WO2024088346A1 - Anticorps anti-ccr8 et leurs utilisations - Google Patents

Anticorps anti-ccr8 et leurs utilisations Download PDF

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Publication number
WO2024088346A1
WO2024088346A1 PCT/CN2023/126829 CN2023126829W WO2024088346A1 WO 2024088346 A1 WO2024088346 A1 WO 2024088346A1 CN 2023126829 W CN2023126829 W CN 2023126829W WO 2024088346 A1 WO2024088346 A1 WO 2024088346A1
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seq
amino acid
cdrs
nos
acid sequences
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PCT/CN2023/126829
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Inventor
Meimei YIN
Huichao LIANG
Yanan GUO
Aki Kawagishi
Takaho TERADA
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Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Liberothera Co., Ltd.
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Publication of WO2024088346A1 publication Critical patent/WO2024088346A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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 [IGs], 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
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This disclosure relates to anti-CCR8 (chemokine (C-C motif) receptor 8) antibodies and uses thereof.
  • Treg Regulatory T cells suppress abnormal/excessive immune responses to self-antigens and nonself-antigens to maintain immune homeostasis.
  • Treg cells are involved in tumor development and progression by inhibiting antitumor immunity.
  • This disclosure relates to anti-CCR8 antibodies, antigen-binding fragment thereof, and the uses thereof.
  • the present disclosure provides full human anti-CCR8 antibodies or antigen binding fragments thereof (e.g., that are produced by genetically modified non-human animals with a humanized heavy chain immunoglobulin locus and a humanized light chain immunoglobulin locus) .
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CCR8 (chemokine (C-C motif) receptor 8) , comprising:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VL light chain variable region
  • the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence
  • the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence
  • the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence
  • VH CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively.
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 72, 73, 74, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 75, 76, 77, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 78, 79, 80, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 81, 82, 83, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 84, 85, 86, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 87, 88, 89, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 90, 91, 92, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 93, 94, 95, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 96, 97, 98, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 99, 100, 101, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 102, 103, 104, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 105, 106, 107, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 144, 145, 146, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 147, 148, 149, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 150, 151, 152, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 153, 154, 155, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 156, 157, 158, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 159, 160, 161, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 162, 163, 164, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 165, 166, 167, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 168, 169, 170, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 171, 172, 173, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 174, 175, 176, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 177, 178, 179, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 219, 220, 221, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 222, 223, 224, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 225, 226, 227, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 228, 229, 230, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 231, 232, 233, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 234, 235, 236, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 237, 238, 239, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 240, 241, 242, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 243, 244, 245, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 246, 247, 248, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 249, 250, 251, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 252, 253, 254, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 255, 256, 257, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 258, 259, 260, respectively;
  • VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 108, 109, 110, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 111, 112, 113, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 114, 115, 116, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 117, 118, 119, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 120, 121, 122, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 123, 124, 125, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 126, 127, 128, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 129, 130, 131, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 132, 133, 134, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 135, 136, 137, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 138, 139, 140, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 141, 142, 143, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 180, 181, 182, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 183, 184, 185, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 186, 187, 188, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 189, 190, 191, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 192, 193, 194, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 195, 196, 197, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 198, 199, 200, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 201, 202, 203, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 204, 205, 206, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 207, 208, 209, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 210, 211, 212, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 213, 214, 215, respectively.
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CCR8 (chemokine (C-C motif) receptor 8) , comprising:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VL light chain variable region
  • the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence
  • the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence
  • the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence
  • VH CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • the selected VH CDR 1 is SYVMH (SEQ ID NO: 99) ;
  • the selected VH CDR2 is VISYX 1 X 2 SDKYYADSVKG (SEQ ID NO: 100) ;
  • the selected VH CDR3 is GRGYRYGQSYYYGMDV (SEQ ID NO: 101) ;
  • the selected VH CDR 1 is SYVVH (SEQ ID NO: 102) ;
  • the selected VH CDR2 is VISYX 1 X 2 DNKFYADSVKG (SEQ ID NO: 103) ;
  • the selected VH CDR3 is GRX 5 YX 6 X 7 YYGLDV (SEQ ID NO: 104) ;
  • the selected VH CDR 1 is TYVMH (SEQ ID NO: 105) ;
  • the selected VH CDR2 is VISYX 1 X 2 NNKYYADSVKG (SEQ ID NO: 106) ;
  • the selected VH CDR3 is GRSYVNYYGLDV (SEQ ID NO: 107) ;
  • the selected VH CDR 1 is GFSFSSY (SEQ ID NO: 171) ;
  • the selected VH CDR2 is SYX 1 X 2 SD (SEQ ID NO: 172) ;
  • the selected VH CDR3 is GRGYRYGQSYYYGMDV (SEQ ID NO: 173) ;
  • the selected VH CDR 1 is GFTFSSY (SEQ ID NO: 174) ;
  • the selected VH CDR2 is SYX 1 X 2 DN (SEQ ID NO: 175) ;
  • the selected VH CDR3 is GRX 5 YX 6 X 7 YYGLDV (SEQ ID NO: 176) ;
  • the selected VH CDR 1 is GFTFSTY (SEQ ID NO: 177) ;
  • the selected VH CDR2 is SYX 1 X 2 NN (SEQ ID NO: 178) ;
  • the selected VH CDR3 is GRSYVNYYGLDV (SEQ ID NO: 179) ;
  • VL CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 135) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 136) ;
  • the selected VL CDR3 is MQSIKLPLT (SEQ ID NO: 137) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 138) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 139) ;
  • the selected VL CDR3 is MQSLKVPPT (SEQ ID NO: 140) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 141) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 142) ;
  • the selected VL CDR3 is MQSVKIPLT (SEQ ID NO: 143) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 207) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 208) ;
  • the selected VL CDR3 is MQSIKLPLT (SEQ ID NO: 209) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 210) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 211) ;
  • the selected VL CDR3 is MQSLKVPPT (SEQ ID NO: 212) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 213) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 214) ;
  • the selected VL CDR3 is MQSVKIPLT (SEQ ID NO: 215) ;
  • X 1 and X 3 in each sequence are independently selected from the group consisting of D, S, and A,
  • X 2 and X 4 in each sequence are independently selected from the group consisting of G and A,
  • X 5 in SEQ ID NO: 104 or 176 is independently selected from the group consisting of N and S,
  • X 6 in SEQ ID NO: 104 or 176 is independently selected from the group consisting of Y and V,
  • X 7 in SEQ ID NO: 104 or 176 is independently selected from the group consisting of Y and N.
  • the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
  • VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively according to Kabat definition;
  • VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23 and 24, respectively according to Kabat definition;
  • VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8 and 9, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26 and 27, respectively according to Kabat definition;
  • VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively
  • VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively according to Chothia definition
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively according to Chothia definition
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively according to Chothia definition
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23 and 24, respectively according to Kabat definition
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26 and 27, respectively according to Kabat definition;
  • VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6 respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively according to Kabat definition;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6 respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively according to Kabat definition;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8 and 9 respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively according to Kabat definition;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9 respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively according to Kabat definition;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively according to Chothia definition;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively according to Chothia definition;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively according to Chothia definition
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively according to Chothia definition
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively according to Chothia definition
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively according to Chothia definition.
  • the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 114, 115, and 116, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 72, 73, and 74, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 114, 115, and 116, respectively;
  • VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 72, 73, and 74, respectively
  • VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 108, 109, and 110, respectively;
  • VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 78, 79, and 80, respectively
  • VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 114, 115, and 116, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 75, 76, and 77, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 111, 112, and 113, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 186, 187, and 188, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 144, 145, and 146, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 186, 187, and 188, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 144, 145, and 146, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 180, 181, and 182, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 150, 151, and 152, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 186, 187, and 188, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 147, 148, and 149, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 183, 184, and 185, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 72, 73, and 74, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 144, 145, and 146, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 219, 220, and 221, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 222, 223, and 224, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 225, 226, and 227, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 228, 229, and 230, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 233, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 234, 235, and 236, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively;
  • VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 237, 238, and 239, respectively
  • VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 240, 241, and 242, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 243, 244, and 245, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 246, 247, and 248, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 249, 250, and 251, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 252, 253, and 254, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 255, 256, and 257, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively;
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 258, 259, and 260, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.
  • the antibody or antigen-binding fragment specifically binds to human CCR8, mouse CCR8 or monkey CCR8.
  • the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody) .
  • the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) , a one-armed antibody, or a multi-specific antibody (e.g., a bispecific antibody) .
  • scFv single-chain variable fragment
  • a one-armed antibody e.g., a one-armed antibody
  • a multi-specific antibody e.g., a bispecific antibody
  • the disclosure is related to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising: an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, and wherein the VH, when paired with a light chain variable region (VL) binds to CCR8,
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively.
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 72, 73, 74, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 75, 76, 77, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 78, 79, 80, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 81, 82, 83, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 84, 85, 86, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 87, 88, 89, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 90, 91, 92, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 93, 94, 95, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 96, 97, 98, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 99, 100, 101, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 102, 103, 104, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 105, 106, 107, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 144, 145, 146, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 147, 148, 149, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 150, 151, 152, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 153, 154, 155, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 156, 157, 158, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 159, 160, 161, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 162, 163, 164, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 165, 166, 167, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 168, 169, 170, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 171, 172, 173, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 174, 175, 176, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 177, 178, 179, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 219, 220, 221, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 222, 223, 224, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 225, 226, 227, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 228, 229, 230, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 231, 232, 233, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 234, 235, 236, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 237, 238, 239, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 240, 241, 242, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 243, 244, 245, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 246, 247, 248, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 249, 250, 251, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 252, 253, 254, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 255, 256, 257, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 258, 259, 260, respectively;
  • VL amino acid sequence is selected from the group consisting of: SEQ ID NOs: 40, 41, 42, 66, 67, and 68.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8 and 9, respectively.
  • the disclosure is related to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising: an immunoglobulin heavy chain or a fragment thereof comprising a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, and wherein the VL, when paired with a heavy chain variable region (VH) binds to CCR8,
  • VL light chain variable region
  • CDRs complementarity determining regions
  • VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 108, 109, 110, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 111, 112, 113, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 114, 115, 116, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 117, 118, 119, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 120, 121, 122, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 123, 124, 125, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 126, 127, 128, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 129, 130, 131, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 132, 133, 134, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 135, 136, 137, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 138, 139, 140, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 141, 142, 143, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 180, 181, 182, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 183, 184, 185, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 186, 187, 188, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 189, 190, 191, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 192, 193, 194, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 195, 196, 197, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 198, 199, 200, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 201, 202, 203, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 204, 205, 206, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 207, 208, 209, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 210, 211, 212, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 213, 214, 215, respectively,
  • VH amino acid sequence is selected from the group consisting of: SEQ ID NOs: 37, 38, 39, 63, 64, 65, 69, 70, 71, 216, 261, 262, 263, 264, 265, 266, and 267.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23 and 24, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26 and 27, respectively.
  • the VH when paired with a VL specifically binds to human CCR8, mouse CCR8 or monkey CCR8, or the VL when paired with a VH specifically binds to human CCR8, mouse CCR8 or monkey CCR8.
  • the immunoglobulin heavy chain or the fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof
  • the immunoglobulin light chain or the fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof.
  • the nucleic acid encodes a single-chain variable fragment (scFv) , a one-armed antibody, or a multi-specific antibody (e.g., a bispecific antibody) .
  • scFv single-chain variable fragment
  • a one-armed antibody e.g., a one-armed antibody
  • a multi-specific antibody e.g., a bispecific antibody
  • the nucleic acid is cDNA.
  • the disclosure is related to a vector comprising one or more of the nucleic acids described herein.
  • the disclosure is related to a vector comprising two of the nucleic acids described herein, wherein the vector encodes the VH region and the VL region that together bind to CCR8.
  • the disclosure is related to a pair of vectors, wherein each vector comprises one of the nucleic acids described herein, wherein together the pair of vectors encodes the VH region and the VL region that together bind to CCR8.
  • the disclosure is related to a cell comprising the vector described herein, or the pair of vectors described herein.
  • the cell is a CHO cell.
  • the disclosure is related to a cell comprising one or more of the nucleic acids described herein.
  • the disclosure is related to a cell comprising two of the nucleic acids described herein.
  • the two nucleic acids together encode the VH region and the VL region that together bind to CCR8.
  • the disclosure is related to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising:
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CCR8 comprising
  • VH heavy chain variable region
  • VL light chain variable region
  • VH sequence is selected from the group consisting of: SEQ ID NOs: 37, 38, 39, 63, 64, 65, 69, 70, 71, 216, 261, 262, 263, 264, 265, 266, and 267,
  • VL sequence is selected from the group consisting of: SEQ ID NOs: 40, 41, 42, 66, 67, and 68.
  • the VH sequence and the VL sequence are one of the following:
  • VH comprises the sequence of SEQ ID NO: 37 and the VL comprises the sequence of SEQ ID NO: 40;
  • VH comprises the sequence of SEQ ID NO: 38 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 39 and the VL comprises the sequence of SEQ ID NO: 42;
  • VH comprises the sequence of SEQ ID NO: 37 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 37 and the VL comprises the sequence of SEQ ID NO: 42;
  • VH comprises the sequence of SEQ ID NO: 38 and the VL comprises the sequence of SEQ ID NO: 40;
  • VH comprises the sequence of SEQ ID NO: 38 and the VL comprises the sequence of SEQ ID NO: 42;
  • VH comprises the sequence of SEQ ID NO: 39 and the VL comprises the sequence of SEQ ID NO: 40;
  • VH comprises the sequence of SEQ ID NO: 39 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 37 and the VL comprises the sequence of SEQ ID NO: 68;
  • the VH comprises the sequence of SEQ ID NO: 63 and the VL comprises the sequence of SEQ ID NO: 68;
  • the VH comprises the sequence of SEQ ID NO: 63 and the VL comprises the sequence of SEQ ID NO: 66;
  • VH comprises the sequence of SEQ ID NO: 65 and the VL comprises the sequence of SEQ ID NO: 68;
  • VH comprises the sequence of SEQ ID NO: 64 and the VL comprises the sequence of SEQ ID NO: 67;
  • VH comprises the sequence of SEQ ID NO: 63 and the VL comprises the sequence of SEQ ID NO: 42;
  • VH comprises the sequence of SEQ ID NO: 216 and the VL comprises the sequence of SEQ ID NO: 42;
  • VH comprises the sequence of SEQ ID NO: 261 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 262 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 263 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 264 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 265 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 266 and the VL comprises the sequence of SEQ ID NO: 41;
  • VH comprises the sequence of SEQ ID NO: 267 and the VL comprises the sequence of SEQ ID NO: 41.
  • the antibody or antigen-binding fragment specifically binds to human CCR8, mouse CCR8 or monkey CCR8.
  • the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) , a one-armed antibody, or a multi-specific antibody (e.g., a bispecific antibody) .
  • scFv single-chain variable fragment
  • a one-armed antibody e.g., a one-armed antibody
  • a multi-specific antibody e.g., a bispecific antibody
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CCR8 comprising a heavy chain variable region (VH) comprising VH CDRs 1, 2, 3, and a light chain variable region comprising VL CDRs 1, 2, 3, wherein the VH CDRs 1, 2, 3 and the VL CDRs 1, 2, 3 are identical to complementarity determining regions in the antibody or antigen-binding fragment thereof described herein.
  • VH heavy chain variable region
  • VL CDRs 1, 2, 3 a light chain variable region comprising VL CDRs 1, 2, 3
  • the VH CDRs 1, 2, 3 are identical to complementarity determining regions in SEQ ID NO: 37, 38, 39, 63, 64, 65, 69, 70, 71, 216, 261, 262, 263, 264, 265, 266, or 267, and the VL CDRs 1, 2, 3 are identical to complementary determining regions in SEQ ID NO: 40, 41, 42, 66, 67, or 68.
  • the disclosure is related to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof described herein.
  • the disclosure is related to an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof described herein covalently bound to a therapeutic agent.
  • the therapeutic agent is a cytotoxic or cytostatic agent.
  • the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, to the subject.
  • the subject has a solid tumor.
  • the cancer is breast cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, colon cancer, or colorectal cancer (e.g., colorectal adenocarcinoma) .
  • the cancer is resistant to a PD-1 pathway inhibitor treatment.
  • the method further comprises administering a PD-1 pathway inhibitor to the subject.
  • the PD-1 pathway inhibitor is an anti-PD-1 antibody (e.g., Pembrolizumab, Nivolumab, or Cemiplimab) .
  • an anti-PD-1 antibody e.g., Pembrolizumab, Nivolumab, or Cemiplimab
  • the PD-1 pathway inhibitor is an anti-PD-L1 antibody (e.g., Atezolizumab, Avelumab, or Durvalumab) .
  • an anti-PD-L1 antibody e.g., Atezolizumab, Avelumab, or Durvalumab
  • the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein.
  • the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein.
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of described herein, and a pharmaceutically acceptable carrier.
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody drug conjugate described herein, and a pharmaceutically acceptable carrier.
  • cancer refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • malignancies of the various organ systems such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine.
  • Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections.
  • a carcinogen s
  • cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene and cancer caused by infections, e.g., viral infections.
  • the term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • an “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin.
  • a hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • a hematologic cancer is a cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer include e.g., leukemia, lymphoma, and multiple myeloma etc.
  • antibody refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope.
  • CDR complementary determining region
  • Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
  • an antibody can contain an Fc region of a human antibody.
  • the term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, heavy chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
  • the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen.
  • the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain) .
  • variable domains include, e.g., Fab, Fab’ , F (ab’ ) 2 , VHH, and Fv fragments.
  • human antibody refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) derived from a human.
  • a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells) .
  • a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line) .
  • a human antibody is produced in a bacterial or yeast cell.
  • a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus) .
  • a transgenic non-human animal e.g., a bovine
  • human immunoglobulin locus e.g., heavy or light chain human immunoglobulin locus
  • chimeric antibody refers to an antibody that contains a sequence present in at least two different species (e.g., antibodies from two different mammalian species such as a human and a mouse antibody) .
  • a non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
  • humanized antibody refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin.
  • humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity.
  • the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues.
  • humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance.
  • the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin.
  • CDRs hypervariable loops
  • the humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc) , typically, that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.
  • single-chain antibody refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen.
  • immunoglobulin variable domains e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain
  • single-chain antibodies are described herein.
  • multimeric antibody refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains.
  • the multimeric antibody is able to crosslink one target molecule (e.g., CCR8) to at least one second target molecule (e.g., PD-L1) on the surface of mammalian cell (e.g., Treg cell and cancer cell) .
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided.
  • Veterinary and non-veterinary applications are contemplated in the present disclosure.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) .
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • equine canine, feline, bovine, and other domestic, farm, and zoo animals.
  • the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., CCR8) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general.
  • An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody.
  • an antibody that specifically binds to a CCR8 molecule may be referred to as a CCR8-specific antibody or an anti-CCR8 antibody.
  • polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
  • nucleic acid molecule As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
  • FIG. 1 shows the effects on tumor growth of the anti-CCR8 antibodies in vivo in a colon cancer model.
  • FIGs. 2A-2C show CDR sequences corresponding to anti-human CCR8 mAbs 11B12, 11F5, and 11F8 and CDR-modified anti-human CCR8 mAbs, as defined by Kabat definition.
  • FIGs. 3A-3C show CDR sequences corresponding to anti-human CCR8 mAbs 11B12, 11F5, and 11F8 and CDR-modified anti-human CCR8 mAbs, as defined by Chothia definition.
  • FIG. 4 shows some of the relevant amino acid sequences discussed in the present disclosure.
  • FIG. 5 shows some of the VH and VL combinations in the present disclosure.
  • FIG. 6 shows the average tumor volume in different groups of B-hPD-1/hPD-L1/hCCR8 mice that were subcutaneously injected with MC38 cells (murine colon cancer cells) , and were treated with PBS (G1) , Pembrolizumab analog (G2) , anti-CCR8 antibody 11F5 (G3) , or a combination of Pembrolizumab analog and 11F5 (G4) .
  • FIG. 7 shows the average tumor volume in different groups of B-hPD-1/hPD-L1/hCCR8 mice that were subcutaneously injected with MC38 cells, and were treated with PBS (G1) , Pembrolizumab analog (G2) , anti-CCR8 antibody 11F5-SI (G3) , a combination of Pembrolizumab analog and 11F5-SI (G4) , anti-CCR8 antibody 11B12-SI (G5) , or a combination of Pembrolizumab analog and 11B12-SI (G6) .
  • PBS Pembrolizumab analog
  • G3 anti-CCR8 antibody 11F5-SI
  • G4 anti-CCR8 antibody 11B12-SI
  • G6 a combination of Pembrolizumab analog and 11B12-SI
  • FIG. 8 shows the blockade of hCCL1 binding to hCCR8 by anti-CCR8 antibodies 11F5 and 11F5’s CDR3-modified antibodies, as tested by calcium (Ca) flux assays using human CCR8-expressing 293T cells.
  • Chemokines and chemokine receptors play an important role in the immune defense system by controlling the migration, activation, differentiation, and survival of leukocytes.
  • the 50 human chemokines are divided into C, CC, CXC, and CX3C classes based on the number and spacing of conserved cysteine residues in their N-terminus region.
  • Chemokine receptors belong to the family A of G-protein coupled receptors (GPCRs) , characterized by a seven transmembrane (7TM) helical domain.
  • C chemokine receptor 1
  • CC CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10
  • CXC CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6
  • CX3C CX3CR1
  • ACKRs ACKR1, ACKR2, ACKR3/CXCR7, and ACKR4 .
  • Chemokine receptors are considered to interact with their chemokine ligands via a two-step binding mechanism in which: (i) the structured C-terminal region of the chemokine first binds the N-terminus region and extracellular loops (ECLs) of the receptor (chemokine recognition site 1, CRS1) , allowing (ii) the unstructured N-terminus of the chemokine to target the 7TM helical bundle (chemokine recognition site 2, CRS2) and stabilize the receptor in an active conformation that facilitates intracellular signal transduction by, e.g., G-proteins or arrestins. Because of their crucial role in cell migration chemokine receptors are important therapeutic targets for inflammatory diseases and cancer.
  • Chemokine (C-C motif) receptor 8 (CCR8, or CDw198) is a seven transmembrane G protein-couple receptor (GPCR) , and belongs to the C-C subfamily of chemokine receptors.
  • CCR8 is one of the most important chemokine receptors and is mainly expressed in Tregs with small proportions of expression in T helper 2 cells and monocytes.
  • CCR8 has four known ligands: CCL1, CCL8, CCL16 and CCL18.
  • Human CCL1 is the dominant ligand for human CCR8 that binds simultaneously to the extracellular loop 2 and the N-terminal domain of the receptor.
  • CCL1 enhances Treg immunosuppressive activity by recruiting CCR8, FOXp3 and IL-10 with a positive feedback mechanism.
  • CCR8 was also reported to be correlated with poor prognosis in non-small cell lung cancer and colorectal cancer.
  • CCR8 is selectively up-regulated in tumor resident Treg cells in multiple solid tumors, but rarely observed on Treg cells in peripheral blood mononuclear cells (PBMC) or normal organs.
  • Treg cells are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. These cells play an important role in maintaining self-tolerance and immune homeostasis.
  • Tumor-resident Treg cells can suppress anti-tumor immunity in the tumor microenvironment.
  • High CCR8 expression is often associated with poor survival in cancer patients.
  • CCR8 is a potential target for Treg cells.
  • Treg cells inhibition and/or depletion by targeting CCR8 have several advantages.
  • CCR8 is more restricted to the Treg cells, not on the CD8 T cells, there is no attenuation of T effector cell antitumor activities.
  • systemic Treg cell depletion can often lead to autoimmunity. There is less risk of autoimmunity as CCR8 is rarely expressed on Treg cells in peripheral blood mononuclear cells (PBMC) or normal organs.
  • PBMC peripheral blood mononuclear cells
  • treatment with rat anti-mouse CCR8 blocking antibodies can generate significant antitumor activity and improve long-term survival in a colorectal tumor mouse model. Therefore, CCR8 is a superior target among various known Treg targets as its expression is found to be highly selected in tumor-resident Treg cells.
  • Anti-CCR8 antibodies can also have single agent activity in a PD-1 resistant setting and can restore PD-1 activity as a combination partner.
  • CCR8 CCR8 and its function can be found, e.g., in Plitas G. et al., "Regulatory T cells exhibit distinct features in human breast cancer. " Immunity 45.5 (2016) : 1122-1134; Villarreal. et al., “Targeting CCR8 induces protective antitumor immunity and enhances vaccine-induced responses in colon cancer. " Cancer research 78.18 (2016) : 5340-5348; Arimont et al., "Stural analysis of chemokine receptor–ligand interactions.
  • the present disclosure provides anti-CCR8 antibodies, antigen-binding fragments thereof, and methods of using these anti-CCR8 antibodies and antigen-binding fragments to inhibit tumor growth and to treat cancers, particularly, the present disclosure provides full human anti-CCR8 antibodies (e.g., that are produced by mice with a humanized heavy chain immunoglobulin locus and a humanized light chain immunoglobulin locus) .
  • the disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CCR8.
  • the antibodies and antigen-binding fragments described herein are capable of binding to CCR8.
  • these antibodies can block the binding of CCL1 to CCR8 thereby inhibiting CCL1-induced suppressive function of tumor resident Treg cells.
  • these antibodies can inhibit CCL1-CCR8-mediated downstream signaling.
  • these antibodies can initiate complement-dependent cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC) .
  • CMC complement-dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • these antibodies and antigen-binding fragments thereof can block the interaction between CCR8 and a CCR8 ligand (e.g., CCL1, CCL8, CCL16 or CCL18) .
  • the disclosure provides e.g., mouse anti-CCR8 antibodies 11B12, 11F5, and 11F8, the chimeric antibodies thereof, and the humanized antibodies thereof.
  • the CDR sequences for 11B12, and 11B12 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 2, 3, and CDRs of the light chain variable domain, SEQ ID NOs: 19, 20, 21 as defined by Kabat definition.
  • the CDRs can also be defined by Chothia definition. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 10, 11, 12, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 28, 29, 30.
  • the CDR sequences for 11F5, and 11F5 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 4, 5, 6, and CDRs of the light chain variable domain, SEQ ID NOs: 22, 23, 24, as defined by Kabat definition.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 13, 14, 15, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 31, 32, 33.
  • the CDR sequences for 11F8, and 11F8 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 7, 8, 9, and CDRs of the light chain variable domain, SEQ ID NOs: 25, 26, 27, as defined by Kabat definition.
  • the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 16, 17, 18, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 34, 35, 36.
  • the amino acid sequence for the heavy chain variable region of 11B12 antibody is set forth in SEQ ID NO: 37.
  • the amino acid sequence for the light chain variable region of 11B12 antibody is set forth in SEQ ID NO: 40.
  • the amino acid sequence for the heavy chain variable region of 11F5 antibody is set forth in SEQ ID NO: 38.
  • the amino acid sequence for the light chain variable region of 11F5 antibody is set forth in SEQ ID NO: 41.
  • the amino acid sequence for the heavy chain variable region of 11F8 antibody is set forth in SEQ ID NO: 39.
  • the amino acid sequence for the light chain variable region of 11F8 antibody is set forth in SEQ ID NO: 42.
  • the amino acid sequences for heavy chain variable regions and light chain variable regions of the humanized antibodies are also provided.
  • the heavy chain and the light chain of an antibody can have more than one version of humanized sequences.
  • the humanized heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 37, 38, or 39.
  • the humanized light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 40, 41, or 42.
  • the heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and together they bind to CCR8.
  • Humanization percentage means the percentage identity of the heavy chain or light chain variable region sequence as compared to human antibody sequences in International Immunogenetics Information System (IMGT) database.
  • the top hit means that the heavy chain or light chain variable region sequence is closer to a particular species than to other species.
  • top hit to human means that the sequence is closer to human than to other species.
  • Top hit to human and Macaca fascicularis means that the sequence has the same percentage identity to the human sequence and the Macaca fascicularis sequence, and these percentages identities are highest as compared to the sequences of other species.
  • humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.
  • a detailed description regarding how to determine humanization percentage and how to determine top hits is known in the art, and is described, e.g., in Jones, et al. "The INNs and outs of antibody nonproprietary names. " MAbs. Vol. 8. No. 1. Taylor &Francis, 2016, which is incorporated herein by reference in its entirety.
  • a high humanization percentage often has various advantages, e.g., more safe and more effective in humans, more likely to be tolerated by a human subject, and/or less likely to have side effects.
  • the antibody is a full human antibody.
  • the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 4-6, SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 13-15, and SEQ ID NOs: 16-18; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 19-21, SEQ ID NOs: 22-24, SEQ ID NOs: 25-27, SEQ ID NOs: 28-30, SEQ ID NOs: 31-33, and SEQ ID NOs: 34-36.
  • the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence.
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • the antibodies can have a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence.
  • VL light chain variable region
  • FIGs. 2A-2C Kabat CDR
  • FIGs. 3A-3C Chothia CDR
  • the disclosure relates to CDR-modified VH or VL.
  • the “DG” in VH CDR2 of an antibody described herein is mutated to “SG, ” “AG, ” or “DA, ” to create CDR-modified VH.
  • the “DG” in VH CDR2 of 11B12 is mutated to “SG, ” and the resulting CDR-modified VH is named 11B12-SG VH.
  • the sequences of some of these CDR-modified VH can be found in FIG. 4.
  • the “DG” in VL CDR1 of an antibody described herein is mutated to “SG, ” “AG, ” or “DA, ” to create CDR-modified VL.
  • the “DG” in VL CDR1 of 11B12 is mutated to SG, and the resulting CDR-modified VL is named 11B12-SG VL.
  • the sequences of some of these CDR-modified VL can be found in FIG. 4.
  • the disclosure relates to framework region modified (FR-modified) VH.
  • the “DNS” in the framework of the VH of an antibody described herein is mutated to “DNA, ” to create FR-modified VH.
  • the “DNS” in the framework of the VH of 11B12 is mutated to “DNA, ” and the resulting FR-modified VH is named 11B12-DNA VH.
  • the sequences of some these FR-modified VH can be found in FIG. 4 (e.g., SEQ ID NOs: 69-71) .
  • the antibody has a S74A mutation (Kabat numbering) .
  • the amino acid at position 74 (Kabat numbering) is A.
  • the VH has both a FR modification (e.g., DNA) and a CDR modification (e.g., SG) .
  • a FR modification e.g., DNA
  • a CDR modification e.g., SG
  • the “DNS” in the framework of the VH of 11B12 is mutated to “DNA, ” and the “DG” in VH CDR2 of 11B12 is mutated to “SG, ” and the resulting FR-modified and CDR-modified VH is named 11B12-SG-DNA VH.
  • 11F5 CDR3-modified VHs can be created.
  • the amino acid “N” at position 3 in VH CDR3 of 11F5 e.g., SEQ ID NO: 6 or 15
  • the amino acid “Y” at position 5 in VH CDR3 of 11F5 is mutated to “V. ”
  • the amino acid “Y”at position 6 in VH CDR3 of 11F5 is mutated to “N.
  • the amino acid “N” at position 3 in VH CDR3 of 11F5 can be mutated to “S” and the resulting CDR-modified VH is named 11F5-N3S VH; the amino acid “Y” at position 5 in VH CDR3 of 11F5 can be mutated to “V” and the resulting CDR-modified VH is named 11F5-Y5V VH; the amino acid “Y” at position 6 in VH CDR3 of 11F5 can be mutated to “N” and the resulting CDR-modified VH is named 11F5-Y6N VH; the amino acid “N” at position 3 and amino acid “Y” at position 5 in VH CDR3 of 11F5 can be mutated to “S” and “V, ” respectively, and the resulting CDR-modified VH is named 11F5-N3S-Y5V VH; the amino acid “N” at position 3 and the amino acid “Y” at position 6 in VH CDR3
  • the VH of any antibody described herein can be paired with the VL of any antibody described herein to form antibodies.
  • the VH of 11B12 is paired with the VL of 11F8, and the resulting antibody is named 11B12-11F8.
  • the VH of 11B12-SG is paired with the VL of 11F8-SG, and the resulting antibody is named 11B12-SG-11F8-SG.
  • the VH of 11B12-SG is paired with the VL of 11B12-SG, and the resulting antibody is named 11B12-SG-11B12-SG.
  • the binding affinities of some of these antibodies are shown in Tables 15, 16, and 19.
  • VH and VL combinations are listed in FIG. 5. As shown in FIG. 5, at least 163 VH and VL combinations are disclosed. In addition, the VH CDRs and VL CDRs in the VH and VL can be combined as well, as shown in FIG. 5.
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CCR8 (chemokine (C-C motif) receptor 8) , comprising:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • the VH CDR1 region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a selected VH CDR1 amino acid sequence
  • the VH CDR2 region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a selected VH CDR2 amino acid sequence
  • the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence
  • VL light chain variable region
  • the VL CDR1 region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a selected VL CDR1 amino acid sequence
  • the VL CDR2 region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a selected VL CDR2 amino acid sequence
  • the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence
  • VH CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively.
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 72, 73, 74, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 75, 76, 77, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 78, 79, 80, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 81, 82, 83, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 84, 85, 86, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 87, 88, 89, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 90, 91, 92, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 93, 94, 95, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 96, 97, 98, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 99, 100, 101, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 102, 103, 104, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 105, 106, 107, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 144, 145, 146, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 147, 148, 149, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 150, 151, 152, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 153, 154, 155, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 156, 157, 158, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 159, 160, 161, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 162, 163, 164, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 165, 166, 167, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 168, 169, 170, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 171, 172, 173, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 174, 175, 176, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 177, 178, 179, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 219, 220, 221, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 222, 223, 224, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 225, 226, 227, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 228, 229, 230, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 231, 232, 233, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 234, 235, 236, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 237, 238, 239, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 240, 241, 242, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 243, 244, 245, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 246, 247, 248, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 249, 250, 251, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 252, 253, 254, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 255, 256, 257, respectively;
  • VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 258, 259, 260, respectively;
  • VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 108, 109, 110, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 111, 112, 113, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 114, 115, 116, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 117, 118, 119, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 120, 121, 122, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 123, 124, 125, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 126, 127, 128, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 129, 130, 131, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 132, 133, 134, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 135, 136, 137, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 138, 139, 140, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 141, 142, 143, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 180, 181, 182, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 183, 184, 185, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 186, 187, 188, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 189, 190, 191, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 192, 193, 194, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 195, 196, 197, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 198, 199, 200, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 201, 202, 203, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 204, 205, 206, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 207, 208, 209, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 210, 211, 212, respectively;
  • VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 213, 214, 215, respectively.
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to CCR8 (chemokine (C-C motif) receptor 8) , comprising:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VL light chain variable region
  • the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence
  • the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence
  • the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence
  • VH CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • the selected VH CDR 1 is SYVMH (SEQ ID NO: 99) ;
  • the selected VH CDR2 is VISYX 1 X 2 SDKYYADSVKG (SEQ ID NO: 100) ;
  • the selected VH CDR3 is GRGYRYGQSYYYGMDV (SEQ ID NO: 101) ;
  • the selected VH CDR 1 is SYVVH (SEQ ID NO: 102) ;
  • the selected VH CDR2 is VISYX 1 X 2 DNKFYADSVKG (SEQ ID NO: 103) ;
  • the selected VH CDR3 is GRX 5 YX 6 X 7 YYGLDV (SEQ ID NO: 104) ;
  • the selected VH CDR 1 is TYVMH (SEQ ID NO: 105) ;
  • the selected VH CDR2 is VISYX 1 X 2 NNKYYADSVKG (SEQ ID NO: 106) ;
  • the selected VH CDR3 is GRSYVNYYGLDV (SEQ ID NO: 107) ;
  • the selected VH CDR 1 is GFSFSSY (SEQ ID NO: 171) ;
  • the selected VH CDR2 is SYX 1 X 2 SD (SEQ ID NO: 172) ;
  • the selected VH CDR3 is GRGYRYGQSYYYGMDV (SEQ ID NO: 173) ;
  • the selected VH CDR 1 is GFTFSSY (SEQ ID NO: 174) ;
  • the selected VH CDR2 is SYX 1 X 2 DN (SEQ ID NO: 175) ;
  • the selected VH CDR3 is GRX 5 YX 6 X 7 YYGLDV (SEQ ID NO: 176) ;
  • the selected VH CDR 1 is GFTFSTY (SEQ ID NO: 177) ;
  • the selected VH CDR2 is SYX 1 X 2 NN (SEQ ID NO: 178) ;
  • the selected VH CDR3 is GRSYVNYYGLDV (SEQ ID NO: 179) ;
  • VL CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 135) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 136) ;
  • the selected VL CDR3 is MQSIKLPLT (SEQ ID NO: 137) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 138) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 139) ;
  • the selected VL CDR3 is MQSLKVPPT (SEQ ID NO: 140) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 141) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 142) ;
  • the selected VL CDR3 is MQSVKIPLT (SEQ ID NO: 143) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 207) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 208) ;
  • the selected VL CDR3 is MQSIKLPLT (SEQ ID NO: 209) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 210) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 211) ;
  • the selected VL CDR3 is MQSLKVPPT (SEQ ID NO: 212) ;
  • the selected VL CDR 1 is KSSQSLLYSX 3 X 4 KTYLY (SEQ ID NO: 213) ;
  • the selected VL CDR2 is EVSNRFS (SEQ ID NO: 214) ;
  • the selected VL CDR3 is MQSVKIPLT (SEQ ID NO: 215) ;
  • X 1 and X 3 in each sequence are independently selected from the group consisting of D, S, and A,
  • X 2 and X 4 in each sequence are independently selected from the group consisting of G and A,
  • X 5 in SEQ ID NO: 104 or 176 is independently selected from the group consisting of N and S,
  • X 6 in SEQ ID NO: 104 or 176 is independently selected from the group consisting of Y and V,
  • X 7 in SEQ ID NO: 104 or 176 is independently selected from the group consisting of Y and N.
  • humanization may involve limited modifications on the CDRs. However, these modifications will have limited impact on the binding affinities.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 33 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
  • the CDR is determined based on Kabat definition. In some embodiments, the CDR is determined based on Chothia definition.
  • the VH is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 37-39, 63-65, 69-71, 216, and 261-267.
  • the VL is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 40-42 and 66-68.
  • the disclosure also provides antibodies or antigen-binding fragments thereof that bind to CCR8.
  • the antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence.
  • VH heavy chain variable region
  • VL light chain variable region
  • the selected VH sequence is SEQ ID NO: 37
  • the selected VL sequence is SEQ ID NO: 40
  • the selected VH sequence is SEQ ID NO: 38 and the selected VL sequence is SEQ ID NO: 41.
  • the selected VH sequence is SEQ ID NO: 39 and the selected VL sequence is SEQ ID NO: 42. In some embodiments, the selected VH sequence is SEQ ID NO: 261 and the selected VL sequence is SEQ ID NO: 41. In some embodiments, the selected VH sequence is SEQ ID NO: 262 and the selected VL sequence is SEQ ID NO: 41. In some embodiments, the selected VH sequence is SEQ ID NO: 263 and the selected VL sequence is SEQ ID NO: 41. In some embodiments, the selected VH sequence is SEQ ID NO: 264 and the selected VL sequence is SEQ ID NO: 41. In some embodiments, the selected VH sequence is SEQ ID NO: 265 and the selected VL sequence is SEQ ID NO: 41.
  • the selected VH sequence is SEQ ID NO: 266 and the selected VL sequence is SEQ ID NO: 41. In some embodiments, the selected VH sequence is SEQ ID NO: 267 and the selected VL sequence is SEQ ID NO: 41.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain.
  • the immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIGs. 2A-2C or FIGs. 3A-3C or have sequences as shown in FIG. 4.
  • the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region)
  • the paired polypeptides bind to CCR8 (e.g., human CCR8) .
  • the anti-CCR8 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments.
  • Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof.
  • the antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass.
  • the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
  • Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody.
  • a fragment of an antibody that binds to CCR8 will retain an ability to bind to CCR8.
  • An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody.
  • Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
  • the polypeptide comprises a linker with the sequence of GGGS (SEQ ID NO: 61) or GGGSGGGS (SEQ ID NO: 62) .
  • the present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment as described herein.
  • the cross-competing assay is known in the art, and is described e.g., in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein. " Journal of virology 70.3 (1996) : 1863-1872, which is incorporated herein reference in its entirety.
  • the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment as described herein.
  • the epitope binning assay is known in the art, and is described e.g., in Estep et al. "High throughput solution-based measurement of antibody-antigen affinity and epitope binning. " MAbs. Vol. 5. No. 2. Taylor &Francis, 2013, which is incorporated herein reference in its entirety.
  • antibodies also called immunoglobulins
  • antibodies are made up of two classes of polypeptide chains, light chains and heavy chains.
  • a non-limiting examples of antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains.
  • the heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.
  • the light chain can be a kappa light chain or a lambda light chain.
  • An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain.
  • the heavy chains which each contain one variable domain (or variable region, V H ) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody.
  • the light chains which each contain one variable domain (or variable region, V L ) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding.
  • the variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound.
  • the variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) .
  • CDRs complementary determining regions
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting the beta-sheet structure, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.
  • the CDRs are important for recognizing an epitope of an antigen.
  • an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody.
  • the minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.
  • the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) .
  • the IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains.
  • the sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions. " Frontiers in immunology 5 (2014) ; Irani, et al.
  • the antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid) .
  • Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide.
  • the term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F (ab') 2 , and variants of these fragments.
  • an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain.
  • Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
  • the Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain.
  • F (ab') 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
  • Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL) .
  • VH and VL polypeptide chain
  • Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions.
  • Linear antibodies can be bispecific or monospecific.
  • Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
  • Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG 1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
  • purified antibody preparations e.g., purified IgG 1 molecules
  • antibody homodimers may be formed through chemical linkage techniques known in the art.
  • heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers.
  • SMCC succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate
  • SATA N-succinimidyl S-acethylthio-acetate
  • An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997) .
  • Antibody homodimers can be converted to Fab’ 2 homodimers through digestion with pepsin. Another way to form antibody homodimers
  • the multi-specific antibody is a bi-specific antibody.
  • Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the interface can contain at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
  • Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin.
  • Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • bi-specific antibodies can be prepared using chemical linkage.
  • Brennan et al. (Science 229: 81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F (ab’ ) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TNB thionitrobenzoate
  • One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bi-specific antibody.
  • any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) .
  • stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) .
  • the conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .
  • the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent.
  • the antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent.
  • the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) .
  • cytotoxic or cytostatic agent e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenopos
  • the antigen binding fragment can form a part of a chimeric antigen receptor (CAR) .
  • the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane-and endodomain.
  • the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) .
  • the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency.
  • the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.
  • the scFv has one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFv has two heavy chain variable domains, and two light chain variable domains.
  • the antibodies or antigen-binding fragment thereof as described herein comprise heavy chain constant regions (e.g., CH1, CH2, and/or CH3) selected from mouse IgG1, IgG2a, and IgG2b.
  • the antibodies or antigen-binding fragment thereof as described herein comprise light chain constant regions (e.g., CL) selected from mouse kappa or lambda CL.
  • the antibodies or antigen-binding fragment thereof as described herein comprise heavy chain constant regions (e.g., CH1, CH2, and/or CH3) selected from human IgG (e.g., human IgG1, IgG2, IgG3, or IgG4) .
  • the antibodies or antigen-binding fragment thereof as described herein comprise light chain constant regions (e.g., CL) selected from human kappa or lambda CL.
  • the antibodies or antigen-binding fragments thereof described herein can block the binding between CCR8 and CCR8 ligands (e.g., CCL1) .
  • CCR8 CCR8 ligands
  • the antibody can inhibit CCL1-induced suppressive function of tumor resident Treg cells.
  • the antibody can upregulate immune response.
  • the antibody can deplete Treg cells (e.g., tumor-resident Treg cells) .
  • the antibody has reduced risk of inducing autoimmunity and reduced risk of inducing systemic Treg cell depletion.
  • the antibodies or antigen-binding fragments thereof as described herein increase antitumor immunity of at least or about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%.
  • the antibodies or antigen-binding fragments thereof as described herein have less risk of inducing autoimmunity or causing systemic Treg cell depletion. In some embodiments, the antibodies or antigen-binding fragments thereof as described herein has a lower risk of inducing autoimmunity by least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%as compared to other therapeutic agents that target Tregs (e.g., anti-CCR4, CTLA4, OX40, or GITR antibodies) .
  • Tregs e.g., anti-CCR4, CTLA4, OX40, or GITR antibodies
  • the antibodies or antigen-binding fragments thereof as described herein can decrease activity or number of Treg cells (e.g., tumor-resident Treg cells and/or at the tumor microenvironment) by least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, e.g., as compared to the activity or number of Treg cells when the antibodies or antigen-binding fragments thereof are not administered.
  • Treg cells e.g., tumor-resident Treg cells and/or at the tumor microenvironment
  • the antibodies or antigen-binding fragments thereof as described herein can decrease activity or number of Treg cells (e.g., tumor-resident Treg cells and/or at the tumor microenvironment) by least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, e.g., as compared to the activity or number of Treg cells when the antibodies or antigen-binding fragments thereof are not administered.
  • the antibodies or antigen-binding fragments thereof as described herein can increase immune response, activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds, e.g., as compared to the case when the antibodies or antigen-binding fragments thereof are not administered.
  • immune cells e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells
  • the antibodies or antigen-binding fragments thereof as described herein can effectively increase Teff: Treg ratio, e.g., by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds, e.g., as compared to the case when the antibodies or antigen-binding fragments thereof are not administered.
  • the antibody specifically binds to CCR8 (e.g., human CCR8, monkey CCR8 (e.g., cynomolgus monkey (Macaca fascicularis) , mouse CCR8, and/or chimeric CCR8) with a dissociation rate (koff) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s -1 .
  • CCR8 e.g., human CCR8, monkey CCR8 (e.g., cynomolgus monkey (Macaca fascicularis) , mouse CCR8, and/or chimeric CCR8) with a dissociation rate (koff) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s -1 .
  • the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 , or greater than 0.000001 s -1 .
  • kinetic association rates (kon) is greater than 1 ⁇ 10 2 /Ms, greater than 1 ⁇ 10 3 /Ms, greater than 1 ⁇ 10 4 /Ms, greater than 1 ⁇ 10 5 /Ms, or greater than 1 ⁇ 10 6 /Ms. In some embodiments, kinetic association rates (kon) is less than 1 ⁇ 10 5 /Ms, less than 1 ⁇ 10 6 /Ms, or less than 1 ⁇ 10 7 /Ms.
  • KD is less than 1 ⁇ 10 -6 M, less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, or less than 1 ⁇ 10 -10 M. In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.
  • KD is greater than 1 ⁇ 10 -7 M, greater than 1 ⁇ 10 -8 M, greater than 1 ⁇ 10 -9 M, greater than 1 ⁇ 10 -10 M, greater than 1 ⁇ 10 -11 M, or greater than 1 ⁇ 10 -12 M.
  • the antibody or an antigen-binding fragment thereof binds to human CCR8 at Y17, F21, D97, W99, F101, G102, C106, G179, C183, W194, K195, I196, and/or L278.
  • the binding affinity of the antibodies or antigen-binding fragments thereof as described herein can be measured by FACS.
  • An exemplary procedure is shown in Example 4.
  • EC50 is less than 25 ⁇ g/mL, less than 15 ⁇ g/mL, less than 10 ⁇ g/mL, less than 5 ⁇ g/mL, less than 2.5 ⁇ g/mL, less than 1 ⁇ g/mL, less than 0.5 ⁇ g/mL, less than 0.4 ⁇ g/mL, less than 0.3 ⁇ g/mL, less than 0.2 ⁇ g/mL, less than 0.15 ⁇ g/mL, less than 0.1 ⁇ g/mL, or less than 0.05 ⁇ g/mL.
  • EC50 is about 0.05-0.5 ⁇ g/mL, about 0.1-1 ⁇ g/mL, about 0.1-0.8 ⁇ g/mL, about 0.1-0.5 ⁇ g/mL, about 0.2-2 ⁇ g/mL, about 0.2-1 ⁇ g/mL, or about 0.3-5 ⁇ g/mL.
  • the blockade of hCCL1 binding to hCCR8 by the antibodies or antigen-binding fragments thereof as described herein can be measured by Fluorescent Imaging Plate Reader (FLIPR) Calcium 6 Assay Kit.
  • FLIPR Fluorescent Imaging Plate Reader
  • EC50 is less than 30 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.5 nM, or 1 nM. In some embodiments, EC50 is about 1-10 nM, 1.5-20 nM, or 2.5-30 nM.
  • the maximum inhibition is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the maximum inhibition is less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • the antibodies can bind to one or more of the following human CCR2, human CCR4, human CCR5, human CCR8, and human CX3CR1. In some embodiments, the antibodies cannot bind to one or more of the following human CCR2, human CCR4, human CCR5, human CCR8, and human CX3CR1.
  • the antibody-dependent cellular cytotoxicity can be determined by a reporter gene bioassay (ADCC reporter bioassay) .
  • EC50 is less than 25 ⁇ g/mL, less than 15 ⁇ g/mL, less than 10 ⁇ g/mL, less than 5 ⁇ g/mL, less than 2.5 ⁇ g/mL, less than 1 ⁇ g/mL, less than 0.5 ⁇ g/mL, less than 0.4 ⁇ g/mL, less than 0.3 ⁇ g/mL, less than 0.2 ⁇ g/mL, less than 0.15 ⁇ g/mL, less than 0.1 ⁇ g/mL, less than 0.05 ⁇ g/mL, less than 0.025 ⁇ g/mL, less than 0.01 ⁇ g/mL, less than 0.005 ⁇ g/mL, or less than 0.0025 ⁇ g/mL.
  • EC50 is about 0.0025-0.05 ⁇ g/mL, about 0.005-0.1 ⁇ g/mL, about 0.005-0.05 ⁇ g/mL, about 0.005-0.01 ⁇ g/mL, about 0.01-0.2 ⁇ g/mL, or about 0.025-0.3 ⁇ g/mL.
  • the antibody binds to human CCR8 (SEQ ID NO: 43) .
  • thermal stabilities are determined.
  • the antibodies or antigen binding fragments as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • the antibody has a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.
  • TGI% tumor growth inhibition percentage
  • TGI% can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts.
  • Ti is the average tumor volume in the treatment group on day i.
  • T0 is the average tumor volume in the treatment group on day zero.
  • Vi is the average tumor volume in the control group on day i.
  • V0 is the average tumor volume in the control group on day zero.
  • the antibodies or antigen-binding fragments thereof as described herein block the binding of CCR8 to its ligand (e.g., CCL1) . In some embodiments, the antibodies or antigen-binding fragments thereof as described herein inhibit CCL-1 induced suppressive function of tumor-resident Treg cells.
  • the antibodies or antigen binding fragments can induce complement-dependent cytotoxicity (CMC) and/or antibody dependent cellular cytotoxicity (ADCC) , and kill the cells that express CCR8 (e.g., Treg cells) .
  • CMC complement-dependent cytotoxicity
  • ADCC antibody dependent cellular cytotoxicity
  • the antibodies or antigen binding fragments have a functional Fc region.
  • effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • the antibodies or antigen binding fragments can induce complement mediated cytotoxicity (CMC) .
  • the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
  • the antibody is a human IgG1 antibody.
  • the antibodies or antigen binding fragments do not have a functional Fc region.
  • the antibodies or antigen binding fragments are Fab, Fab’ , F (ab’ ) 2, and Fv fragments.
  • the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
  • the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
  • the antibody is a human IgG1 antibody, optionally with SI mutations, LALA mutations, N297A mutation, YTE mutations, and/or FLAA mutations.
  • the antibody is a human IgG4 antibody, optionally with SI mutations, LALA mutations, N297A mutation, YTE mutations, and/or FLAA mutations.
  • the antibodies or antigen binding fragments do not have a functional Fc region.
  • the antibodies or antigen binding fragments are Fab, Fab’ , F (ab’ ) 2, and Fv fragments.
  • the Fc region has LALA mutations (L234A and L235A mutations according to EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations according to EU numbering) .
  • the Fc region has FLAA mutations (F234A and L235A according to EU numbering) .
  • the Fc has SI mutations (S239D and I332E mutations according to EU numbering) .
  • the Fc has N297A mutation according to EU numbering.
  • the Fc has YTE mutations (M252Y, S254T and T256E according to EU numbering) .
  • An isolated membrane protein of human CCR8 can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation.
  • Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein.
  • the antigenic peptide or protein is injected with at least one adjuvant.
  • the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .
  • the full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens.
  • the antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of CCR8 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
  • the full length sequence of human CCR8 is known in the art (SEQ ID NO: 43) .
  • An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus) .
  • An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., a fragment of human CCR8) .
  • the preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a CCR8 polypeptide, or an antigenic peptide thereof (e.g., part of CCR8) as an immunogen.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized CCR8 polypeptide or peptide.
  • ELISA enzyme-linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) , or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Lis
  • Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
  • Antibodies disclosed herein can be derived from any species of animal, including mammals.
  • Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) , including transgenic rodents genetically engineered to produce human antibodies.
  • Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs.
  • a humanized antibody typically has a human framework (FR) grafted with non-human CDRs.
  • a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., Jones et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse. " Nature 321.6069 (1986) : 522; Riechmann et al.
  • humanized antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.
  • humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s) , is achieved.
  • amino acid sequence variants of the human, humanized, or chimeric anti-CCR8 antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%percent identity with a sequence present in the light or heavy chain of the original antibody.
  • a mouse e.g., RenMab TM mouse with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used to generate antibodies.
  • the heavy chain immunoglobulin locus is a region on the chromosome that contains genes for the heavy chains of antibodies.
  • the locus can include e.g., human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes.
  • the kappa chain immunoglobulin locus is a region on the chromosome that contains genes that encode the light chains of antibodies (kappa chain) .
  • the kappa chain immunoglobulin locus can include e.g., human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes.
  • human IGKV variable
  • human IGKJ joining
  • mouse light chain constant domain genes e.g., RenMab TM mice.
  • RenMab TM mice can be found in PCT/CN2020/075698 or US20200390073A1, which is incorporated herein by reference in its entirety.
  • a mouse e.g., RenLite TM mouse with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used to generate antibodies.
  • the heavy chain immunoglobulin locus is a region on the chromosome that contains genes for the heavy chains of antibodies.
  • the locus can include e.g., human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes.
  • the kappa chain immunoglobulin locus is a region on the chromosome that contains genes that encode a common light chain.
  • the kappa chain immunoglobulin locus can include e.g., a human IGKV (variable) gene, a human IGKJ (joining) gene, and mouse light chain constant domain genes.
  • IGKV variable
  • IGKJ joining
  • mouse light chain constant domain genes e.g., a human IGKV (variable) gene, a human IGKJ (joining) gene, and mouse light chain constant domain genes.
  • RenLite TM mice can be found in PCT/CN2021/097652, which is incorporated herein by reference in its entirety.
  • the antibodies generated by the mice have a full human VH, a full human VL, and mouse constant regions.
  • the human VH and human VL is linked to a human IgG constant regions (e.g., IgG1, IgG2, IgG3, and IgG4) .
  • Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric anti-CCR8 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • a cysteine residue can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo.
  • Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. Wolff et al. (“Monoclonal antibody homodimers: enhanced antitumor activity in nude mice. " Cancer research 53.11 (1993) : 2560-2565) .
  • an antibody can be engineered which has dual Fc regions.
  • a covalent modification can be made to the anti-CCR8 antibody or antigen-binding fragment thereof.
  • These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage.
  • Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody composition may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function.
  • the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
  • the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) .
  • S228P serine at position 228
  • a detailed description regarding S228 mutation is described, e.g., in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. " Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleo
  • a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly-A tail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus.
  • viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y.
  • the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan.
  • the promoter is a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors can include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
  • Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) , which is incorporated herein by reference in its entirety.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • the antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.
  • the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
  • the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) , e.g., breast cancer (e.g., triple-negative breast cancer) , carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, stomach cancer, testis cancer, gallbladder cancer, bile duct cancer, esophagus cancer, thymic carcinoma, acute myeloid leukemia, lymphoma lymphoblastic, transitional cell carcinoma or hematologic malignancy.
  • breast cancer e.g., triple-negative breast cancer
  • the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, or metastatic hormone-refractory prostate cancer.
  • the subject has a solid tumor.
  • the cancer is squamous cell carcinoma of the head and neck (SCCHN) , renal cell carcinoma (RCC) , triple-negative breast cancer (TNBC) , or colorectal carcinoma.
  • the subject has Hodgkin's lymphoma.
  • the subject has triple-negative breast cancer (TNBC) , gastric cancer, urothelial cancer, Merkel-cell carcinoma, or head and neck cancer.
  • the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.
  • the subject has a PD-1 inhibitor resistant cancer. In some embodiments, the subject is not responsive to an anti-PD-1 antibody or an anti-PD-L1 antibody treatment.
  • compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer.
  • Patients with cancer can be identified with various methods known in the art.
  • the disclosure provides methods for treating, preventing, or reducing the risk of developing disorders associated with an abnormal or unwanted immune response, e.g., an autoimmune disorder.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • an effective amount of an antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.
  • Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal.
  • a typical daily dosage of an effective amount of an antibody is 0.01 mg/kg to 100 mg/kg (mg per kg of patient weight) .
  • the dosage can be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg.
  • the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg.
  • the dosage is about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.
  • the at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein
  • at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
  • at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition) .
  • At least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) .
  • the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent) .
  • the at least one additional therapeutic agent is administered as a pill, tablet, or capsule.
  • the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
  • the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) .
  • the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.
  • the subject can be administered the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) .
  • a skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) .
  • a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .
  • one or more additional therapeutic agents can be administered to the subject.
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) .
  • the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase-1) (IDO1)
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, prala
  • therapeutic agents
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.
  • TNF tumor necrosis factor
  • carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.
  • the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-B7-H3 antibody, an anti-CLDN18 antibody, an anti-SIGLEC15 antibody, an anti-41BB antibody, an anti-CD40 antibody, or an anti-GITR antibody.
  • the additional therapeutic agent is a PD-1 pathway inhibitor, e.g., an anti-PD-1 antibody or anti-PD-L1 antibody.
  • exemplary anti-PD-1 antibodies include e.g., Pembrolizumab, Nivolumab, Cemiplimab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Toripalimab, and Dostarlimab.
  • Exemplary anti-PD-L1 antibodies include e.g., Atezolizumab, Avelumab, Durvalumab, and KN035.
  • compositions that contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination.
  • the pharmaceutical compositions may be formulated in any manner known in the art.
  • compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) .
  • the compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) .
  • an agent that delays absorption e.g., aluminum monostearate and gelatin
  • controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) .
  • biodegradable, biocompatible polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid
  • compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .
  • parenteral e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal
  • dosage unit form i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration) .
  • Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen.
  • antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) .
  • Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) .
  • Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
  • a therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) .
  • any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .
  • Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject’s weight (e.g., about 1 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 50 mg/kg; about 10 ⁇ g/kg to about 5 mg/kg; about 10 ⁇ g/kg to about 0.5 mg/kg; about 1 ⁇ g/kg to about 50 ⁇ g/kg; about 1 mg/kg to about 10 mg/kg; or about 1 mg/kg to about 5 mg/kg) .
  • weight e.g., about 1 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 50 mg/kg; about 10 ⁇ g/kg to about 5 mg/kg; about 10 ⁇ g/kg to about 0.5 mg/kg; about 1 ⁇ g/kg to about 50 ⁇ g/kg; about 1
  • therapeutic agents including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art.
  • relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained.
  • the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.
  • RenMice TM i.e., engineered mice comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions, for example, RenMab TM mice, RenLite TM mice
  • the antibody immune response was monitored by an antigen-specific immunoassay.
  • FACS Fluorescence-Activated Cell Sorting
  • Spleen tissues were then collected and grinded.
  • Spleen cells were first selected by CD3 ⁇ microbeads and anti-mouse IgM Microbeads, and then fused with SP2/0 cells (to generate hybridoma cells) .
  • the cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.
  • HAT hypoxanthine-aminopterin-thymidine
  • antigen-specific immune cells were isolated from the immunized mice to further obtain anti-CCR8 antibodies or to obtain the light chain and heavy chain variable region sequences of the anti-CCR8 antibodies.
  • single cell technology for example, using Optofluidic System, Berkeley Lights Inc.
  • reverse transcription and PCR sequencing were used to obtain antibody variable region sequences.
  • the obtained variable region sequences were used for antibody expression.
  • the binding affinity to CCR8 was determined using FACS.
  • phage display was performed to screen and find monoclonal antibodies that are antigen-specific.
  • Exemplary antibodies obtained by this method included: 11B12, 11F8 and 11F5.
  • the “DG” in VH CDR2 of the antibodies described herein was mutated to “SG, ” “AG” or “DA” to create CDR-modified VHs.
  • the “DG” in VH CDR2 of 11B12 was mutated to “SG, ” and the resulting CDR-modified VH is named 11B12-SG VH.
  • the sequences of some of these CDR-modified VHs can be found in FIG. 4.
  • the “DG” in VL CDR1 of the antibodies described herein was mutated to “SG, ” “AG, ” or “DA, ” to create CDR-modified VLs.
  • the “DG” in VL CDR1 of 11B12 was mutated to SG, and the resulting CDR-modified VL is named 11B12-SG VL.
  • the sequences of some of these CDR-modified VLs can be found in FIG. 4.
  • the “DNS” in the VH framework region of the antibodies described herein was mutated to “DNA, ” to create FR-modified VHs.
  • the “DNS” in the framework of the VH of 11B12 was mutated to “DNA, ” and the resulting FR-modified VH is named 11B12-DNA VH.
  • the sequences of some of these FR-modified VHs can be found in FIG. 4 (e.g., SEQ ID NOs: 69-71) .
  • the antibody has a S74A mutation (Kabat numbering) .
  • the VH was modified by both a FR modification (e.g., DNA) and a CDR modification (e.g., SG) .
  • a FR modification e.g., DNA
  • a CDR modification e.g., SG
  • the “DNS” in the framework of the VH of 11B12 is mutated to “DNA, ” and the “DG” in VH CDR2 of 11B12 is mutated to “SG, ” and the resulting FR-modified and CDR-modified VH is named 11B12-SG-DNA VH.
  • the sequence of the resulting FR-modified and CDR-modified VH can be found in FIG. 4 (e.g., SEQ ID NO: 216) .
  • the amino acid “N” at position 3 in VH CDR3 of the 11F5 was mutated to “S, ” the amino acid “Y” at position 5 in VH CDR3 of the 11F5 was mutated to “V, ” and/or the amino acid “Y” at position 6 in VH CDR3 of the 11F5 was mutated to “N” to create 11F5 CDR3-modified VHs.
  • the amino acid “N” at position 3 in VH CDR3 of the 11F5 was mutated to “S” and the resulting CDR-modified VH is named 11F5- N3S VH.
  • the amino acid “Y” at position 5 in VH CDR3 of the 11F5 was mutated to “V” and the resulting CDR-modified VH is named 11F5-Y5V VH.
  • the amino acid “Y” at position 6 in VH CDR3 of the 11F5 was mutated to “N” and the resulting CDR-modified VH is named 11F5-Y6N VH.
  • the amino acid “N” at position 3 and amino acid “Y” at position 5 in VH CDR3 of the 11F5 were mutated to “S” and “V, ” respectively, and the resulting CDR-modified VH is named 11F5-N3S-Y5V VH.
  • the amino acid “N” at position 3 and the amino acid “Y” at position 6 in VH CDR3 of the 11F5 were mutated to “S” and “N, ” respectively, and the resulting CDR-modified VH is named 11F5-N3S-Y6N VH.
  • the amino acid “Y” at position 5 and the amino acid “Y” at position 6 in VH CDR3 of the 11F5 were mutated to “V” and “N, ” respectively, and the resulting CDR-modified VH is named 11F5-Y5V-Y6N VH.
  • the VH of an antibody described herein was paired with the VL of another antibody described herein to form antibodies.
  • the VH of 11B12 was paired with the VL of 11F8, and the resulting antibody is named 11B12-11F8.
  • the VH of 11B12-SG was paired with the VL of 11F8-SG, and the resulting antibody is named 11B12-SG-11F8-SG.
  • the VH of 11B12-SG was paired with the VL of 11B12-SG, and the resulting antibody is named 11B12-SG-11B12-SG.
  • the VH of 11B12-SG-DNA was paired with the VL of 11F8, and the resulting antibody is named 11B12-SG-DNA-11F8.
  • FIG. 5 The sequences of the heavy and light chain variable regions of these antibodies are shown in FIG. 4.
  • FIG. 2A and FIG. 3A show the heavy and light chain CDR sequences of 11B12, 11F8, and 11F5 according to Kabat definition and Chothia definition, respectively.
  • IgG1, IgG2 and IgG4 antibodies were made. If the VH and VL of 11B12 are connected to IgG1 constant regions, the antibody is named as 11B12. If the VH and VL of 11B12 are connected to IgG4 constant regions, the antibody is named as 11B12-IgG4.
  • the constant region can also include some mutations. For example, when SI mutations (EU numbering: S239D and I332E mutations) are introduced into the Fc region of 11B12, the resulting antibody is named as 11B12-SI.
  • CHO-S cells expressing human CCR8 (CHO-S-hCCR8) , CHO-S cells expressing mouse CCR8 (CHO-S-mCCR8) , HEK293T cells expressing human CCR8 (293T-hCCR8) and HEK293T cells expressing cynomolgus CCR8 (293T-cynCCR8) were plated in a 96-well plate at a density of 5 ⁇ 10 4 cells/well. Diluted sample anti-CCR8 antibodies (1 ⁇ g/mL) were added to the 96-well plate, and incubated at 4°C for 30 minutes.
  • the cells were incubated with the secondary antibody anti-hIgG-Fc-Alexa 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170) at 4°C in the dark for 15 minutes before flow cytometry analysis.
  • RL1-H secondary antibody anti-hIgG-Fc-Alexa 647
  • CHO-S-hCCR8 cells were obtained by transfecting CHO-S cells with a vector encoding human CCR8 (hCCR8, SEQ ID NO: 43) .
  • CHO-S-mCCR8 cells were obtained by transfecting CHO-S cells with a vector encoding mouse CCR8 (mCCR8, SEQ ID NO: 44) .
  • 293T-hCCR8 cells were obtained by transfecting HEK293T cells (ATCC, Cat#: CRL-3216 TM ) with a vector encoding human CCR8.
  • 293T-cynCCR8 cells were obtained by transfecting HEK293T cells with a vector encoding a construct containing the amino acid sequence of cynomolgus (monkey) CCR8 (cynCCR8, SEQ ID NO: 45) .
  • the CHO cells were transfected to express human CCR8 (CHO-S-hCCR8) , human CCR2 (hCCR2, SEQ ID NO: 46) (CHO-S-hCCR2) , human CCR4 (hCCR4, SEQ ID NO: 47) (CHO-S-hCCR4) , human CCR5 (hCCR5, SEQ ID NO: 48) (CHO-S-hCCR5) , or human CX3CR1 (hCX3CR1, SEQ ID NO: 49) (CHO-S-hCX3CR1) .
  • CHO-S-hCCR8, CHO-S-hCCR2, CHO-S-hCCR4, CHO-S-hCCR5 and CHO-S-hCX3CR1 were plated in a 96-well plate at a density of 5 ⁇ 10 4 cells/well. Diluted sample anti-CCR8 antibodies (1 ⁇ g/mL) were added to the 96-well plate, and incubated at 4°C for 30 minutes. Then, the cells were incubated with the secondary antibody Anti-hIgG-Fc-Alexa 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170) at 4°C in the dark for 15 minutes before flow cytometry analysis.
  • RL1-H secondary antibody Anti-hIgG-Fc-Alexa 647
  • Plozalizuzumab (MLN1202) is a humanized immunoglobulin G1 (IgG1) anti-CCR2 monoclonal antibody developed by Millenium Pharmaceuticals (Takeda) .
  • the VH and VL sequences of Plozalizuzumab analog are shown as SEQ ID NO: 50 and SEQ ID NO: 51, respectively.
  • Mogamulizumab (KW-0761) is a humanized immunoglobulin G1 (IgG1) anti-CCR4 monoclonal antibody developed by Kyowa Kirin.
  • the VH and VL sequences of Mogamulizumab analog-SI are shown as SEQ ID NO: 52 and SEQ ID NO: 53, respectively.
  • Leronlimab is a fully humanized IgG4 monoclonal antibody targeting CCR5, developed by CytoDyn.
  • the VH and VL sequences of Leronlimab analog are shown as SEQ ID NO: 54 and SEQ ID NO: 55, respectively.
  • BII-00313 is a single heavy chain variable domain (VHH) antibody targeting human CX3C chemokine receptor 1 (CX3CR1) variants, developed by Boehringer-Ingelheim (VH SEQ ID NO: 56) .
  • VHH variable chain variable domain
  • CX3CR1 CX3C chemokine receptor 1
  • BII-00313-FC-LALA was constructed by linking BII-00313 variable domain to IgG1 Fc with LALA mutations (EU numbering: L234A and L235A mutations) .
  • anti-CCR8 antibodies 11B12, 11F8, 11F5 and 7B16 analog
  • 293T-hCCR8 cells HEK293T cells expressing human CCR8
  • 293T-cynCCR8 HEK293T cells expressing cynomolgus CCR8
  • Alexa 647 anti human IgG Fc ⁇ Jackson ImmunoResearch Laboratories, Inc., Cat#: 109-606-170
  • EC50 was determined using serially diluted sample anti-CCR8 antibodies. The results are shown in the table below.
  • 7B16 is a humanized immunoglobulin G1 (IgG1) anti-CCR8 monoclonal antibody developed by JOUNCE THERAPEUTICS (see WO2021163064A2) .
  • the VH and VL sequences of 7B16 analog are shown as SEQ ID NO: 59 and SEQ ID NO: 60, respectively.
  • 10A11 is a humanized immunoglobulin G1 (IgG1) anti-CCR8 monoclonal antibody developed by Shionogi (see WO2020138489A1) .
  • the VH and VL sequences of 10A11 analog are shown as SEQ ID NO: 57 and SEQ ID NO: 58, respectively.
  • Blockade of calcium flux on hCCR8-expressing cells by anti-hCCR8 mAbs Blockade of hCCL1 binding to hCCR8 by human IgG1 anti-CCR8 antibodies and CDR-modified and FR-modified anti-CCR8 antibodies were tested by conducting calcium (Ca) flux assays using human CCR8-expressing 293T cells. Fluorescent Imaging Plate Reader (FLIPR) Calcium 6 Assay Kit (Molecular Devices, Cat#: R8190) was used. Cells were seeded the day prior to the experiment and incubated overnight at 37°C, 5%CO 2 .
  • FLIPR Fluorescent Imaging Plate Reader
  • the cells were then loaded with FLIPR Calcium 6 dye (Molecular Devices) including probenecid (Invitrogen) and PowerLoad (Invitrogen) , and incubated for 30 minutes at 37°C. Different anti-CCR8 mAbs were added to the cells and further incubated at 37°C for 30 minutes. After incubation, the Ca flux fluorescent signal induced by 1 ⁇ g/mL of recombinant human CCL1 (BioLegend, Cat#: 582706) was measured using iD3 (Molecular Devices) for 1 minute. The results are showed in the table below.
  • FLIPR Calcium 6 dye Molecular Devices
  • probenecid Invitrogen
  • PowerLoad Invitrogen
  • 11B12, 11F8, 11B12-11F8, 11F8-SG-11F8-SG, 11F5-SG-11F5-SG, 11B12-SG-11F8 and 11B12-DNA-11B12 showed almost complete inhibition.
  • the inhibition of 10A11 analog was partial even at 10 ⁇ g/mL.
  • Three anti-CCR8 antibodies 11B12, 11F8 and 11F5 were buffer exchanged into pH 6.0 (3 mg/mL histidine, 80 mg/mL sucrose, and 0.2 mg/mL Tween 80) .
  • the antibodies were kept in sealed Eppendorf tubes at 40 ⁇ 2 °C, 60% ⁇ 5%RH (hereinafter referred to as 40°C) and 4 ⁇ 3°C (hereinafter referred to as 4°C) for 14 days, and their thermal stability were evaluated.
  • Three anti-CCR8 antibodies 11B12, 11F8 and 11F5 were loaded into protein A column and eluted with a buffer (0.1 mol/L HAc) at pH 3.5. Half of the antibodies were added into 2 M Tris buffer to make the pH into 7.5 immediately. The remain half were kept at pH 3.5 for 6 hours and then adjusted the pH into 7.5. The diluted antibodies were kept in sealed Eppendorf tubes at pH 3.5 ⁇ 0.1, 25 ⁇ 2°C (hereinafter referred to as pH 3.5) for 6 hours to test stability at low pH.
  • Three anti-CCR8 antibodies 11B12, 11F8 and 11F5 were buffer exchanged into pH 6.0 (3 mg/mL histidine, 80 mg/mL sucrose, and 0.2 mg/mL 80) and added NH 4 HCO 3 stock to final of 0.94%NH 4 HCO 3.
  • Samples were kept in sealed Eppendorf tubes at 40 ⁇ 2°C, 60% ⁇ 5%RH for 6 hours (hereinafter referred to as NH 4 HCO 3 6h) , or 24 hours (hereinafter referred to as NH 4 HCO 3 24h) to test stability.
  • the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatograph system (connected with XBridge Protein BEH SEC column ( Waters Corporation) ) was used.
  • the following parameters were used: mobile phase: 0.1M phosphate buffer (PB) +10%ACN, pH 7.4; flow rate: 1.8 mL/min; column temperature: 25°C; detection wavelength: 280 nm, 220 nm; injection volume: 10 ⁇ L; sample tray temperature: about 4°C; and running time: 7 minutes.
  • a Maurice TM cIEF Method Development Kit (ProteinSimple, Cat#: PS-MDK01-C) was used for sample preparation. Specifically, 8 ⁇ L, 30 ug protein sample was mixed with the following reagents in the kit: 1 ⁇ L Maurice TM cIEF pI Marker-7.05, 1 ⁇ L Maurice TM cIEF pI Marker-10.10, 35 ⁇ L 1%Methyl Cellulose Solution, 2 ⁇ L Maurice TM cIEF 500 mM Arginine, 1.33 ⁇ L Ampholytes (Pharmalyte pH ranges 3-10) , and 6.66 ⁇ L Ampholytes (Pharmalyte pH ranges 8-10.5) water (added to make a final volume of 100 ⁇ L) .
  • Maurice TM analyzer ProteinSimple, Santa Clara, CA
  • Maurice TM cIEF Cartridges PS-MC02-C
  • CE-SDS NR
  • 30 ⁇ L Sample Buffer 30 ⁇ L, 30 ⁇ g antibody sample, 1.5 ⁇ L 25 ⁇ internal standard, 3 ⁇ L 250 nM Iodoacetamide (SIGMA, Cat#: 16125) were add to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 minute and heating in a 70°C water bath for 10 minutes. The samples were then cooled to room temperature followed by centrifugation at 10000 rpm for 3 minutes. Supernatant sample preparations were then transferred to a 96-well plate and tested in Maurice TM . The following parameters were used: injection voltage 4.6 kV, injection time 20 sec, separation voltage 5.75 kV, and separation time 40 min.
  • ADCC reporter bioassay engineered Jurkat cells were used as effector cells (Jurkat-Luc-hCD16A-158V) , and H293T cells with hCCR8 surface antigen were used as target cells. The cells were incubated with each antibody at a ratio of 4: 1 for 6 hours to determine ADCC activity. The results are shown in the table below.
  • Anti-CCR8 antibodies were tested for their effects on tumor growth in vivo in a colon cancer model.
  • About 5 ⁇ 10 5 MC38 cells (murine colon cancer cells) were injected subcutaneously in each B-hCCR8 mouse (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Beijing, China; Cat#: 110096) .
  • B-hCCR8 mouse Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Beijing, China; Cat#: 110096) .
  • the tumors in the mice reached a volume of about 100 mm 3
  • the mice were randomly placed into different groups based on tumor volumes.
  • the mice were then injected with phosphate buffer saline (PBS) or anti-human CCR8 antibodies by intraperitoneal (i. p. ) administration.
  • PBS phosphate buffer saline
  • anti-human CCR8 antibodies intraperitoneal (i. p. ) administration.
  • the injected volume was calculated based on the weight of the mouse and desired dosage of 10 mg/kg.
  • the length of the long axis and the short axis of the tumor were measured and the volume of the tumor was calculated as 0.5 ⁇ (long axis) ⁇ (short axis) 2 .
  • TGI tumor growth inhibition percentage
  • mice in different groups all increased. On the day of grouping (Day 0) , the average weight of each group was in the range of 19.8 g-20.3 g. At the end of the experiment (Day 21) , the average weight of each group was in the range of 21.6 g-24.6 g, the average weight of each group was in the range of 111.4%-121.8%. The results showed that the tested antibodies were well tolerated and were not obviously toxic to the mice.
  • the tumor size in groups treated with the anti-CCR8 antibodies are shown in FIG. 1.
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 7 days after the grouping (Day 7) , 14 days after the grouping (Day 14) , and at the end of the experiment (Day 21) ; the survival rate of the mice; Tumor Growth Inhibition value (TGI) ; and the statistical differences (P value) of tumor volume and body weight between the treatment and control groups.
  • anti-CCR8 antibodies 11F5, 11F5-SI and/or 11F8-SI were tested for their effects on tumor growth in vivo in the colon cancer model at a dosage of 3 mg/kg.
  • Table 13 and Table 14 summarize the tumor volumes on the day of grouping (Day 0) , 14 days after the grouping (Day 14) , and 21 days after the grouping (Day 21) ; Tumor Growth Inhibition value (TGI) ; and the statistical differences (P value) of tumor volume and body weight between the treatment and control groups in two different experiments.
  • 11F8-SI and 11B12-SI were also tested for there effects on tumor growth in a colon cancer model.
  • About 5 ⁇ 10 5 MC38 cells were injected subcutaneously in B-hPD-1/hPD-L1/hCCR8 mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Beijing, China; Cat#: 131764) .
  • the tumors in the mice reached a volume of about 100-150 mm 3
  • the mice were randomly placed into different groups based on tumor volumes (6 mice in each group) .
  • the mice were then injected with PBS (G1, control) , 3 mg/kg 11F8-SI (G2) , and 3 mg/kg 11B12-SI (G3) by intraperitoneal injection.
  • the antibody was administered on the first day and the fourth day of each week for 3 weeks (6 injections in total) .
  • the binding activity of anti-CCR8 antibodies to 293T-hCCR8 cells was verified by flow cytometry.
  • the secondary antibody used in the experiment was: PE Goat ⁇ -Human IgG Fc (SouthernBiotech, Cat#: 2014-09) .
  • EC50 was determined using serially diluted sample anti-CCR8 antibodies. The results are shown in the table below.
  • anti-CCR8 antibodies were administered to mice with another therapeutic agent Pembrolizumab analog.
  • Pembrolizumab analog is a humanized immunoglobulin G1 (IgG1) anti-PD-1 monoclonal antibody.
  • the VH and VL sequences of Pembrolizumab analog are shown as SEQ ID NO: 217 and SEQ ID NO: 218, respectively.
  • mice About 5 ⁇ 10 5 MC38 cells (murine colon cancer cells) were injected subcutaneously in B-hPD-1/hPD-L1/hCCR8 mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Beijing, China; Cat#: 131764) .
  • the tumors in the mice reached a volume of about 100-150 mm 3
  • the mice were randomly placed into different groups based on tumor volumes (6 mice in each group) .
  • the mice were then injected with PBS (G1, control) , 0.3 mg/kg Pembrolizumab analog (G2) , 3 mg/kg 11F5 (G3) , or a combination of 0.3 mg/kg Pembrolizumab analog and 3 mg/kg 11F5 (G4) by intraperitoneal injection.
  • the antibody was administered on the first day and the fourth day of each week for 3 weeks (6 injections in total) .
  • mice The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased. On the day of grouping (Day 0) , the average body weight of each group was in the range of 18.2 g-19.2 g. At the end of the experiment (Day 28) , the average body weight of each group was in the range of 21.7 g-23.5 g, and the average body weight of each group was in the range of 114.4%-123.3%. The results showed that the tested antibodies were well tolerated and were not obviously toxic to the mice.
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after the grouping (Day 14) and at the end of the experiment (Day 28) ; the survival rate of the mice; Tumor Growth Inhibition value (TGI) ; and the statistical differences (P value) of tumor volume and body weight between the treatment and control groups.
  • the tumor size for each treatment group is also shown in FIG. 6.
  • the results showed that, as compared to the control group (G1) , the treatment group (G2-G4) exhibited different anti-tumor effects.
  • the treatment group (G2-G4) exhibited different anti-tumor effects.
  • combination of 11F5 and Pembrolizumab analog (G4) inhibited tumor growth with a higher TGI%than that of 11F5 (G3) or Pembrolizumab analog (G2) .
  • the tumor size for each treatment group is shown in FIG. 7.
  • the results showed that the combination of 11F5-SI and Pembrolizumab analog (G4) inhibited tumor growth with a significantly higher TGI% (77.49%) than that of 11F5-SI (G3) or Pembrolizumab analog (G2) .
  • the combination of 11B12-SI and Pembrolizumab analog (G6) inhibited tumor growth with a significantly higher TGI% (74.52%) than that of 11B12-SI (G5) or Pembrolizumab analog (G2) .
  • engineered Jurkat cells were used as effector cells (Jurkat-Luc-hCD16A-158V)
  • H293T cells with hCCR8 surface antigen were used as target cells.
  • the cells were incubated with each antibody at a ratio of 4: 1 for 6 hours to determine ADCC activity. The results are shown in the table below.
  • the biophysical properties of 11F5 and its CDR3-modified antibodies were evaluated. Similar experiments described in Example 6 were carried out. In addition, the colloidal stability detection using Standup Monolayer Chromatography (SMAC) method (indicated as the retention time of the main peak (SMAC, min) and cross-interaction detection with human serum polyclonal antibodies using Cross-Interaction Chromatography (CIC) method (indicated as the retention time of the main peak (CIC, min) were performed.
  • SMAC Standup Monolayer Chromatography
  • CIC Cross-Interaction Chromatography
  • Example 15 Blockade of calcium flux on hCCR8-expressing cells by 11F5 and its CDR3-modified antibodies
  • Blockade of hCCL1 binding to hCCR8 by 11F5 and its CDR3-modified antibodies were tested by conducting calcium (Ca) flux assays using human CCR8-expressing 293T cells.
  • Fluorescent Imaging Plate Reader (FLIPR) Calcium 6 Assay Kit (Molecular Devices, Cat#: R8190) was used. Cells were seeded the day prior to the experiment and incubated overnight at 37°C, 5%CO 2 . The cells were then loaded with FLIPR Calcium 6 dye (Molecular Devices) including probenecid (Invitrogen) and PowerLoad (Invitrogen) , and incubated for 30 minutes at 37°C. Anti-CCR8 antibodies were added to the cells and further incubated at 37°C for 30 minutes.
  • 11F5 and its CDR3-modified antibodies were tested for their effects on tumor growth in vivo in a colon cancer model.
  • About 5 ⁇ 10 5 MC38 cells were injected subcutaneously in each B-hCCR8 mouse.
  • the mice were randomly placed into different groups based on tumor volumes.
  • the mice were then injected with phosphate buffer saline (PBS) , 11F5 or its CDR3-modified antibodies by intraperitoneal (i. p. ) administration.
  • PBS phosphate buffer saline
  • the injected volume was calculated based on the weight of the mouse and desired dosage of 3 mg/kg (Details are shown in the table below) .
  • the table below summarizes the results for this experiment, including the tumor volumes on the day of grouping (Day 0) , 14 days after the grouping (Day 14) , and on the 21 days of the experiment (Day 21) ; the survival rate of the mice; Tumor Growth Inhibition value (TGI) ; and the statistical differences (P value) of tumor volume and body weight between the treatment and control groups.

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Abstract

L'invention concerne des anticorps anti-CCR8 (récepteur 8 de chémokine (motif C-C)), des fragments de liaison à l'antigène de ceux-ci et leurs utilisations.
PCT/CN2023/126829 2022-10-28 2023-10-26 Anticorps anti-ccr8 et leurs utilisations WO2024088346A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007044756A2 (fr) * 2005-10-11 2007-04-19 Icos Corporation Anticorps monoclonaux reconnaissant le ccr8 humain
CN103857421A (zh) * 2011-06-13 2014-06-11 Ith免疫治疗控股股份公司 治疗癌症
CN110835371A (zh) * 2018-08-19 2020-02-25 普米斯生物技术(苏州)有限公司 抗ccr8单克隆抗体及其应用
WO2021152186A2 (fr) * 2020-06-26 2021-08-05 Bayer Aktiengesellschaft Anticorps ccr8 pour applications thérapeutiques
WO2021194942A1 (fr) * 2020-03-23 2021-09-30 Bristol-Myers Squibb Company Anticorps anti-ccr8 pour le traitement du cancer
CN115052892A (zh) * 2020-10-16 2022-09-13 礼新医药科技(上海)有限公司 抗ccr8单克隆抗体及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007044756A2 (fr) * 2005-10-11 2007-04-19 Icos Corporation Anticorps monoclonaux reconnaissant le ccr8 humain
CN103857421A (zh) * 2011-06-13 2014-06-11 Ith免疫治疗控股股份公司 治疗癌症
CN110835371A (zh) * 2018-08-19 2020-02-25 普米斯生物技术(苏州)有限公司 抗ccr8单克隆抗体及其应用
WO2021194942A1 (fr) * 2020-03-23 2021-09-30 Bristol-Myers Squibb Company Anticorps anti-ccr8 pour le traitement du cancer
WO2021152186A2 (fr) * 2020-06-26 2021-08-05 Bayer Aktiengesellschaft Anticorps ccr8 pour applications thérapeutiques
WO2021260210A2 (fr) * 2020-06-26 2021-12-30 Bayer Aktiengesellschaft Thérapie par anticorps anti-ccr8 : biomarqueurs et polythérapies
CN115052892A (zh) * 2020-10-16 2022-09-13 礼新医药科技(上海)有限公司 抗ccr8单克隆抗体及其用途

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