WO2024109657A1 - 抗ccr8抗体及其用途 - Google Patents

抗ccr8抗体及其用途 Download PDF

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Publication number
WO2024109657A1
WO2024109657A1 PCT/CN2023/132346 CN2023132346W WO2024109657A1 WO 2024109657 A1 WO2024109657 A1 WO 2024109657A1 CN 2023132346 W CN2023132346 W CN 2023132346W WO 2024109657 A1 WO2024109657 A1 WO 2024109657A1
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antibody
amino acid
ccr8
seq
antigen
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English (en)
French (fr)
Inventor
苏云鹏
李勇
庄伟亮
姜殿东
梁绍勤
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Hc Biopharma Inc
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Hc Biopharma Inc
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Priority to EP23893747.8A priority Critical patent/EP4624492A1/en
Priority to JP2025530347A priority patent/JP2025538606A/ja
Priority to KR1020257020600A priority patent/KR20250130296A/ko
Priority to CN202380080563.3A priority patent/CN120239710A/zh
Publication of WO2024109657A1 publication Critical patent/WO2024109657A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the field of monoclonal antibodies and/or engineered antibodies, and in particular, provides antibodies or antigen-binding fragments thereof that specifically bind to CCR8 and compositions comprising the same. Also provided are nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof of the present invention, vectors and host cells for expressing the antibodies or antigen-binding fragments thereof of the present invention, and treatment and diagnosis/detection methods and uses of the antibodies or antigen-binding fragments thereof of the present invention.
  • the body's immune system is the core defense system against tumor generation and progression, and the inability of the immune system to recognize and eliminate malignant cells plays an important role in the pathogenesis of cancer.
  • high expression of multiple immune checkpoints reduces immune activation and inhibits anti-tumor immunity.
  • Monoclonal antibodies targeting inhibitory immune checkpoints CTLA-4 and PD-1/PD-L1 produce significant anti-tumor responses by upregulating immune activation in the tumor microenvironment.
  • cancer immunotherapy has brought significant improvements to patients in terms of survival rate and quality of life (K Esfahani et al., A review of cancer immunotherapy: from the past, to the present, to the future, Curr Oncol. 2020 Apr; 27(Suppl 2):S87-S97.).
  • immune checkpoint inhibitors have been clinically approved for the treatment of a variety of solid tumors, their response rates vary greatly among tumor types, and the overall response rate is low.
  • the overall response rate (ORR) of immune checkpoint inhibitors in non-small cell lung cancer (NSCLC), urothelial carcinoma, and head and neck cancer is only about 20%, while it is less than 10% in solid tumors such as gastric cancer (Schoenfeld AJ and Hellmann MD. Acquired Resistance to Immune Checkpoint Inhibitors. Cancer Cell. 2020 Apr 13; 37(4): 443-455. doi: 10.1016/j.ccell.2020.03.017.).
  • the low overall response rate, acquired resistance, and hyperprogression of immune checkpoint inhibitors are related to multiple mechanisms in the internal signaling pathways of tumor cells and the tumor microenvironment (Baxter MA et al. Resistance to immune checkpoint inhibitors in advanced gastro-oesophageal cancers. Br J Cancer. 2021 Oct; 125(8): 1068-1079. doi: 10.1038/s41416-021-01425-7.).
  • Regulatory T cells play an important role in immune regulation and are related to the occurrence and development of tumors.
  • Treg Compared with the periphery, the number of Treg in the tumor microenvironment increases and the function is enhanced; Treg inhibits anti-tumor immunity through mechanisms such as secretion of inhibitory cytokines such as IL-10, consumption of IL-2, and direct contact inhibition, which is closely related to the natural and acquired resistance of immune checkpoint inhibitors (Baxter MA et al.).
  • PD-1 blockade can induce the recovery of dysfunctional PD-1 + CD8 + T cells, but it can also enhance PD-1 + Treg cell-mediated immunosuppression.
  • Tregs expressing PD-1 in the tumor microenvironment may lead to hyperprogression of the tumor (Kumagai S et al. The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies. Nat Immunol. 2020 Nov; 21(11): 1346-1358. doi: 10.1038/s41590-020-0769-3.). Preclinical animal models and clinical studies have shown that eliminating Tregs in the tumor microenvironment can promote anti-tumor immunity and increase the therapeutic effect of PD-1/PD-L1.
  • chemokine receptor CCR8 is highly expressed on Treg cells in human tumors (including breast cancer, non-small cell lung cancer, colorectal cancer, melanoma, hepatocellular carcinoma and pancreatic ductal adenocarcinoma), but is not expressed or is lowly expressed on Treg and effector T cells in peripheral and normal tissues (Plitas G et al. Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity. 2016 Nov 15; 45(5): 1122-1134. doi: 10.1016/j.immuni.2016.10.032, and De Simone M et al.
  • the present invention uses cells overexpressing human CCR8 and mice immunized with CCR8 nucleic acid, and obtains anti-human CCR8 specific antibodies through hybridoma technology screening.
  • Anti-CCR8 antibodies have high affinity to both human and monkey CCR8, can block the signal pathway of binding/induction of CCL1 and CCR8, and can also eliminate CCR8 positive cells (for example, Treg cells) through ADCC, and effectively inhibit tumor growth in various models in mice (such as colorectal cancer, lung cancer, breast cancer, melanoma, etc.).
  • the present invention provides an anti-CCR8 antibody or an antigen-binding fragment thereof, wherein the anti-CCR8 antibody or the antigen-binding fragment thereof comprises:
  • the present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, which comprises a heavy chain variable region and/or a light chain variable region, wherein
  • the heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO:1, SEQ ID NO:2 or 10 or 11 or 41 and SEQ ID NO:3; or HCDR1, HCDR2 and HCDR3 which are respectively identical to the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 or 10 or 11 or 41 and SEQ ID NO:3 or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acids addition, substitution or deletion or any combination thereof); or
  • HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences are identical or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acids addition, substitution or deletion or any combination thereof); or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; or HCDR1, HCDR2 and HCDR3 which are identical to the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acids addition, substitution or deletion or any combination thereof);
  • the light chain variable region comprises:
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14; or LCDR1, LCDR2 and LCDR3 which are identical to the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acids addition, substitution or deletion or any combination thereof); or
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17; or LCDR1, LCDR2 and LCDR3 which are identical to the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acids addition, substitution or deletion or any combination thereof); or
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; or LCDR1, LCDR2 and LCDR3 which are identical to the amino acid sequences shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acids addition, substitution or deletion or any combination thereof).
  • the present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in any combination of the following tables:
  • the present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, which comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein:
  • (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 21-40; or
  • (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 21-40; or
  • amino acid change comprising an amino acid sequence having at least one addition, substitution or deletion of an amino acid or any combination thereof (e.g., 1, 2 or 3 additions, substitutions or deletions or any combination thereof) compared to an amino acid sequence selected from any one of SEQ ID NOs: 21-40, preferably, the amino acid change occurs in a CDR region, preferably, the amino acid change occurs in a FR region;
  • (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 42-54; or
  • amino acid change comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to the amino acid sequence shown in any one of SEQ ID NOs:42-54, preferably, the amino acid change does not occur in the CDR region, preferably, the amino acid change occurs in the FR region.
  • the present invention provides antibodies or antigen-binding fragments thereof that bind to CCR8. It comprises a heavy chain variable region VH and a light chain variable region VL as shown in any combination of the following tables:
  • the present invention provides an anti-CCR8 antibody or an antigen-binding fragment thereof, which comprises a heavy chain and/or a light chain, wherein
  • (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 55-74; or
  • amino acid change comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to an amino acid sequence selected from any one of SEQ ID NOs: 55-74, preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the amino acid change does not occur in the heavy chain variable region, most preferably, the amino acid change occurs in the heavy chain constant region;
  • (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 75-82; or
  • amino acid change comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to the amino acid sequence selected from any one of SEQ ID NOs:75-82, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change does not occur in the light chain variable region, most preferably, the amino acid change occurs in the light chain constant region.
  • the antibody or antigen-binding fragment thereof of the present invention wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region and/or a light chain constant region, preferably, the light chain constant region is a ⁇ chain or a ⁇ chain constant region; the heavy chain constant region is selected from mouse mIgG2a, human IgG1, human IgG2, human IgG3 or IgG4. In some preferred embodiments, the heavy chain constant region is human IgG1 or human IgG4 with S228P mutation.
  • the antibody or antigen-binding fragment thereof of the present invention wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, and the sequence of the heavy chain constant region has one or more amino acid substitutions compared to the sequence of the natural human heavy chain constant region, preferably, the one or more amino acid substitutions enhance the ADCC effect of the antibody.
  • the one or more amino acid substitutions occur at positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 3 01, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439.
  • the one or more amino acid substitutions occur in one or more of the heavy chain constant region sequence positions L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, One or more of R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339, and P396.
  • the one or more amino acid substitutions are selected from one or more of G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L substitutions according to the EU numbering system.
  • the one or more amino acid substitutions are selected from one or more of N297A substitution, N297Q substitution, L235A substitution together with L237A substitution, L234A substitution together with L235A substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, D265A substitution, A327Q substitution and P329A substitution according to the EU numbering system.
  • the one or more amino acid substitutions occur in one or more of the heavy chain constant region sequence positions 235, 239, 243, 292, 300, 330, 332, 396 according to the EU numbering system.
  • the one or more amino acid substitutions are selected from at least one of S239D, L235V, F243L, R292P, Y300L, A330L, I332E and P396L according to the EU numbering system.
  • the heavy chain constant region has one or more sets of mutations occurring simultaneously at a combination of positions selected from the following according to the EU numbering system: (1) L235/F243/R292/Y300/P396, (2) F243/R292/Y300/V305/P396, (3) D270/K326/A330/K334, (4) S239/A330/I332, (5) S298/E333/K334, (6) L234/L235/G236/S239/H268/D270/S298, (7) M252/S254/T256, (8) L234/L235/D265, (9) G236/S239/I332 and (10) S239/I332.
  • the heavy chain constant region has one or more mutations selected from the following combinations of mutations according to the EU numbering system: (1) L235V/F243L/R292P/Y300L/P396L, (2) F243L/R292P/Y300L/V305I/P396L, (3) D270E/K326D/A330M/K334E, (4) S239D/A330L/I332E, (5) S298A/E333A/K334A, (6) L234Y/L235Q/G236W/S239M/H268D/D270E/S298A, (7) M252Y/S254T/T256E, (8) L234A/L235A/D265A, (9) L234F/L235E/D265A, (10) G236A/S239D/I332E and (11) S239D/I332E.
  • the antibody or antigen-binding fragment thereof that binds to CCR8 of the present invention comprises a heavy chain (HC) and a light chain (LC) as shown in any combination of the following tables:
  • the constant regions of the antibodies of the invention are afucosylated or have reduced fucosylation.
  • the antibodies of the invention are monoclonal antibodies.
  • the antibodies of the invention are murine antibodies, chimeric antibodies, or humanized antibodies or human antibodies.
  • antigen-binding fragments of the invention include the following antibody fragments: Fab, Fab', Fab'-SH, Fv, Fd, single chain antibody (eg, scFv) or (Fab') 2 , single domain antibody, diabody (dAb), or linear antibody.
  • the present invention provides an isolated anti-CCR8 antibody or antigen-binding fragment thereof having one or more of the following properties:
  • the antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:
  • (1) binds to human/cynomolgus monkey CCR8 with high affinity, for example, exhibiting an EC 50 value of no higher than 1000 ng/mL, an EC 50 value of no higher than 950 ng/mL, an EC 50 value of no higher than 900 ng/mL, an EC 50 value of no higher than 850 ng/mL, an EC 50 value of no higher than 800 ng/mL, an EC 50 value of no higher than 750 ng/mL, an EC 50 value of no higher than 700 ng/mL, an EC 50 value of no higher than 650 ng/mL, an EC 50 value of no higher than 600 ng/mL, an EC 50 value of no higher than 550 ng/mL, an EC 50 value of no higher than 500 ng/mL, an EC 50 value of no higher than 450 ng/mL, an EC 50 value of no higher than 400 ng/mL, an EC 50 value of no higher than 350 ng
  • 50 value no higher than 60ng/ mL , no higher than 55ng/mL, no higher than 50ng /mL, no higher than 45ng/mL, no higher than 40ng / mL , no higher than 35ng/mL, no higher than 30ng /mL, no higher than 25ng /mL, no higher than 20ng /mL, no higher than 18ng /mL, no higher than 16ng /mL, no higher than 15ng /mL, no higher than 14ng /mL, no higher than 13ng/mL, no higher than 12ng /mL, no higher than 11ng/mL, no higher than 10ng/mL, no higher than 8ng/mL EC 50 value .
  • an EC 50 value no higher than 6 ng/mL, an EC 50 value no higher than 4 ng/mL, an EC 50 value no higher than 2 ng/mL, an EC 50 value no higher than 1 ng/mL, or an EC 50 value lower .
  • IC 50 value IC 50 value not higher than 250ng/mL, IC 50 value not higher than 200ng/mL, IC 50 value not higher than 180ng/mL, IC 50 value not higher than 160ng/mL, IC 50 value not higher than 150ng/mL, IC 50 value not higher than 140ng/mL, IC 50 value not higher than 130ng/mL, IC 50 value not higher than 120ng/mL, IC 50 value not higher than 110ng/mL
  • IC 50 value no higher than 18 ng / mL , no higher than 16 ng/mL, no higher than 15 ng/mL, no higher than 14 ng /mL, no higher than 13 ng/ mL , no higher than 12 ng/mL, no higher than 11 ng/mL, no higher than 10 ng/mL, no higher than 8 ng/mL, no higher than 6 ng / mL , no higher than 4 ng/mL, no higher than 2 ng/mL, no higher than 1 ng/mL, or an IC 50 value of less.
  • 50 value no more than 3000ng/mL, no more than 2500ng /mL, no more than 2000ng/mL, no more than 1800ng /mL, no more than 1600ng/ mL , no more than 1500ng/mL, no more than 1400ng /mL, no more than 1300ng /mL, no more than 1200ng /mL, no more than 1100ng /mL, no more than 1000ng/mL, no more than 950ng /mL, no more than 900ng /mL, no more than 850ng /mL, no more than 800ng /mL, no more than 750ng/mL IC 50 value 50 value, no more than 700 ng/mL, no more than 650 ng/mL, no more than 600 ng/mL, no more than 550 ng/mL, no more than 500 ng/ mL , no more than 450 ng/mL, no more than 400 ng/mL, no more than 350
  • IC 50 value no higher than 120 ng/mL, no higher than 110 ng /mL, no higher than 100 ng/mL, no higher than 80 ng/mL, no higher than 60 ng/ mL , no higher than 40 ng/mL, no higher than 20 ng/mL, no higher than 10 ng/mL, or lower IC 50 value .
  • the present invention also provides a multispecific antibody comprising the antibody or antigen-binding fragment thereof described herein. light chain variable region and/or heavy chain variable region.
  • the present invention also provides a single-chain antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
  • the present invention also provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof described herein conjugated to a therapeutic agent or a diagnostic agent.
  • the present invention provides a polynucleotide molecule encoding any of the anti-CCR8 antibodies described herein or any fragment thereof.
  • the present invention provides an expression vector comprising the polynucleotide molecule of the present invention.
  • the vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising the polynucleotide molecule of the present invention or the expression vector of the present invention.
  • the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the present invention provides a method for preparing the anti-CCR8 antibody or antigen-binding fragment thereof described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-CCR8 antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate described herein, and optionally at least one pharmaceutically acceptable carrier or excipient.
  • the present invention provides a drug combination comprising an antibody or antigen-binding fragment thereof, a polynucleotide, a vector, a host cell, an immunoconjugate or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.
  • the additional therapeutic agent is a chemotherapeutic agent.
  • the additional therapeutic agent is other antibodies.
  • the additional therapeutic agent is other monoclonal antibodies.
  • the additional therapeutic agent is a monoclonal antibody targeting an immune checkpoint.
  • the additional therapeutic agent is a monoclonal antibody targeting PD-1.
  • the additional therapeutic agent is a monoclonal antibody targeting PD-L1.
  • the additional therapeutic agent is a monoclonal antibody targeting CTLA4.
  • the present invention provides a method for eliminating CCR8-positive Treg cells in vitro or in vivo, the method comprising contacting an anti-CCR8 antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate or pharmaceutical composition described herein with a cell population comprising CCR8-positive Treg cells or administering it into a subject.
  • the present invention provides the use of the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition or pharmaceutical combination described herein in the preparation of a medicament for treating and/or preventing a tumor, an autoimmune disease, or an infectious disease, wherein the tumor is preferably a melanoma.
  • the present invention provides an antibody or antigen-binding fragment thereof, a polynucleotide, a vector, a host cell, an immunoconjugate or a pharmaceutical composition described herein in combination with other therapeutic agents in the preparation of a drug for treating and/or preventing a tumor, an autoimmune disease, or an infectious disease, preferably a tumor is melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer.
  • the additional therapeutic agent is a chemotherapeutic agent.
  • the additional therapeutic agent is another antibody.
  • the additional therapeutic agent is another monoclonal antibody. In some preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting an immune checkpoint. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-L1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting CTLA4.
  • the present invention provides the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, host cells, immunoconjugates, pharmaceutical compositions or drug combinations described herein for use in treating and/or preventing tumors, autoimmune diseases, or infectious diseases, preferably the tumor is melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer.
  • the present invention provides a method for treating and/or preventing tumors, autoimmune diseases, or infectious diseases, comprising administering to a subject in need thereof a therapeutically or preventively effective amount of the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition or drug combination described herein, wherein the tumor is preferably melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition or pharmaceutical combination described herein, preferably further comprising a drug delivery device.
  • the present invention provides a method for detecting the presence of CCR8 in a sample using the antibody, antigen-binding fragment or immunoconjugate described herein, or a detection composition containing the antibody, antigen-binding fragment or immunoconjugate.
  • Figure 1 shows the binding activity of the anti-CCR8 antibody of the present invention to human CCR8.
  • A-D are the binding curves of the antibody in the culture supernatant of the purified and quantified monoclonal hybridoma cells to 293F-hCCR8.
  • Figure 2 shows the binding activity of the anti-CCR8 antibody of the present invention to cynomolgus monkey CCR8.
  • AB is the binding curve of the antibody in the purified and quantified monoclonal hybridoma cell culture supernatant and 293T-cynoCCR8.
  • FIG3 shows that multiple antibody clones of the present invention can block the binding of CCL1 and CCR8.
  • A-C are inhibition curves of antibodies in purified and quantified monoclonal hybridoma cell culture supernatants blocking 10 nM AlexaFlour-647-labeled human CCL1 and 293F-hCCR8.
  • Figure 4 shows the binding activity of the anti-CCR8 chimeric antibody of the present invention to human CCR8.
  • A-B and C are the binding curves of hIgG1 and mIgG2a chimeric antibodies to 293F-hCCR8, respectively.
  • Figure 5 shows the binding activity of the anti-CCR8 chimeric antibody of the present invention to cynomolgus monkey CCR8.
  • a and B are the binding curves of hIgG1 and mIgG2a chimeric antibodies to 293T-cynoCCR8, respectively.
  • Figure 6 shows the activity of the anti-CCR8 chimeric antibody of the present invention in blocking the binding of CCL1.
  • A-B and C are the inhibition curves of hIgG1 and mIgG2a chimeric antibodies blocking 10nM AlexaFlour-647-labeled human CCL1 and 293F-hCCR8, respectively.
  • Figure 7 shows that the CCR8 chimeric antibody of the present invention blocks CCL1-induced ⁇ -Arrestin recruitment.
  • a and B are the inhibition curves of hIgG1 and mIgG2a chimeric antibodies blocking human CCL1-induced ⁇ -Arrestin recruitment, respectively.
  • Figure 8 shows the binding activity of the humanized anti-CCR8 antibodies of the present invention to human CCR8.
  • A-B, C-D and E-G are the binding curves of humanized antibodies of clones 27B9-1G3, 559E1B10 and 563E10E12 to 293F-hCCR8, respectively.
  • FIG9 shows the binding activity of the humanized anti-CCR8 antibodies of the present invention to cynomolgus monkey CCR8, as reflected by the binding curves of 559E1B10 and 563E10E12 humanized antibodies to 293T-cynoCCR8.
  • FIG10 shows the binding activity of the humanized anti-CCR8 antibody of the present invention in blocking CCL1.
  • A-B, C-D and E-G are the inhibition curves of 27B9-1G3, 559E1B10 and 563E10E12 humanized antibodies blocking 10 nM AlexaFlour-647 labeled human CCL1 and 293F-hCCR8, respectively.
  • Figure 11 shows the experiment of blocking CCL1-induced ⁇ -Arrestin recruitment by anti-CCR8 humanized antibodies of the present invention.
  • A-B and C-E are the inhibition curves of 559E1B10 and 563E10E12 humanized antibodies blocking CCL1-induced ⁇ -Arrestin recruitment, respectively.
  • Figure 12 shows that the chimeric antibody and humanized antibody of 559E1B10 can activate Jurkat-human Fc ⁇ RIIIa(158V)-NFAT in a dose-dependent manner.
  • Figure 13 shows that the chimeric antibody and humanized antibody of 563E10E12 can activate Jurkat-human Fc ⁇ RIIIa(158V)-NFAT in a dose-dependent manner.
  • FIG. 14 shows the binding activity of the anti-CCR8 antibody of the present invention to HuT78.
  • Figure 15 shows the specific binding of the anti-CCR8 antibody of the present invention to hCCR8. Binding activity of the anti-CCR8 antibody to CHOK1-hCCR8 (A) and CHO-K1 (B) cells.
  • FIG16 shows the CCR8-dependent activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT by the anti-CCR8 antibody of the present invention.
  • the target cells corresponding to panels A and B are CHOK1-hCCR8 and CHOK1-Blank, respectively.
  • FIG. 17 shows the binding of the anti-CCR8 antibody of the present invention to 293F cells transfected with hCCR4. Binding of anti-CCR8 antibody to 293F cells transfected with hCCR4-GFP (A, GFP positive cells) and empty cells (B, all live cells).
  • A GFP positive cells
  • B empty cells
  • Figure 18 shows the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT by incubation of 559E1B10 and 563E10E12 humanized antibodies with Fc mutation or reduced fucosylation with 293F-humanCCR8.
  • A-C are the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT by incubation of 559E1B10_hzH1L1(A), 563E10E12_hzH1L1_hIgG1(B) and 563E10E12_hzH1L0_hIgG1(C) with Fc mutation or reduced fucosylation with 293F-humanCCR8, respectively.
  • Figure 19 shows the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT by incubation of 563E10E12_hzH1L0 with Fc mutation or reduced fucosylation and HuT78.
  • Figure 20 shows the ADCC effect induced by healthy human PBMC and anti-CCR8 antibodies.
  • IL-2 activated healthy human PBMC, anti-CCR8 antibodies and CHOK1-hCCR8 were co-incubated for about 5 hours, and the proportion of PI-positive CHOK1-hCCR8 was detected. Its relationship with different antibodies and their concentrations is shown in the curve.
  • Figure 21 shows that the anti-CCR8 antibody of the present invention eliminates Tregs in peripheral PBMCs. After IL-2-activated PBMCs were incubated with anti-CCR8 antibodies for 96 hours, the ratio of Foxp3-positive cells to CD4 + T cells (A) and the ratio of CD8 + T cells (CD3 + CD4 ⁇ ) to CD3 + T cells (B).
  • FIG22 shows that the anti-CCR8 antibody of the present invention inhibits MC38 tumor growth.
  • CCR8 humanized mice B-hCCR8 were inoculated with colon cancer MC38 cells.
  • 10 mg/kg of antibody or negative control (hIgG1) or equal volume of vehicle (PBS) control were subcutaneously injected twice a week. The changes in tumor volume are shown in the curve.
  • FIG. 23 shows the changes in body weight of mice in the MC38 tumor model treated with the anti-CCR8 antibody of the present invention.
  • FIG24 shows that anti-CCR8 antibodies with Fc mutation and reduced fucosylation inhibit MC38 tumor growth.
  • CCR8 humanized mice B-hCCR8 were inoculated with colon cancer MC38 cells to form tumors.
  • 10 mg/kg of anti-CCR8 antibody or an equal volume of vehicle (PBS) control were subcutaneously injected twice a week. The changes in tumor volume are shown in the curve.
  • PBS vehicle
  • FIG. 25 shows the changes in body weight of mice in the MC38 tumor model treated with Fc-mutated and fucosylated anti-CCR8 antibodies.
  • Figure 26 shows that the anti-CCR8 antibody of the present invention alone and in combination with the anti-mPD-1 antibody inhibits breast cancer EMT-6 tumor growth in mice.
  • Each group was treated with 5-10 mg/kg of antibody (i.v.), anti-mPD-1 antibody (i.p.) or an equal volume of vehicle (PBS) control (i.v.), and was administered twice a week. The changes in tumor volume are shown in the curve.
  • FIG. 27 shows the changes in the body weight of mice during the treatment of the EMT-6 tumor model with the CCR8 antibody of the present invention.
  • the term "or” should be understood to have the same meaning as “and/or” as defined above.
  • “or” or “and/or” should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when terms are clearly indicated to the contrary, such as “only one” or “exactly one” or when “consisting of" is used in the claims, it will refer to only one number listed or one element of the list.
  • CCR8 refers to any native CCR8 produced by expression of CCR8 in a cell. Unless otherwise indicated, the term includes CCR8 from any vertebrate source, such as mammals (e.g., primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats)).
  • mammals e.g., primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats)
  • rodents e.g., mice and rats
  • CCR8 includes variants, subtypes, species homologs of human CCR8, CCR8 of other species, and analogs with at least one common epitope of CCR8, unless otherwise specified.
  • the term includes unprocessed full-length CCR8 and any form of CCR8 produced due to processing in cells.
  • the term encompasses "full-length” unprocessed CCR8 and any form of CCR8 or any fragment thereof produced by intracellular processing, such as splice variants or allelic variants.
  • CCR8 refers to the full length or fragments thereof (such as mature fragments lacking signal peptides) from humans or cynomolgus monkeys.
  • CCL1 and CC motif chemokine ligand 1 refer to any native CCL1 produced by expression of the CCL1 gene in a cell.
  • CCL1 belongs to the CC chemokine family and is secreted by activated monocytes/macrophages, T lymphocytes, and endothelial cells.
  • the term includes all chemokines from CCL1 of any vertebrate origin, such as mammalian (eg, primate (eg, human and cynomolgus monkey) and rodent (eg, mouse and rat)).
  • immune response refers to the actions of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines and complement), which result in the selective damage, destruction or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.
  • Treg and "regulatory T cells” refer to a class of T cells that exhibit immunosuppressive effects in immune responses, generally inhibiting or downregulating the induction and proliferation of effector T cells.
  • the effect of Treg on cancer is complex, but since it has been observed that Treg cells tend to be upregulated in individuals with cancer, and they appear to be recruited to tumor sites, and multiple studies suggest that the presence of a large number of Tregs in the tumor microenvironment indicates a poor prognosis, it is generally believed that Treg cells inhibit human anti-tumor immunity.
  • Various immunotherapies are studying methods of targeting Treg to treat cancer.
  • the tumor contains tumor-infiltrating Treg cells.
  • the tumor contains cells expressing CCR8.
  • the cells expressing CCR8 are Treg cells. In some embodiments of the present invention, T effector cells do not express or substantially do not express CCR8. In some embodiments, the antibodies of the present invention inhibit/eliminate Treg cells in whole/partially. In some embodiments, the antibodies of the present invention inhibit/eliminate Treg cells in whole/partially by means of an ADCC mechanism, and/or block the binding of CCL1 to CCR8, inhibiting CCL1-induced signaling pathways. In some embodiments, the antibodies of the present invention treat cancer by inhibiting/clearing Treg cells in whole/partially.
  • the use of the antibodies of the present invention to inhibit/clear Treg cells in whole/partially is better and/or more efficient than other methods of inhibiting/clearing Treg cells.
  • the antibodies of the present invention treat cancer by blocking the combination of CCL1 and CCR8 in whole/partially.
  • the use of the antibodies of the present invention to block the combination of CCL1 and CCR8 in whole/partially is better and/or more efficient than other methods of blocking the combination of CCL1 and CCR8.
  • the effects include: enhanced anti-tumor immunity, enhanced immune response to tumor antigens, slowed tumor growth, reduced tumor volume, increased secretion of anti-tumor cytokines, increased number/enhanced function of T effector cells infiltrating in tumors, enhanced long-term memory effect of anti-tumor immunity, or any combination of the foregoing.
  • the use of the antibodies of the present invention to inhibit/clear Treg cells in whole/partially has fewer adverse reactions than other methods of inhibiting/clearing Treg cells.
  • the adverse reaction is immune disorder, such as autoimmune disorder, such as autoimmune disease.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical cause and effect relationship, usually initiated by protein-protein interactions such as the binding of a growth factor to a receptor, such as the binding of CCL1 (ligand) to CCR8 (receptor), which results in the transmission of a signal from one part of a cell to another part of the cell.
  • the transmission includes specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions that cause signal transduction.
  • the penultimate process usually includes nuclear events, resulting in changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological characteristic” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, the ability to neutralize or antagonize CCR8 activity in vivo or in vitro, IC50 , the in vivo stability of the antibody, and the immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the targeted peptide, or with other proteins or tissues in general), and the ability to maintain high expression levels of the protein in mammalian cells.
  • antibody refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium.
  • isolated does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule that contains at least four peptide chains when naturally present: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2, and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementary determining regions (CDRs) and regions separated by more conservative regions called framework regions (FRs).
  • CDRs highly variable complementary determining regions
  • FRs framework regions
  • Each VH or VL region consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • antigen-binding fragment of an antibody includes fragments or derivatives of an antibody, generally comprising at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody, which retains the Maintain at least some binding specificity of the parent antibody.
  • binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments well known in the art; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.
  • the antigen-binding fragment of the present invention is selected from Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.
  • the binding fragment or derivative When the binding activity of the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • the antigen-binding fragment of the antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as “conservative variants” or “functional conservative variants” of antibodies).
  • binding compound refers to both antibodies and binding fragments thereof.
  • single-chain Fv or "scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide generally also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain.
  • two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • bivalent antibody contains two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific.
  • diabody refers to a small antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen binding sites.
  • hybridoma antibody refers to a monoclonal antibody against CCR8 prepared according to the knowledge and skills in the field. During preparation, the test subject is injected with CCR8 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated. Hybridoma technology is achieved by fusing two cells while maintaining the main characteristics of both. The two cells are mouse spleen cells immunized with antigens and mouse myeloma cells.
  • mouse spleen cells (B lymphocytes) immunized with specific antigens is its antibody secretion function, but they cannot be cultured continuously in vitro, while mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, they have so-called immortality.
  • the selective culture medium only hybrid cells fused with B cells and myeloma cells can have the ability to be continuously cultured, forming cell clones that have both antibody secretion function and cell immortality.
  • the present invention obtains hybridoma cells that can express positive antibodies by immunizing mice with CCR8 protein, and then obtaining spleen cells of mice and fusing them with myeloma cells.
  • chimeric antibody is an antibody having the variable domain of a first antibody and the constant domain of a second antibody.
  • the first antibody and the second antibody are from different species.
  • the variable domains are obtained from antibodies of rodents or the like ("parent antibodies"), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibodies are less likely to induce adverse immune responses in human subjects than the parent rodent antibodies.
  • the rodent is a mouse or a rat.
  • the affinity of the chimeric antibody for the antigen is not less than or almost not less than that of the parent mouse antibody.
  • humanized antibody refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies.
  • a humanized antibody comprises substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat sugar chains. Similarly, a “mouse antibody” refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat sugar chains. Similarly, a "rat antibody” refers to an antibody that contains only rat immunoglobulin sequences.
  • the light chain of an antibody can be classified into one of two types (called kappa ( ⁇ ) and lambda ( ⁇ )) based on the amino acid sequence of its constant domain.
  • the heavy chain of an antibody can be divided into mainly 5 different types according to the amino acid sequence of its heavy chain constant region: IgA, IgD, IgE, IgG and IgM, and several of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.
  • An "isotype” antibody refers to the antibody species (e.g., IgM, IgE, IgG such as IgG1, IgG2 or IgG4) provided by the heavy chain constant region gene. Isotypes also include modified forms of one of these species, in which modifications have been produced to change Fc function, such as to enhance or reduce effector function or the binding to Fc receptors.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
  • effector function refers to biological activities mediated by the Fc region of an antibody, which vary with the antibody isotype.
  • Antibody effector functions include, but are not limited to: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; opsonization; downregulation of cell surface receptors; and B cell activation, etc.
  • epitope refers to a protein determinant that can specifically bind to an antibody.
  • Epitopes are usually composed of various chemically active surface molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that in the presence of denaturing solvents, the former Rather than the loss of union with the latter.
  • cross-reaction refers to the combination of antigen fragments of the same target molecule of human, monkey, and/or mouse (mouse or rat). Therefore, “cross-reaction” should be understood as the inter-species reaction between antigen binding molecules (e.g., antibodies) and similar molecules (e.g., CCR8) expressed in different species.
  • antigen binding molecules e.g., antibodies
  • CCR8 similar molecules
  • the cross-reaction specificity of the monoclonal antibodies recognizing human CCR8, monkey, and/or mouse CCR8 (mouse or rat) can be determined by FACS analysis.
  • affinity refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant ( KD ), which is the ratio of the dissociation rate constant and the association rate constant ( kdis and kon , respectively). Affinity can be measured by common methods known in the art. In some embodiments of the invention, affinity, such as affinity between an antibody of the invention and an antigen, is measured using surface plasmon resonance (SPR) technology.
  • SPR surface plasmon resonance
  • affinity refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant ( KD ), which is the ratio of the dissociation rate constant and the association rate constant ( kdis and kon , respectively). Affinity can be measured by common methods known in the art. In some embodiments of the invention, affinity, such as affinity between an antibody of the invention and an antigen, is measured using surface plasmon resonance (SPR) technology.
  • SPR surface plasmon resonance
  • the term "does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the EC50 for binding to the protein or cell is 1.0 ⁇ 10-7 M or higher, more preferably 1.0 ⁇ 10-6 M or higher, more preferably 1.0 ⁇ 10-5 M or higher, more preferably 1.0 ⁇ 10-4 M or higher, more preferably 1.0 ⁇ 10-3 M or higher.
  • high affinity means, for IgG antibodies, an EC 50 for an antigen of 1.0 ⁇ 10 -7 M or less, preferably 5.0 ⁇ 10 -8 M or less, more preferably 1.0 ⁇ 10 -8 M or less, more preferably 5.0 ⁇ 10 -9 M or less, more preferably 1.0 ⁇ 10 -9 M or less, more preferably 5.0 ⁇ 10 -10 M or less, more preferably 1.0 ⁇ 10 -10 M or less.
  • "high affinity” binding may vary.
  • "high affinity” binding for the IgM subtype means an EC 50 of 10 -7 M or less, preferably 10 -8 M or less, more preferably 10 -9 M or less.
  • blocking for IgG antibodies to receptors refers to the administration of the antibody so that the antibody competes with the intended ligand of the receptor, inhibiting the binding and interaction between the receptor and the ligand. This inhibition can occur through a variety of mechanisms, including, for example, altering ligand affinity due to overlapping binding sites on the receptor and/or receptor conformational changes induced by the antibody.
  • Antibodies and antibody fragments referred to as "functional" are characterized by having such properties.
  • the ability to "block” refers to an EC 50 value of 5.0 ⁇ 10 -5 M or less, 1.0 ⁇ 10 -5 M or less, 5.0 ⁇ 10 -6 M or less, 1.0 ⁇ 10 -6 M or less, preferably 5.0 ⁇ 10 -7 M or less, more preferably 1.0 ⁇ 10 -7 M or less, more preferably 5.0 ⁇ 10 -8 M or less, more preferably 1.0 ⁇ 10 -8 M or less.
  • antibody-dependent cellular cytotoxicity refers to a cell-mediated immune defense in which immune system effector cells actively lyse target cells that have antibody-bound cell surface antigens.
  • complement-dependent cytotoxicity refers to the effector function of IgG and IgM antibodies that, when bound to surface antigens, initiate the classical complement pathway, including formation of the membrane attack complex and target cell lysis.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form.
  • RNA ribonucleic acid
  • nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.
  • a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as explicitly indicated sequences.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), which can be derived from any source, capable of integrating with the genome or autonomously replicating, and contains one or more polynucleotide molecules that have been linked (i.e., operably linked) in a functionally operational manner.
  • a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule which can be derived from any source, capable of integrating with the genome or autonomously replicating, and contains one or more polynucleotide molecules that have been linked (i.e., operably linked)
  • the recombinant construct comprises a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence, which drives and/or directs transcription of the polynucleotide of the present invention in a host cell.
  • a transcription initiation regulatory sequence which drives and/or directs transcription of the polynucleotide of the present invention in a host cell.
  • Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to drive and/or direct the expression of the polynucleotide of the present invention.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be connected.
  • viral vector in which additional DNA segments can be connected to the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors).
  • vectors After introduction into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule that is capable of replicating and expressing a target gene when transformed, transfected or transduced into a host cell.
  • the expression vector comprises one or more phenotypic selection markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host when necessary.
  • the expression vector of the present invention comprises a construct of the present invention and/or a polynucleotide of the present invention.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and cells derived therefrom. Progeny.
  • Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as E. coli cells.
  • Host cells include cultured cells, and also include cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.
  • Activation can have the same meaning, such as a cell or receptor is activated, stimulated or treated with a ligand, unless the context otherwise or clearly dictates.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins and binding compounds derived from antibodies.
  • Ligand also includes small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies.
  • Activation can refer to cell activation regulated by internal mechanisms as well as external or environmental factors.
  • Response/reaction such as the response of a cell, tissue, organ or organism, includes changes in biochemical or physiological behavior (such as concentration, density, adhesion or migration within a biological compartment, gene expression rate or differentiation state), where the change is related to activation, stimulation or treatment, or to internal mechanisms such as genetic programming.
  • tumor is intended to emphasize malignant tumors
  • cancer and “tumor” are used interchangeably to refer to any abnormal, uncontrolled cell or tissue growth or proliferation in an animal.
  • cancer and “tumor” include solid tumors and hematological tumors, and also include precancerous lesions.
  • tumors include breast cancer, squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, lung cancer (such as non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, colon cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, thyroid cancer, testicular cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, thymoma and various head and neck cancers.
  • lung cancer such as non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma
  • peritoneal cancer hepatocellular carcinoma
  • gastric cancer intestinal cancer
  • hematologic neoplasms include mixed B-cell and T-cell leukemia, B-cell lymphoma, granulocytic leukemia (acute and chronic), lymphocytic leukemia (acute and chronic), childhood/juvenile lymphocytic leukemia, myelomonocytic leukemia, diffuse large B-cell lymphoma (DLBC), Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), multiple myeloma, myelodysplastic syndrome, etc.
  • the tumor is a benign tumor.
  • treatment refers in one embodiment to slowing down, interrupting, blocking, alleviating, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical signs of a disease, alleviating symptoms, or preventing or inhibiting the further development of a disease, condition, or illness (i.e., slowing down or preventing or reducing at least one of the progression of a disease or its clinical symptoms).
  • treatment refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient.
  • treatment refers to regulating a disease or condition physically (e.g., the stabilization of discernible symptoms), physiologically (e.g., the stabilization of physical parameters), or in both aspects.
  • methods for assessing the treatment and/or prevention of a disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, such as mammals. Mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
  • cyno or “cynomolgus monkey” refers to or derived from the cynomolgus monkey.
  • Administration "in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) administration and consecutive administration in either order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to the amount of the CCR8 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to cells, tissues or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions or the development of the disease or condition.
  • a therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to cause symptom improvement, such as an amount to treat, cure, prevent or improve a related medical condition or to increase the rate of treatment, cure, prevention or improvement of such a condition.
  • the therapeutically effective dose refers only to that ingredient.
  • the therapeutically effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of a therapeutic agent will result in an increase in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.
  • “Pharmaceutically acceptable carrier” refers to a component of a pharmaceutical preparation or composition other than the active ingredient that is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CCR8.
  • the term "anti-CCR8 antibody”, “anti-CCR8”, “CCR8 antibody” or “antibody that binds to CCR8” refers to an antibody that can bind to a CCR8 protein or a fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting CCR8.
  • the antibodies of the invention bind to human or cynomolgus monkey CCR8 proteins. In some embodiments, the antibodies of the invention bind to CHOK1-humanCCR8 cells or 293F-cynoCCR8 cells. In some embodiments, the antibodies of the invention inhibit/block the binding of CCR8 to its ligand CCL1. In some embodiments, the antibodies of the invention inhibit/block the recruitment of induced ⁇ -Arrestin.
  • Any suitable method for producing antibodies can be used to produce antibodies of the present invention.
  • Any suitable form of CCR8 can be used as an immunogen (antigen) to produce antibodies.
  • any CCR8 variant or fragment thereof can be used as an immunogen.
  • hybridoma cells producing monoclonal anti-CCR8 antibodies of mouse origin can be produced by methods known in the art.
  • Antibodies derived from rodents may cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use may lead to an immune response against the therapeutic antibody in the human body, which may result in at least a loss of therapeutic efficacy and, in severe cases, potentially lethal allergic reactions.
  • One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which the mouse variable region is fused to the human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, the completeness of the chimeric antibody is not necessarily the same as that of the human constant region.
  • the antibodies of the invention are chimeric antibodies. In some preferred embodiments, the antibodies of the invention are humanized antibodies.
  • chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal hybridoma antibody prepared as described above.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric CCR8 antibody of the present invention can be prepared by effectively linking the variable regions of the heavy and light chains of immunoglobulins derived from hybridomas to the constant regions of human IgG using methods known in the art (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy chains and chimeric light chains.
  • the constant region contained in the chimeric antibody of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG1.
  • the chimeric CCR8 antibodies of the present invention can be obtained by transfecting expression cells with chimeric light chain and chimeric heavy chain expression plasmids by "mixing and matching", and the CCR8 binding of such "mixed and matched" antibodies can be tested using the above-mentioned binding assays and other conventional binding assays (e.g., ELISA).
  • CDR regions are regions of an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen contact residues.
  • CDR (“antigen contact point") region.
  • CDR is primarily responsible for binding to the antigen epitope.
  • the CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the variability of antibody sequences (Kabat et al., Sequen ces of Proteins of Immunological Interest, 4th ed., U.S.
  • the residues of each CDR are as follows.
  • CDR or “CDR sequence” encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). In some embodiments, the HCDRs and LCDRs in the antibodies of the present invention are determined according to the Kabat scheme, respectively.
  • the boundaries of the CDRs of the antibodies of the invention can be determined by one skilled in the art according to any scheme in the art (eg, different assignment systems or combinations).
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but due to the application of different schemes (e.g. different assignment systems or combinations) that result in their declared CDR boundaries being different from the specific CDR boundaries defined in the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding.
  • the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of a CDR.
  • the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.
  • the present invention mixes and pairs various chimeric heavy chain and light chain expression plasmids and transfects expression cells to produce anti-CCR8 chimeric antibodies.
  • the humanized antibodies of the present invention can be prepared by inserting mouse CDR regions into human germline framework regions using methods known in the art, such as Winter et al. U.S. Pat. No. 5,225,539 and Queen et al. U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370.
  • amino acid changes include amino acid deletions, additions or substitutions, etc.
  • the anti-CCR8 antibodies or antigen-binding fragments thereof of the present invention include those having amino acid sequences that have been mutated by amino acid deletions, additions or substitutions, but are still at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the above antibodies (particularly in the CDR regions depicted in the above sequences).
  • the antibodies of the present invention have at least one, for example, 1, 2, 3, 4 or 5 amino acid mutations in the CDR regions that have been deleted, added or substituted by amino acids when compared to the CDR regions described in the specific sequences.
  • the antibodies of the present invention have at least one, for example, 1, 2, 3, 4 or 5 amino acid mutations in the framework regions that have been deleted, added or substituted by amino acids when compared to the framework regions in the specific sequences.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after the amino acid sequences are aligned (and gaps introduced where necessary) to obtain maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity.
  • Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software/algorithms.
  • One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.
  • the antibody or antigen-binding fragment thereof of the present invention wherein the antibody or antigen-binding fragment thereof
  • the original binding fragment comprises a heavy chain constant region and/or a light chain constant region, preferably, the light chain constant region is a ⁇ chain or a ⁇ chain constant region; the heavy chain constant region is selected from mouse mIgG2a, human IgG1, human IgG2, human IgG3 or IgG4 type, or a modified form thereof.
  • the heavy chain constant region is a human IgG1 type or a human IgG4 type with an S228P mutation.
  • the modified form of the constant region includes amino acid sequence modification and/or glycosylation modification.
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., addition, deletion or substitution, preferably substitution) at one or more amino acid positions.
  • the antibody comprises at least one modification that enhances cell killing.
  • the enhanced cell killing is enhanced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
  • the modification is defucosylation/reduced fucosylation.
  • the modification is at a site selected from 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 330, 331, 332, 333, 334, 335 09,312,315,320,322,324,326,327,328,329,330,331,333,334,335,337,338,340,360,373,376,378,382,388,389,398,414,416,419
  • the mutation in the amino acid sequence can be named as follows: the single letter code of the parent amino acid, followed by the position number, followed by the single letter code of the amino acid after the mutation.
  • L 234th leucine
  • A alanine
  • C236 deletion the deletion of cysteine (C) at position 236 can be represented as "C236 deletion”.
  • the modification is one or more heavy chain constant region mutations at one or more positions selected from L234, L235, G236, S239, F243, D265, H268, D270, R292, S298, Y300, V305, K326, A330, I332, E333, K334 and P396 according to the EU numbering system.
  • the one or more heavy chain constant region mutations are selected from N297A substitution, N297Q substitution, L235A substitution together with L237A substitution, L234A substitution together with L235A substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, D265A substitution, A327Q substitution and P329A substitution according to the EU numbering system.
  • the modification is selected from one or more heavy chain constant region mutations of G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L according to the EU numbering system.
  • the one or more heavy chain constant region mutations are selected from L235V, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L, A330M, I332E, E333A, according to the EU numbering system.
  • One or more heavy chain constant region mutations of K334A, K334E and P396L are one or more heavy chain constant region mutations selected from M252Y, S254T and T256E according to the EU numbering system.
  • the heavy chain constant region has one or more groups of mutations selected from the following combinations of positions where mutations occur simultaneously: (1) L235/F243/R292/Y300/P396, (2) F243/R292/Y300/V305/P396, (3) D270/K326/A330/K334, (4) S239/A330/I332, (5) S298/E333/K334, (6) L234/L235/G236/S239/H268/D270/S298, (7) M252/S254/T256, (8) L234/L235/D265, (9) G236/S239/I332, and (10) S239/I332.
  • the heavy chain constant region has one or more mutations selected from the following mutation combinations: (1) L235V/F243L/R292P/Y300L/P396L, (2) F243L/R292P/Y300L/V305I/P396L, (3) D270E/K326D/A330M/K334E, (4) S239D/A330L/I332E, (5) S298A/E 333A/K334A, (6) L234Y/L235Q/G236W/S239M/H268D/D270E/S298A, (7) M252Y/S254T/T256E, (8) L234A/L235A/D265A, (9) L234F/L235E/D265A, (10) G236A/S239D/I332E and (11) S239D/I332E.
  • mutations selected from the following mutation combinations: (1) L235V/F243L/R292P/Y
  • cysteine engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • one, two, or more mutations e.g., amino acid substitutions
  • one, two, or more mutations are introduced into the hinge region (CH1 domain) to alter the number of cysteine residues in the hinge region (e.g., increase or decrease).
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties that are known in the art and readily available.
  • the present invention provides a polynucleotide molecule encoding an anti-CCR8 antibody or any fragment thereof as described herein.
  • the polynucleotide molecule may comprise a polynucleotide molecule encoding an amino acid sequence of a light chain variable region and/or a heavy chain variable region of an antibody or at least a portion thereof, or a polynucleotide molecule encoding an amino acid sequence of a light chain and/or a heavy chain of an antibody or at least a portion thereof.
  • the polynucleotide molecule of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-95, or comprises a nucleic acid encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1-95.
  • the polynucleotide molecules encoding the antibodies of the present invention include polynucleotide molecules that have been mutated by nucleotide deletion, addition or substitution, but still have at least about 60, 70, 80, 90, 95 or 100% identity with the CDR corresponding coding regions depicted in the sequences described above.
  • the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably, the vector is a eukaryotic expression vector.
  • the polynucleotide molecule as described herein is contained in one or more expression vectors.
  • the present invention provides a host cell comprising the polynucleotide molecule as described herein or the expression vector as described herein, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the present invention provides a method for preparing an anti-CCR8 antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof in a host cell as described herein under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.
  • the present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention or any fragment thereof, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These include Chinese hamster ovary (CHO) cells, NS0, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has a high expression level.
  • ATCC American Type Culture Collection
  • the present invention provides a method for preparing an anti-CCR8 antibody, wherein the method comprises, when an expression vector is introduced into a mammalian host cell, producing the antibody by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell, or more preferably, secreted into the culture medium in which the host cell is grown.
  • Standard protein purification methods can be used to recover antibodies from the culture medium.
  • Antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these are obvious to those skilled in the art.
  • the purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.
  • antibodies expressed by different cell lines or expressed in transgenic animals have different glycosylation from one another.
  • all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies.
  • non-fucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their sugar structures are normal components of native human serum IgG.
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CCR8 antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or an immunoconjugate as described herein, and a pharmaceutically acceptable carrier or excipient.
  • the anti-CCR8 antibody or pharmaceutical composition thereof provided by the present invention can be integrated with suitable carriers, excipients and other agents in the formulation for combined administration, thereby providing improved transfer, delivery, tolerance, etc.
  • composition refers to a preparation which permits the active ingredient contained therein to exist in biologically effective form, and which contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.
  • compositions containing the anti-CCR8 antibodies described herein can be prepared by mixing the anti-CCR8 antibodies of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized preparation.
  • pharmaceutical excipients Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)
  • the pharmaceutical composition or formulation of the present invention may also include one or more other active ingredients that are required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the pharmaceutical composition of the present invention further comprises a composition of polynucleotide molecules encoding anti-CCR8 antibodies.
  • the additional therapeutic agent is a monoclonal antibody targeting PD-1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-L1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting CTLA4.
  • the present invention provides a pharmaceutical combination comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the immunoconjugate described herein, or the pharmaceutical composition described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the immunoconjugate described herein, the pharmaceutical composition described herein, or the pharmaceutical combination described herein.
  • the present invention also provides a combination product comprising an anti-CCR8 antibody or fragment thereof of the present invention and one or more additional therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, anti-infective agents, small molecule drugs or immunomodulators, etc.).
  • additional therapeutic agents e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, anti-infective agents, small molecule drugs or immunomodulators, etc.
  • the combination product is used to prevent or treat diseases associated with CCR8/immune checkpoints and/or diseases mediated by CCR8 and/or immune checkpoints.
  • the additional therapeutic agent is an existing standard therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent is other antibodies. In some embodiments, the additional In some preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting an immune checkpoint. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-L1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting CTLA4.
  • two or more components of the combination product may be co-administered to a subject sequentially, separately or simultaneously.
  • the present invention also provides a kit comprising an anti-CCR8 antibody or fragment thereof, a pharmaceutical composition or a combination product of the present invention, and optionally a package insert directing administration.
  • the present invention also provides a pharmaceutical product comprising the anti-CCR8 antibody or fragment thereof, pharmaceutical composition or combination product of the present invention, optionally further comprising a package insert for guiding administration.
  • anti-CCR8 antibody or corresponding immunoconjugate can be used in a therapeutic method. It should also be understood that when discussing "antibodies", compositions containing antibodies are also included.
  • the anti-CCR8 antibodies of the present invention can be used in a therapeutic or preventive method described in any embodiment of the present invention in a therapeutically effective amount or a preventive effective amount.
  • the present invention provides the use of antibodies or antigen-binding fragments thereof, polynucleotides, expression vectors, host cells, immunoconjugates, or pharmaceutical compositions described herein in the preparation of drugs.
  • the present invention also provides the use of the drug combinations described herein in the preparation of drugs.
  • the present invention substantially provides an effective amount of antibodies or antigen-binding fragments thereof, polynucleotides, expression vectors, host cells, immunoconjugates, or pharmaceutical compositions described herein in combination with other therapeutic agents for the preparation of drugs.
  • the drug is used to prevent and/or treat tumors, autoimmune diseases or infectious diseases in a subject
  • the tumor is preferably, for example, melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer and its metastatic cancer.
  • the present invention provides an antibody or antigen-binding fragment thereof, a polynucleotide, an expression vector, a host cell, an immunoconjugate, a pharmaceutical composition or a pharmaceutical combination as described herein for use in treating and/or preventing a tumor, an autoimmune disease, or an infectious disease, wherein the tumor is preferably, for example, melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer and its metastatic cancer.
  • the present invention provides a method for treating and/or preventing tumors, autoimmune diseases, or infectious diseases, comprising administering to a subject in need thereof a therapeutically or preventively effective amount of the antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, immunoconjugate, pharmaceutical composition or drug combination described herein, wherein the tumor is preferably, for example, melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer and its metastatic cancer.
  • the tumor is preferably, for example, melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer and its metastatic cancer.
  • the administration methods of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or local administration to the lesion, etc.
  • treatment refers to a clinical intervention intended to change the natural course of a disease in an individual being treated.
  • the desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any adverse feelings or direct or indirect pathological consequences of the disease, reducing the rate of progression of the disease, improving or alleviating the disease state, and alleviating or improving prognosis.
  • the present invention also provides for co-administering a therapeutically effective amount of one or more therapies (e.g., treatment modalities and/or other therapeutic agents) to a subject.
  • therapies e.g., treatment modalities and/or other therapeutic agents
  • the antibodies of the present invention can be used alone or in combination with other therapeutic agents in therapy. In some embodiments, the antibodies of the present invention are co-administered with at least one additional therapeutic agent.
  • the present invention provides a method for detecting the presence of CCR8 in a sample using an antibody or antigen-binding fragment thereof as described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum or other liquid samples of biological origin.
  • the biological sample comprises cells or tissues.
  • CCR8 is human CCR8 or cynomolgus monkey CCR8.
  • the method includes the step of contacting an antibody or antigen-binding fragment thereof as described herein or a detection composition containing the antibody or antigen-binding fragment thereof with a sample, and the step of detecting whether there is a conjugate or a binding signal generated by the combination of the antibody or antigen-binding fragment thereof with CCR8.
  • the antibody or antigen-binding fragment thereof as described herein may be labeled to indicate whether the conjugate has been formed.
  • the method may be an in vitro or in vivo method.
  • CCR8 is detected prior to treatment, for example, prior to initiation of treatment or prior to a treatment after a treatment interval.
  • an anti-CCR8 antibody or antigen-binding fragment thereof for use in a diagnostic or detection method is provided.
  • the compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention. example.
  • Example 1 Animal immunization and preparation of mouse hybridoma antibodies producing anti-human CCR8
  • the full-length human CCR8 gene (DNA or mRNA) and the engineered cell line overexpressing human CCR8 are used to immunize mice.
  • the full-length monkey CCR8 gene (DNA or mRNA) and cells overexpressing monkey CCR8 are also used for immunization or shock immunization. SP2/0 cells are fused and positive clones are screened by flow cytometry. The specific method is as follows:
  • mice Animal immunization: The gene encoding the full length of human CCR8 (UniProtKB/Swiss-Prot: P51685) was constructed into PCDNA3.1 or PCDNA3.4 plasmid (constructed by Shanghai Ruizhi Chemical Research Co., Ltd.) as an immunogen, and DNA immunization was performed on Balb/c female mice, 4 ⁇ g/mouse. DNA immunization was repeated 6-7 times at intervals of 2 weeks. Ten days after the last immunization, 293F cells expressing human CCR8 (constructed by Shanghai Ruizhi Chemical Research Co., Ltd., 293F-hCCR8) were injected intraperitoneally for shock immunization. Three days later, the spleen of the mice was taken for cell fusion.
  • PCDNA3.1 or PCDNA3.4 plasmid constructed by Shanghai Ruizhi Chemical Research Co., Ltd.
  • 293F cells expressing human CCR8 (293F-hCCR8) were used as immunogens to immunize Balb/c female mice intraperitoneally (2-5x10 6 cells/mouse). Cell immunization was repeated 3 times at intervals of 2 weeks. Ten days after the last immunization, 293F-hCCR8 cells (2-5x10 6 cells/mouse) were injected intraperitoneally again for impact immunization. Three days later, the spleen of the mice was taken for cell fusion.
  • the full-length mRNA encoding human CCR8 (UniProtKB/Swiss-Prot: P51685) was used as an immunogen (prepared by Shanghai Hongcheng Pharmaceutical Co., Ltd., 50 ⁇ g/mouse) to immunize Balb/c female mice with mRNA. mRNA immunization was repeated 3 or 4 times at intervals of 3 weeks, and 293F-hCCR8 cells were injected intraperitoneally 10 days after the final immunization for shock immunization. The spleen of the mice was taken for cell fusion 3 days later.
  • the clones in Table 1 have a higher mean fluorescence intensity (MFI) for binding to human CCR8 (CHOK1-hCCR8) and cynomolgus monkey CCR8 (CHOK1-cynoCCR8) than the blank control cells (CHOK1), indicating that these clones have specific binding to human and cynomolgus monkey CCR8.
  • MFI mean fluorescence intensity
  • the hybridoma cells with binding activity to human CCR8 and cynomolgus monkey CCR8 in Table 1 were cultured in serum-free medium, and the culture supernatant was collected after 10 days.
  • the mouse monoclonal antibody was purified using a Protein A column (Borgron (Shanghai) Biotechnology Co., Ltd., Catalog No.: AA0272) to obtain purified monoclonal antibodies.
  • the binding activity of the anti-human CCR8 antibody and the blocking activity of the CCL1 binding were detected by flow cytometry.
  • the cell concentration of 293F cell line expressing human CCR8 (293F-hCCR8, constructed by Shanghai Ruizhi Chemical Research Co., Ltd.) or 293T cell line expressing cynomolgus monkey CCR8 (293T-cyno CCR8, constructed by Kangyuan Broad Group Co., Ltd.) was adjusted to 1 ⁇ 10 6 cells/mL, 100 ⁇ L/well was placed in a 96-well U-bottom plate, and the supernatant was discarded after centrifugation.
  • Anti-human CCR8 antibody, control antibody (anti-human CCR8 antibody 433H, clone number: 433H, BD Pharmingen, catalog number: 624092; 10A11 is the sequence in patent WO2020138489 (light chain variable region sequence number: 59, heavy chain variable region sequence number: 41), fused with the human and mouse constant region sequences in Table 5 of this article to obtain 10A11_hIgG1 and 10A11_mIgG2a antibodies), IgG1isotype (Bai Ying Biotechnology Co., Ltd., catalog number: B117901) were diluted to the starting working concentration in FACS buffer (PBS solution containing 1% FBS), and then gradiently diluted with FACS buffer solution.
  • FACS buffer PBS solution containing 1% FBS
  • the mean fluorescence intensity (MFI) was read by flow cytometer (BECKMAN COULTER cytoFLEX), and the experimental data were analyzed by Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis and the corresponding MFI value was used as the y-axis. The four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC 50 .
  • antibody clones such as 27B9-1G3, 559E1B10, 563E10E12, 569D11B5 and 589D7C7 have strong binding activity to 293F-hCCR8, with EC50 of 107.0 to 360.6 ng/mL; as controls, the EC50s of 433H and 10A11_mIgG2a binding to 293F-hCCR8 were 206.0 and 185.1 ng/mL, respectively.
  • Antibody clones 27B9-1G3, 559E1B10, 563E10E12, 569D11B5 and 589D7C7 all had strong binding activity to cynomolgus monkey CCR8, with EC 50 of 146.1 to 590.8 ng/mL, while the control antibodies 433H and 10A11_mIgG2a had very weak binding activity to cynomolgus monkey CCR8, with EC 50 of 15312.5 and 5770.5 ng/mL, respectively.
  • CCL1 is the main ligand of CCR8, and CCR8 is the only known receptor of CCL1.
  • High expression of CCL1 in tumor tissue is negatively correlated with tumor prognosis.
  • CCL1 can induce Treg migration through CCR8, and blocking the binding of CCL1 to CCR8 can inhibit Treg migration and tumor growth (see Klarquist J et al. Ccl22 Diverts T Regulatory Cells and Controls the Growth of Melanoma. Cancer Res. 2016 Nov 1; 76 (21): 6230-6240 and Xu Y et al. Sox2 Communicates with Tregs Through CCL1 to Promote the Stemness Property of Breast Cancer Cells. Stem Cells. 2017 Dec; 35 (12): 2351-2365.).
  • Anti-CCR8 antibodies with the function of blocking the binding of CCL1 to CCR8 may have stronger anti-tumor activity.
  • a competitive binding assay was used to detect the activity of anti-CCR8 antibodies in blocking the binding of AlexaFlour-647-labeled human CCL1 to 293F-hCCR8. The specific method is as follows:
  • the 293F cell line expressing human CCR8 (293F-hCCR8, constructed by Shanghai Ruizhi Chemical Research Co., Ltd.) was adjusted to a cell concentration of 1 ⁇ 10 6 cells/mL, and 100 ⁇ L/well was placed in a 96-well U-bottom plate. The supernatant was discarded after centrifugation.
  • Anti-CCR8 antibody, control antibody (anti-human CCR8 antibody, clone number: 433H, BD Pharmingen, catalog number: 624092), and hIgG1isotype (Bai Ying Biotechnology Co., Ltd., catalog number: B117901) were diluted to 2 times the initial working concentration with FACS buffer (PBS solution containing 1% FBS), and then gradiently diluted with FACS buffer solution. Resuspend the cells with 50 ⁇ L/well of gradient diluted antibodies and mix them by pipetting; then add 50 ⁇ L/well of 20nM human CCL1-AlexaFlour-647 (Almac, CAF-7) solution, mix them by pipetting, and incubate at 4°C for 1 hour.
  • FACS buffer PBS solution containing 1% FBS
  • the mean fluorescence intensity (MFI) was read by flow cytometer (BECKMAN COULTER cytoFLEX), and the experimental data were analyzed by Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis, and the corresponding MFI value was used as the y-axis.
  • the four-parameter regression model was used to fit the antibody dose-effect curve and calculate the IC 50 .
  • antibody clones 27B9-1G3, 559E1B10, 563E10E12, 569D11B5 and 589D7C7 can block the binding of CCL1 to CCR8, with IC50 of blocking activity ranging from 75.0 to 131.7 ng/mL, while the IC50 of control 433H blocking activity was 94.3 ng/mL.
  • sequenced light and heavy chain variable regions were constructed onto human constant regions (IgG1/K, see Table 5) or mouse constant regions (mIgG2a) for in vitro functional identification or in vivo efficacy studies.
  • the constructed hIgG1 and mIgG2a chimeric antibodies were confirmed to be consistent with the sequences in Tables 4 and 5 by sequencing.
  • Expi293F cells (Thermofisher catalog number: A1452) were used for expression, and Protein Purification was performed by column A.
  • the specific method is as follows:
  • Expi293 cells expressing antibodies One day before transfection, dilute the Expi293 cell density to 1.5 ⁇ 10 6 cells/mL and culture in a 37°C, 8% CO 2 shaker at 120 rpm. The next day, measure the viable cell density and viability. The cell transfection density should be 3 ⁇ 10 6 cells/mL and the cell viability should be greater than 95%.
  • Prepare PEI/plasmid complex Mix PEI (1 mg/mL, polysciences, catalog number: 24765-1) by inversion. Dilute the plasmid with OPM-293 CD05 Medium (OPM, catalog number: 81075-001). The plasmid dosage is 1 ⁇ g/mL transfection volume.
  • the volume of the culture medium for diluting the plasmid is 1/20 of the transfection volume. Mix gently. The heavy chain to light chain ratio is 1:1.5. Dilute the PEI reagent with OPM-293 CD05 Medium, the amount of PEI is twice the amount of plasmid, the volume of the medium for diluting PEI is 1/20 of the transfection volume, gently invert to mix, and incubate at room temperature for 5 minutes. Add the diluted PEI reagent to the diluted plasmid and gently invert to mix. Incubate the PEI/plasmid complex at room temperature for 15 minutes, then slowly add the solution dropwise to the transfer shake flask, gently rotating the shake flask during the addition process.
  • Protein A column purification of antibodies Prepare a gravity chromatography column, open the upper cover of the gravity chromatography column, place the gasket at the bottom of the gravity column and press it tightly.
  • equilibration buffer If the target pH is not reached, continue to add equilibration buffer until the target pH is reached. Slowly add a certain volume of sample to the gravity chromatography column. Add at least 10CV of elution buffer to the gravity chromatography column. Slowly add 5CV of elution buffer (10-50mM NaAc, pH3.0-pH3.5) to the gravity chromatography column and incubate for 3-5 minutes to collect the eluate. Repeat the elution step as needed.
  • Neutralization Adjust pH to target pH with neutralization buffer (1M Tris). Determine protein concentration using Nanodrop. Replace the buffer containing the antibody with PBS by ultrafiltration.
  • Example 2-2.1 The saturation binding assay described in Example 2-2.1 was used to detect the binding activity of anti-CCR8 chimeric antibodies to human and cynomolgus monkey CCR8.
  • the results are shown in Figures 4 and 5 and Table 6.
  • hIgG1 chimeric antibody 27B9-1G3 mIgG2a chimeric antibodies 563E10E12, 569D11B, 589D7C7 and 559E1B10 had strong binding activity to cynomolgus monkey CCR8, with EC 50 of 66.2-303.5 ng/ml, while 433H and 10A11_hIgG1 had weaker binding activity to cynomolgus monkey CCR8, with EC 50 of 14580.5 and 6309.0 ng/ml.
  • Example 2-2.2 The competition binding experiment method in Example 2-2.2 was used to detect the competition of anti-CCR8 chimeric antibodies for CCL1 binding. The results are shown in Figure 6 and Table 7. Both hIgG1 and mIgG2a chimeric antibodies can dose-dependently block the binding of CCL1 to 293F-hCCR8, and the IC50 of their blocking activity is 29.5-273.4 ng/mL. The IC50 of 433H and 10A11_hIgG1 blocking is 70.5 and 54.6 ng/mL.
  • Anti-CCR8 chimeric antibody blocks CCL1-induced ⁇ -Arrestin recruitment
  • CCL1 induces chemotaxis and receptor endocytosis through CCR8, the latter of which depends on the expression and recruitment of ⁇ -arrestin.
  • ⁇ -arrestin analysis is used to detect the activation function of CCL1 on CCR8 (James M Fox et al. Structure/function relationships of CCR8 agonists and antagonists. Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist. J Biol Chem. 2006 Dec 1; 281(48): 366 52-61.doi:10.1074/jbc.M605584200. and Libao Liu et al.
  • Tango-H_CCR8-CHO-K1 cells were inoculated into a cell culture flask, and Doxycycline (Selleck, S4163-100mg) was added to the culture medium at a final concentration of 10 ⁇ g/mL. The culture was continued for about 48 hours to induce human CCR8 expression using Doxycycline. The induced Tango-H_CCR8-CHO-K1 cells were digested with trypsin (Gibco, 25200072) containing 0.25% EDTA, centrifuged at 200 ⁇ g for 5 minutes, and the supernatant was removed.
  • trypsin Gibco, 25200072
  • the cells were resuspended in fresh F12K complete medium (Gibco, 21127022), the cell viability was detected and counted, and the cell concentration was adjusted to 5 ⁇ 10 5 cells/mL with fresh medium, and 100 ⁇ L/well was inoculated in a white opaque plate, and placed in a 37°C, 5% CO 2 incubator for overnight culture to allow the cells to adhere to the wall.
  • Anti-CCR8 antibody, positive control antibody, and hIgG1isotype (Baiying Biotechnology Co., Ltd., B117901) were diluted to 2 times the starting working mass concentration with F12K complete medium, and then gradient dilution was performed with F12K complete medium.
  • Graphpad Prism 8.0 software was used to analyze the experimental data, with the logarithm of the antibody concentration as the x-axis and the corresponding RLU value as the y-axis. A four-parameter regression model was selected to fit the antibody dose-effect curve.
  • hIgG1 or mIgG2a chimeric antibodies 27B9-1G3, 563E10E12, 569D11B5, 589D7C7, 559C12G12 and 559E1B10 were able to block CCL1-induced ⁇ -Arrestin recruitment with IC50s of 260.2-1757.0 ng/mL, and 433H, 10A11_hIgG1 and 10A11_mIgG2a had IC50s of 1291.5, 3268.0 and 3250.0 ng/mL, respectively.
  • the CDR was determined using Kabat numbering, and the human germline gene with the highest homology to the mouse sequence was selected as the receptor framework, and the CDR of the mouse sequence was transplanted into the human framework. According to the importance of the amino acids, back mutation was performed, that is, some key amino acids in the transplanted framework region were backmutated into the corresponding mouse amino acids, and several variants were designed for the heavy and light chains respectively.
  • mice variable region sequences and the selected human germline gene amino acid sequences are shown in Table 9 below; the humanized heavy and light chain sequences designed therefrom are shown in Table 10.
  • humanized heavy and light chains were combined to obtain the following humanized antibodies:
  • mice variable region sequences and the selected human germline gene amino acid sequences are shown in Table 11 below; the humanized heavy and light chain sequences designed therefrom are shown in Table 12.
  • humanized heavy and light chains were combined to obtain the following humanized antibodies:
  • the sequences of the three antibody clones 563E10E12, 569D11B5 and 589D7C7 are similar and are considered to be a family of sequences. When humanizing, these three antibodies are considered to be integrated together for humanization.
  • the mouse variable region sequence and the selected human germline gene amino acid sequence are shown in Table 13 below; the humanized heavy and light chain sequences designed thereby are shown in Table 14.
  • humanized heavy and light chains were combined to obtain the following humanized antibodies:
  • the light and heavy chain variable regions of the humanized antibodies 27B9-1G3, 559E1B10 and 563E10E12 were constructed onto the human constant region (hIgG1/K, sequence shown in Table 5), and gene synthesis was performed. Sequencing confirmed that they were consistent with the designed sequence.
  • the method in Example 3 was used to express them in Expi293 cells and purified using a Protein A column. Binding experiments with human and cynomolgus monkey CCR8, binding experiments blocking CCL1, and blocking CCL1-induced ⁇ -Arrestin recruitment experiments were performed to identify their functions.
  • the binding activity of anti-CCR8 humanized antibodies to human CCR8 and cynomolgus monkey CCR8 was detected by the method in Example 2-2.1.
  • the binding activity of the humanized antibody of 27B9-1G3 to 293F-hCCR8 is basically equivalent to the activity of its parent chimeric antibody.
  • the binding activity of most of the humanized antibodies of 559E1B10 to 293F-hCCR8 is basically equivalent to the activity of its parent chimeric antibody.
  • the binding activity of most of the humanized antibodies of 563E10E12 to 293F-hCCR8 is basically equivalent to the activity of its parent chimeric antibody.
  • the humanized antibodies 559E1B10_hzH1L1, 563E10E12_hzH1L0 and 563E10E12_hzH1L1 tested had strong binding activity with 293T-cynoCCR8, while 433H and 10A11_hIgG1 had almost no binding activity with cynomolgus monkey CCR8.
  • Table 15 EC 50 values of anti-CCR8 humanized antibodies binding to human CCR8 (293F-hCCR8) and cynomolgus monkey CCR8 (293T-cynoCCR8)
  • Example 2-2.2 The competition binding assay method in Example 2-2.2 was used to detect the humanized anti-CCR8 antibody competition for CCL1 binding. The results are shown in Figure 10 and Table 16.
  • All humanized antibodies of 563E10E12 were The blocking activity was comparable to that of its parent chimeric antibody.
  • 559E1B10 humanized antibodies 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH0L1 and 559E1B10_hzH0L2 and all humanized antibodies of 563E10E12 were better than or similar to that of 433H.
  • N/A The binding activity was poor and no valid EC50 value was obtained.
  • Anti-CCR8 humanized antibody blocks CCL1-induced ⁇ -Arrestin recruitment
  • Example 3-3.4 The method in Example 3-3.4 was used to detect the anti-CCR8 humanized antibody blocking CCL1-induced ⁇ -Arrestin recruitment experiment. The results are shown in Figure 11 and Table 17.
  • the blocking activity of some 27B9-1G3 humanized antibodies, such as 27B9-1G3_hzH3L1 and 27B9-1G3_hzH3L2 is basically equivalent to the blocking activity of its parent chimeric antibody.
  • the blocking activity of most 559E1B10 humanized antibodies is equivalent to the blocking activity of its parent chimeric antibody.
  • the blocking activities of all humanized antibodies of 563E10E12 were comparable to those of their parent chimeric antibodies.
  • 559E1B10 humanized antibodies 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH0L1 and 559E1B10_hzH0L2 and all humanized antibodies of 563E10E12 were better than or similar to those of 433H.
  • N/A Not applicable, no blocking activity.
  • the Fab end of the CCR8 antibody binds to the target on the target cell, and its Fc end binds to the Fc ⁇ RIIIa receptor on the effector cell, thereby activating the NFAT signaling pathway in the effector cell.
  • the luciferase produced by the activation of the NFAT pathway is quantified to reflect the ADCC activity of the antibody.
  • Digest 293F-human CCR8 (Shanghai Ruizhi Chemical Research Co., Ltd., 293F-hCCR8) cells with trypsin (Gibco, 25200072) containing 0.25% EDTA, centrifuge at 300g for 5 minutes, and adjust the cell concentration to 3 ⁇ 10 5 cells/mL with DMEM medium. Inoculate 100 ⁇ L per well in a white opaque plate Incubate overnight in a 37°C, 5% CO 2 incubator to allow cells to adhere to the wall and be used as target cells.
  • Anti-CCR8 antibody, positive control antibody, and hIgG1isotype (Bai Ying Biotechnology Co., Ltd., B117901) were diluted to 2 times the initial working mass concentration in 1640 medium, and then diluted 5 times with medium.
  • the collected Jurkat-human Fc ⁇ RIIIa(158V)-NFAT (Jiman Biotechnology (Shanghai) Co., Ltd., GM-C05619) cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. 1640 medium was added to adjust the cell concentration to 3 ⁇ 10 6 cells/mL.
  • the experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of the anti-CCR8 antibody concentration as the x-axis and the corresponding RLU value as the y-axis.
  • a four-parameter regression model was used to fit the dose-effect curve of the anti-CCR8 antibody.
  • chimeric and humanized antibodies of 559E1B10 and 563E10E12 can activate Jurkat-human Fc ⁇ RIIIa(158V)-NFAT in a dose-dependent manner.
  • the activities of humanized antibodies 559E1B10_hzH0L1, 559E1B10_hzH1L1, 559E1B10_hzH2L1, 559E1B10_hzH3L1, 559E1B10_hzH0L2, 559E1B10_hzH1L2, 559E1B10_hzH2L2, and 559E1B10_hzH3L2 were comparable to the activity of their parent chimeric antibody 559E1B10_hIgG1.
  • Table 18 EC 50 values of 559E1B10 humanized antibody for activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT
  • Human T cell lymphoma cell line HuT78 and activated human Treg both express CCR8 (James M Fox et al. and Yiftah Barsheshet et al. CCR8+FOXp3+T reg cells as master drivers of immune regulation. Proc Natl Acad Sci U S A. 2017Jun 6; 114(23):6086-6091. doi:10.1073/pnas.1621280114.), and FACS was used to detect the binding activity of anti-CCR8 antibody to endogenously expressed CCR8.
  • HuT78 (Cobioer, CBP60267) cells were adjusted to a cell concentration of 1 ⁇ 10 6 cells/mL, and 100 ⁇ L was placed in each well of a 96-well U-bottom plate, and the supernatant was removed by centrifugation.
  • Anti-CCR8 antibody, control antibody 433H, 10A11_hIgG1, B16_hIgG1:7-B16.001 (sequence in patent US20210277129A1, light chain variable region sequence number: 81, heavy chain variable region sequence number: 80) and hIgG1isotype (Baiying Biotechnology Co., Ltd., B117901) were gradient diluted with PBS solution containing 2% FBS (FACS buffer), starting at 10 ⁇ g/mL, 4-fold gradient dilution. Resuspend the cells with 100 ⁇ L/well of gradient diluted antibody, pipet and mix, incubate at 4°C for 1 hour, and wash three times with FACS buffer after incubation.
  • the experimental data were analyzed using Graphpad Prism 8.0 software.
  • the logarithm of the anti-CCR8 antibody concentration was used as the x-axis and the corresponding MFI value was used as the y-axis.
  • a four-parameter regression model was used to fit the dose-effect curve of the anti-CCR8 antibody and calculate the EC 50 .
  • Anti-CCR8 antibodies specifically bind to CHO-K1 cells expressing human CCR8
  • CHO-K1 cells were used in the mouse immunization and screening process of CCR8 antibodies.
  • the binding of anti-CCR8 antibodies to cell surface CCR8 was detected using a CHO-K1 cell line overexpressing human CCR8 (constructed by Ruizhi Chemical, CHOK1-hCCR8) and a wild-type CHO-K1 cell line (Jiman Bio, GM-15570) and the experimental method in Example 2-2.1.
  • 559E1B10_hzH1L1, 563E10E12_hzH1L0, and 563E10E12_hzH1L1 all bound to CHOK1-hCCR8 in a dose-dependent manner, but did not bind to CHOK1 null cells.
  • Table 21 EC 50 of anti-CCR8 antibodies specifically binding to hCCR8 on the cell surface
  • the CHO-K1 cell line overexpressing human CCR8 (constructed by Ruizhi Chemical, CHOK1-hCCR8) and the CHO-K1 blank cell line (Jiman Bio, GM-15570) were used to determine that the activation of Jurkat-hu man Fc ⁇ RIIIa(158V)-NFAT by anti-CCR8 antibodies depends on CCR8.
  • CHOK1-hCCR8 cells and CHOK1 blank cells were adjusted to a cell concentration of 3 ⁇ 10 5 cells/mL with 1640 complete medium, and the method in Example 5-5.4 was used to detect the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NF AT by anti-CCR8 antibodies.
  • 4A19 is the sequence in patent WO2021194942A1, the light chain variable region sequence number: 18, the heavy chain variable region sequence number: 6.
  • anti-CCR8 antibodies dose-dependently activated Jurkat-human Fc ⁇ RIIIa(158V)-NFAT; however, when CHO-K1 blank cells were present, anti-CCR8 antibodies could not activate Jurkat-human Fc ⁇ RIIIa(158V)-NFAT, indicating that anti-CCR8 antibodies can specifically recognize CCR8 and mediate CCR8-dependent ADCC effects.
  • Table 22 EC 50 of CCR8-dependent activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT by anti-CCR8 antibodies
  • CCR4 The gene with the highest homology to CCR8 in humans is CCR4 (Antonio Recchiuti et al. Pro-Resolving Lipid Mediators (SPMs) and Their Actions in Regulating miRNA in Novel Resolution Circuits in Inflammation. Front Immunol. 2012 Oct 22; 3:298. doi:10.3389/fimmu.2012.00298.eCollection 2012.), but the homology between CCR4 and CCR8 is only 46%. Homology (NCBI blast results). The following method was used to detect the binding activity of anti-CCR8 antibody to human CCR4.
  • 293F cells were transiently transfected with hCCR4-GFP (Sino, HG13064-ACG).
  • the cell concentration of the transfected cells was adjusted to 1 ⁇ 10 6 cells/mL, and 100 ⁇ L was placed in each well of a 96-well U-bottom plate, and the cells were obtained by centrifugation.
  • the anti-CCR8 antibody was diluted to the starting working mass concentration with PBS solution containing 2% FBS (FACS buffer), and the starting concentration was 30 ⁇ g/mL, and then diluted 4 times with FACS buffer solution.
  • the chimeric and humanized antibodies of 563E10E12 did not bind to cells transfected with hCCR4 (GFP-positive cells) nor to 293F cells, whereas the chimeric and humanized antibodies of 559E1B10 weakly bound to hCCR4 at high concentrations.
  • CCR8 is not expressed in other immune cells in healthy human peripheral blood except for a small amount of expression on Treg (George Plitas et al. Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity. 2016 Nov 15; 45(5): 1122-1134. doi: 10.1016/j.immuni.2016.10.032.).
  • PBMCs from healthy donors were used to detect whether anti-CCR8 antibodies had non-specific binding to peripheral immune cells.
  • PBMCs from healthy donors (Allcells, catalog number Z0060 and Y1446) were resuscitated and adjusted to a cell concentration of 2 ⁇ 10 6 cells/mL.
  • the anti-CCR8 antibody and hIgG1 isotype (Bio-Ying Biotechnology Co., Ltd., B117901) labeled with AF647 labeling kit (Invitrogen, A20186) were diluted with FACS buffer to a starting concentration of 100 ⁇ g/mL, and then diluted 5-fold with FACS buffer solution. 10 ⁇ L was added to the above cells and incubated at 4°C for 1 hour. After incubation, the cells were washed three times with FACS buffer and resuspended in 100 ⁇ L FACS buffer per well. The mean fluorescence intensity (MFI) was read using a flow cytometer.
  • MFI mean fluorescence intensity
  • anti-CCR8 antibodies 559E1B10_hzH1L1_hIgG1, 563E10E12_hzH1L0_hIgG1 and 563E10E12_hzH1L1_hIgG1 had no significant binding to CD8 + , CD4 + (CD3 + CD8 - ) T cells, CD14 + monocytes, CD19 + B cells and other CD3 - CD14 - CD19 - immune cells.
  • Example 8 Fc mutation and reduced fucosylation enhance ADCC effect mediated by anti-CCR8 antibody
  • Antibody constant region (Fc) mutations S239D/I332E, S239D/A330L/I332E or L235V/F243L/R292P/Y300L/P396L can improve affinity with Fc ⁇ RIIIa and increase antibody-mediated ADCC effect (Greg A Lazar et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S A. 2006 Mar 14; 103(11):4005-10. doi:10.1016/0300-0100. 0.1073/pnas.0508123103. and Jeffrey L Nordstrom et al.
  • the Fc of humanized antibodies against CCR8 were mutated to S239D/I332E (DE), S239D/A330L/I332E (DLE) or L235V/F243L/R292P/Y300L/P396L (VLPLL) to enhance the ADCC effect induced by them.
  • Defucosylation or reduced fucosylation can increase the ADCC effect of antibodies.
  • Many antibodies with defucosylation or reduced fucosylation have been approved for marketing or are in the late stage of clinical research (Natasha A Pereira et al. The "less-is-more" in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity. MAbs. 2018 Jul; 10 (5): 693-711.
  • ExpiCHO cells were subcultured and expanded using the growth medium ExpiCHO Expression Medium (Gibco, Catalog No.: A2910001) containing 100 ⁇ M 2F-peracetyl-fucose (MILLIPORE, Catalog No.: 344827-10MGCN) before transient transfection.
  • ExpiCHO Expression Medium Gibco, Catalog No.: A2910001
  • MILLIPORE MILLIPORE, Catalog No.: 344827-10MGCN
  • the cells were diluted to 6 ⁇ 10 6 cells/mL and transiently transfected using the Expi CHO transfection kit (Gibco, Catalog No.: A29129) at a concentration of 1 ⁇ g plasmid/mL cell suspension.
  • the transiently transfected cells were fed (Gibco, Catalog No.: A29129) and continued to be cultured in a shaker at 37°C, 8% CO 2 , and 100 rpm.
  • the cell culture medium was harvested 7 days after transient transfection, and the supernatant was obtained by 0.45 ⁇ m filtration after centrifugation.
  • the antibody was purified using Protein A filler.
  • Example 5-5.4 The method in Example 5-5.4 was used to detect the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT by incubation of anti-CCR8 antibody with 293F-hCCR8.
  • Figure 18 and Table 24 compared with 559E1B10_hzH1L1_hIgG1, the Fc mutants 559E1B10_hzH1L1_DE and 559E1B10_hzH1L1_VLPLL enhanced the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT, and the EC 50 was reduced by 9-21 times.
  • the Fc mutants 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and 563E10E12_hzH1L0_VLPLL and the reduced fucosylation 563E10E12_hzH1L0_AF enhanced the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT with a 7-18-fold decrease in EC 50.
  • the Fc mutants 563E10E12_hzH1L1_DE and 563E10E12_hzH1L1_VLPLL enhanced the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT with a 6-14-fold decrease in EC 50 .
  • the activities of Fc-mutated 559E1B10 and 563E10E12 humanized antibodies or reduced fucosylation 563E10E12_hzH1L0 were comparable to that of reduced fucosylated B16 (B16_AF).
  • the anti-CCR8 antibodies with different Fc mutations or reduced fucosylation were incubated with Hut78 and Jurkat-human Fc ⁇ RIIIa(158V)-NFAT cells to detect the effects of S239D/I332E, S239D/A330L/I332E, L235V/F243L/R292P/Y300L/P396L mutations or reduced fucosylation on the activation of Jurkat-human Fc ⁇ RIIIa( 158V )-NFAT by anti-CCR8 antibodies and HuT78.
  • HuT78 cells were used as target cells, and the method in Example 5-5.4 was used to detect the activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT by anti-CCR8 antibodies.
  • Table 25 EC 50 of 563E10E12_hzH1L0 with Fc mutation or reduced fucosylation and Hut78 for activation of Jurkat-human Fc ⁇ RIIIa(158V)-NFAT
  • Example 9 ADCC effect induced by healthy human PBMC and anti-CCR8 antibody
  • PBMCs were used as effector cells and CHOK1-hCCR8 as target cells to detect the ADCC effect of anti-CCR8 antibodies.
  • PBMCs Allcells
  • 1640 complete medium containing 200 IU/mL IL-2 (Peprotech, catalog number: 200-02), and cultured overnight in a 37°C, 5% CO 2 incubator.
  • non-adherent PBMCs were aspirated, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 1640 medium was added to adjust the cell concentration to about 3 ⁇ 10 6 cells/mL.
  • 1640 medium was used to dilute anti-CCR8 antibodies and hIgG1 isotype (Bai Ying Biotechnology Co., Ltd., catalog number: B117901) to 2 times the working mass concentration, with a starting concentration of 0.8 ⁇ g/mL and 5-fold gradient dilution.
  • the collected CHOK1-hCCR8 cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. The cell concentration was adjusted to 2 ⁇ 10 6 cells/mL with DPBS. CellTrace TM Violet (Invitrogen, Cat.
  • No.: 006990-50 dye was added to each well, mixed and reacted in the dark for 5 minutes at room temperature to label dead cells.
  • the experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of the anti-CCR8 antibody concentration as the x-axis and the corresponding lysis% value as the y-axis.
  • a four-parameter regression model was used to fit the dose-effect curve of the anti-CCR8 antibody.
  • the humanized antibody 563E10E12_hzH1L0_AF with reduced fucosylation and the antibodies 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE with Fc mutations all increased the ADCC effect induced by them and PBMC.
  • the ADCC activity induced by humanized antibody 563E10E12_hzH1L0_hIgG was comparable to that induced by B16_hIgG1.
  • the ADCC effect induced by humanized antibody 563E10E12_hzH1L0 with reduced fucosylation and Fc mutations and PBMC was similar to the activity induced by B16 with reduced fucosylation.
  • PBMC Use healthy human PBMC to detect the specific elimination of Treg cells expressing CCR8 in PBMC by anti-CCR8 antibodies. Since the proportion of Treg cells expressing CCR8 in healthy human PBMC is low, IL-2 is used in this experiment to promote the proliferation and activation of Treg. Resuscitate PBMC (Allcells) from healthy donors, adjust the cell concentration to 2 ⁇ 10 6 cells/mL with 1640 complete medium containing 200IU/mL IL-2 (Peprotech, catalog number: 200-02), add 100 ⁇ L per well to 96-well U-bottom plate, and incubate at 37°C, 5% CO 2 for 48 hours.
  • FcX reagent Biolegend, Catalog No.: 422302
  • 50 ⁇ L of FcX reagent diluted 1:25 was added to each well and incubated at room temperature for 15 minutes.
  • 50 ⁇ L of AF700 Anti-Human CD3 Biolegend, Catalog No.: 317340
  • Percp cy5.5 Anti-Human CD4 Biolegend, Catalog No.: 300530
  • mixed staining solution diluted 1:50 was added to each well, incubated at 4°C for 1 hour, and washed three times with FACS buffer.
  • Anti-CCR8 antibody inhibits the growth of MC38 colon cancer in a CCR8 humanized mouse model
  • the MC38 (Beijing Biocytogen Pharmaceuticals Co., Ltd.) colon cancer animal model was established using humanized CCR8 mice (C57BL/6-Ccr8 tm1(CCR8) /Bcgen, B-hCCR8, Biocytogen Pharmaceuticals Co., Ltd., Cat. No. 110096) and the in vivo efficacy of anti-CCR8 antibodies was tested.
  • MC38 cells (5 ⁇ 10 6 cells/mL) resuspended in PBS were inoculated into B-hCCR8 mice at a concentration of 100 ⁇ L/mouse. The right side of the mouse was subcutaneously injected.
  • mice When the average tumor volume reached about 100 mm 3 , 6-8 mice were divided into groups and subcutaneously injected with 10 mg/kg of anti-CCR8 antibody or negative control (hIgG1) (Bai Ying Biotechnology Co., Ltd., catalog number: B117901) or an equal volume of solvent (PBS) control, twice a week. After administration, the mice were raised normally, the survival status of the mice was observed, and the weight and tumor volume of the mice were recorded.
  • hIgG1 anti-CCR8 antibody or negative control
  • PBS solvent
  • anti-CCR8 antibodies 559E1B10_hzH1L1_mIgG2a, 563E10E12_hzH1L0_mIgG2a, 563E10E12_hzH1L1_mIgG2a and 10A11_mIgG2a all inhibited the growth of MC38 colon cancer cells (Figure 22).
  • the tumor inhibition rates TGI (%) of anti-CCR8 antibodies 559E1B10_hzH1L1_mIgG2a, 563E10E12_hzH1L0_mIgG2a, 563E10E12_hzH1L1_mIgG2a and 10A11_mIgG2a were 40.1%, 54.0%, 22.5% and 53%, respectively.
  • all animals were active and ate well during the administration period, and their body weight increased to a certain extent, indicating that the animals tolerated the anti-CCR8 antibody well ( FIG. 23 ).
  • the MC38 colon cancer model was established in CCR8 humanized mice B-hCCR8 using the method in Example 11-11.1.
  • the average tumor size was about 100 mm 3
  • 5 mice were divided into groups and subcutaneously injected with 10 mg/kg of anti-CCR8 antibody or an equal volume of solvent (PBS) as a control, twice a week. After administration, the mice were raised normally, the survival status of the mice was observed, and the weight and tumor volume of the mice were recorded.
  • PBS solvent
  • the anti-CCR8 antibody 563E10E12_hzH1L0_hIgG1 As shown in Figure 24, compared with the vehicle, the anti-CCR8 antibody 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the antibody 563E10E12_hzH1L0_AF with reduced fucosylation all inhibited the growth of MC38 colon cancer cells.
  • the tumor inhibition rates TGI (%) of 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the antibody 563E10E12_hzH1L0_AF with reduced fucosylation were 60.1%, 57.8%, 60.2% and 52.9%, respectively.
  • Mice in all drug-treated groups showed no behavioral abnormalities or weight loss, indicating that tumor-bearing mice had good tolerance to the anti-CCR8 antibody at this dose (Figure 25).
  • Human CD34 + HSC humanized mice (Jicui Yaokang) were obtained by transplanting human hematopoietic cells hCD34 + HSC into irradiated NCG mice for immune reconstruction.
  • Human non-small cell lung cancer A549 cells were inoculated subcutaneously on the right side of human CD34 + HSC humanized mice, with an inoculation volume of 0.2 mL/mouse, containing 30% Matrigel. When the average tumor volume was about 100 mm 3 , the mice were randomly divided into groups and given anti-CCR8 antibodies (1-20 mg/kg), pembrolizumab (1-20 mg/kg), a combination of CCR8 antibodies (1-20 mg/kg) and pembrolizumab (1-20 mg/kg), and negative control (PBS).
  • the drug was administered twice a week for 4 weeks. After administration, the mice were fed normally and the tumor volume was recorded. . Tumor samples were harvested at the end of the experiment, minced with dissecting laboratory scissors, and enzymatically dissociated with the human tumor dissociation kit (Miltenyi Biotech) combined with the gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 ⁇ m MACS smart filter to obtain a single cell suspension. Immune cell subsets (Treg and CD8 + T cells) were analyzed by flow cytometry.
  • 563E10E12_hzH1L0_hIgG1 its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.
  • mice Human non-small cell lung cancer NCI-H1299 cells were inoculated subcutaneously on the right side of human PBMC humanized mice (Jicui Yaokang), with an inoculation volume of 0.2 mL/mouse, containing 30% Matrigel.
  • the mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg/kg), pembrolizumab (1-20 mg/kg), a combination of CCR8 antibody (1-20 mg/kg) and pembrolizumab (1-20 mg/kg), and negative control (PBS), twice a week for 4 weeks. After administration, the mice were raised normally and the tumor volume was recorded.
  • Tumor samples were harvested at the end of the experiment, minced with dissecting laboratory scissors, and enzymatically dissociated tumor tissue using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions, and the cell suspension was filtered through a 70 ⁇ m MACS smart filter to obtain a single cell suspension.
  • Immune cell subsets (Treg and CD8 + T cells) were analyzed by flow cytometry.
  • 563E10E12_hzH1L0_hIgG1 its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.
  • mice Human non-small cell lung cancer NCI-H292 cells were inoculated subcutaneously on the right side of human PBMC humanized mice (Jicui Yaokang), with an inoculation volume of 0.2 mL/mouse, containing 30% Matrigel.
  • the mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg/kg), pembrolizumab (1-20 mg/kg), a combination of CCR8 antibody (1-20 mg/kg) and pembrolizumab (1-20 mg/kg) and negative control (PBS), twice a week for 3 weeks. After administration, the mice were raised normally and the tumor volume was recorded.
  • Tumor samples were harvested at the end of the experiment, minced with dissection laboratory scissors, and enzymatically dissociated tumor tissue using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions, and the cell suspension was filtered through a 70 ⁇ m MACS smart filter to obtain a single cell suspension.
  • Immune cell subsets and functional biomarkers were analyzed by flow cytometry.
  • Immune cell subsets (Treg and CD8 + T cells) were analyzed by flow cytometry.
  • 563E10E12_hzH1L0_hIgG1 its Fc mutant antibodies 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the antibody with reduced fucosylation 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.
  • Human rectal cancer HT29 cells were inoculated subcutaneously on the right side of human PBMC humanized mice (Shanghai Model Organisms) with an inoculation volume of 0.1 mL/mouse containing 30% Matrigel.
  • the mice were randomly divided into groups and given anti-CCR8 antibody (10 mg/kg), pembrolizumab (3.25 mg/kg), a combination of CCR8 antibody (10 mg/kg) and pembrolizumab (3.25 mg/kg), and negative control (PBS) twice a week for 4 weeks. After administration, the mice were raised normally and the tumor volume was recorded.
  • the tumor samples were harvested, minced with dissecting laboratory scissors, and the tumor tissue was enzymatically dissociated using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions.
  • the cell suspension was filtered through a 70 ⁇ m MACS smart filter to obtain a single cell suspension.
  • Immune cell subsets (Treg and CD8 + T cells) were analyzed by flow cytometry.
  • 563E10E12_hzH1L0_hIgG1 its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.
  • Mouse breast cancer EMT-6 cells were inoculated subcutaneously on the right side of human CCR8 humanized mice (Ncapturing Model Organisms) with an inoculation volume of 0.1 mL/mouse (in PBS).
  • the mice were randomly divided into groups and given anti-CCR8 antibodies (1-20 mg/kg) and negative controls (PBS) twice a week for 3 weeks. After administration, the mice were raised normally and the tumor volume was recorded.
  • Tumor samples were harvested at the end of the experiment, minced with dissecting laboratory scissors, and enzymatically dissociated tumor tissue using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions, and the cell suspension was filtered through a 70 ⁇ m MACS smart filter to obtain a single cell suspension.
  • Immune cell subsets (Treg and CD8 + T cells) were analyzed by flow cytometry.
  • 563E10E12_hzH1L0_hIgG1 its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.
  • mice The mouse breast cancer EMT-6 cells were inoculated subcutaneously on the right side of human CCR8 humanized mice (Shanghai Model Organisms) with an inoculation volume of 0.1 mL/mouse (in PBS).
  • PBS positive control
  • Tumor samples were harvested at the end of the experiment, minced with dissecting laboratory scissors, and Tumor tissue was enzymatically dissociated using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions, and the cell suspension was filtered through a 70 ⁇ m MACS smart filter to obtain a single cell suspension.
  • Immune cell subsets (Treg and CD8 + T cells) were analyzed by flow cytometry.
  • Murine melanoma B16F10 cells were inoculated subcutaneously on the right side of humanized CCR8 mice (Biocytogen) at a volume of 0.1 mL/mouse (in PBS).
  • the mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg/kg), anti-mouse PD1 monoclonal antibody (1-20 mg/kg), anti-CCR8 antibody (1-20 mg/kg) and anti-mouse PD1 monoclonal antibody (1-20 mg/kg) and negative control (PBS) twice a week for 3 weeks. After administration, the mice were fed normally and the tumor volume was recorded.
  • Tumor samples were harvested at the end of the experiment, minced with dissection laboratory scissors, and enzymatically dissociated using the human tumor dissociation kit (Miltenyi Biotech) combined with the gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions, and the cell suspension was filtered through a 70 ⁇ m MACS smart filter to obtain a single cell suspension.
  • Immune cell subsets (Treg and CD8 + T cells) were analyzed by flow cytometry.
  • 563E10E12_hzH1L0_hIgG1 its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.
  • Mouse colorectal cancer CT26 cells were inoculated subcutaneously on the right side of human CCR8 humanized mice (Shanghai Model Organisms) with an inoculation volume of 0.1 mL/mouse (in PBS).
  • the mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg/kg), anti-mouse PD1 monoclonal antibody (1-20 mg/kg), and a combination of anti-CCR8 antibody (1-20 mg/kg) and anti-mouse PD1 monoclonal antibody (1-20 mg/kg).
  • Drug and negative control (PBS) were administered twice a week for 3 weeks. After administration, mice were fed normally and tumor volume was recorded.
  • Tumor samples were harvested at the end of the experiment, minced with dissecting laboratory scissors, and enzymatically dissociated tumor tissue using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions.
  • the cell suspension was filtered through a 70 ⁇ m MACS smart filter to obtain a single cell suspension.
  • Immune cell subsets (Treg and CD8 + T cells) were analyzed by flow cytometry.
  • 563E10E12_hzH1L0_hIgG1 its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.

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Abstract

提供了一种靶向CCR8的抗体或其抗原结合片段及其应用。所述靶向CCR8的抗体或其抗原结合片段特异性结合CCR8。进一步提供了所述抗体用于阻断CCL1与CCR8的结合/诱导的信号通路，通过ADCC作用清除CCR8阳性Treg细胞，增强抗肿瘤免疫的用途。

Description

抗CCR8抗体及其用途 技术领域
本发明涉及单克隆抗体和/或工程化抗体领域，具体地，本发明提供了与CCR8特异性结合的抗体或其抗原结合片段和包含其的组合物。还提供了编码本发明抗体或其抗原结合片段的核酸分子，用于表达本发明抗体或其抗原结合片段的载体和宿主细胞，以及本发明抗体或其抗原结合片段的治疗和诊断/检测方法和用途。
背景技术
机体的免疫系统是抵抗肿瘤产生和进展的核心防御系统，而免疫系统不能识别和清除恶性细胞在癌症的发病机制中起着重要作用。在肿瘤微环境中，多种免疫检查点的高表达降低免疫激活，抑制抗肿瘤免疫。针对抑制性免疫检查点CTLA-4和PD-1/PD-L1的单克隆抗体通过在肿瘤微环境中上调免疫激活，产生显著的抗肿瘤反应。目前临床上多个针对CTLA-4和PD-1/PD-L1的单克隆抗体获批用于多种实体瘤的治疗，包括抗CTLA-4抗体伊匹单抗(ipilimumab)，抗PD-1抗体纳武单抗(nivolumab)和帕博利珠单抗(pembrolizumab)和抗PD-L1抗体阿替利珠单抗(atezolizumab)等。与传统的治疗方法(包括化疗、放疗和手术)相比，癌症免疫治疗在生存率和生活质量方面为患者带来了显著改善(K Esfahani等，A review of cancer immunotherapy:from the past,to the present,to the future,Curr Oncol.2020 Apr；27(Suppl 2):S87-S97.)。
虽然免疫检查点抑制剂在临床上获批用于多种实体瘤的治疗，但是其在各个瘤种的响应率差别较大，而且总响应率较低。免疫检查点抑制剂在非小细胞肺癌(NSCLC)，尿路上皮癌，和头颈癌的总响应率(ORR)只有20％左右，而胃癌等实体瘤中低于10％(Schoenfeld AJ和Hellmann MD.Acquired Resistance to Immune Checkpoint Inhibitors.Cancer Cell.2020 Apr 13；37(4):443-455.doi:10.1016/j.ccell.2020.03.017.)。部分病人对免疫检查点抑制剂的反应具有持久性，但也有许多最初有反应的患者后来产生获得性耐药，例如超过50％的NSCLC和胃癌患者会产生获得性耐药(Schoenfeld AJ和Hellmann MD)。甚至部分实体瘤患者非但未从免疫检查点抑制剂中获益，反而更为不幸地发生肿瘤超进展(Hyperprogressive)，这在多种实体瘤患者经免疫检查点治疗后以不同比例出现，例如在NSCLC中有14％的患者会发生超进展(Champiat S等.Hyperprogressive disease:recognizing a novel pattern to improve patient management.Nat Rev Clin Oncol.2018 Dec；15(12):748-762.doi:10.1038/s41571-018-0111-2.)。目前仍需开发新的免疫检查点抑制剂或免疫调节剂来补充或克服当下临床上使用的免疫检查点抑制剂的不足和存在的问题。
免疫检查点抑制剂的总响应率低、获得性耐药和产生超进展与肿瘤细胞内部信号通路和肿瘤微环境中的多种机制有关(Baxter MA等.Resistance to immune checkpoint inhibitors in advanced gastro-oesophageal cancers.Br J Cancer.2021 Oct；125(8):1068-1079.doi:10.1038/s41416-021-01425-7.)。调节性T细胞(Treg)在免疫调节中起着重要作用，与肿瘤的发生、发展有关。与外周相比，肿瘤微环境中Treg数量增多和功能增强；Treg通过分泌IL-10等抑制性细胞因子、消耗IL-2和直接接触抑制等机制抑制抗肿瘤免疫，与免疫检查点抑制剂的天然和获得性耐药有着重要的关系(Baxter MA等.)。PD-1阻断可诱导功能失调的PD-1+CD8+T细胞的恢复，但是也能增强PD-1+Treg细胞介导的免疫抑制。如果肿瘤微环境中表达PD-1的Treg的数量多于PD-1+CD8+T细胞，PD-1阻断可能导致肿瘤的超进展(Kumagai S等.The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies.Nat Immunol.2020 Nov；21(11):1346-1358.doi:10.1038/s41590-020-0769-3.)。临床前的动物模型中和临床上的研究表明清除肿瘤微环境中的Treg可以促进抗肿瘤免疫，增加PD-1/PD-L1的治疗效果。临床上发现，抗CTLA-4抗体伊匹单抗(ipilimumab)和抗PD-1抗体纳武单抗(nivolumab)的联合应用对黑色素瘤和肾细胞癌患者的生存更有利(Hayashi H和Nakagawa K.Combination therapy with PD-1 or PD-L1 inhibitors for cancer.Int J Clin Oncol.2020 May；25(5):818-830.doi:10.1007/s10147-019-01548-1.)
目前临床上在研的具有清除Treg的功能的抗体，包括针对CTLA-4、CD25、协同刺激因子OX40和GITR等靶点的抗体，在临床上取得了一定的进展(Togashi Y等.Regulatory T cells in cancer immunosuppression-implications for anticancer therapy.Nat Rev Clin Oncol.2019 Jun；16(6):356-371.doi:10.1038/s41571-019-0175-7.)。但是这些靶点在外周Treg和效应T细胞上均具有较高水平的表达，因而对外周Treg和效应T细胞的同时清除降低了其临床疗效并增加毒性，这样就限制了其在临床上的应用。近年发现趋化因子受体CCR8在人类肿瘤(包括乳腺癌、非小细胞肺癌，结直肠癌、黑色素瘤、肝细胞癌和胰腺导管腺癌等)中的Treg细胞上高度表达，而在外周的和正常组织中的Treg与效应T细胞上不表达或低表达(Plitas G等.Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer.Immunity.2016Nov 15；45(5):1122-1134.doi:10.1016/j.immuni.2016.10.032，以及De Simone M等.Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.Immunity.2016 Nov 15；45(5):1135-1147.doi:10.1016/j.immuni.2016.10.021.)。CCR8高表达与乳腺癌、非小细胞肺癌和大肠癌预后不良相关。与CCR8阴性的Treg细胞相比，肿瘤微环境中CCR8阳性的Treg细胞对效应T细胞增殖的抑制能力更强(Gang,Yi等.Identification and functional analysis of heterogeneous FOXP3+Treg cell subpopulations  in human pancreatic ductal adenocarcinoma[J].Science Bulletin,2018.Doi:10.1016/j.scib.2018.05.028)。在动物模型中发现，通过CCR8抗体可以特异的清除肿瘤微环境中的Treg，抑制肿瘤的生长，并与PD-1抗体具有协同作用(美国专利申请号US20190071508以及Van Damme H等.Therapeutic depletion of CCR8+tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.J Immunother Cancer.2021 Feb；9(2):e001749.doi:10.1136/jitc-2020-001749.)。开发针对人CCR8特异性的抗体，可能在临床上增加免疫检查点的治疗效果，具有重要的应用价值。
发明概述
本发明采用超表达人CCR8的细胞以及CCR8核酸免疫小鼠，通过杂交瘤技术筛选得到抗人CCR8特异性抗体。抗CCR8抗体与人和猴CCR8均具有高亲和力，可以阻断CCL1与CCR8的结合/诱导的信号通路，还可以通过ADCC作用清除CCR8阳性细胞(例如，Treg细胞)，在小鼠体内多种模型(如结直肠癌、肺癌、乳腺癌、黑色素瘤等)中均有效抑制肿瘤的生长。
在一个方面，本发明提供了一种抗CCR8抗体或其抗原结合片段，其中所述抗CCR8抗体或其抗原结合片段包含：
(i)如SEQ ID NO:21-40任一项所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，和/或如SEQ ID NO:42-54任一项所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
(ii)如(i)所述的CDR组合，其中相比HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和/或LCDR3，在所述序列上包含至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)，优选地氨基酸取代，优选地保守性取代，并保持与CCR8的亲和力。
在一些实施方案中，本发明提供了结合CCR8的抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中
所述重链可变区包含：
(1)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2或10或11或41和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:1、SEQ ID NO:2或10或11或41和SEQ ID NO:3所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的HCDR1、HCDR2和HCDR3；或
(2)氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6 所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的HCDR1、HCDR2和HCDR3；或
(3)氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的HCDR1、HCDR2和HCDR3；
和/或
所述轻链可变区包含：
(1)氨基酸序列分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的LCDR1、LCDR2和LCDR3；或
(2)氨基酸序列分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的LCDR1、LCDR2和LCDR3；或
(3)氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明提供了结合CCR8的抗体或其抗原结合片段，其包含如下表任一组合所示的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3:

在一些实施方案中，本发明提供了结合CCR8的抗体或其抗原结合片段，其包含重链可变区VH和/或轻链可变区VL，其中，
(a)重链可变区VH
(i)包含与选自SEQ ID NO:21-40任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者
(ii)包含选自SEQ ID NO:21-40任一项所示的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO:21-40任一项所示的氨基酸序列相比具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的氨基酸序列，优选地，所述氨基酸改变发生在CDR区中，优选地，所述氨基酸改变发生在FR区中；
和/或
(b)轻链可变区VL
(i)包含与选自SEQ ID NO:42-54任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO:42-54任一项所示的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO:42-54任一项所示的氨基酸序列相比具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中。
在一些实施方案中，本发明提供了结合CCR8的抗体或其抗原结合片段， 其包含如下表任一组合所示的重链可变区VH和轻链可变区VL:
在一些实施方案中，本发明提供了抗CCR8抗体或其抗原结合片段，其包含重链和/或轻链，其中
(a)重链
(i)包含与选自SEQ ID NO:55-74任一项的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO:55-74任一项的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO:55-74任一项的氨基酸序列相比具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中，最优选地，所述氨基酸改变发生在重链恒定区中；
和/或
(b)轻链
(i)包含与选自SEQ ID NO:75-82任一项的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO:75-82任一项的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO:75-82任一项的氨基酸序列相比具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中，最优选的，所述氨基酸改变发生在轻链恒定区中。
在一些实施方案中，本发明的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链恒定区和/或轻链恒定区，优选地，所述轻链恒定区是λ链或κ链恒定区；所述重链恒定区选自鼠mIgG2a、人IgG1、人IgG2、人IgG3或IgG4型。在一些优选的实施方案中，所述重链恒定区是人IgG1型或具有S228P突变的人IgG4型。
在一些实施方案中，本发明的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链恒定区，且所述重链恒定区的序列与天然人重链恒定区序列相比具有一个或多个氨基酸取代，优选地，所述一个或多个氨基酸取代使得抗体的ADCC效应增强。在一些优选的实施方案中，所述一个或多个氨基酸取代发生在根据EU编号系统的重链恒定区序列位点238、239、248、249、252、254、255、256、258、265、267、268、269、270、272、276、278、280，283、285、286、289、290、292、293、294、295、296、298、301、303、305、307、309、312、315、320、322、324、326、327、328、329、330、331、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、434、435、437、438和439中的一个或多个。在一些优选的实施方案中，所述一个或多个氨基酸取代发生在根据EU编号系统的重链恒定区序列位点L234、L235、G236、S239、F243、T256、D265、H268、D270、K290、 R292、S298、Y300、V305、K326、A330、I332、E333、K334、A339和P396中的一个或多个。在一些优选的实施方案中，所述一个或多个氨基酸取代是选自根据EU编号系统的G236A、S239D、F243L、T256A、K290A、R292P、S298A、Y300L、V305I、A330L、I332E、E333A、K334A、A339T和P396L取代的一个或多个。在一些优选的实施方案中，所述一个或多个氨基酸取代是选自根据EU编号系统的N297A取代、N297Q取代、L235A取代连同L237A取代、L234A取代连同L235A取代、E233P取代、L234V取代、L235A取代、C236缺失、P238A取代、D265A取代、A327Q取代和P329A取代的一个或多个。在一些优选的实施方案中，所述一个或多个氨基酸取代发生在根据EU编号系统的重链恒定区序列位点235、239、243、292、300、330、332、396中的一个或多个。在一些优选的实施方案中，所述一个或多个氨基酸取代是选自根据EU编号系统的S239D、L235V、F243L、R292P、Y300L、A330L、I332E和P396L中的至少一个。
在一些优选的实施方案中，所述重链恒定区具有在选自以下根据EU编号系统的位置的组合上同时发生的一组或多组突变：(1)L235/F243/R292/Y300/P396、(2)F243/R292/Y300/V305/P396、(3)D270/K326/A330/K334、(4)S239/A330/I332、(5)S298/E333/K334、(6)L234/L235/G236/S239/H268/D270/S298、(7)M252/S254/T256、(8)L234/L235/D265、(9)G236/S239/I332和(10)S239/I332。
在一些优选的实施方案中，所述重链恒定区具有选自以下根据EU编号系统的突变组合的一组或多组突变：(1)L235V/F243L/R292P/Y300L/P396L、(2)F243L/R292P/Y300L/V305I/P396L、(3)D270E/K326D/A330M/K334E、(4)S239D/A330L/I332E、(5)S298A/E333A/K334A、(6)L234Y/L235Q/G236W/S239M/H268D/D270E/S298A、(7)M252Y/S254T/T256E、(8)L234A/L235A/D265A、(9)L234F/L235E/D265A、(10)G236A/S239D/I332E和(11)S239D/I332E。
在一些实施方案中，本发明的结合CCR8的抗体或其抗原结合片段，其包含如下表任一组合所示的重链(HC)和轻链(LC):

在一些实施方案中，本发明的抗体的恒定区是去岩藻糖基化的或降低岩藻糖基化的。
在一些实施方案中，本发明的抗体是单克隆抗体。
在一些实施方案中，本发明的抗体是鼠源抗体、嵌合抗体、或人源化抗体或人抗体。
在一些实施方案中，本发明的抗原结合片段包括以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、Fd、单链抗体(例如scFv)或(Fab’)2、单结构域抗体、双体抗体diabody(dAb)或线性抗体。
在另一个方面，本发明提供了一种分离的抗CCR8抗体或其抗原结合片段，其具有以下特性中的一种或多种：
(1)与本发明的任何一种抗CCR8抗体或其抗原结合片段结合相同或者完全或部分重叠的人CCR8蛋白的表位；
(2)与本发明的任何一种抗CCR8抗体或其抗原结合片段竞争结合人CCR8蛋白的表位；
(3)显示与本发明抗体对CCR8相同或相似的结合亲和力和/或特异性；
(4)具有本发明抗体的一个或多个生物学特性。
在一些实施方案中，本发明的抗体或其抗原结合片段，其具有以下特性中的一种或多种：
(1)以高亲和力与人/食蟹猴CCR8结合，例如，表现出不高于1000ng/mL的EC50值、不高于950ng/mL的EC50值、不高于900ng/mL的EC50值、不高于850ng/mL的EC50值、不高于800ng/mL的EC50值、不高于750ng/mL的EC50值、不高于700ng/mL的EC50值、不高于650ng/mL的EC50值、不高于600ng/mL的EC50值、不高于550ng/mL的EC50值、不高于500ng/mL的EC50值、不高于450ng/mL的EC50值、不高于400ng/mL的EC50值、不高于350ng/mL的EC50值、不高于300ng/mL的EC50值、不高于250ng/mL的EC50值、不高于200ng/mL的EC50值、不高于180ng/mL的EC50值、不高于160ng/mL的EC50值、不高于150ng/mL的EC50值、不高于140ng/mL的EC50值、不高于130ng/mL的EC50值、不高于120ng/mL的EC50值、不高于110ng/mL的EC50值、不高于100ng/mL的EC50值、不高于95ng/mL的EC50值、不高于90ng/mL的EC50值、不高于85ng/mL的EC50值、不高于80ng/mL的EC50值、不高于75ng/mL的EC50值、不高于70ng/mL的EC50值、不高于65ng/mL的EC50值、不高于60ng/mL的EC50值、不高于55ng/mL的EC50值、不高于50ng/mL的EC50值、不高于45ng/mL的EC50值、不高于40ng/mL的EC50值、不高于35ng/mL的EC50值、不高于30ng/mL的EC50值、不高于25ng/mL的EC50值、不高于20ng/mL的EC50值、不高于18ng/mL的EC50值、不高于16ng/mL的EC50值、不高于15ng/mL的EC50值、不高于14ng/mL的EC50值、不高于13ng/mL的EC50值、不高于12ng/mL的EC50值、不高于11ng/mL的EC50值、不高于10ng/mL的EC50值、不高于8ng/mL的EC50值、不高于6ng/mL的EC50值、不高于4ng/mL的EC50值、不高于2ng/mL的EC50值、不高于1ng/mL的EC50值、或更低的EC50值。
(2)阻断人/食蟹猴CCR8与其配体(例如，CCL1)的结合，例如，表现出不高于1000ng/mL的IC50值、不高于950ng/mL的IC50值、不高于900ng/mL的IC50值、不高于850ng/mL的IC50值、不高于800ng/mL的IC50值、不高于750ng/mL的IC50值、不高于700ng/mL的IC50值、不高于650ng/mL的IC50值、不高于600ng/mL的IC50值、不高于550ng/mL的IC50值、不高于500ng/mL的IC50值、不高于450ng/mL的IC50值、不高于400ng/mL的IC50值、不高于350ng/mL的IC50值、不高于300ng/mL的IC50值、不高于250ng/mL的IC50值、不高于200ng/mL的IC50值、不高于180ng/mL的IC50值、不高于160ng/mL的IC50值、不高于150ng/mL的IC50值、不高于140ng/mL的IC50值、不高于130ng/mL的IC50值、不高于120ng/mL的IC50值、不高于110ng/mL 的IC50值、不高于100ng/mL的IC50值、不高于95ng/mL的IC50值、不高于90ng/mL的IC50值、不高于85ng/mL的IC50值、不高于80ng/mL的IC50值、不高于75ng/mL的IC50值、不高于70ng/mL的IC50值、不高于65ng/mL的IC50值、不高于60ng/mL的IC50值、不高于55ng/mL的IC50值、不高于50ng/mL的IC50值、不高于45ng/mL的IC50值、不高于40ng/mL的IC50值、不高于35ng/mL的IC50值、不高于30ng/mL的IC50值、不高于25ng/mL的IC50值、不高于20ng/mL的IC50值、不高于18ng/mL的IC50值、不高于16ng/mL的IC50值、不高于15ng/mL的IC50值、不高于14ng/mL的IC50值、不高于13ng/mL的IC50值、不高于12ng/mL的IC50值、不高于11ng/mL的IC50值、不高于10ng/mL的IC50值、不高于8ng/mL的IC50值、不高于6ng/mL的IC50值、不高于4ng/mL的IC50值、不高于2ng/mL的IC50值、不高于1ng/mL的IC50值、或更低的IC50值。
(3)阻断CCL1诱导的β-Arrestin募集，例如，表现出不高于10000ng/mL的IC50值、不高于9500ng/mL的IC50值、不高于9000ng/mL的IC50值、不高于8500ng/mL的IC50值、不高于8000ng/mL的IC50值、不高于7500ng/mL的IC50值、不高于7000ng/mL的IC50值、不高于6500ng/mL的IC50值、不高于6000ng/mL的IC50值、不高于5500ng/mL的IC50值、不高于5000ng/mL的IC50值、不高于4500ng/mL的IC50值、不高于4000ng/mL的IC50值、不高于3500ng/mL的IC50值、不高于3000ng/mL的IC50值、不高于2500ng/mL的IC50值、不高于2000ng/mL的IC50值、不高于1800ng/mL的IC50值、不高于1600ng/mL的IC50值、不高于1500ng/mL的IC50值、不高于1400ng/mL的IC50值、不高于1300ng/mL的IC50值、不高于1200ng/mL的IC50值、不高于1100ng/mL的IC50值、不高于1000ng/mL的IC50值、不高于950ng/mL的IC50值、不高于900ng/mL的IC50值、不高于850ng/mL的IC50值、不高于800ng/mL的IC50值、不高于750ng/mL的IC50值、不高于700ng/mL的IC50值、不高于650ng/mL的IC50值、不高于600ng/mL的IC50值、不高于550ng/mL的IC50值、不高于500ng/mL的IC50值、不高于450ng/mL的IC50值、不高于400ng/mL的IC50值、不高于350ng/mL的IC50值、不高于300ng/mL的IC50值、不高于250ng/mL的IC50值、不高于200ng/mL的IC50值、不高于180ng/mL的IC50值、不高于160ng/mL的IC50值、不高于150ng/mL的IC50值、不高于140ng/mL的IC50值、不高于130ng/mL的IC50值、不高于120ng/mL的IC50值、不高于110ng/mL的IC50值、不高于100ng/mL的IC50值、不高于80ng/mL的IC50值、不高于60ng/mL的IC50值、不高于40ng/mL的IC50值、不高于20ng/mL的IC50值、不高于10ng/mL的IC50值、或更低的IC50值。
本发明还提供了一种多特异性抗体，其包含本文所述抗体或其抗原结合片段 的轻链可变区和/或重链可变区。
本发明还提供了一种单链抗体，其包含本文所述抗体或其抗原结合片段的轻链可变区和重链可变区。
本发明还提供了一种免疫缀合物，其包含与治疗剂或诊断剂缀合的本文所述抗体或其抗原结合片段。
在又一个方面，本发明提供了多核苷酸分子，其编码本文所述的任一抗CCR8抗体或其任何片段。
在又一个方面，本发明提供了表达载体，其包含本发明所述的多核苷酸分子，优选地，所述载体为真核表达载体。
在又一个方面，本发明提供了宿主细胞，其包含本发明所述的多核苷酸分子或本发明所述的表达载体，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了制备本文所述的抗CCR8抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下在本文所述的宿主细胞中表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
在又一个方面，本发明提供了药物组合物，其包含本文所述的抗CCR8抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、免疫缀合物，和任选地至少一种药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了药物组合，其包含如本文所述的抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、免疫缀合物或药物组合物，以及一种或多种另外的治疗剂。在一些实施方案中，所述另外的治疗剂是化疗剂。在一些实施方案中，所述另外的治疗剂是其他抗体。在一些实施方案中，所述另外的治疗剂是其他单克隆抗体。在一些优选的实施方案中，所述另外的治疗剂是靶向免疫检查点的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向PD-1的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向PD-L1的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向CTLA4的单克隆抗体。
在又一个方面，本发明提供了一种在体外或体内清除CCR8阳性的Treg细胞的方法，所述方法包括将本文所述的抗CCR8抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、免疫缀合物或药物组合物接触包含CCR8阳性的Treg细胞的细胞群或施用于受试者体内。
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、免疫缀合物、药物组合物或药物组合在制备用于治疗和/或预防肿瘤、自身免疫性疾病、或感染性疾病的药物中的用途，优选所述肿瘤为黑色素 瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌。
在一些实施方案中，本发明提供了本文所述的抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、免疫缀合物或药物组合物与其他治疗剂联合在制备用于治疗和/或预防肿瘤、自身免疫性疾病、或感染性疾病的药物中的用途，优选所述肿瘤为黑色素瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌。在一些实施方案中，所述另外的治疗剂是化疗剂。在一些实施方案中，所述另外的治疗剂是其他抗体。在一些实施方案中，所述另外的治疗剂是其他单克隆抗体。在一些优选的实施方案中，所述另外的治疗剂是靶向免疫检查点的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向PD-1的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向PD-L1的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向CTLA4的单克隆抗体。
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、免疫缀合物、药物组合物或药物组合，其用于治疗和/或预防肿瘤、自身免疫性疾病、或感染性疾病，优选所述肿瘤为黑色素瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌。
在又一个方面，本发明提供了一种治疗和/或预防肿瘤、自身免疫性疾病、或感染性疾病的方法，其包括向有需要的受试者施用治疗或预防有效量的本文所述的抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、免疫缀合物、药物组合物或药物组合，优选所述肿瘤为黑色素瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌。
在又一个方面，本发明提供了一种试剂盒，其包括本文所述的抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、免疫缀合物、药物组合物或药物组合，优选其进一步包括给药装置。
在又一个方面，本发明提供了一种使用本文所述的抗体或其抗原结合片段或免疫缀合物或含有所述抗体或其抗原结合片段或免疫缀合物的检测组合物检测CCR8在样品中的存在的方法。
附图说明
图1所示为本发明的抗CCR8抗体与人CCR8的结合活性。A-D为经纯化定量的单克隆杂交瘤细胞培养上清中的抗体与293F-hCCR8的结合曲线。
图2所示为本发明的抗CCR8抗体对食蟹猴CCR8的结合活性。A-B为经纯化定量的单克隆杂交瘤细胞培养上清中的抗体与293T-cynoCCR8的结合曲线。
图3所示为本发明的多个抗体克隆可以阻断CCL1与CCR8的结合。A-C为经纯化定量的单克隆杂交瘤细胞培养上清中的抗体阻断10nM AlexaFlour-647标记的人CCL1与293F-hCCR8的抑制曲线。
图4所示为本发明的抗CCR8嵌合抗体与人CCR8的结合活性。A-B和C分别为hIgG1和mIgG2a嵌合抗体与293F-hCCR8的结合曲线。
图5所示为本发明的抗CCR8嵌合抗体与食蟹猴CCR8的结合活性。A和B分别为hIgG1和mIgG2a嵌合抗体与293T-cynoCCR8的结合曲线。
图6所示为本发明的抗CCR8嵌合抗体阻断CCL1结合的活性。A-B和C分别为hIgG1和mIgG2a嵌合抗体阻断10nM AlexaFlour-647标记的人CCL1与293F-hCCR8的抑制曲线。
图7所示为本发明的CCR8嵌合抗体阻断CCL1诱导的β-Arrestin募集。A和B分别为hIgG1和mIgG2a嵌合抗体阻断人CCL1诱导的β-Arrestin募集的抑制曲线。
图8所示为本发明的人源化抗CCR8抗体与人CCR8的结合活性。A-B，C-D和E-G分别为克隆27B9-1G3、559E1B10和563E10E12的人源化抗体与293F-hCCR8的结合曲线。
图9所示为本发明的人源化抗CCR8抗体对食蟹猴CCR8的结合活性，通过559E1B10和563E10E12人源化抗体与293T-cynoCCR8的结合曲线所反映。
图10所示为本发明的人源化抗CCR8抗体阻断CCL1的结合活性。A-B，C-D和E-G分别为27B9-1G3、559E1B10和563E10E12人源化抗体阻断10nM AlexaFlour-647标记的人CCL1与293F-hCCR8的抑制曲线。
图11所示为本发明的抗CCR8人源化抗体阻断CCL1诱导的β-Arrestin募集实验。A-B和C-E分别为559E1B10和563E10E12人源化抗体的阻断人CCL1诱导的β-Arrestin募集的抑制曲线。
图12所示为559E1B10的嵌合抗体及人源化抗体可以以剂量依赖的方式激活Jurkat-human FcγRⅢa(158V)-NFAT。
图13所示为563E10E12的嵌合抗体及人源化抗体可以以剂量依赖的方式激活Jurkat-human FcγRⅢa(158V)-NFAT。
图14所示为本发明的抗CCR8抗体与HuT78的结合活性。
图15所示为本发明的抗CCR8抗体与hCCR8特异性结合。抗CCR8抗体与CHOK1-hCCR8(A)和CHO-K1(B)细胞的结合活性。
图16所示为本发明的抗CCR8抗体CCR8依赖的对Jurkat-human FcγRⅢa(158V)-NFAT激活。小图A和B所对应的靶细胞分别为CHOK1-hCCR8和CHOK1-Blank。
图17所示为本发明的抗CCR8抗体与转染了hCCR4的293F细胞的结合。 抗CCR8抗体与转染了hCCR4-GFP(A，GFP阳性细胞)和空白(B，所有活细胞)293F细胞的结合。
图18所示为Fc突变或降低岩藻糖基化的559E1B10和563E10E12人源化抗体与293F-humanCCR8孵育对Jurkat-human FcγRⅢa(158V)-NFAT的激活。A-C分别为Fc突变或降低岩藻糖基化的559E1B10_hzH1L1(A)，563E10E12_hzH1L1_hIgG1(B)和563E10E12_hzH1L0_hIgG1(C)与293F-humanCCR8孵育对Jurkat-human FcγRⅢa(158V)-NFAT的激活。
图19所示为Fc突变或降低岩藻糖基化的563E10E12_hzH1L0与HuT78孵育对Jurkat-human FcγRⅢa(158V)-NFAT的激活。
图20所示为健康人PBMC和抗CCR8抗体诱导的ADCC效应。IL-2活化的健康人PBMC、抗CCR8抗体和CHOK1-hCCR8共同孵育约5小时，检测PI阳性的CHOK1-hCCR8的比例，其与不同抗体及其浓度的关系如曲线所示。
图21所示为本发明的抗CCR8抗体清除外周PBMC中的Treg。IL-2活化的PBMC和抗CCR8抗体孵育96小时后，Foxp3阳性细胞占CD4+T细胞的比例(A)和CD8+T细胞(CD3+CD4-)占CD3+T细胞的比例(B)。
图22所示为本发明的抗CCR8抗体抑制MC38肿瘤生长。CCR8人源化小鼠B-hCCR8接种结肠癌MC38细胞。皮下注射10mg/kg的抗体或阴性对照(hIgG1)或等体积的溶媒(PBS)对照，每周给药2次。肿瘤体积变化如曲线所示。
图23所示为本发明的抗CCR8抗体治疗MC38肿瘤模型中小鼠体重的变化。
图24所示为Fc突变和降低岩藻糖基化的抗CCR8抗体抑制MC38肿瘤生长。CCR8人源化小鼠B-hCCR8接种结肠癌MC38细胞成瘤。皮下注射10mg/kg的抗CCR8抗体或等体积的溶媒(PBS)对照，每周给药2次。肿瘤体积变化如曲线所示。
图25所示为Fc突变和降低岩藻糖基化的抗CCR8抗体治疗MC38肿瘤模型中小鼠体重的变化。
图26所示为本发明的抗CCR8抗体单药和联合抗mPD-1抗体在小鼠中抑制乳腺癌EMT-6肿瘤生长。各组用药方案为5-10mg/kg的抗体(i.v.)，抗mPD-1抗体(i.p.)或等体积的溶媒(PBS)对照(i.v.)，每周给药2次。肿瘤体积变化如曲线所示。
图27所示为本发明的CCR8抗体治疗EMT-6肿瘤模型过程中，小鼠体重的变化。
具体实施方式
定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、 细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语或表述都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员至少部分地可以具体参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写遵循本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值，包括但不限于±5％、±2％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。
如本文所用，术语“或”应被理解为与如上定义的“和/或”具有相同的含义。例如，当分离列表中的项目时，“或”或“和/或”应被解释为包容性的，即，包括数量或元素列表中的至少一个，但也包括多于一个，以及任选地，额外的未列出的项目。只有明确指出相反的术语下，例如“只有一个”或“的确一个”或者在权利要求中使用“由...组成”时，将指的是仅列出的一个数字或列表的一个元素。
除非上下文明确指出相反的情况，否则如本文所用，词“一”和“一个”应理解为“至少一个”。
本文中的“CCR8”、“CC基序趋化因子受体8型”和“趋化因子(CC基序)受体8”等术语是指由细胞中CCR8的表达产生的任何天然CCR8。除非另有说明，否则该术语包括来自任何脊椎动物来源的CCR8，例如哺乳动物(例如灵长类动物(例如人类和食蟹猴)和啮齿动物(例如小鼠和大鼠))。示例性人CCR8蛋白参见UniProt记录P51685，示例性食蟹猴CCR8蛋白参见UniProt记录G7NYJ2，示例性小鼠CCR8蛋白参见UniProt记录P56484。
术语“CCR8”包括人CCR8的变体、亚型、物种同源物或其他物种的CCR8和具有CCR8的至少一个共同表位的类似物，除非另有说明。该术语包括未处理的全长CCR8以及由于在细胞中处理而产生的任何形式的CCR8。该术语涵盖“全长”未加工的CCR8以及由细胞内加工产生的任何形式的CCR8或其任何片段，例如剪接变体或等位基因变体。在一个实施方案中，CCR8是指来自人或食蟹猴CCR8全长或其片段(诸如其缺乏信号肽的成熟片段)。
如本文所用，“CCL1”和“CC基序趋化因子配体1”是指由细胞中CCL1基因表达产生的任何天然CCL1。如其命名，CCL1属于CC趋化因子家族，由活化的单核/巨噬细胞、T淋巴细胞和内皮细胞分泌。除非另有说明，否则该术语包括来自 任何脊椎动物来源的CCL1，例如哺乳动物(例如灵长类动物(例如人类和食蟹猴)和啮齿动物(例如小鼠和大鼠))。
术语“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
术语“Treg”、“调节性T细胞”指在免疫应答中表现出免疫抑制作用、通常抑制或下调效应T细胞的诱导和增殖的一类T细胞。Treg对癌症的影响是复杂的，但由于观察到Treg细胞在患有癌症的个体中趋于上调，并且它们似乎被招募到肿瘤部位，而且多项研究提示肿瘤微环境中存在大量的Treg表明预后不良，因此一般倾向于认为Treg细胞抑制人体抗肿瘤免疫。多种免疫疗法正在研究靶向Treg以治疗癌症的方法。在本发明的一些实施方案中，肿瘤中含有肿瘤浸润性Treg细胞。在一些实施方案中，肿瘤中含有表达CCR8的细胞。在一些实施方案中，表达CCR8的细胞是Treg细胞。在本发明的一些实施方案中，T效应细胞不表达或基本不表达CCR8。在一些实施方案中，本发明的抗体全部/部分地抑制/清除Treg细胞。在一些实施方案中，本发明的抗体借助ADCC机制以全部/部分地抑制/清除Treg细胞，和/或阻断CCL1与CCR8的结合，抑制CCL1诱导的信号通路。在一些实施方案中，本发明的抗体通过全部/部分地抑制/清除Treg细胞以治疗癌症。在一些实施方案中，利用本发明的抗体全部/部分地抑制/清除Treg细胞比其他抑制/清除Treg细胞的方法的效果更好和/或效率更高。在一些实施方案中，本发明的抗体通过全部/部分地阻断CCL1与CCR8的结合以治疗癌症。在一些实施方案中，利用本发明的抗体全部/部分地阻断CCL1与CCR8的结合比其他阻断CCL1与CCR8的结合的方法的效果更好和/或效率更高。在一些更具体实施方案中，所述效果包括：抗肿瘤免疫增强、针对肿瘤抗原的免疫应答增强、肿瘤生长减缓、肿瘤体积缩小、抗肿瘤细胞因子分泌增加、肿瘤中浸润的T效应细胞数量增多/功能增强、抗肿瘤免疫长期记忆效应增强，或前述任意组合。在一些实施方案中，利用本发明的抗体全部/部分地抑制/清除Treg细胞比其他抑制/清除Treg细胞的方法的不良反应更少。在一些更具体实施方案中，所述不良反应是免疫失调，例如自身免疫失调，例如自身免疫病。
术语“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子对受体的结合启动的生化因果关系，例如诸如CCL1(配体)对CCR8(受体)的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导致基因表达的变化。
术语“活性”或“生物活性”，或术语“生物性质”或“生物特征”此处可互换使用，包括但不限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗CCR8活性的能力、IC50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“全长抗体”，是指在天然存在时包含至少四条肽链的免疫球蛋白分子：两条重(H)链和两条轻(L)链通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保 持亲代抗体的至少一些结合特异性。抗体的结合片段的实例包括但不限于本领域所熟知的Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。在本发明的一些优选实施方案中，本发明的抗原结合片段选自Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。
术语“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
术语“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
术语“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。
术语“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
术语“鼠源抗体”或“杂交瘤抗体”在本发明中为根据本领域知识和技能制备的抗CCR8的单克隆抗体。制备时用CCR8抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。杂交瘤技术是通过融合两种细胞，并且同时还保持两者的主要特征。这两种细胞分别是经抗原免疫的小鼠脾细胞和小鼠骨髓瘤细胞。被特异性抗原免疫的小鼠脾细胞(B淋巴细胞)的主要特征是它的抗体分泌功能，但不能在体外连续培养，小鼠骨髓瘤细胞则可在培养条件下无限分裂、增殖，即具有所谓永生性。在选择培养基的作用下，只有B细胞与骨髓瘤细胞融合的杂交细胞才能具有持续培养的能力，形成同时具备抗体分泌功能和保持细胞永生性两种特征的细胞克隆。在一些实施方案中，本发明通过使用CCR8蛋白免疫小鼠，再获取小鼠的脾细胞和骨髓瘤细胞融合，获得能够表达阳性抗体的杂交瘤细胞。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗 体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。在本发明的一些实施方案中，所述啮齿动物是小鼠或大鼠。在本发明的一些优选的实施方案中，嵌合抗体对抗原的亲和力不低于或几乎不低于亲本鼠抗体。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
抗体的轻链可以基于其恒定结构域的氨基酸序列归入两种类型(称为kappa(κ)和lambda(λ))中的一种。抗体的重链可以根据其重链恒定区的氨基酸序列而划分为主要5种不同的种类：IgA、IgD、IgE、IgG和IgM，并且这些类型中的几种可以进一步划分成亚类，如，IgG1、IgG2、IgG3和IgG4、IgA1以及IgA2。“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
本文术语“Fc区”用于定义包含至少一部分恒定区的免疫球蛋白重链的C端区域。该术语包括天然序列Fc区和变异Fc区。在一些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羧基末端。但是，Fc区的C端赖氨酸(Lys447)可能存在或不存在(此段中的编号是根据EU编号系统，也称为EU索引，如Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991)。
本文术语“效应子功能”是指由抗体Fc区介导生物活性，其随抗体同种型的不同而不同。抗体效应子功能包括但不限于：C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；调理作用；细胞表面受体的下调；和B细胞活化等等。
术语“表位”指能够与抗体特异性结合的蛋白质决定簇。表位通常由各种化学活性表面分子诸如氨基酸或糖侧链组成，并且通常具有特定三维结构特征以及特定电荷特征。构象性表位和非构象性表位的区别在于在变性溶剂存在下，与前者 而非与后者的结合丧失。
本文中所描述的术语“交叉反应”指的是对人类、猴、和/或鼠源(小鼠或大鼠)相同靶分子的抗原片段的结合。因此，“交叉反应”应被理解为抗原结合分子(例如，抗体)与在不同物种中表达的同类分子(例如，CCR8)的种属间反应。识别人CCR8、猴、和/或鼠CCR8(小鼠或大鼠)的单克隆抗体的交叉反应特异性可通过FACS分析确定。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。在本发明的一些实施方案中，利用表面等离子共振(SPR)技术测量亲和力，例如本发明的抗体与抗原之间的亲和力。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。在本发明的一些实施方案中，利用表面等离子共振(SPR)技术测量亲和力，例如本发明的抗体与抗原之间的亲和力。
术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的EC50为1.0×10-7M或更高，更优选1.0×10-6M或更高，更优选1.0×10-5M或更高，更优选1.0×10-4M或更高、更优选1.0×10-3M或更高。
术语“高亲和性”对于IgG抗体而言，是指对于抗原的EC50为1.0×10-7M或更低，优选5.0×10-8M或更低，更优选1.0×10-8M或更低，更优选5.0×10-9M或更低，更优选1.0×10-9M或更低，更优选5.0×10-10M或更低，更优选1.0×10-10M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指EC50为10-7M或更低，优选10-8M或更低，更优选10-9M或更低。
术语“阻断”对于受体的IgG抗体而言，是指通过施用所述抗体，使得抗体与受体的原定配体产生竞争，抑制受体与配体之间的结合和相互作用。这种抑制可以通过多种机制发生，包括例如由于受体上的重叠结合位点、和/或由抗体诱导的受体构象变化改变配体亲和力等。被称为“功能性的”抗体和抗体片段以具有这样的性质为特征。在一些实施方案中，“阻断”的能力指EC50值为5.0×10-5M或更低，1.0×10-5M或更低，5.0×10-6M或更低，1.0×10-6M或更低，优选5.0×10-7M或更低，更优选1.0×10-7M或更低，更优选5.0×10-8M或更低，更优选1.0×10-8M或更低。
术语“抗体依赖的细胞毒性”、“抗体依赖的细胞介导的细胞毒性”或“ADCC”是指细胞介导的免疫防御，其中免疫系统效应细胞主动地将细胞膜表面抗原与抗体结合的靶细胞裂解。
术语“补体依赖的细胞毒性”或“CDC”是指IgG和IgM抗体的效应功能，当与表面抗原结合时引发典型的补体途径，包括形成膜攻击复合体以及靶细胞裂解。。
术语“核酸”、“多核苷酸”、“核酸分子”以及“多核苷酸分子”在本文中可互换地使用(除非上下文另有指明)，并且是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸(参见，属于Kariko等人的美国专利No.8,278,036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(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的序列来实现(Batzer等人，Nucleic Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，其可衍生自任何来源，能够与基因组整合或自主复制，其中包含已经以功能操作的方式连接(即，可操作地连接)的一或多个多核苷酸分子。在本发明的一些优选的实施方案中，重组构建体包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会驱动和/或导引本发明的多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者驱动和/或导引本发明的多核苷酸的表达。
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到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及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引被操作性连接的基因的表达。本文将此类载体称为“表达载体”。
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。在本发明的一些优选的实施方案中，本发明的表达载体包含本发明的构建体和/或本发明的多核苷酸。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的 子代。宿主细胞是可以用来产生本发明抗体分子的任何类型的细胞系统，包括真核细胞，例如，哺乳动物细胞、昆虫细胞、酵母细胞；和原核细胞，例如，大肠杆菌细胞。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
用于细胞或受体的操作“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
在本发明的一些实施方案中，术语“肿瘤”意在强调恶性肿瘤，并且术语“癌症”和“肿瘤”可互换使用，指动物中的任何异常、不受控的细胞或组织生长或增殖。如本文所用，术语“癌症”和“肿瘤”包括实体瘤和血液系统肿瘤，还包括癌前病变。肿瘤的具体的非限制性实例包括乳腺癌、鳞状细胞癌、小细胞肺癌、垂体癌、食道癌、星形细胞瘤、软组织肉瘤、肺癌(如非小细胞肺癌、肺腺癌、肺鳞状细胞癌)、腹膜癌、肝细胞癌、胃癌、肠癌、胰腺癌、胶质母细胞瘤、宫颈癌、卵巢癌、肝癌、结肠癌、结直肠癌、子宫内膜癌、肾癌、前列腺癌、膀胱癌、甲状腺癌、睾丸癌、胆管癌、胆囊癌、黑色素瘤、间皮瘤、胸腺瘤和各种头颈癌。在一些实施方案中，血液系统肿瘤包括B细胞和T细胞混合白血病、B细胞淋巴瘤、粒细胞白血病(急性和慢性)、淋巴细胞白血病(急性和慢性)、儿童/青少年淋巴细胞白血病、粒单核细胞白血病、弥漫性大B细胞淋巴瘤(DLBC)、霍奇金淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤(MCL)、多发性骨髓瘤、骨髓增生异常综合征等等。在一些实施方案中，肿瘤是良性肿瘤。
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方案中是指减缓、中断、阻滞、缓解、停止、降低、或逆转疾病的症状、并发症、或生化征的发作、缓解症状或阻止或抑制疾病、病状或病症的进一步发展(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一个实施方案中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方案中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺 乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴或源自食蟹猴的。
“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的CCR8抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
“药学上可接受的载体”是指药物制剂或组合物中除活性成分以外的对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲剂，赋形剂，稳定剂或防腐剂。
抗CCR8抗体
在一个方面，本发明提供了特异性结合CCR8的抗体或其抗原结合片段。术语“抗CCR8抗体”、“抗CCR8”、“CCR8抗体”或“结合CCR8的抗体”是指能够以足够的亲合力结合CCR8蛋白或其片段以致所述抗体可以用作靶向CCR8的诊断剂和/或治疗剂。
在一些实施方案中，本发明抗体结合人或食蟹猴CCR8蛋白。在一些实施方案中，本发明抗体结合CHOK1-humanCCR8细胞或293F-cynoCCR8细胞。在一些实施方案中，本发明抗体抑制/阻断CCR8与其配体CCL1的结合。在一些实施方案中，本发明抗体抑制/阻断诱导的β-Arrestin募集。
可采用用于产生抗体的任何合适方法来产生本发明的抗体。任何合适形式的CCR8都可用作产生抗体的免疫原(抗原)。作为举例而非限制，任何CCR8变体或其片段都可用作免疫原。在一些实施方案中，产生鼠源的单克隆抗CCR8抗体的杂交瘤细胞可通过本领域公知的方法产生。
来源于啮齿动物(如小鼠)的抗体在体内用作治疗药物时可能引起不需要的抗体免疫原性，重复使用导致人体产生针对治疗性抗体的免疫应答，这类免疫应答至少导致丧失治疗功效，而严重的则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84:3439-43)。然而，嵌合抗体中的完 整啮齿动物可变区的保留仍可能在患者中引起有害的免疫原性。将啮齿动物可变结构域的互补决定区(CDR)环移植到人框架上(即人源化)已被用于进一步将啮齿动物序列减至最低(Jones等(1986)Nature 321:522；Verhoeyen等(1988)Science 239:1534)。在一些实施方案中，本发明的抗体是嵌合抗体。在一些优选的实施方案中，本发明的抗体是人源化抗体。
在一些实施方案中，本发明的嵌合或人源化抗体可基于所述制备的鼠单克隆杂交瘤抗体的序列来制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
在一些实施方案中，本发明所述的嵌合CCR8抗体，可使用本领域已知的方法将杂交瘤来源的免疫球蛋白重链和轻链可变区与人IgG恒定区有效连接(参见例如属于Cabilly等人的美国专利No.4,816,567)，获得嵌合型重链和嵌合型轻链来制备。在一些实施方案中，本发明的嵌合抗体包含的恒定区可选自任何人IgG亚型，如IgG1、IgG2、IgG3、IgG4，优选IgG1。
在一些实施方案中，本发明的嵌合CCR8抗体可由嵌合型轻链与嵌合型重链表达质粒“混合和匹配”转染表达细胞获得，此类“混合和匹配”的抗体的CCR8结合可使用上述结合测定和其它常规结合测定(例如，ELISA)来进行测试。
如本文所述定义的，“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基
(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health (1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
例如，根据不同的CDR确定方案，每一个CDR的残基如下所述。
以下为采用kabat、AbM、Chothia、Contact和IMGT方案定义的CDR的区域范围。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991)中的Kabat编号系统的编号位置。在一些实施方案中，本发明抗体中的HCDR和LCDR分别按照Kabat方案确定。
除非另有说明，否则本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例 如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat、Chothia、AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基取代。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
本发明将各种嵌合型重链和轻链表达质粒混合配对转染表达细胞，其抗CCR8嵌合抗体。
本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源CDR区插入人种系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101；5,585,089；5,693,762和6,180,370。
在一些实施方案中，氨基酸改变包括氨基酸缺失、添加或取代等。在一些实施方案中，本发明的抗CCR8抗体或其抗原结合片段包括具有已通过氨基酸缺失、添加或取代突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方案中，本发明的抗体与具体序列中描述的CDR区相比较时，在CDR区中已通过氨基酸缺失、添加或取代的氨基酸突变具有至少一个，例如，1、2、3、4或5个。在一些实施方案中，本发明的抗体与具体序列中框架区相比较时，在框架区中已通过氨基酸缺失、添加或取代的氨基酸突变具有至少一个，例如，1、2、3、4或5个。
术语“百分比(％)氨基酸序列同一性”或简称“同一性”定义为在将氨基酸序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守性取代视为序列同一性的部分之后，候选氨基酸序列中的氨基酸残基与参比氨基酸序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件/算法。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
在一些实施方案中，本发明的抗体或其抗原结合片段，其中所述抗体或其抗 原结合片段包含重链恒定区和/或轻链恒定区，优选地，所述轻链恒定区是λ链或κ链恒定区；所述重链恒定区选自鼠mIgG2a、人IgG1、人IgG2、人IgG3或IgG4型，或其修饰形式。在一些优选的实施方案中，所述重链恒定区是人IgG1型或具有S228P突变的人IgG4型。在一些优选的实施方案中，所述恒定区的修饰形式包括氨基酸序列修饰和/或糖基化修饰。
在一些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如添加、缺失或取代，优选地，取代)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。
在一些实施方案中，抗体包含至少一种增强细胞杀伤的修饰。在一些实施方案中，增强的细胞杀伤是增强的抗体依赖性细胞毒性(ADCC)和/或补体依赖性细胞毒性(CDC)。在一些实施方案中，所述修饰是去岩藻糖基化/降低的岩藻糖基化。在一些实施方案中，所述修饰是在选自根据EU编号系统的位点238、239、248、249、252、254、255、256、258、265、267、268、269、270、272、276、278、280，283、285、286、289、290、292、293、294、295、296、298、301、303、305、307、309、312、315、320、322、324、326、327、328、329、330、331、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、434、435、437、438和439的一个或多个位置处的一个或多个重链恒定区突变。在本文中，如无上下文另外规定，则涉及恒定区氨基酸位置编号时，参考EU编号系统。在本文中，氨基酸序列中的突变可如下命名：亲本氨基酸的单字母代码，接着是位置编号，之后是突变后氨基酸的单字母代码。例如，将第234位的亮氨酸(L)突变为丙氨酸(A)表示为“L234A”。有时，在序列中，使用斜线(/)来并列可替换的多种选项。例如，将第236位的半胱氨酸(C)缺失可表示为“C236缺失”。在一些实施方案中，所述修饰是在选自根据EU编号系统的L234、L235、G236、S239、F243、D265、H268、D270、R292、S298、Y300、V305、K326、A330、I332、E333、K334和P396的一个或多个位置处的一个或多个重链恒定区突变。在一些实施方案中，一种或多种重链恒定区突变是选自根据EU编号系统的N297A取代、N297Q取代、L235A取代连同L237A取代、L234A取代连同L235A取代、E233P取代、L234V取代、L235A取代、C236缺失、P238A取代、D265A取代、A327Q取代和P329A取代的一个或多个重链恒定区突变。在一些实施方案中，所述修饰是选自根据EU编号系统的G236A、S239D、F243L、T256A、K290A、R292P、S298A、Y300L、V305I、A330L、I332E、E333A、K334A、A339T和P396L的一个或多个重链恒定区突变。在一些实施方案中，一种或多种重链恒定区突变是选自根据EU编号系统的L235V、S239D、S239M、F243L、H268D、D270E、R292P、S298A、Y300L、V305I、K326D、A330L、A330M、I332E、E333A、 K334A、K334E和P396L的一个或多个重链恒定区突变。在一些实施方案中，一种或多种重链恒定区突变是选自根据EU编号系统的M252Y、S254T和T256E的一个或多个重链恒定区突变。
在一些实施方案中，重链恒定区具有选自以下同时发生突变的位置组合的一组或多组突变：(1)L235/F243/R292/Y300/P396、(2)F243/R292/Y300/V305/P396、(3)D270/K326/A330/K334、(4)S239/A330/I332、(5)S298/E333/K334、(6)L234/L235/G236/S239/H268/D270/S298、(7)M252/S254/T256、(8)L234/L235/D265、(9)G236/S239/I332和(10)S239/I332。
在一些实施方案中，重链恒定区具有选自以下突变组合的一组或多组突变：(1)L235V/F243L/R292P/Y300L/P396L、(2)F243L/R292P/Y300L/V305I/P396L、(3)D270E/K326D/A330M/K334E、(4)S239D/A330L/I332E、(5)S298A/E333A/K334A、(6)L234Y/L235Q/G236W/S239M/H268D/D270E/S298A、(7)M252Y/S254T/T256E、(8)L234A/L235A/D265A、(9)L234F/L235E/D265A、(10)G236A/S239D/I332E和(11)S239D/I332E。
在一些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基取代。在一些实施方案中，将一个、两个或更多个突变(例如氨基酸取代)引入到铰链区(CH1结构域)中，从而改变铰链区中半胱氨酸残基的数量(例如增加或减少)。
在一些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。
抗体表达
在又一个方面，本发明提供了一种多核苷酸分子，其编码本文所述的抗CCR8抗体或其任何片段。所述多核苷酸分子可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列或其至少一部分的多核苷酸分子，或包含编码抗体的轻链和/或重链的氨基酸序列或其至少一部分的多核苷酸分子。
例如，本发明的多核苷酸分子包含编码选自SEQ ID NO:1-95中任一项所示氨基酸序列的核酸，或包含编码与选自SEQ ID NO:1-95中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。
在一些实施方案中，编码本发明抗体的多核苷酸分子包括已通过核苷酸缺失、添加或取代突变的，但仍然与上文中所述的序列中描绘的CDR对应编码区具有至少约60、70、80、90、95或100％同一性的多核苷酸分子。
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸分子，优选地，所述载体为真核表达载体。在一些实施方案中，如本文所述的多核苷酸分子包含在一个或多个表达载体中。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸分子或如本文所述的表达载体，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种用于制备如本文所述的抗CCR8抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下在本文所述的宿主细胞中表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
本发明提供用于表达本发明的重组抗体或其任何片段的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。
在一个实施方案中，本发明提供制备抗CCR8抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选抗体分泌到宿主细胞生长的培养基中，来产生抗体。
可采用标准蛋白质纯化方法从培养基中回收抗体。如本文所述制备的抗体分子可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括尺寸排阻层析、凝胶电泳、高效液相色谱等。
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方案中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗CCR8抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞或本文所述的免疫缀合物，和药学上可接受的载体或赋形剂。应理解，本发明提供的抗CCR8抗体或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗CCR8抗体与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编辑(1980))混合来制备包含本文所述的抗CCR8抗体的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方案中，本发明的药物组合物还包含编码抗CCR8抗体的多核苷酸分子的组合物。
本发明的药物组合物还可以包含一种或多种另外的治疗剂，所述治疗剂是被治疗的特定适应证所需的，涵盖在预防或治疗肿瘤(例如癌症)中有效的任何物质，优选具有不会不利地影响彼此活性的那些治疗剂。例如，化疗剂，激素，等等。所述治疗剂以对于目的应用有效的量合适地组合存在。在一些实施方案中，所述另外的治疗剂是化疗剂。在一些实施方案中，所述另外的治疗剂是其他抗体。在一些实施方案中，所述另外的治疗剂是其他单克隆抗体。在一些优选的实施方案中，所述另外的治疗剂是靶向免疫检查点的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向PD-1的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向PD-L1的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向CTLA4的单克隆抗体。
在又一个方面，本发明提供了一种药物组合，其包含本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的免疫缀合物、或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括本文所述的抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的免疫缀合物、本文所述的药物组合物或本文所述的药物组合。
组合产品或试剂盒
在一些实施方案中，本发明还提供了组合产品，其包含本发明的抗CCR8抗体或其片段，以及一种或多种另外的治疗剂(例如化疗剂、其他抗体、细胞毒性剂、抗感染活性剂、小分子药物或免疫调节剂等)。
在一些实施方案中，所述组合产品用于预防或治疗与CCR8/免疫检查点相关的疾病和/或由CCR8和/或免疫检查点介导的疾病。在一些实施方案中，所述另外的治疗剂是已有的标准治疗剂。在一些实施方案中，所述另外的治疗剂是化疗剂。在一些实施方案中，所述另外的治疗剂是其他抗体。在一些实施方案中，所述另 外的治疗剂是其他单克隆抗体。在一些优选的实施方案中，所述另外的治疗剂是靶向免疫检查点的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向PD-1的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向PD-L1的单克隆抗体。在一些更优选的实施方案中，所述另外的治疗剂是靶向CTLA4的单克隆抗体。
在一些方案中，所述组合产品中的两种或多种成分可以依次、分开或同时联合施用给受试者。
在一些实施方案中，本发明还提供了包含本发明的抗CCR8抗体或其片段、药物组合物或组合产品的试剂盒，以及任选的指导施用的包装插页。
在一些实施方案中，本发明还提供了包含本发明的抗CCR8抗体或其片段、药物组合物或组合产品的药物制品，任选地，所述药物制品还包括指导施用的包装插页。
医药用途及治疗方法
本文提供的任何抗CCR8抗体或相应的免疫缀合物均可用于治疗方法。还应当理解，在讨论“抗体”时，也包括包-含抗体的组合物。本发明的抗CCR8抗体可以治疗有效量或预防有效量用于本发明任一实施方案所述的治疗或预防方法中。
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、免疫缀合物、或药物组合物在制备药物中的用途。本发明还提供了本文所述的药物组合在制备药物中的用途。从而，本发明实质上提供了有效量的本文所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、免疫缀合物、或药物组合物与其它治疗剂联合在制备药物中的用途。其中，所述药物用于在受试者中预防和/或治疗肿瘤、自身免疫性疾病或感染性疾病，所述肿瘤优选为，例如黑色素瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌及其转移性癌症。
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、免疫缀合物、药物组合物或药物组合，其用于治疗和/或预防肿瘤、自身免疫性疾病、或感染性疾病，所述肿瘤优选为，例如黑色素瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌及其转移性癌症。
在又一个方面，本发明提供了一种治疗和/或预防肿瘤、自身免疫性疾病、或感染性疾病的方法，其包括向有需要的受试者施用治疗或预防有效量的本文所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、免疫缀合物、药物组合物或药物组合，所述肿瘤优选为，例如黑色素瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌及其转移性癌症。
在一些实施方案中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或病灶局部给予等。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何不良感受或直接或间接病理学后果、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。对于本发明的抗体而言，能够从一个或多个方面降低病情的严重程度，即发挥治疗作用。在某些实施方案中，表现为以下情形中的一项或多项：患者的平均寿命(存活期)延长；疾病进展延迟；对医疗照护的需求下降。
本发明还提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体。在一些实施方案中，本发明抗体与至少一种另外的治疗剂共施用。
用于诊断和检测的方法
在又一个方面，本发明提供了一种使用本文所述的抗体或其抗原结合片段检测CCR8在样品中的存在的方法。术语“检测”用于本文中时，包括定量或定性检测。在一些实施方案中，所述样品是生物样品。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。在某些实施方案中，CCR8是人CCR8或食蟹猴CCR8。所述方法包括使本文所述的抗体或其抗原结合片段或含有所述抗体或其抗原结合片段的检测组合物与样品接触的步骤，以及检测是否存在所述抗体或其抗原结合片段与CCR8结合产生的结合物或结合信号的步骤。用于检测用途时，本文所述的抗体或其抗原结合片段可被标记，以指示是否形成了所述结合物。在某些实施方案中，方法可以是体外或体内方法。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测CCR8。在一个实施方案中，提供用于诊断或检测方法的抗CCR8抗体或其抗原结合片段。
本发明包括所述特定实施方案的所有组合。本发明的进一步实施方案及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方案，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施 例。
实施例
通过以下实施例对本发明进行说明，但并不旨在对本发明作出任何限制。本文已经详细描述了本发明，其中也公开了其具体实施方式。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1、动物免疫及制备产生抗人CCR8的小鼠杂交瘤抗体
采用人CCR8全长的基因(DNA或mRNA)和超表达人CCR8的工程细胞株免疫小鼠，免疫或冲击免疫也会采用猴CCR8全长的基因(DNA或mRNA)和超表达猴CCR8的细胞，SP2/0细胞融合，流式细胞术筛选阳性克隆。具体方法如下：
动物免疫：将编码人CCR8(UniProtKB/Swiss-Prot：P51685)全长的基因构建到PCDNA3.1或PCDNA3.4质粒(上海睿智化学研究有限公司构建)中作为免疫原，对Balb/c雌性小鼠进行DNA免疫，4μg/小鼠。DNA免疫以2周的时间间隔反复进行6-7次，最后一次免疫10天后腹腔内注射表达人CCR8的293F细胞(上海睿智化学研究有限公司构建，293F-hCCR8)进行冲击免疫，3天后取小鼠脾脏进行细胞融合。
将表达人CCR8的293F细胞(293F-hCCR8)作为免疫原，对Balb/c雌性小鼠进行腹腔免疫(2-5x106细胞/小鼠)。细胞免疫以2周的时间间隔反复免疫3次，最后一次免疫10天后再次于腹腔内注射293F-hCCR8细胞(2-5x106细胞/小鼠)进行冲击免疫，3天后取小鼠脾脏进行细胞融合。
将编码人CCR8(UniProtKB/Swiss-Prot：P51685)全长的mRNA作为免疫原(上海宏成药业有限公司制备，50μg/小鼠)，对Balb/c雌性小鼠进行mRNA免疫。mRNA免疫以3周的时间间隔反复进行3次或4次，最终免疫10天后腹腔内注射293F-hCCR8细胞进行冲击免疫，3天后取小鼠脾脏进行细胞融合。
细胞融合：将小鼠脾细胞与SP2/0细胞(ATCC No.CRL-1581)按2:1比例电融合(BTX电融合仪：ECM2001+)，在96孔培养板中以HAT(GIBCO，货号：H0262)培养基培养，10天后进行杂交瘤细胞上清抗体筛选。
筛选人CCR8和食蟹猴CCR8特异性阳性克隆：将表达人CCR8(CHOK1-hCCR8，上海睿智化学研究有限公司构建)及食蟹猴CCR8的细胞(CHOK1-cynoCCR8，上海睿智化学研究有限公司构建)按5x107细胞/mL密度，100μL/孔铺于96孔U型检测板。加入100μL杂交瘤细胞培养上清于4℃孵育1小时。用FACS buffer(含有1％FBS的PBS)300g离心洗涤细胞2次。加入Alexa488标记的抗小鼠IgG抗体4℃孵育1小时。用FACS buffer 300g离心 洗涤细胞3次。流式细胞仪(BECKMAN COULTER cytoFLEX)读取平均荧光强度(Median Fluorescence Intensity，MFI)。通过与阴性对照(不表达CCR8的CHOK1细胞)比较，筛选出阳性克隆。表1中的克隆对人CCR8(CHOK1-hCCR8)和食蟹猴CCR8(CHOK1-cynoCCR8)结合的平均荧光强度(MFI)高于空白对照细胞(CHOK1)，说明这些克隆对人和食蟹猴CCR8有特异性结合。
表1阳性克隆杂交瘤上清在人CCR8(CHOK1-hCCR8)、食蟹猴CCR8(CHOK1-cynoCCR8)和空白细胞CHOK1上的结合(平均荧光强度，MFI)
实施例2、抗人CCR8的鼠源单克隆抗体的制备及鉴定
将表1中与人CCR8和食蟹猴CCR8具有结合活性的杂交瘤细胞用无血清培养基培养，10天后收集培养上清液，用Protein A柱(博格隆(上海)生物技术有限公司，货号：AA0272)纯化鼠源单克隆抗体，获得纯化的单克隆抗体。采用流式细胞术检测抗人CCR8抗体的结合活性和对CCL1结合的阻断活性。
2.1抗人CCR8抗体与人CCR8和食蟹猴CCR8的结合活性
抗CCR8抗体与表达人CCR8或食蟹猴CCR8细胞的饱和结合实验:
将表达人CCR8的293F细胞系(293F-hCCR8，上海睿智化学研究有限公司构建)、或表达食蟹猴CCR8的293T细胞系(293T-cyno CCR8，康源博创公司构建)调整细胞浓度至1×106细胞/mL，100μL/孔置于96孔U底孔板，离心后舍弃上清。FACS buffer(含1％FBS的PBS溶液)将抗人CCR8抗体、对照抗体(抗人CCR8抗体433H，克隆号：433H，BD Pharmingen，货号：624092；10A11为专利WO2020138489中序列(轻链可变区序列编号：59，重链可变区序列编号为：41)，与本文表5中的人和鼠恒定区序列分别融合得到10A11_hIgG1和10A11_mIgG2a抗体)、IgG1isotype(百英生物科技有限公司，货号：B117901)稀释至起始工作浓度，再用FACS buffer溶液进行梯度稀释。将梯度稀释的抗体100μL/孔重悬细胞，吹打混匀，4℃孵育1小时。孵育后细胞经离心，用FACS buffer溶液清洗三次。每孔加入100μL二抗(Goat anti-Human IgG(H+L)Cross-Adsorbed Secondary Antibody,Alexa FluorTM647，Invitrogen，货号：A21445)稀释液重悬细胞团块，吹打混匀，于4℃孵育约45分钟。孵育后细胞 经离心，清洗三次后，FACS buffer 100μL/孔重悬细胞。流式细胞仪(BECKMAN COULTER cytoFLEX)读取平均荧光强度(Median Fluorescence Intensity，MFI)，利用Graphpad Prism 8.0软件分析试验数据，以抗体浓度对数为x轴，对应的MFI值为y轴，选用四参数方程回归模型，拟合抗体剂量效应曲线，计算出EC50。
如图1、2和表2所示，抗体克隆如27B9-1G3、559E1B10、563E10E12、569D11B5和589D7C7对293F-hCCR8具有强结合活性，EC50为107.0～360.6ng/mL；作为对照，433H和10A11_mIgG2a与293F-hCCR8结合的EC50分别为206.0和185.1ng/mL。抗体克隆27B9-1G3、559E1B10、563E10E12、569D11B5和589D7C7均与食蟹猴CCR8具有较强的结合活性，EC50为146.1～590.8ng/mL，而对照抗体433H和10A11_mIgG2a对食蟹猴CCR8的结合活性很弱，EC50分别为15312.5和5770.5ng/mL。
表2抗CCR8抗体与人CCR8和食蟹猴CCR8的结合活性
N/A：不适用，没有得到有效的EC50
2.2抗CCR8抗体阻断CCL1结合实验
CCL1是CCR8的主要配体，而CCR8是CCL1唯一的已知受体。CCL1在肿瘤组织中高表达与肿瘤的预后负相关。CCL1可以通过CCR8诱导Treg的迁移，阻断CCL1与CCR8的结合可以抑制Treg的迁移和肿瘤的生长(参见Klarquist J等Ccl22 Diverts T Regulatory Cells and Controls the Growth of Melanoma.Cancer Res.2016 Nov 1；76(21):6230-6240和Xu Y等Sox2 Communicates with Tregs Through CCL1to Promote the Stemness Property of Breast Cancer Cells.Stem Cells.2017 Dec；35(12):2351-2365.)。具有阻断CCL1与CCR8结合功能的抗CCR8抗体可能具有更强抗肿瘤活性。采用竞争结合实验，检测抗CCR8抗体阻断AlexaFlour-647标记的人CCL1与293F-hCCR8的结合的活性， 具体方法如下：
将表达人CCR8的293F细胞系(293F-hCCR8，上海睿智化学研究有限公司构建)调整细胞浓度调整至1×106细胞/mL，100μL/孔置于96孔U底孔板，离心后舍弃上清。FACS buffer(含1％FBS的PBS溶液)将抗CCR8抗体、对照抗体(抗人CCR8抗体，克隆号：433H，BD Pharmingen，货号：624092)、hIgG1isotype(百英生物科技有限公司，货号：B117901)稀释至2倍起始工作浓度，再用FACS buffer溶液进行梯度稀释。将梯度稀释的抗体50μL/孔重悬细胞，吹打混匀；随后加入50μL/每孔20nM人CCL1-AlexaFlour-647(Almac，CAF-7)溶液，吹打混匀，于4℃孵育1小时。孵育后细胞经离心，清洗三次后，FACS buffer 100μL/孔重悬细胞。流式细胞仪(BECKMAN COULTER cytoFLEX)读取平均荧光强度(Median Fluorescence Intensity，MFI)，利用Graphpad Prism 8.0软件分析试验数据，以抗体浓度对数为x轴，对应的MFI值为y轴，选用四参数方程回归模型，拟合抗体剂量效应曲线，计算出IC50。
如图3和表3所示，抗体克隆27B9-1G3、559E1B10、563E10E12、569D11B5和589D7C7均可以阻断CCL1与CCR8的结合，阻断活性的IC50为75.0～131.7ng/mL，对照433H阻断活性的IC50为94.3ng/mL。
表3抗CCR8抗体阻断CCL1的结合活性
N/A：无阻断活性或阻断活性较差，没有得到有效的IC50值。
实施例3、抗CCR8抗体测序及嵌合抗体功能鉴定
3.1抗CCR8抗体测序及嵌合抗体构建、表达和纯化
将在上述活性鉴定中表现较好的杂交瘤克隆进行测序，序列见表4。
将测序得到的轻、重链可变区(见表4)分别构建到人源恒定区(IgG1/K，参见表5)或鼠源恒定区(mIgG2a)上，用于体外功能鉴定或体内药效研究。经测序确认构建的hIgG1和mIgG2a嵌合抗体与表4和表5的序列一致。
采用Expi293F细胞(Thermofisher货号：A1452)进行表达，并使用Protein A柱进行纯化，具体方法如下：
Expi293细胞表达抗体：在转染前一天，将Expi293细胞密度稀释到1.5×106细胞/mL，放于37℃，8％CO2摇床中120rpm进行培养。第二天，测定活细胞密度和存活率，细胞转染密度应在3×106细胞/mL，细胞活率大于95％。制备PEI/质粒复合物：将PEI(1mg/mL，polysciences，货号：24765-1)颠倒混匀。用OPM-293 CD05 Medium(奥浦迈，货号：81075-001)稀释质粒，质粒用量为1μg/mL转染体积，稀释质粒的培养基的体积为转染体积的1/20，轻轻混匀，重轻链比例为1：1.5。用OPM-293 CD05 Medium稀释PEI试剂，PEI用量为质粒用量的2倍，稀释PEI的培养基的体积为转染体积的1/20，轻轻颠倒混匀，在室温下孵育5分钟。将稀释的PEI试剂加入到稀释的质粒中，轻轻颠倒混匀。将PEI/质粒复合物在室温下孵育15分钟，然后将溶液缓慢滴加到转移摇瓶中，在加入过程中轻轻旋转摇瓶。转染完毕后将摇瓶放于37℃，8％CO2的摇床中120rpm培养。在转染后第二天(转染后24小时)，在摇瓶中加入10％OPM-293ProFeed(奥浦迈，货号：F081918)，在加入过程中轻轻旋转烧瓶，之后将摇瓶放回摇床中继续培养5-7天，收获上清。
Protein A柱纯化抗体：准备重力层析柱，打开重力层析柱上盖，将垫片置于重力柱底部，压紧。准备填料Protein A(Cytiva，货号：17549801)，由目标填料体积和填料的悬浊比例精确计算出所需填料悬浊液体积，所需填料悬浊液体积＝目标填料体积/填料的悬浊比例。将填料充分涡旋震荡，确保填料完全悬浮。填料悬浊液加入重力层析柱底部。添加至少10CV的平衡缓冲液PBS到重力层析柱，平衡结束后，检测出口pH。如未至目标pH，继续加入平衡缓冲液直至平衡至目标pH。缓慢加入一定体积的样品至重力层析柱中。添加至少10CV的淋洗缓冲液到重力层析柱。缓慢加入5CV的洗脱缓冲液(10-50mM NaAc，pH3.0-pH3.5)到重力层析柱，孵育3-5分钟对洗脱液进行收集。根据需要重复洗脱步骤。中和：用中和缓冲液(1M Tris)调节pH至目标pH。用Nanodrop测定蛋白浓度。通过超滤换液将保存抗体的缓冲液置换成PBS。
表4抗CCR8抗体序列

表5嵌合抗体及人源化抗体恒定区序列

3.2抗CCR8嵌合抗体对人CCR8和食蟹猴CCR8的结合活性
采用实施例2-2.1中的饱和结合实验方法检测抗CCR8嵌合抗体与人和食蟹猴CCR8的结合活性。结果如图4、5和表6所示，27B9-1G3、559E1B10、563E10E12、569D11B5和589D7C7hIgG1或mIgG2a嵌合抗体与293F-hCCR8 细胞结合活性的EC50为27.3-273.4ng/mL，433H与293F-hCCR8细胞结合活性的EC50为153.4ng/mL。hIgG1嵌合抗体27B9-1G3，mIgG2a嵌合抗体563E10E12，569D11B，589D7C7和559E1B10与食蟹猴CCR8具有较强的结合活性，EC50为66.2-303.5ng/ml，而433H和10A11_hIgG1与食蟹猴CCR8的结合活性较弱，EC50为14580.5和6309.0ng/ml。
表6抗CCR8嵌合抗体对人CCR8和食蟹猴CCR8的结合活性
NT：没有检测；N/A：无结合活性或结合活性较差，没有得到有效的EC50值。
3.3抗CCR8嵌合抗体竞争CCL1结合实验
采用实施例2-2.2中的竞争结合实验方法检测抗CCR8嵌合抗体竞争CCL1结合实验，结果如图6和表7所示，hIgG1或mIgG2a嵌合抗体均能剂量依赖性地阻断CCL1与293F-hCCR8的结合，其阻断活性的IC50为29.5-273.4ng/mL，433H和10A11_hIgG1阻断的IC50为70.5和54.6ng/mL。
表7抗CCR8嵌合抗体阻断CCL1结合的活性

N/A：无结合活性，没有得到有效的EC50值
3.4抗CCR8嵌合抗体阻断CCL1诱导的β-Arrestin募集
CCL1通过CCR8诱导趋化作用和受体内吞，后者依赖于β-arrestin的表达和募集，β-arrestin分析用于检测CCL1对CCR8的活化功能(James M Fox等.Structure/function relationships of CCR8agonists and antagonists.Amino-terminal extension of CCL1by a single amino acid generates a partial agonist.J Biol Chem.2006Dec 1；281(48):36652-61.doi:10.1074/jbc.M605584200.和Libao Liu等.Biological characterization of ligands targeting the human CC chemokine receptor 8(CCR8)reveals the biased signaling properties of small molecule agonists.Biochem Pharmacol.2021 Jun；188:114565.doi:10.1016/j.bcp.2021.114565.)。采用Tango-H_CCR8-CHO-K1细胞(吉满生物科技(上海)有限公司，GM-C09028)检测抗CCR8抗体阻断CCL1诱导的β-arrestin募集。根据吉满生物科技(上海)有限公司提供的实验方法检测抗CCR8抗体阻断CCL1诱导的β-arrestin的募集，具体方法如下：
将Tango-H_CCR8-CHO-K1细胞接种至细胞培养瓶中，培养液中添加Doxycycline(Selleck，S4163-100mg)，终浓度为10μg/mL，持续培养约48小时，利用Doxycycline诱导人CCR8表达。用含0.25％EDTA的胰蛋白酶(Gibco，25200072)将诱导完成的Tango-H_CCR8-CHO-K1细胞进行消化，200×g离心5分钟，去除上清。以新鲜F12K完全培养基(Gibco，21127022)重悬细胞，检测细胞活力并计数，再以新鲜培养基调整细胞浓度为5×105个细胞/mL，以100μL/孔接种于白色不透明板中，置于37℃、5％CO2培养箱中培养过夜，使细胞贴壁。用F12K完全培养基将抗CCR8抗体、阳性对照抗体、hIgG1isotype(百英生物科技有限公司，B117901)稀释至2倍的起始工作质量浓度，再用F12K完全培养基进行梯度稀释。轻轻吸尽Tango-H_CCR8-CHO-K1细胞培养板里的培养基后，加入梯度稀释抗体，每孔50μL。随后每孔加入50μL F12K完全培养基稀释的终浓度为20nM的人CCL1(R&D，272-I)溶液。置于37℃、5％CO2条件下孵育6小时后，室温条件下平衡至少15分钟。每孔加入100μL荧光素酶底物溶液(Vazyme，DD1203)，混合后在室温条件下避光反应5分钟，用酶标仪在细胞培养板上读取相对光单位(relative light unit，RLU)数值。利用Graphpad Prism 8.0软件分析试验数据，以抗体浓度对数为x轴，对应的RLU值为y轴，选用四参数方程回归模型，拟合抗体的剂量效应曲线。
如图7和表8所示，hIgG1或mIgG2a嵌合抗体27B9-1G3、563E10E12、569D11B5、589D7C7、559C12G12和559E1B10均能阻断CCL1诱导的β-Arrestin募集，IC50为260.2-1757.0ng/mL，433H、10A11_hIgG1和10A11_mIgG2a的IC50为1291.5、3268.0和3250.0ng/mL。
表8抗CCR8嵌合抗体阻断CCL1诱导的β-Arrestin募集
N/A：无阻断活性或阻断活性较弱，没有得到有效的IC50值
实施例4、抗CCR8抗体人源化
采用Kabat编号确定CDR，选择与鼠源序列同源性最高的人源种系基因作为受体框架，将鼠源序列的CDR移植至人源框架中。根据氨基酸重要性，进行回复突变，即将移植后框架区部分关键氨基酸回复突变成相对应的鼠源氨基酸，重、轻链分别设计数个变体。
4.1抗CCR8抗体27B9-1G3人源化
鼠源可变区序列和选择的人种系基因氨基酸序列如下表9所示；由此设计的人源化重轻链序列如表10所示。
将人源化重链和轻链组合，得到以下人源化抗体：

4.2抗CCR8抗体559E1B10人源化
鼠源可变区序列和选择的人种系基因氨基酸序列如下表11所示；由此设计的人源化重轻链序列如表12所示。
将人源化重链和轻链组合，得到以下人源化抗体：
4.3抗CCR8抗体563E10E12，569D11B5和589D7C7的人源化
563E10E12，569D11B5和589D7C7三个抗体克隆的序列相似，被认为是一族序列，人源化时考虑将这三个抗体综合在一起进行人源化。鼠源可变区序列和选择的人种系基因氨基酸序列如下表13所示；由此设计的人源化重轻链序列如表14所示。
将人源化重链和轻链组合，得到以下人源化抗体：






实施例5、抗CCR8人源化抗体功能鉴定
将27B9-1G3，559E1B10和563E10E12人源化抗体的轻、重链可变区分别构建到人源恒定区(hIgG1/K，序列见表5)上，进行基因合成，测序确定与设计序列一致。采用实施例3中的方法在Expi293细胞中进行表达，并使用Protein A柱进行纯化。分别进行与人和食蟹猴CCR8的结合实验、阻断CCL1的结合实验和阻断CCL1诱导的β-Arrestin募集实验鉴定其功能。
5.1抗CCR8人源化抗体饱和结合实验
采用实施例2-2.1中的方法检测抗CCR8人源化抗体与人CCR8和食蟹猴CCR8的结合活性。结果如图8和9及表15所示，27B9-1G3的人源化抗体与293F-hCCR8的结合活性与其母本嵌合抗体的活性基本相当。大部分559E1B10的人源化抗体与293F-hCCR8的结合活性与其母本嵌合抗体的活性基本相当。大部分563E10E12的人源化抗体与293F-hCCR8的结合活性与其母本嵌合抗体的活性基本相当。检测的人源化抗体559E1B10_hzH1L1、563E10E12_hzH1L0和563E10E12_hzH1L1与293T-cynoCCR8具有较强的结合活性，而433H和10A11_hIgG1与食蟹猴CCR8几乎没有结合活性。
表15抗CCR8人源化抗体与人CCR8(293F-hCCR8)、食蟹猴CCR8(293T-cynoCCR8)结合的EC50值

NT：没有检测；N/A：没有结合活性或结合活性较差，没有得到有效的EC50值。
5.2抗CCR8人源化抗体竞争CCL1结合实验
采用实施例2-2.2中的竞争结合实验方法检测人源化抗CCR8抗体竞争CCL1结合实验，结果如图10和表16所示，部分27B9-1G3的人源化抗体，如27B9-1G3_hzH3L1和27B9-1G3_hzH3L2的阻断活性与其母本嵌合抗体的阻断活性基本相当。大部分559E1B10_的人源化抗体，如559E1B10_hzH2L1、559E1B10_hzH3L1、559E1B10_hzH1L2、559E1B10_hzH3L2、559E1B10_hzH0L1和559E1B10_hzH0L2的阻断活性与其母本嵌合抗体的阻断活性相当。所有563E10E12的人源化抗体的 阻断活性与其母本嵌合抗体的阻断活性相当。559E1B10的人源化抗体559E1B10_hzH3L1、559E1B10_hzH1L2、559E1B10_hzH0L1和559E1B10_hzH0L2以及所有563E10E12的人源化抗体的阻断活性优于433H或与433H的阻断活性类似。
表16抗CCR8人源化抗体在人CCR8(293F-hCCR8)上阻断CCL1结合的IC50值

N/A：结合活性较差，没有得到有效的EC50值。
5.3抗CCR8人源化抗体阻断CCL1诱导的β-Arrestin募集实验
采用实施例3-3.4中的方法检测抗CCR8人源化抗体阻断CCL1诱导的β-Arrestin募集实验。结果如图11和表17所示，部分27B9-1G3的人源化抗体，如27B9-1G3_hzH3L1和27B9-1G3_hzH3L2的阻断活性与其母本嵌合抗体的阻断活性基本相当。大部分559E1B10的人源化抗体，如559E1B10_hzH2L1、559E1B10_hzH3L1、559E1B10_hzH1L2、559E1B10_hzH3L2、559E1B10_hzH0L1和559E1B10_hzH0L2的阻断活性与其母本嵌合抗体的阻断活性相当。所有563E10E12的人源化抗体的阻断活性与其母本嵌合抗体的阻断活性相当。559E1B10的人源化抗体559E1B10_hzH3L1、559E1B10_hzH1L2、559E1B10_hzH0L1和559E1B10_hzH0L2以及所有563E10E12的人源化抗体的阻断活性优于433H或与433H的阻断活性类似。
表17抗CCR8人源化抗体阻断CCL1诱导的β-Arrestin募集的IC50值

N/A:不适用，没有阻断活性。
5.4用Jurkat-human FcγRⅢa(158V)-NFAT测定人源化抗CCR8抗体依赖的FcγRⅢa激活
在小鼠模型的研究中发现抗CCR8抗体主要通过ADCC作用清除肿瘤组织中的Treg抑制肿瘤的增长(Helena Van Damme等.Therapeutic depletion of CCR8+tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1therapy.J Immunother Cancer.2021Feb；9(2):e001749.doi:10.1136/jitc-2020-001749.)。采用Jurkat-human FcγRⅢa(158V)-NFAT与表达CCR8的细胞孵育测定抗CCR8抗体依赖的FcγRⅢa激活。CCR8抗体的Fab端与靶细胞上的靶点结合，其Fc端与效应细胞上的FcγRIIIa受体结合，进而激活效应细胞内NFAT信号通路，对NFAT通路活化产生的萤光素酶定量来反映抗体的ADCC活性，具体方法如下:
含0.25％EDTA的胰蛋白酶(Gibco，25200072)消化293F-human CCR8(上海睿智化学研究有限公司，293F-hCCR8)细胞，300g离心5分钟，用DMEM培养基调整细胞浓度至3×105细胞/mL。每孔100μL接种于白色不透明板 中，置于37℃、5％CO2培养箱中培养过夜，使细胞贴壁，作为靶细胞待用。1640培养基稀释抗CCR8抗体、阳性对照抗体、hIgG1isotype(百英生物科技有限公司，B117901)至2倍起始工作质量浓度，再用培养基进行5倍稀释。收集的Jurkat-human FcγRⅢa(158V)-NFAT(吉满生物科技(上海)有限公司，GM-C05619)细胞300g离心5分钟后弃去上清，加入1640培养基调整细胞浓度至3×106细胞/mL。取出293F-hCCR8细胞培养板，轻轻将上清吸尽，每孔加入50μL Jurkat-human FcγRⅢa(158V)-NFAT细胞和50μL梯度稀释的抗CCR8抗体。37℃、5％CO2条件下诱导6小时后，室温条件下平衡至少15分钟。每孔加入100μL荧光素酶底物溶液(Vazyme，DD1203)，混合后在室温条件下避光反应5分钟，用酶标仪在细胞培养板上读取相对光单位(relative light unit，RLU)数值。利用Graphpad Prism 8.0软件分析试验数据，以抗CCR8抗体浓度对数为x轴，对应的RLU值为y轴，选用四参数方程回归模型，拟合抗CCR8抗体的剂量效应曲线。
如图12和13，表18和19所示，559E1B10和563E10E12的嵌合抗体及人源化抗体可以以剂量依赖的方式激活Jurkat-human FcγRⅢa(158V)-NFAT。人源化抗体559E1B10_hzH0L1、559E1B10_hzH1L1、559E1B10_hzH2L1、559E1B10_hzH3L1、559E1B10_hzH0L2、559E1B10_hzH1L2、559E1B10_hzH2L2和559E1B10_hzH3L2的活性与其母本嵌合抗体559E1B10_hIgG1的活性相当。所有检测的563E10E12的人源化克隆如563E10E12_hzH0L0、563E10E12_hzH1L0、563E10E12_hzH0L1等与其母本嵌合抗体的活性相当。
表18 559E1B10人源化抗体对Jurkat-human FcγRⅢa(158V)-NFAT激活的EC50值

表19 563E10E12人源化抗体对Jurkat-human FcγRⅢa(158V)-NFAT激活的EC50
实施例6、抗CCR8抗体与具有内源性CCR8表达的HuT78细胞的结合活性
人T细胞淋巴瘤细胞系HuT78和活化的人Treg均表达CCR8(James M Fox等.和Yiftah Barsheshet等.CCR8+FOXp3+T reg cells as master drivers of immune regulation.Proc Natl Acad Sci U S A.2017Jun 6；114(23):6086-6091.doi:10.1073/pnas.1621280114.)，采用FACS检测抗CCR8抗体对内源性表达的CCR8的结合活性。将HuT78(Cobioer，CBP60267)细胞调整细胞浓度至1×106个细胞/mL，每孔100μL置于96孔U底孔板，离心去掉上清。用含2％FBS的PBS溶液(FACS buffer)将抗CCR8抗体，对照抗体433H、10A11_hIgG1、B16_hIgG1:7-B16.001(专利US20210277129A1中序列，轻链可变区序列编号：81，重链可变区序列编号为：80)和hIgG1isotype(百英生物科技有限公司，B117901)进行梯度稀释，起始浓度10μg/mL，4倍梯度稀释。将梯度稀释的抗体100μL/孔重悬细胞，吹打混匀，于4℃孵育1小时，孵育后用FACS buffer清洗三次。用FACS buffer 1：1000稀释的二抗AF647-Goat anti-Human IgG(H+L)(Invitrogen，A21445)或AF647-Donkey anti-Mouse IgG(H+L)(Invitrogen，货号:A31571；检测鼠源抗体433H)100μL/孔重悬细胞，吹打混匀，于4℃孵育约45分钟。孵育后细胞经离心，用FACS buffer清洗三次。100μL/孔FACS buffer 重悬细胞，用流式细胞仪读取平均荧光强度(Median Fluorescence Intensity，MFI)，利用Graphpad Prism 8.0软件分析试验数据，以抗CCR8抗体浓度对数为x轴，对应的MFI值为y轴，选用四参数方程回归模型，拟合抗CCR8抗体的剂量效应曲线，计算出EC50。
如图14和表20所示，人源化抗体563E10E12_hzH1L0和563E10E12_hzH1L1以剂量依赖的方式与具有内源性CCR8表达的HuT78细胞结合，其结合活性的EC50与433H、B16_hIgG1和10A11_hIgG1相当，并且最大结合MFI高于B16_hIgG1和10A11_hIgG1，但是559E1B10_hzH1L1与HuT78的基本没有结合活性。
表20抗CCR8抗体与HuT78的结合活性的EC50
N/A:没有活性或没有得到有效的EC50值。
实施例7、抗CCR8抗体特异性地结合人CCR8
7.1抗CCR8抗体特异性地结合表达人CCR8的CHO-K1细胞
在CCR8抗体的小鼠免疫和筛选过程中均使用到CHO-K1细胞，采用过表达人CCR8的CHO-K1细胞系(睿智化学构建，CHOK1-hCCR8)和野生型CHO-K1细胞系(吉满生物，GM-15570)和实施例2-2.1中的实验方法检测抗CCR8抗体对细胞表面CCR8的结合。
如图15和表21所示，559E1B10_hzH1L1、563E10E12_hzH1L0和563E10E12_hzH1L1均以剂量依赖的方式与CHOK1-hCCR8结合，而与CHOK1空白细胞不结合。
表21抗CCR8抗体与细胞表面的hCCR8特异性结合的EC50

N/A:没有活性或没有得到有效的EC50值。
7.2抗CCR8抗体介导CCR8依赖的对Jurkat-human FcγRⅢa(158V)-NFAT激活
采用过表达人CCR8的CHO-K1细胞系(睿智化学构建，CHOK1-hCCR8)和CHO-K1空白细胞系(吉满生物，GM-15570)确定抗CCR8抗体对Jurkat-hu man FcγRⅢa(158V)-NFAT的激活依赖于CCR8。分别将CHOK1-hCCR8细胞和CHOK1空白细胞用1640完全培养基调整细胞浓度至3×105个细胞/mL，采用-实施例5-5.4中的方法检测抗CCR8抗体对Jurkat-human FcγRⅢa(158V)-NF AT的激活。4A19为专利WO2021194942A1中序列，轻链可变区序列编号：18，重链可变区序列编号：6。如图16和表22所示，在CHOK1-hCCR8细胞存在时，抗CCR8抗体剂量依赖性地激活Jurkat-human FcγRⅢa(158V)-NFAT；而当CHO-K1空白细胞存在时，抗CCR8抗体不能激活Jurkat-human FcγRⅢa(158V)-NFAT，表明抗CCR8抗体可以特异地识别CCR8并介导CCR8依赖的ADCC效应。
表22抗CCR8抗体CCR8依赖的对Jurkat-human FcγRⅢa(158V)-NFAT激活的EC50
N/A:没有活性或没有得到有效的EC50值。
7.3抗CCR8抗体不与人CCR4结合
人类中与CCR8同源性最高的基因是CCR4(Antonio Recchiuti等.Pro-Resolving Lipid Mediators(SPMs)and Their Actions in Regulating miRNA in Novel Resolution Circuits in Inflammation.Front Immunol.2012Oct 22；3:298.doi:10.3389/fimmu.2012.00298.eCollection 2012.)，但是CCR4与CCR8也只有46％ 的同源性(NCBI blast的结果)。采用如下方法检测抗CCR8抗体与人CCR4结合的结合活性。按照ExpiFectamineTM293转染试剂盒(Gibco，A14524)的步骤，293F细胞瞬转hCCR4-GFP(Sino，HG13064-ACG)。将转染后的细胞调整细胞浓度至1×106个细胞/·mL，每孔100μL置于96孔U底孔板，离心获取细胞。用含2％FBS的PBS溶液(FACS buffer)将抗CCR8抗体稀释至起始工作质量浓度，起始浓度为30μg/mL，再用FACS buffer溶液进行4倍稀释。每孔100μL重悬细胞，吹打混匀，于4℃孵育1小时后用FACS buffer清洗三次。将FACS buffer 1:1000稀释的二抗AF647-Goat anti-Human IgG(H+L)(Invitrogen，A21445)每孔100μL稀释液重悬细胞，吹打混匀，于4℃孵育40分钟。孵育后细胞经离心，用FACS buffer清洗三次。根据LIVE/DEADTMFixable Near IR (780)Viability Kit(Invitrogen，L34992)说明书标记死细胞后，每孔100μL FACS buffer重悬细胞，用流式细胞仪分别读取平均的荧光强度(Median Fluorescence Intensity，MFI)。转染了hCCR4-GFP的细胞用抗CCR4抗体(Biolegend，359412)确定CCR4的表达。分别分析转染了hCCR4-GFP中GFP阳性(表达CCR4的细胞)的细胞和转染空白质粒中的活细胞与抗CCR8抗体的结合。如图17所示，563E10E12的嵌合抗体和人源化抗体不与转染了hCCR4的细胞(GFP阳性细胞)结合也不与293F细胞结合，而559E1B10的嵌合抗体和人源化抗体在高浓度下与hCCR4有较弱的结合。
7.4抗CCR8抗体与PBMC中免疫细胞的结合
CCR8除了在Treg上有少量表达外，在健康人类外周血中其他免疫细胞中没有表达(George Plitas等.Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer.Immunity.2016 Nov 15；45(5):1122-1134.doi:10.1016/j.immuni.2016.10.032.)。采用健康供者PBMC检测抗CCR8抗体是否与外周免疫细胞有非特异性结合。复苏冻存的健康供者的PBMC(Allcells,货号Z0060和Y1446)调整细胞浓度至2×106个细胞/mL，每孔100μL置于96孔U底孔板，离心去上清。根据LIVE/DEADTMFixable Near IR(780)Viability Kit(Invitrogen，L34992)说明书标记死细胞。每孔加入50μL FACS buffer 1：25稀释的FcX试剂(Biolegend，422302)阻断抗体对Fc受体的非特异性染色，室温孵育15分钟。用FACS buffer配制PE anti-human CD3 Antibody(Biolegend-317308，1:50稀释)、FITC-anti-human CD8(Biolegend-301006，1:50稀释)、Pacific BlueTManti-human CD19(Biolegend-363036，1:50稀释)和Brilliant Violet605TManti-human CD14(Biolegend-367126，1:50稀释)的混合液，每孔40μL加入细胞中，混匀。用FACS buffer稀释AF647标记试剂盒(Invitrogen，A20186)标记的抗CCR8抗体及hIgG1isotype(百英生物科技有限公司，B117901)至起始浓度100μg/mL，再用FACS buffer溶液进行5倍稀释，每孔 10μL加入上述细胞中，于4℃孵育1小时。孵育后用FACS buffer清洗三次，每孔100μL FACS buffer重悬细胞。用流式细胞仪读取平均荧光强度(Median Fluorescence Intensity，MFI)。
如表23所示，抗CCR8抗体559E1B10_hzH1L1_hIgG1、563E10E12_hzH1L0_hIgG1和563E10E12_hzH1L1_hIgG1对CD8+,CD4+(CD3+CD8-)T细胞、CD14+单核细胞、CD19+B细胞和其他CD3-CD14-CD19-的免疫细胞没有明显的结合。
表23抗CCR8抗体与PBMC中免疫细胞的结合强度(平均荧光强度，MFI)

实施例8、Fc突变及降低岩藻糖基化增强抗CCR8抗体介导的ADCC效应
8.1 S239D/I332E、S239D/A330L/I332E、L235V/F243L/R292P/Y300L/P396L突变和降低岩藻糖基化增强抗CCR8抗体与293F-humanCCR8对Jurkat-human FcγRⅢa(158V)-NFAT的激活
抗体恒定区(Fc)S239D/I332E、S239D/A330L/I332E或L235V/F243L/R292P/Y300L/P396L突变可以提高与FcγRⅢa的亲和力，增加抗体介导的ADCC效应(Greg A Lazar等.Engineered antibody Fc variants with enhanced effector function.Proc Natl Acad Sci U S A.2006 Mar 14；103(11):4005-10.doi:10.1073/pnas.0508123103.和Jeffrey L Nordstrom等.Anti-tumor activity and toxicokinetics analysis of MGAH22,an anti-HER2 monoclonal antibody with enhanced Fcγreceptor binding properties.Breast Cancer Res.2011；13(6):R123.doi:10.1186/bcr3069.)。这几组突变已经在临床上用于增强抗体的ADCC效应(Rena Liu等.Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment.Antibodies(Basel).2020Nov 17；9(4):64.doi:10.3390/antib9040064.)。
分别对抗CCR8的人源化抗体的Fc进行S239D/I332E(DE)、S239D/A330L/I332E(DLE)或L235V/F243L/R292P/Y300L/P396L(VLPLL)的突变来增强其诱导的ADCC效应。去岩藻糖基化或降低岩藻糖基化可以增加抗体的ADCC效应，已有多款采用去岩藻糖基化或降低岩藻糖基化的抗体获批上市或处于临床研究的后期(Natasha A Pereira et al.The"less-is-more"in therapeutic antibodies:Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity.MAbs.2018Jul；10(5):693-711.doi: 10.1080/19420862.2018.1466767.)。为了抑制岩藻糖基化，在瞬转前用含有100μM的2F-过乙酰基-岩藻糖(MILLIPORE，货号：344827-10MGCN)的生长培养基ExpiCHO Expression Medium(Gibco，货号：A2910001)对ExpiCHO细胞进行传代扩增培养。瞬转当天将细胞稀释至6×106细胞/mL，根据1μg质粒/mL细胞悬液的浓度采用Expi CHO转染试剂盒(Gibco，货号：A29129)进行瞬转。瞬转后18-22小时对瞬转细胞进行补料(Gibco，货号：A29129)，继续于37℃，8％CO2，100rpm的摇床内培养。瞬转7天后收获细胞培养液，离心后0.45μm过滤得到上清液，使用ProteinA填料进行纯化，收获抗体。。
采用实施例5-5.4中的方法检测抗CCR8抗体与293F-hCCR8孵育对Jurkat-human FcγRⅢa(158V)-NFAT的激活。如图18和表24所示，与559E1B10_hzH1L1_hIgG1相比，Fc突变的559E1B10_hzH1L1_DE和559E1B10_hzH1L1_VLPLL增强对Jurkat-human FcγRⅢa(158V)-NFAT的激活，EC50降低了9-21倍。与563E10E12_hzH1L0_hIgG1相比，Fc突变的563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和563E10E12_hzH1L0_VLPLL和降低岩藻糖基化的563E10E12_hzH1L0_AF增强对Jurkat-human FcγRⅢa(158V)-NFAT的激活，EC50降低了7-18倍。与563E10E12_hzH1L1_hIgG1相比，Fc突变的563E10E12_hzH1L1_DE和563E10E12_hzH1L1_VLPLL增强对Jurkat-human FcγRⅢa(158V)-NFAT的激活，EC50降低了6-14倍。Fc突变的559E1B10和563E10E12人源化抗体或降低岩藻糖基化的563E10E12_hzH1L0的活性与降低岩藻糖基化的B16(B16_AF)的活性相当。
表24 Fc突变或降低岩藻糖基化的抗CCR8抗体与293F-humanCCR8孵育对Jurkat-human FcγRⅢa(158V)-NFAT的激活(EC50,μg/mL)

N/A:没有活性或没有得到有效的EC50值。
8.2 Fc突变和降低岩藻糖基化增强抗CCR8抗体与HuT78对Jurkat-human FcγRⅢa(158V)-NFAT的激活
将具体不同Fc突变的或降低岩藻糖基化的抗CCR8抗体与Hut78和Jurkat-human FcγRⅢa(158V)-NFAT细胞一起孵育，检测S239D/I332E、S239D/A330L/I332E、L235V/F243L/R292P/Y300L/P396L突变或降低岩藻糖基化对抗CCR8抗体与HuT78对Jurkat-human FcγRⅢa(158V)-NFAT的激活的影响。HuT78细胞用DMEM培养基调整细胞浓度至3×105个细胞/mL作为靶细胞，采用实施例5-5.4中的方法检测抗CCR8抗体对Jurkat-human FcγRⅢa(158V)-NFAT的激活。
如图19和表25所示，与HuT78孵育Fc突变的抗体563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE、563E10E12_hzH1L0_VLPLL和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF均能增加563E10E12_hzH1L0对Jurkat-human FcγRⅢa(158V)-NFAT的激活，EC50降低了2-6倍，最大信号增加了4-5倍。Fc突变或降低岩藻糖基化的563E10E12_hzH1L0对Jurkat-human FcγRⅢa(158V)-NFAT的激活与降低岩藻糖基化的B16相当。
表25 Fc突变或降低岩藻糖基化的563E10E12_hzH1L0与Hut78孵育对Jurkat-human FcγRⅢa(158V)-NFAT激活的EC50
N/A:没有活性或没有得到有效的EC50值。
实施例9、健康人PBMC和抗CCR8抗体诱导的ADCC效应
以PBMC作为效应细胞，CHOK1-hCCR8作为靶细胞来检测抗CCR8抗体的ADCC效应。复苏冻存健康供者的PBMC(Allcells)，用含200IU/mL IL-2(Peprotech，货号：200-02)的1640完全培养基调整细胞浓度至2×106个细胞/mL，置于37℃、5％CO2培养箱中培养过夜。第二天吸取未贴壁的PBMC，300g离心5分钟后弃去上清，加入1640培养基调整细胞浓度至约3×106个细胞/mL。1640培养基稀释抗CCR8抗体和hIgG1 isotype(百英生物科技有限公司，货号：B117901)至2倍的工作质量浓度，起始浓度为0.8μg/mL，5倍梯度稀释。将收集的CHOK1-hCCR8细胞300g离心5分钟后弃去上清，用DPBS调整细胞浓度至2×106个细胞/mL，按说明书加入CellTraceTMViolet(Invitrogen，货号：C34557)标记CHOK1-hCCR8，并将CellTraceTMViolet标记的CHO-hCCR8用1640培养基调整细胞浓度至约1.5×105个细胞/mL。U底96孔板每孔分别加入50μL CellTraceTMViolet标记的CHOK1-hCCR8、50μL PBMC和100μL稀释的抗CCR8抗体，混匀后置37℃、5％CO2的培养箱中孵育约5小时。每孔加入2μL PI(Invitrogen，货号：006990-50)染料，混合后在室温条件下避光反应5分钟标记死细胞。用流式细胞仪读取PI阳性细胞占CellTraceTMViolet阳性的百分数，采用lysis％＝CellTraceTMViolet标记的PI阳性细胞的比例，计算抗体诱导的ADCC效应。利用Graphpad Prism 8.0软件分析试验数据，以抗CCR8抗体浓度对数为x轴，对应的lysis％值为y轴，选用四参数方程回归模型，拟合抗CCR8抗体的剂量效应曲线。
如图20和表26所示，与563E10E12_hzH1L0_hIgG1相比，降低岩藻糖基化的人源化抗体563E10E12_hzH1L0_AF和Fc突变的抗体563E10E12_hzH1L0_DE和563E10E12_hzH1L0_DLE均增加了其和PBMC诱导的ADCC效应。人源化抗体563E10E12_hzH1L0_hIgG与B16_hIgG1诱导的ADCC活性相当。降低岩藻糖基化和Fc突变的人源化抗体563E10E12_hzH1L0与PBMC诱导的ADCC效应与降低岩藻糖基化的B16诱导的活性类似。
表26人PBMC和抗CCR8抗体诱导的ADCC效应的EC50
N/A:不适用
实施例10、抗CCR8抗体特异性清除外周PBMC中的CCR8阳性的Treg
使用健康人PBMC检测抗CCR8抗体特异性的清除PBMC中表达CCR8的Treg细胞。由于健康人PBMC中Treg表达CCR8的细胞比例较低，本实验中采用IL-2促进Treg的增殖和活化。复苏健康供者的PBMC(Allcells)，用含有200IU/mL IL-2(Peprotech，货号：200-02)的1640完全培养基调整细胞浓度至2×106个细胞/mL，每孔100μL加入96孔U底孔板，在37℃，5％CO2孵育48小时。1640完全培养基稀释抗CCR8抗体和hIgG1isotype(百英生物科技有限公司，货号：B117901)，起始浓度20μg/mL，4倍梯度稀释。将稀释的抗体每孔100μL加入上述96孔板中，吹打混匀，在37℃，5％CO2孵育96小时。离心获取细胞，根据LIVE/DEADTMFixable Near IR(780)Viability Kit(Invitrogen，货号：L34992)说明书标记死细胞。每孔加入50μL经1：25稀释的FcX试剂(Biolegend，货号：422302)，室温孵育15分钟。每孔加入50μL经1:50稀释的AF700Anti-Human CD3(Biolegend，货号：317340)和Percp cy5.5Anti-Human CD4(Biolegend，货号：300530)混合染色液，于4℃孵育1小时，用FACS buffer清洗三次。按照Foxp3/Transcription Factor Staining Kit(Invitrogen，货号：00-5523-00)说明书，固定细胞和标记PE anti human Foxp3(Biolegend，货号：320208)，用FACS buffer清洗三次。每孔100μL FACS buffer重悬细胞，用流式细胞仪读取相关信息，利用Graphpad Prism 8.0软件分析试验数据。在实验结束时检测Treg(Foxp3+CD4+)细胞上CCR8的表达，Treg中CCR8阳性细胞的比例为31.5％。
如图21所示，降低岩藻糖基化的563E10E12_hzH1L0_AF和Fc突变的563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE、563E10E12_hzH1L0_VLPLL和降低岩藻糖基化的B16_AF剂量依赖性地部分清除PBMC中的Treg，但是对不表达CCR8的CD8+T(CD3+CD4-)细胞没有影响。563E10E12_hzH1L0_hIgG1和B16_hIgG1在较高浓度下仅少量的清除Treg。这表明人外周PBMC经IL-2活化后，Treg具有一定水平的CCR8的表达，可以被抗CCR8抗体清除，而不表达CCR8的CD8T细胞不会被清除。
实施例11、抗CCR8抗体在小鼠荷瘤模型中抑制实验
11.1抗CCR8抗体在CCR8人源化小鼠模型中抑制MC38结肠癌的生长
利用人CCR8人源化小鼠(C57BL/6-Ccr8tm1(CCR8)/Bcgen,B-hCCR8，百奥赛图(北京)医药科技股份有限公司，货号：110096)小鼠建立MC38(百奥赛图(北京)医药科技股份有限公司)结肠癌动物模型并检测抗CCR8抗体的体内药效。将PBS重悬的MC38细胞(5×106个细胞/mL)100μL/只接种于B-hCCR8小 鼠的右侧皮下。当平均肿瘤体积达到约100mm3时，按每组6-8只小鼠分组并分别皮下注射10mg/kg的抗CCR8抗体或阴性对照(hIgG1)(百英生物科技有限公司，货号：B117901)或等体积的溶媒(PBS)对照，每周给药2次。给药后小鼠正常饲养，观察小鼠的生存状况，记录小鼠体重和肿瘤体积。
与溶媒对照和阴性对照(hIgG1)相比，抗CCR8抗体559E1B10_hzH1L1_mIgG2a、563E10E12_hzH1L0_mIgG2a、563E10E12_hzH1L1_mIgG2a和10A11_mIgG2a均抑制了MC38结肠癌细胞生长(图22)。在给药后的第20天，抗CCR8抗体559E1B10_hzH1L1_mIgG2a、563E10E12_hzH1L0_mIgG2a、563E10E12_hzH1L1_mIgG2a和10A11_mIgG2a的肿瘤抑制率TGI(％)分别是40.1％、54.0％、22.5％和53％。在实验过程中，所有动物在给药期间活动和进食状态良好，体重均有一定程度的上升，表明动物对抗CCR8抗体耐受良好(图23)。
11.2 Fc突变和降低岩藻糖基化的抗CCR8抗体在CCR8人源化小鼠模型中抑制MC38结肠癌的生长
采用实施例11-11.1中方法在CCR8人源化小鼠B-hCCR8中建立MC38结肠癌模型，当肿瘤平均值为约100mm3，按每组5只小鼠进行分组并分别皮下注射10mg/kg的抗CCR8抗体或等体积的溶媒(PBS)对照，每周给药2次。给药后小鼠正常饲养，观察小鼠的生存状况，记录小鼠体重和肿瘤体积。
如图24所示，与溶媒相比，抗CCR8抗体563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF均抑制了MC38结肠癌细胞生长。在给药后的第25天，563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF的肿瘤抑制率TGI(％)分别是60.1％、57.8％、60.2％和52.9％。所有给药组的小鼠均无行为异常和体重下降，表明荷瘤鼠对该剂量下的抗CCR8抗体具有良好的耐受性(图25)
11.3在人CD34+HSC人源化小鼠和人PBMC人源化小鼠中，抗CCR8抗体抑制人非小细胞肺癌细胞生长
11.3.1抗CCR8抗体在人CD34+HSC人源化小鼠中抑制非小细胞肺癌细胞系A549的生长
用人的造血细胞hCD34+HSC移植到经过辐照的NCG鼠体内做免疫重建得到人CD34+HSC人源化小鼠(集萃药康)。人非小细胞肺癌A549细胞接种于人CD34+HSC人源化小鼠的右侧皮下，接种体积0.2mL/只小鼠，含30％Matrigel。当平均肿瘤体积约为约100mm3时，将小鼠随机分组，分别给予抗CCR8抗体(1-20mg/kg)、帕博利珠单抗(1-20mg/kg)、CCR8抗体(1-20mg/kg)与帕博利珠单抗(1-20mg/kg)的联合用药、以及阴性对照(PBS)， 每周给药2次，给药4周。给药后小鼠正常饲养，记录肿瘤体积。。实验终点收获肿瘤样本，用解剖实验室剪刀将其切碎，并根据制造商的说明，使用人类肿瘤解离试剂盒(Miltenyi Biotech)结合gentleMACS解离器(Miltenyi Biotec)酶解肿瘤组织，细胞悬液通过70μm MACS智能过滤器过滤得到单细胞悬液。用流式细胞仪对免疫细胞亚群(Treg和CD8+T细胞)进行分析。
563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_VLPLL、563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF相对于阴性对照组有效抑制肿瘤生长。
11.3.2 CCR8抗体在人PBMC人源化小鼠模型中抑制人非小细胞肺癌细胞系NCI-H1299的生长
人非小细胞肺癌NCI-H1299细胞接种于人PBMC人源化小鼠(集萃药康)右侧皮下，接种体积0.2mL/只小鼠，含30％Matrigel。当平均肿瘤体积约为约100mm3时，将小鼠随机分组，分别给予的抗CCR8抗体(1-20mg/kg)、帕博利珠单抗(1-20mg/kg)、CCR8抗体(1-20mg/kg)与帕博利珠单抗(1-20mg/kg)的联合用药，和阴性对照(PBS)，每周给药2次，给药4周。给药后小鼠正常饲养，记录肿瘤体积。实验终点收获肿瘤样本，用解剖实验室剪刀将其切碎，并根据制造商的说明，使用人类肿瘤解离试剂盒(Miltenyi Biotech)结合gentleMACS解离器(Miltenyi Biotec)酶解肿瘤组织，细胞悬液通过70μm MACS智能过滤器过滤得到单细胞悬液。用流式细胞仪对免疫细胞亚群(Treg和CD8+T细胞)进行分析。
563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_VLPLL、563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF相对于阴性对照组有效抑制肿瘤生长。
11.3.3 CCR8抗体在人PBMC人源化小鼠模型中抑制人非小细胞肺癌NCI-H292的生长
人非小细胞肺癌NCI-H292细胞接种于人PBMC人源化小鼠(集萃药康)右侧皮下，接种体积0.2mL/只小鼠，含30％Matrigel。当平均肿瘤体积约为约100mm3时，将小鼠随机分组，分别给予抗CCR8抗体(1-20mg/kg)、帕博利珠单抗(1-20mg/kg)、CCR8抗体(1-20mg/kg)与帕博利珠单抗(1-20mg/kg)的联合用药和阴性对照(PBS)，每周给药2次，给药3周。给药后小鼠正常饲养，记录肿瘤体积。实验终点收获肿瘤样本，用解剖实验室剪刀将其切碎，并根据制造商的说明，使用人类肿瘤解离试剂盒(Miltenyi Biotech)结合gentleMACS解离器(Miltenyi Biotec)酶解肿瘤组织，细胞悬液通过70μm MACS智能过滤器过滤得到单细胞悬液。用流式细胞仪检测对免疫细胞亚群和功能生物标志物进行分析。用流式细胞仪检测对免疫细胞亚群(Treg和CD8+T细胞)进行分析。
563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF相对于阴性对照组有效抑制肿瘤生长。
11.4 CCR8抗体与帕博利珠单抗联合用药在人PBMC人源化小鼠模型中抑制人结肠癌HT29的生长
人直肠癌HT29细胞接种于人PBMC人源化小鼠(南模生物)右侧皮下，接种体积0.1mL/只小鼠，含30％Matrigel。当平均肿瘤体积约为约100mm3时，将小鼠随机分组，分别给予抗CCR8抗体(10mg/kg)、帕博利珠单抗(3.25mg/kg)、CCR8抗体(10mg/kg)与帕博利珠单抗(3.25mg/kg)的联合用药和阴性对照(PBS)，每周给药2次，给药4周。给药后小鼠正常饲养，记录肿瘤体积。实验终点收获肿瘤样本，用解剖实验室剪刀将其切碎，并根据制造商的说明，使用人类肿瘤解离试剂盒(Miltenyi Biotech)结合gentleMACS解离器(Miltenyi Biotec)酶解肿瘤组织，细胞悬液通过70μm MACS智能过滤器过滤得到单细胞悬液。用流式细胞仪对免疫细胞亚群(Treg和CD8+T cells)进行分析。
563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_VLPLL、563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF相对于阴性对照组有效抑制肿瘤生长。
11.5抗CCR8抗体在人CCR8人源化小鼠乳腺癌EMT-6肿瘤模型中的药效
鼠的乳腺癌EMT-6细胞接种于人CCR8人源化小鼠(南模生物)右侧皮下，接种体积0.1mL/只小鼠(在PBS)。当平均肿瘤体积约为约50-100mm3时，将小鼠随机分组，分别给予抗CCR8抗体(1-20mg/kg)和阴性对照(PBS)，每周给药2次，给药3周。给药后小鼠正常饲养，记录肿瘤体积。实验终点收获肿瘤样本，用解剖实验室剪刀将其切碎，并根据制造商的说明，使用人类肿瘤解离试剂盒(Miltenyi Biotech)结合gentleMACS解离器(Miltenyi Biotec)酶解肿瘤组织，细胞悬液通过70μm MACS智能过滤器过滤得到单细胞悬液。用流式细胞仪对免疫细胞亚群(Treg和CD8+T细胞)进行分析。
563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_VLPLL、563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF相对于阴性对照组有效抑制肿瘤生长。
继续将鼠的乳腺癌EMT-6细胞接种于人CCR8人源化小鼠(南模生物)右侧皮下，接种体积0.1mL/只小鼠(在PBS)。当平均肿瘤体积约为约50-100mm3时，将小鼠随机分组，分别给予抗CCR8抗体(1-20mg/kg)，抗鼠PD-1(mPD-1)抗体(Leinco Technologies,P372)和阴性对照(PBS)，每周给药2次。给药后小鼠正常饲养，记录肿瘤体积。实验终点收获肿瘤样本，用解剖实验室剪刀将其切碎，并 根据制造商的说明，使用人类肿瘤解离试剂盒(Miltenyi Biotech)结合gentleMACS解离器(Miltenyi Biotec)酶解肿瘤组织，细胞悬液通过70μm MACS智能过滤器过滤得到单细胞悬液。用流式细胞仪对免疫细胞亚群(Treg和CD8+T cells)进行分析。
结果如图26所示，与PBS对照组相比，563E10E12_hzH1L0_DLE以5mg/kg和10mg/kg的剂量单独给药均对EMT-6细胞移植的B-hCCR8人源化小鼠显示出明显的抗肿瘤活性，肿瘤体积的增加被明显抑制(p<0.05)(图26)。而563E10E12_hzH1L0_DLE在同样5mg/kg剂量下，与抗mPD-1抗体(5mg/kg)联合给药后的肿瘤抑制率(TGI＝94.2％)优于563E10E12_hzH1L0_DLE单药(TGI＝53.8％)和抗mPD-1抗体单药(TGI＝76.4％)。
试验期间，各组小鼠未发现明显的临床症状。与PBS对照组相似，给药组动物的体重逐渐增加，表示动物对受试物耐受性良好(图27)。实验结束时，与PBS对照组相比，5mg/kg的563E10E12_hzH1L0_DLE组、抗mPD-1抗体组、联合给药组以及10mg/kg的563E10E12_hzH1L0_DLE组肿瘤中的Treg清除率分别为26.3％、24.9％、23.4％和27.3％；及其CD8+T cells/Treg比值增幅分别为32.5％、22.6％、46.8％和11.5％。
11.6抗CCR8抗体在人CCR8人源化小鼠黑色素癌B16F10肿瘤模型中的药效
鼠的黑色素癌B16F10细胞接种于人CCR8人源化小鼠(百奥赛图)右侧皮下、接种体积0.1mL/只小鼠(在PBS)。当平均肿瘤体积约为约50-100mm3时，将小鼠随机分组，分别给予抗CCR8抗体(1-20mg/kg)、anti-mouse PD1单抗(1-20mg/kg)、抗CCR8抗体(1-20mg/kg)与anti-mouse PD1单抗(1-20mg/kg)的联合用药和阴性对照(PBS)，每周给药2次，给药3周。给药后小鼠正常饲养，记录肿瘤体积。实验终点收获肿瘤样本，用解剖实验室剪刀将其切碎，并根据制造商的说明，使用人类肿瘤解离试剂盒(Miltenyi Biotech)结合gentleMACS解离器(Miltenyi Biotec)酶解肿瘤组织，细胞悬液通过70μm MACS智能过滤器过滤得到单细胞悬液。用流式细胞仪对免疫细胞亚群(Treg和CD8+T cells)进行分析。
563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_VLPLL、563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF相对于阴性对照组有效抑制肿瘤生长。
11.7抗CCR8抗体在人CCR8人源化小鼠结直肠癌CT26肿瘤模型中的药效
鼠的结直肠癌CT26细胞接种于人CCR8人源化小鼠(南模生物)右侧皮下，接种体积0.1mL/只小鼠(在PBS)。当平均肿瘤体积约为约50-100mm3时，将小鼠随机分组，分别给予抗CCR8抗体(1-20mg/kg)、anti-mouse PD1单抗(1-20mg/kg)、抗CCR8抗体(1-20mg/kg)与anti-mouse PD1单抗(1-20mg/kg)的联合用 药和阴性对照(PBS)，每周给药2次，给药3周。给药后小鼠正常饲养，记录肿瘤体积。实验终点收获肿瘤样本，用解剖实验室剪刀将其切碎，并根据制造商的说明，使用人类肿瘤解离试剂盒(Miltenyi Biotech)结合gentleMACS解离器(Miltenyi Biotec)酶解肿瘤组织，细胞悬液通过70μm MACS智能过滤器过滤得到单细胞悬液。用流式细胞仪对免疫细胞亚群(Treg和CD8+T cells)进行分析。
563E10E12_hzH1L0_hIgG1、其Fc突变抗体563E10E12_hzH1L0_VLPLL、563E10E12_hzH1L0_DE、563E10E12_hzH1L0_DLE和降低岩藻糖基化的抗体563E10E12_hzH1L0_AF相对于阴性对照组有效抑制肿瘤生长。
本发明所述方法和系统的多种修改和变化对于本领域技术人员将显而易见，而不脱离本发明的范围和精神。虽然已经联系具体的优选实施方案描述本发明，应当理解如要求的本发明不应不当地受限于此类具体的实施方案。实际上，用于实施本发明的所述模式的多种修改，对于分子生物学、免疫学或相关领域的技术人员显而易见，意在属于所附权利要求的范围内。
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Claims (39)

  1. 结合CCR8的抗体或其抗原结合片段，其具有以下一种或多种特性：
    (1)以不高于500ng/mL的EC50与人CCR8和/或食蟹猴CCR8特异性结合；
    (2)阻断人和/或食蟹猴CCR8与配体CCL1的结合；
    (3)阻断CCL1诱导的β-Arrestin募集；
    (4)清除CCR8+Treg细胞；
    (5)抑制肿瘤生长，优选地不影响体重。
  2. 结合CCR8的抗体或其抗原结合片段，其包含
    (i)如SEQ ID NO:21-40任一项所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，和/或如SEQ ID NO:42-54任一项所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
    (ii)如(i)所述的CDR组合，其中相比HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和/或LCDR3，在所述序列上包含至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)，优选地氨基酸取代，优选地保守性取代，并保持与CCR8的亲和力。
  3. 结合CCR8的抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中
    所述重链可变区包含：
    (1)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2或10或11或41和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:1、SEQ ID NO:2或10或11或41和SEQ ID NO:3所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的HCDR1、HCDR2和HCDR3；或
    (2)氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的HCDR1、HCDR2和HCDR3；或
    (3)氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的HCDR1、HCDR2和HCDR3；
    和/或
    所述轻链可变区包含：
    (1)氨基酸序列分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的LCDR1、LCDR2和LCDR3；或
    (2)氨基酸序列分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的LCDR1、LCDR2和LCDR3；或
    (3)氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示氨基酸序列分别相同或具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的LCDR1、LCDR2和LCDR3。
  4. 结合CCR8的抗体或其抗原结合片段，其包含如下表任一组合所示的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3:

  5. 结合CCR8的抗体或其抗原结合片段，其包含重链可变区VH和/或轻链可变区VL，其中，
    (a)重链可变区VH
    (i)包含与选自SEQ ID NO:21-40任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者
    (ii)包含选自SEQ ID NO:21-40任一项所示的氨基酸序列或由其组成；或者
    (iii)包含与选自SEQ ID NO:21-40任一项所示的氨基酸序列相比具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中；
    和/或
    (b)轻链可变区VL
    (i)包含与选自SEQ ID NO:42-54任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
    (ii)包含选自SEQ ID NO:42-54任一项所示的氨基酸序列或由其组成；或者
    (iii)包含与选自SEQ ID NO:42-54任一项所示的氨基酸序列相比具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中。
  6. 结合CCR8的抗体或其抗原结合片段，其包含如下表任一组合所示的重链可变区VH和轻链可变区VL:
  7. 权利要求1-6中任一项的抗体或其抗原结合片段，其是小鼠抗体、嵌合抗 体、人源化抗体或全人抗体。
  8. 权利要求1至7中任一项的抗体或其抗原结合片段，其包含重链恒定区和/或轻链恒定区，优选地，所述轻链恒定区是λ链或κ链恒定区；所述重链恒定区选自鼠mIgG2a、人IgG1、人IgG2、人IgG3或IgG4型；更优选地，所述重链恒定区是人IgG1型或具有S228P突变的人IgG4型。
  9. 权利要求1至7中任一项的抗体或其抗原结合片段，其包含重链和/或轻链，其中
    (a)重链
    (i)包含与选自SEQ ID NO:55-74任一项的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
    (ii)包含选自SEQ ID NO:55-74任一项的氨基酸序列或由其组成；或者
    (iii)包含与选自SEQ ID NO:55-74任一项的氨基酸序列相比具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中，最优选地，所述氨基酸改变发生在重链恒定区中；
    和/或
    (b)轻链
    (i)包含与选自SEQ ID NO:75-82任一项的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
    (ii)包含选自SEQ ID NO:75-82任一项的氨基酸序列或由其组成；或者
    (iii)包含与选自SEQ ID NO:75-82任一项的氨基酸序列相比具有至少一个氨基酸的添加，取代或缺失或其任意组合(例如1个，2个或3个的氨基酸的添加，取代或缺失或其任意组合)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中，最优选地，所述氨基酸改变发生在轻链恒定区中。
  10. 如权利要求9所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链恒定区，且所述重链恒定区的序列与天然人重链恒定区序列相比具有一个或多个氨基酸取代，优选地，所述一个或多个氨基酸取代使得抗体的ADCC效应增强，更优选地，所述一个或多个氨基酸取代发生在根据EU编号系统的重链恒定区序列位点238、239、248、249、252、254、255、256、258、265、267、268、269、270、272、276、278、280，283、285、286、289、290、292、293、294、295、296、298、301、303、305、307、309、312、315、320、322、324、326、327、328、329、330、331、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、434、435、437、438和439中的一个或多个。
  11. 如权利要求10所述的抗体或其抗原结合片段，其中所述一个或多个氨基酸取代发生在根据EU编号系统的重链恒定区序列位点L234、L235、G236、S239、F243、T256、D265、H268、D270、K290、R292、S298、Y300、V305、K326、A330、I332、E333、K334、A339和P396中的一个或多个。
  12. 如权利要求11所述的抗体或其抗原结合片段，其中所述一个或多个氨基酸取代是选自根据EU编号系统的G236A、S239D、F243L、T256A、K290A、R292P、S298A、Y300L、V305I、A330L、I332E、E333A、K334A、A339T和P396L取代的一个或多个。
  13. 如权利要求10所述的抗体或其抗原结合片段，其中所述一个或多个氨基酸取代是选自根据EU编号系统的N297A取代、N297Q取代、L235A取代连同L237A取代、L234A取代连同L235A取代、E233P取代、L234V取代、L235A取代、C236缺失、P238A取代、D265A取代、A327Q取代和P329A取代的一个或多个。
  14. 如权利要求10所述的抗体或其抗原结合片段，其中所述一个或多个氨基酸取代发生在根据EU编号系统的重链恒定区序列位点235、239、243、292、300、330、332、396中的一个或多个。
  15. 如权利要求14所述的抗体或其抗原结合片段，其中一个或多个氨基酸取代是选自根据EU编号系统的S239D、L235V、F243L、R292P、Y300L、A330L、I332E和P396L中的至少一个。
  16. 如权利要求10所述的抗体或其抗原结合片段，其中所述重链恒定区具 有在选自以下根据EU编号系统的位置的组合上同时发生的一组或多组突变：(1)L235/F243/R292/Y300/P396、(2)F243/R292/Y300/V305/P396、(3)D270/K326/A330/K334、(4)S239/A330/I332、(5)S298/E333/K334、(6)L234/L235/G236/S239/H268/D270/S298、(7)M252/S254/T256、(8)L234/L235/D265、(9)G236/S239/I332和(10)S239/I332。
  17. 如权利要求10所述的抗体或其抗原结合片段，其中所述重链恒定区具有选自以下根据EU编号系统的突变组合的一组或多组突变：(1)L235V/F243L/R292P/Y300L/P396L、(2)F243L/R292P/Y300L/V305I/P396L、(3)D270E/K326D/A330M/K334E、(4)S239D/A330L/I332E、(5)S298A/E333A/K334A、(6)L234Y/L235Q/G236W/S239M/H268D/D270E/S298A、(7)M252Y/S254T/T256E、(8)L234A/L235A/D265A、(9)L234F/L235E/D265A、(10)G236A/S239D/I332E和(11)S239D/I332E。
  18. 结合CCR8的抗体或其抗原结合片段，其包含如下表任一组合所示的重链HC和轻链LC:

  19. 如权利要求1-18一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段的恒定区是去岩藻糖基化的或降低岩藻糖基化的。
  20. 权利要求1-19中任一项的结合CCR8的抗体或其抗原结合片段，其中所述抗体是单克隆抗体。
  21. 权利要求1至19中任一项的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fd、Fab’-SH、Fv、单链抗体(例如scFv)或(Fab’)2、单结构域抗体、diabody(dAb)或线性抗体。
  22. 分离的多核苷酸，其编码权利要求1至21中任一项的结合CCR8的抗体或其抗原结合片段的任一条或多条链。
  23. 载体，其包含权利要求22的多核苷酸，优选地所述载体是表达载体。
  24. 宿主细胞，其包含权利要求22的核酸或权利要求23的载体，优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞(例如所述宿主细胞是CHO细胞，例如CHO-K1细胞或expiCHO细胞，或所述宿主细胞是293细胞，例如HEK293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。
  25. 制备结合CCR8的抗体或其抗原结合片段的方法，所述方法包括在适于表达抗体的情况下，培养包含编码权利要求1-21中任一项的抗体或其抗原结合片段的核酸的宿主细胞，任选地所述方法还包括从所述宿主细胞回收所述抗体或其抗原结合片段。
  26. 免疫缀合物，其包含权利要求1-21中任一项的结合CCR8的抗体或其抗原结合片段和其它物质，例如治疗剂或标记。
  27. 药物组合物，其包含权利要求1-21中任一项的结合CCR8的抗体或其抗原结合片段或权利要求20的免疫缀合物，以及任选地一种或多种其它治疗 剂，以及任选地药用辅料。
  28. 药物组合，其包含权利要求1-21中任一项的结合CCR8的抗体或其抗原结合片段或权利要求20的免疫缀合物，以及一种或多种其它治疗剂，优选地，所述其它治疗剂是单克隆抗体，更优选地，所述其它治疗剂是靶向免疫检查点的单克隆抗体，最优选地，所述其它治疗剂是靶向PD-1的单克隆抗体。
  29. 在受试者中增强免疫反应(例如抗肿瘤免疫反应)的方法，其包括向受试者施用有效量的权利要求1-21中任一项的抗体或其抗原结合片段、或权利要求22的多核苷酸、或权利要求23的载体、或权利要求24的宿主细胞、或权利要求26的免疫缀合物、或权利要求27的药物组合物，或权利要求28的组合产品。
  30. 一种在受试者中预防和/或治疗肿瘤、自身免疫性疾病或感染性疾病的方法，所述方法包括向所述受试者施用有效量的权利要求1-21中任一项的抗体或其抗原结合片段、或权利要求22的多核苷酸、或权利要求23的载体、或权利要求24的宿主细胞、或权利要求26的免疫缀合物、或权利要求27的药物组合物，或权利要求28的组合产品，优选地，所述疾病为肿瘤，例如黑色素瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌及其转移性癌症。
  31. 权利要求30所述的方法，其还包括向所述受试者联合施用一种或多种疗法，例如治疗方式和/或其它治疗剂，优选地，治疗方式包括手术治疗和/或放射疗法，。
  32. 有效量的权利要求1-21中任一项的抗CCR8抗体或其抗原结合片段、或权利要求22的多核苷酸、或权利要求23的载体、或权利要求24的宿主细胞、或权利要求26的免疫缀合物、或权利要求27的药物组合物，或权利要求28的药物组合在制备药物中的用途，所述药物用于在受试者中预防和/或治疗肿瘤、自身免疫性疾病或感染性疾病。
  33. 有效量的权利要求1-21中任一项的抗CCR8抗体或其抗原结合片段、或权利要求22的多核苷酸、或权利要求23的载体、或权利要求24的宿主细胞、或权利要求26的免疫缀合物、或权利要求27的药物组合物与其它治疗剂联合在制备药物中的用途，所述药物用于在受试者中预防和/或治疗肿瘤、自身免疫性疾病或感染性疾病，优选地，所述其它治疗剂是单克隆抗体，更优选地，所述其 它治疗剂是靶向免疫检查点的单克隆抗体，最优选地，所述其它治疗剂是靶向PD-1的单克隆抗体。
  34. 权利要求32或33所述的用途，其中所述肿瘤是实体肿瘤。
  35. 权利要求34所述的用途，其中所述肿瘤是黑色素瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、胃癌、食管癌、头颈癌、肺癌、卵巢癌、肾癌、膀胱癌、肝癌或胸腺癌及其转移性癌症。
  36. 权利要求32-35中任一项所述的用途，其还包括向所述受试者联合施用一种或多种其它疗法。
  37. 权利要求36所述的用途，其中所述疗法包括治疗方式和/或其它治疗剂。
  38. 检测样品中CCR8的方法，所述方法包括，包括使用权利要求1～21任一项所述的抗体或其抗原结合片段或权利要求26所述的免疫缀合物的步骤。
  39. 检测试剂盒，其包含权利要求1-21中任一项的抗体或其抗原结合片段或权利要求26的免疫缀合物。
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WO2025145207A1 (en) 2023-12-29 2025-07-03 Bristol-Myers Squibb Company Combination therapy of kras inhibitor and treg-depleting agent

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