5. 根据权利要求1至4任一项所述的抗人CCR8抗体，所述抗体还包含恒定区；
    可选地，所述抗体的重链恒定区选自IgG1、IgG2、IgG3和IgG4的重链恒定区，所述轻链恒定区选自κ或λ链恒定区；
    可选地，所述恒定区的种属来源为鼠或人；
    可选地，所述重链恒定区为鼠IgG2a恒定区、鼠IgG1恒定区或人IgG1恒定区，和/或轻链恒定区为鼠κ恒定区或人κ恒定区；
    可选地，所述重链恒定区为人IgG1(DE)或人IgG1(DLE)恒定区；
    可选地，所述重链恒定区包括SEQ ID NO.39、41或42的氨基酸序列，轻链恒定区包括SEQ ID NO.40或43的氨基酸序列；
    可选地，所述抗人CCR8抗体重链包含SEQ ID NO.46的氨基酸序列，轻链包括SEQ ID NO.47的氨基酸序列；或者
    所述抗人CCR8抗体重链包含SEQ ID NO.44的氨基酸序列，轻链包括SEQ ID NO.45的氨基酸序列；或者
    所述抗人CCR8抗体重链包含SEQ ID NO.48的氨基酸序列，轻链包括SEQ ID NO.49的氨基酸序列。
  6. 根据权利要求1至4任一项所述的抗人CCR8抗体，所述抗体为全长抗体或为选自F(ab’)2、Fab’-SH、Fab’、Fab、scFab、dsFv、(dsFv)2、Fv和scFv中的任意一种抗原结合片段。
  7. 与权利要求1至6任一项所述的抗人CCR8抗体竞争性结合人CCR8的抗体，或与权利要求1至6任一项所述的抗人CCR8抗体结合相同表位的抗体。
  8. 根据权利要求1至7任一项所述的抗人CCR8抗体，其中所述抗人CCR8抗体具有以下特性中的至少一种：
    A.能与人CCR8特异性结合；可选地，其能以≤5nM的EC50值与表达人CCR8的Raji细胞结合，其中，所述EC50值通过流式细胞分析法测定；
    B.具有抑制肿瘤生长功能；
    C.具有ADCC活性。
  9. 一种多特异性抗体，所述多特异性抗体包含权利要求1至8任一项所述的抗人CCR8抗体。
  10. 一种抗体偶联物，所述抗体偶联物包括权利要求1至8任一项所述的抗人CCR8抗体。
  11. 一种嵌合抗原受体(CAR)，所述嵌合抗原受体包括抗原结合结构域，所述抗原结合结构域包括权利要求1至8任一项所述的抗人CCR8抗体。
  12. 一种CAR-免疫细胞，所述CAR-免疫细胞表达权利要求11所述的嵌合抗原受体。
  13. 一种分离的核酸，其编码权利要求1至8任一项所述的抗人CCR8抗体。
  14. 一种细胞，其含有权利要求13所述的核酸。
  15. 一种药物组合物，其包含权利要求1至8任一项所述的抗人CCR8抗体、权利要求9所述的多特异性抗体、权利要求10所述的抗体偶联物、权利要求12所述的细胞或权利要求13所述的核酸；可选地，其还包含一种或多种药学上可接受的载体、稀释剂或赋形剂。
  16. 如权利要求1至8任一项所述的抗人CCR8抗体、权利要求9所述的多特异性抗体、权利要求10所述的抗体偶联物、权利要求12所述的细胞、权利要求13所述的核酸或权利要求15所述的药物组合物在制备具有以下至少一种用途的产品中的应用：诊断人CCR8表达异常的相关疾病、治疗人CCR8表达异常的相关疾病或检测人CCR8的表达；可选地，所述人CCR8表达异常的相关疾病为肿瘤；可选地，所述肿瘤为乳腺癌、卵巢癌、肾癌、胰腺癌、膀胱癌、胃癌、***、结肠癌、肉瘤、肝癌或肺癌。
  17. 一种治疗疾病的方法，所述方法包括向有需要的受试者施用治疗有效量的权利要求1至8任一项所述的抗人CCR8抗体、权利要求9所述的多特异性抗体、权利要求10所述的抗体偶联物、权利要求12所述的细胞、权利要求13所述的核酸或权利要求15所述的药物组合物；可选地，所述疾病为人CCR8表达异常的相关疾病；可选地，所述疾病为肿瘤；可选地，所述肿瘤为乳腺癌、卵巢癌、肾癌、胰腺癌、膀胱癌、胃癌、***、结肠癌、肉瘤、肝癌或肺癌。
  18. 用作药物的权利要求1至8任一项所述的抗人CCR8抗体、权利要求9所述的 多特异性抗体、权利要求10所述的抗体偶联物、权利要求12所述的细胞、权利要求13所述的核酸或权利要求15所述的药物组合物；可选地，所述药物用于治疗疾病；可选地，所述疾病为人CCR8表达异常的相关疾病；可选地，所述疾病为肿瘤；可选地，所述肿瘤为乳腺癌、卵巢癌、肾癌、胰腺癌、膀胱癌、胃癌、***、结肠癌、肉瘤、肝癌或肺癌。
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