WO2023174396A1 - 一种新型免疫调节剂的开发和应用 - Google Patents

一种新型免疫调节剂的开发和应用 Download PDF

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WO2023174396A1
WO2023174396A1 PCT/CN2023/082082 CN2023082082W WO2023174396A1 WO 2023174396 A1 WO2023174396 A1 WO 2023174396A1 CN 2023082082 W CN2023082082 W CN 2023082082W WO 2023174396 A1 WO2023174396 A1 WO 2023174396A1
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variable region
chain variable
variant
seq
amino acid
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PCT/CN2023/082082
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French (fr)
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黄鹂
陈博
王常玉
徐刚
刘瑞勤
张斌
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北京天诺健成医药科技有限公司
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Priority to AU2023233627A priority Critical patent/AU2023233627A1/en
Publication of WO2023174396A1 publication Critical patent/WO2023174396A1/zh

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the present disclosure relates to an immunomodulator and its application, in particular to an anti-CCR8 antibody and its application.
  • Chemokine receptor 8 (CCR8) is a member of the CC chemokine receptor superfamily. Its expression is up-regulated in activated type II Th cells. Its ligand CCL1/I-309 can effectively recruit activated type II Th cells. CCL1 and The interaction of CCR8 (also known as the CCL1/CCR8 axis) is associated with type II Th cell-mediated immune responses (D'Ambrosio, D., et al. 1998.
  • chemokine receptors CCR4 and CCR8 upon activation of polarized human type 2 Th cells.
  • the chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells.
  • CCL1 can activate the CCR8-mediated RAS/MAPK signaling pathway, promote the release of intracellular calcium flux, and inhibit apoptosis (Denis, C., et al. 2012.
  • C-terminal clipping of chemokine CCL1/I-309 enhances CCR8- mediated intracellular calcium release and anti-apoptotic activity.
  • CCR8 also plays an important role in maintaining regulatory T cell (Treg) survival and inhibiting graft-versus-host response (GVHD) (Coghill, JM, et al.
  • CCR8 + Treg are considered to be key cells driving immunosuppression and are potential therapeutic targets for autoimmune diseases (Barsheshet, Y., et al. 2017. CCR8(+)FOXp3(+)T(reg) cells as master drivers of immune regulation. Proc Natl Acad Sci U S A 114(23):6086-6091).
  • Treg cells are also considered to be a key factor in suppressing tumor immunity.
  • tumor patients with low Treg ratios have higher survival rates, including ovarian cancer, breast cancer, non-small cell lung cancer, and non-small cell lung cancer.
  • cell lung cancer hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, cervical cancer and colorectal cancer (Bates, GJ, et al. 2006. Quantification of regulatory T cells enables the identification of high-risk breast cancer patients and those at risk of late relapse.J Clin Oncol 24(34):5373-80; Curiel, et al. 2004; Gao, Q., et al. 2007.
  • Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection.J Clin Oncol 25(18):2586-93; Griffiths,RW,et al 2007. Frequency of regulatory T cells in renal cell carcinoma patients and investigation of correlation with survival.Cancer Immunol Immunother 56(11):1743- 53; Hiraoka, N., et al. 2006. Prevalence of FOXP3+regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions.
  • tumor-infiltrating Treg have significant commonalities, including CCR8 and other series that are related to Treg differentiation.
  • Related genes are up-regulated, and anti-CCR8 antibodies can cause tumor regression in mice after treatment (Magnuson, AM, et al. 2018 Identification and validation of a tumor-infiltrating Treg transcriptional signature conserved across species and tumor types. Proc Natl Acad Sci U S A 115 (45):E10672-e10681). Villarreal D et al.
  • anti-CCR8 antibodies can significantly inhibit tumor growth in colorectal cancer mouse models, with increased anti-tumor activity and tumor-specific T cells, enhanced CD4+ and CD8+ T cell infiltration, and CD4+CCR8+ within the tumor.
  • Tregs are significantly reduced (Villarreal, DO, et al. 2018 Targeting CCR8 Induces Protective Antitumor Immunity and Enhances Vaccine-Induced Responses in Colon Cancer. Cancer Res 78(18):5340-5348).
  • CCR8 is significantly increased on tumor-infiltrating Treg cells, which is beneficial for antibodies targeting CCR8 to distinguish tumor-infiltrating Treg cells from normal T cells. Therefore, the development of monoclonal antibodies targeting CCR8 to effectively eliminate tumor-infiltrating Treg cells or simultaneously inhibit their activity is an urgently needed immunotherapy in clinical practice.
  • the present disclosure relates to new high-affinity CCR8 monoclonal antibodies and humanized antibodies, which have neutralizing activity that blocks the CCL1/CCR8 axis, can inhibit CCL1-induced cell activation, and at the same time target low-affinity Fc ⁇ RIIIA 158F allele carriers in the population. , the ADCC effect is enhanced through Fc optimization.
  • the new humanized antibody can effectively eliminate CCR8+ cells and significantly inhibit the growth of MC38, B16/F10 and MDA-MB-231 tumors in the humanized mouse subcutaneous transplant tumor model.
  • Outstanding efficacy of anti-PD-1 antibody combination Compared with the existing technology, the CCR8 antibody in the present disclosure has a completely new sequence and significant biological activity advantages.
  • the present disclosure relates to novel anti-CCR8 antibodies or antigen-binding portions thereof for inhibiting CCL1-induced calcium flux and improving the ADCC killing activity of effector cells. Further, the present disclosure relates to novel anti-CCR8 antibodies or antigen-binding portions thereof for the treatment of cancer.
  • the present disclosure discloses a monoclonal antibody, or antigen-binding portion thereof, that binds CCR8.
  • the present disclosure provides nucleic acids encoding the aforementioned CCR8-binding antibodies or antigen-binding portions thereof.
  • the present disclosure provides vectors comprising the aforementioned nucleic acids.
  • the present disclosure provides cells comprising the aforementioned nucleic acid or the aforementioned vector.
  • compositions comprising the aforementioned antibodies or antigen-binding portions thereof, encoding nucleic acids, vectors, and cells.
  • kits comprising the aforementioned antibodies or antigen-binding portions thereof, encoding nucleic acids, vectors, cells, and compositions.
  • the present disclosure provides a method for treating cancer-related diseases, comprising the steps of: administering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof, nucleic acid, or vector of any of the foregoing aspects, Cells and/or compositions.
  • the disclosure provides the use of the antibody or antigen-binding portion thereof, nucleic acid, vector, cell and/or pharmaceutical composition of any of the preceding aspects in the preparation of a medicament for treating cancer in a subject.
  • Figure 1 shows the FACS detection results of cell lines stably expressing CCR8 or CCR4, where:
  • Human CCR8 is stably transfected into the HEK293 cell line HEK293-hCCR8;
  • Figure 2 shows the FACS detection results of anti-CCR8 chimeric antibodies, showing high affinity binding of anti-CCR8 chimeric antibodies to human or cynomolgus monkey CCR8+ cell lines.
  • Figure 3 shows the ADCC activity of anti-CCR8 chimeric antibodies.
  • Figure 4 shows that the anti-CCR8 chimeric antibody has significant inhibitory activity on CCL1-induced calcium flux in target cells.
  • Figure 5 shows the binding of the anti-CCR8 chimeric antibody CDR sequence to CCR8+ cells after optimization, where:
  • Figure 6 shows high affinity binding of humanized anti-CCR8 antibodies to human CCR8+ cells.
  • Figure 7 shows high affinity binding of humanized anti-CCR8 antibodies to cynomolgus monkey CCR8+ cells.
  • Figure 8 shows the ADCC killing activity of humanized anti-CCR8 antibodies using NK92 cells as effector cells.
  • FIG 9 shows the ADCC killing activity of humanized anti-CCR8 antibodies with enhanced Fc function on human CCR8+ cells with different expression levels, wherein:
  • Figure 10 shows the inhibitory activity of humanized anti-CCR8 antibodies on CCL1-induced Ca 2+ flux signals.
  • Figure 11 shows high affinity binding of humanized anti-CCR8 antibodies to rabbit CCR8+ cells.
  • Figure 12 shows that humanized anti-CCR8 antibodies significantly inhibit the growth of MC38 subcutaneous xenograft tumors in humanized mice.
  • Figure 13 shows that the humanized anti-CCR8 antibody significantly inhibits the growth of B16/10 subcutaneous transplanted tumors in humanized mice.
  • the humanized anti-CCR8 antibody can more effectively inhibit the growth of B16/10 subcutaneous xenograft tumors in humanized mice. Tumor growth.
  • Figure 14 shows that humanized anti-CCR8 antibodies significantly inhibit the growth of MDA-MB-231 subcutaneous xenograft tumors in humanized mice.
  • antibodies that specifically bind CCR8 and antigen-binding fragments thereof.
  • antibodies that specifically bind CCR8 eg, human CCR8.
  • anti-CCR8 antibodies comprising modifications in one or more amino acid residues (e.g., 1-3 amino acid substitutions in the framework region of the heavy chain variable region), versus no such modifications It retains its affinity for the antigen compared to the parent antibody.
  • CCR8 refers to any CCR8 receptor known to those skilled in the art.
  • the CCR8 can be from a mammal, for example, the CCR8 can be from a human or a cynomolgus monkey.
  • the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where an integer is required, the term means rounding up or down to the nearest integer within plus or minus 10% of a given value or range.
  • the phrase "substantially identical” is understood to mean exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains with 97%, 98%, 99% or more sequence identity.
  • a nucleic acid sequence the term is understood to mean exhibiting at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or higher sequence identity.
  • Sequence "identity” or “identity” has art-recognized meanings, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Pres, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
  • identity is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • substitution variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its same position.
  • the substitution may be single, where only one amino acid is substituted in the molecule, or multiple, where two or more amino acids of the same molecule are substituted. Multiple substitutions can be located at consecutive positions.
  • an amino acid may be substituted by multiple residues, where such variants include both substitutions and insertions.
  • “Insertional" variants are those in which one or more amino acids are inserted into the immediate vicinity of an amino acid at a specific position in a native sequence. Immediately adjacent to an amino acid is meant to be connected to the alpha-carboxy or alpha-amino functionality of that amino acid.
  • a “deletion” variant is a variant in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted from a specific region of their molecule.
  • variable domains of antibodies refers to portions of related molecules that differ widely in sequence between antibodies and are used for specific recognition and binding of a particular antibody against its specific target.
  • variability is not evenly distributed throughout the variable domains of an antibody.
  • the variability is concentrated in three segments called complementarity-determining regions (CDRs; namely CDR1, CDR2 and CDR3) or hypervariable regions, which are located within the variable domains of the light and heavy chains.
  • CDRs complementarity-determining regions
  • FR framework regions or framework sequences.
  • Each variable domain of the native heavy and light chains includes four FR regions, which mainly adopt a ⁇ -sheet configuration and are connected by three CDRs that form a loop that connects the ⁇ -sheet structure and In some cases a partial ⁇ -sheet structure is formed.
  • the CDRs of each chain are often linked in proximity by FR regions and contribute to the formation of the antibody target binding site (epitope or determinant) with the help of CDRs from other chains (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)).
  • the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise stated.
  • a CDR can have the ability to specifically bind to a cognate epitope.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region (e.g., one or more CDR and/or one or more antibody binding sites), and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as heavy Derivatives produced by the group.
  • Antibodies include antibody fragments.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments may include multiple chains linked together, for example by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by displacing the corresponding region), results in an antibody that immunospecifically binds (i.e., exhibits a Ka of at least or at least about 10 7 -10 8 M-1) for the antigen .
  • a "functional fragment” or “analog of an anti-CCR8 antibody” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction.
  • functional fragments generally have the same meaning as "antibody fragments” and, with respect to antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, e.g., Fv, Fab , F(ab') 2 and so on.
  • "Fv" fragments consist of a dimer (V H -V L dimer) formed by a non-covalent combination of the variable domain of a heavy chain and the variable domain of a light chain. In this configuration, the three CDRs of each variable domain interact to determine the target binding site on the surface of the VH - VL dimer, as is the case with intact antibodies. The six CDRs collectively confer the target binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv including only 3 target-specific CDRs) may still have the ability to recognize and bind a target.
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in the population of monoclonal antibodies is identical to every other antibody molecule. This property is in contrast to that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences.
  • Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J. Clin. Pathol (2000), 53, 111-117).
  • monoclonal antibodies can be produced by immortalizing B cells, such as by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV.
  • Recombinant techniques can also be used to prepare antibodies in vitro from clonal populations of host cells by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.
  • a full-length antibody is one that has two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region
  • VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4 and VL-CL full-length light chains
  • VL-CL full-length light chains
  • Antibodies such as those produced naturally by antibody-secreting B cells, as well as synthetically produced antibodies with the same domain.
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as where the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.
  • Humanized antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab') 2 or Other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody), wherein the residues of the complementarity determining region (CDR) of the recipient antibody are derived from a non-human species ( Donor antibody) such as mouse, rat or rabbit CDR residue substitution.
  • the humanized antibodies described in the present disclosure also include antibodies containing two amino acid mutations of 1 or 2 within the CDR.
  • Constant amino acid substitutions may involve replacing a native amino acid residue with a non-native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • conservative substitutions include replacing one non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, norleucine, alanine or methionine with another; Substitution of one polar (hydrophilic) amino acid residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; A basic amino acid residue such as lysine, arginine or histidine is substituted for another; or an acidic residue such as aspartic acid or glutamic acid is substituted for another.
  • conservative amino acid substitution also includes the replacement of a non-derivatized residue with a chemically derivatized residue, provided that the polypeptide exhibits the desired biological activity.
  • Other exemplary amino acid substitutions that may be used according to the present invention are shown in Table 1 below.
  • CDR refers to a complementarity-determining region
  • antibody molecules are known to have 3 CDRs per heavy and light chain.
  • CDRs are also called hypervariable regions and are present in the variable regions of each heavy and light chain of an antibody, with very high variability sites in the primary structure of the CDRs.
  • the CDR of the heavy chain is represented by CDR1, CDR2, and CDR3 derived from the amino terminus of the amino terminus sequence of the heavy chain
  • CDR of the light chain is represented by CDR1, CDR2, and CDR3 derived from the amino terminus of the amino terminus sequence of the light chain.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitope determinants typically comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and often have specific three-dimensional structural features as well as specific charge characteristics.
  • telomere binding binds As used herein, “specifically binds” or “immunospecifically binds” with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refers to the passage of the antibody or antigen-binding fragment between the antibody-binding site of the antibody and the antigen The ability of non-covalent interactions to form one or more non-covalent bonds with the same antigen.
  • the antigen may be an isolated antigen or present on tumor cells.
  • an antibody that immunospecifically binds (or specifically binds) an antigen does so with an affinity constant Ka (or 1 ⁇ 10 -7 M or a dissociation constant (Kd) of 1 ⁇ 10 -8 M or less) binds the antigen.
  • Affinity constants can be determined by standard kinetic methods of antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619 which describes exemplary SPR and ITC methods for calculating binding affinity of antibodies).
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • nucleic acid molecules refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including those linked together usually by a phosphodiester bond.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid molecule is intended to include DNA molecules as well as RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
  • mutations can be introduced randomly along all or a portion of the anti-CCR8 antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-CCR8 antibodies can be screened for improved binding activity.
  • expression refers to the process of producing a polypeptide through transcription and translation of a polynucleotide.
  • the expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from the host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • the host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. References to vectors also include those containing a nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, to amplify nucleic acids, or to express/display polypeptides encoded by nucleic acids. Vectors usually remain episomal, but can be designed to integrate the gene or part thereof into the chromosome of the genome. Vectors for artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.
  • vectors also include “viral vectors” or “viral vectors.”
  • viral vectors are engineered viruses that are operably linked to foreign genes to transfer (either as a vehicle or shuttle) the foreign genes into cells.
  • expression vector includes a vector capable of expressing DNA operably linked to regulatory sequences, such as a promoter region, that are capable of affecting the expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. An expression vector thus refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which when introduced into an appropriate host cell results in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of the host cell.
  • treating an individual suffering from a disease or disease condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged following treatment. Treatment therefore includes prevention, treatment and/or cure. Prevention means preventing underlying disease and/or preventing symptoms from worsening or disease progression. Treatment also includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof provided and the compositions provided herein.
  • therapeutic effect means an effect resulting from treatment of an individual that alters, generally ameliorates, or ameliorates the symptoms of a disease or disease condition, or cures a disease or disease condition.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a substance, compound, material or composition containing a compound that is at least sufficient to produce a therapeutic effect upon administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or condition.
  • a prophylactically effective amount or “prophylactically effective dose” refers to an amount of a substance, compound, material or composition containing a compound that, when administered to a subject, will have a desired prophylactic effect, e.g., prevent or delay a disease or symptom occurrence or recurrence and reduce the possibility of occurrence or recurrence of the disease or symptoms.
  • Complete prevention of an effective dose does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a prophylactically effective amount may be administered in one or more administrations.
  • the term "patient” or “subject” refers to a mammal, such as a human.
  • the present disclosure provides an antibody, or antigen-binding portion thereof, that binds CCR8, comprising a heavy chain variable region and a light chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof includes a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region contains:
  • X 1 is A or T
  • X 2 is F or Y
  • X 3 is N or Y
  • X 4 is Y or H
  • X 5 is Y or S
  • X 6 is I or K
  • X 7 is D or G
  • X 8 is A or does not exist
  • X 9 is Y, T or A
  • X 10 is Y or N
  • X 11 is A or Y
  • X 12 is M or A
  • X 13 is M or does not exist
  • the light chain variable region contains:
  • RSSKSLLHSX 14 X 15 NTYLY (SEQ ID NO.105), where:
  • X 14 is N, A, I, V, Y or R;
  • X 15 is G, I, V, Y, T or R;
  • RX 16 SNLAS (SEQ ID NO.106), where:
  • X 16 is M, A, G or T;
  • X 17 is F or L.
  • the aforementioned antibody or antigen-binding portion thereof includes a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region contains:
  • X 18 is A or S
  • the light chain variable region contains:
  • X 19 is R, T or S
  • X 20 is E, K or S
  • X 21 is V or I
  • X 22 is E or S
  • X 23 is Y, T or not present
  • X 24 is Y, S or not present
  • X 25 is G or absent
  • X 26 is T, Y, or S
  • X 27 is S or N
  • X 28 is L or Y
  • X 29 is L or M
  • X 30 is Q or H
  • X 31 is R, A, L or S
  • X 32 is A or T
  • X 33 is N or T
  • X 34 is L or V
  • X 35 is A or E;
  • X 36 is Q or H
  • X 37 is Q or H
  • X 38 is S, Y or G
  • X 39 is R, S, H or T
  • X 40 is E, K, G, R, V or S
  • X 41 is Y, V, L, S or I
  • X 42 is L or absent
  • X 43 is L, Y, P, R or G.
  • the aforementioned antibody or antigen-binding portion thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 6, 16, 28 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 7, 17 , 22, 82 or the heavy chain CDR2 of any variant thereof, selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO.8, 29, 49 or any variant thereof; and selected from the amino acid sequence SEQ ID NO.11, 32,
  • the light chain CDR1 of 39, 44, 52, 62, 91 or any variant thereof is selected from the amino acid sequence SEQ ID NO. 12, 33, 40, 45, 58, 63 or the light chain CDR2 of any variant thereof, selected from The light chain CDR3 of the amino acid sequence SEQ ID NO. 13, 25, 34, 41, 46, 53, 59, 64 or any variant thereof.
  • the aforementioned antibodies or antigen-binding portions thereof comprise the heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO.6, 7 and 8, and the sequences comprising SEQ ID NO.11, 12 and 13 respectively.
  • CDR combinations of heavy and light chains of light chain CDR1, CDR2 and CDR3 sequences comprise the heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO.6, 7 and 8, and the sequences comprising SEQ ID NO.11, 12 and 13 respectively.
  • the aforementioned antibodies or antigen-binding portions thereof comprise heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO.16, 17 and 8, and respectively comprising SEQ ID NO.11, 12 and 13. CDR combination of light chain CDR1, CDR2 and CDR3 sequences.
  • the aforementioned antibodies or antigen-binding portions thereof comprise the heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO.16, 22 and 8, and the sequences comprising SEQ ID NO.11, 12 and 25 respectively.
  • the aforementioned antibodies or antigen-binding portions thereof comprise heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO. 28, 22 and 29, and respectively comprising SEQ ID NO. 32, 33 and 34.
  • the aforementioned antibodies or antigen-binding portions thereof comprise heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO. 28, 22 and 29, and respectively comprising SEQ ID NO. 39, 40 and 41.
  • the aforementioned antibodies or antigen-binding portions thereof comprise the heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO. 28, 22 and 29, and the sequences comprising SEQ ID NO. 44, 45 and 46 respectively.
  • the aforementioned antibodies or antigen-binding portions thereof comprise heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO. 28, 22 and 49, and respectively comprising SEQ ID NO. 52, 45 and 53.
  • the aforementioned antibodies or antigen-binding portions thereof comprise heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO. 28, 22 and 49, and respectively comprising SEQ ID NO. 44, 58 and 59.
  • the aforementioned antibodies or antigen-binding portions thereof comprise the heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO. 28, 22 and 29, and the sequences comprising SEQ ID NO. 62, 63 and 64 respectively.
  • the aforementioned antibodies or antigen-binding portions thereof comprise the heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO.6, 7 and 8, and the sequences comprising SEQ ID NO.11, 12 and 15 respectively.
  • the aforementioned antibodies or antigen-binding portions thereof comprise heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO.16, 82 and 8, and respectively comprising SEQ ID NO.11, 12 and 25.
  • the aforementioned antibodies or antigen-binding portions thereof comprise heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO.6, 82 and 8, and respectively comprising SEQ ID NO.11, 12 and 25.
  • the aforementioned antibodies or antigen-binding portions thereof comprise the heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO. 16, 7 and 8, and the sequences comprising SEQ ID NO. 91, 63 and 25 respectively.
  • the aforementioned antibody or antigen-binding portion thereof comprises an amino acid sequence selected from SEQ ID NO. 4, 14, 20, 26, 35, 47, 54, 80, 83, 85, 92, 96, 100 or
  • the heavy chain variable region of any variant thereof and/or is selected from the amino acid sequence SEQ ID NO. 9, 18, 23, 30, 37, 42, 50, 56, 60, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 87, 89, 94, 98 or the light chain variable region of any variant thereof.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 9 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 67 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 68 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 69 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 70 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 71 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 72 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises a heavy chain of the amino acid sequence SEQ ID NO. 4 or any variant thereof. variable region, and the light chain variable region of the amino acid sequence SEQ ID NO. 73 or any variant thereof.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 74 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 75 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 76 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 77 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 78 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 79 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 14 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 18 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 20 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 23 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 26 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 30 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 35 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 37 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 35 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 42 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 47 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 50 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 54 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 56 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 35 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 60 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 65 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 80 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 87 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 83 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 87 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 85 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 89 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 92 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 94 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 96 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 98 or any variant thereof. chain variable region.
  • the aforementioned antibody or antigen-binding portion thereof comprises the heavy chain variable region of the amino acid sequence SEQ ID NO. 100 or any variant thereof, and the light chain variable region of the amino acid sequence SEQ ID NO. 98 or any variant thereof. chain variable region.
  • the aforementioned antibodies or antigen-binding portions thereof are humanized.
  • the present disclosure also provides nucleic acids encoding the aforementioned antibodies or antigen-binding portions thereof.
  • the nucleic acid comprises an antibody heavy chain variable region selected from SEQ ID NO. 5, 15, 21, 27, 36, 48, 55, 81, 84, 86, 93, 97, 101 or any variant thereof Nucleic acid sequence, and/or an antibody light chain variable selected from SEQ ID NO. 10, 19, 24, 31, 38, 43, 51, 57, 61, 66, 88, 90, 95, 99 or any variant thereof Region nucleic acid sequence;
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.5 or any variant thereof, and the nucleotide sequence SEQ ID NO.10 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.5 or any variant thereof, and the nucleotide sequence SEQ ID NO.10 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 15 or any variant thereof, and the nucleotide sequence SEQ ID NO. 19 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 21 or any variant thereof, and the nucleotide sequence SEQ ID NO. 24 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 27 or any variant thereof, and the nucleotide sequence SEQ ID NO. 31 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 36 or any variant thereof, and the nucleotide sequence SEQ ID NO. 38 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 36 or any variant thereof, and the nucleotide sequence SEQ ID NO. 43 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 48 or any variant thereof, and the nucleotide sequence SEQ ID NO. 51 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 55 or any variant thereof, and the nucleotide sequence SEQ ID NO. 57 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 36 or any variant thereof, and the nucleotide sequence SEQ ID NO. 61 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 36 or any variant thereof, and the nucleotide sequence SEQ ID NO. 61 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.5 or any variant thereof, and the nucleotide sequence SEQ ID NO.66 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.5 or any variant thereof, and the nucleotide sequence SEQ ID NO.66 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.81 or any variant thereof, and the nucleotide sequence SEQ ID NO.88 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.81 or any variant thereof, and the nucleotide sequence SEQ ID NO.88 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 84 or any variant thereof, and the nucleotide sequence SEQ ID NO. 88 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 84 or any variant thereof, and the nucleotide sequence SEQ ID NO. 88 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 86 or any variant thereof, and the nucleotide sequence SEQ ID NO. 90 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 86 or any variant thereof, and the nucleotide sequence SEQ ID NO. 90 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 93 or any variant thereof, and the nucleotide sequence SEQ ID NO. 95 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 93 or any variant thereof, and the nucleotide sequence SEQ ID NO. 95 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 97 or any variant thereof, and the nucleotide sequence SEQ ID NO. 99 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 97 or any variant thereof, and the nucleotide sequence SEQ ID NO. 99 or any variant thereof.
  • the aforementioned nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 101 or any variant thereof, and the nucleotide sequence SEQ ID NO. 99 or any variant thereof.
  • the nucleotide sequence of the light chain variable region of the body comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 101 or any variant thereof, and the nucleotide sequence SEQ ID NO. 99 or any variant thereof.
  • the present disclosure also provides a vector comprising the nucleic acid encoding the aforementioned.
  • the present disclosure also provides cells comprising the aforementioned nucleic acid or the aforementioned vector.
  • the present disclosure also provides a composition comprising the aforementioned antibody or antigen-binding portion thereof, nucleic acid, vector and/or cell.
  • kits comprising the aforementioned antibody or antigen-binding portion thereof, nucleic acid molecule, vector, cell and/or composition.
  • the method of treating cancer-related diseases with the CCR8 antibody of the present disclosure includes the following steps: administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof or nucleic acid molecule or vector or cell or drug of any of the foregoing aspects. combination.
  • Cancer-related diseases include breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach (gastric gland) cancer, non-small cell lung cancer, pancreatic cancer, head and neck squamous cell cancer, esophageal cancer, bladder cancer Cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin's lymphoma, urothelial cancer, sarcoma, blood cell cancer (leukemia, lymphoma, etc.), cholangiocarcinoma, gallbladder cancer, thyroid cancer, testicular cancer, thymus cancer, Cancers such as liver cancer are preferably breast cancer, uterine cancer, ovarian cancer, lung cancer, colorectal cancer, kidney cancer, and sarcoma, and more preferably are breast cancer, colorectal cancer, kidney cancer, and sarcoma.
  • the disease preferably associated with cancer is colon cancer or rectal cancer.
  • the antibody is administered in a single dose or in multiple doses.
  • the antibody is administered in multiple doses according to the present disclosure, preferably at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses
  • the antibody is administered in at least 10 doses and preferably up to 30 doses, 25 doses, 20 doses, 15 doses or 10 doses.
  • doses of the antibody are administered at intervals of at least 7 days, at least 10 days, at least 14 days, or at least 20 days.
  • Doses of the antibody are preferably administered at intervals of 7 to 30 days, 10 to 20 days, and preferably about 14 days.
  • the present disclosure provides the use of a therapeutic agent selected from a single therapeutic agent or a combination therapeutic agent in the preparation of a medicament for preventing or treating a cancer-related disease.
  • the single therapeutic agent is selected from the aforementioned antibodies or antigen-binding portions thereof, nucleic acids, vectors, cells, and/or compositions.
  • the combination therapeutic agent includes the single therapeutic agent and an immune checkpoint modulator.
  • the immune checkpoint modulator is an immune checkpoint selected from the group consisting of PD-1, CTLA4, PD-L1, PD-L2, CD47, TIGIT, GITR, TIM3, LAG3, 4-1BB, CD27, and B7H4 Inhibitors of dot proteins.
  • the inhibitor of an immune checkpoint protein is an antibody or antigen-binding fragment thereof.
  • the immune checkpoint protein is PD-1 or CTLA4.
  • the inhibitor of the immune checkpoint protein is selected from anti-PD-1 antibodies or antigen-binding fragments thereof or anti-CTLA-4 antibodies or antigen-binding fragments thereof.
  • the present disclosure provides methods of treating a cancer-related disease in a subject using a therapeutic agent selected from a single therapeutic agent or a combination therapeutic agent.
  • the single therapeutic agent is selected from the aforementioned antibodies or antigen-binding portions thereof, nucleic acids, vectors, cells, and/or compositions.
  • the combination therapeutic agent includes the single therapeutic agent and an immune checkpoint modulator.
  • the immune checkpoint modulator is an immune checkpoint selected from the group consisting of PD-1, CTLA4, PD-L1, PD-L2, CD47, TIGIT, GITR, TIM3, LAG3, 4-1BB, CD27, and B7H4 Inhibitors of dot proteins.
  • the inhibitor of an immune checkpoint protein is an antibody or antigen-binding fragment thereof.
  • the immune checkpoint protein is PD-1 or CTLA4.
  • the inhibitor of immune checkpoint protein Selected from anti-PD-1 antibodies or antigen-binding fragments thereof or anti-CTLA-4 antibodies or antigen-binding fragments thereof.
  • the present disclosure provides a therapeutic agent for treating a cancer-related disease in a subject, the therapeutic agent being selected from a single therapeutic agent or a combination therapeutic agent.
  • the single therapeutic agent is selected from the aforementioned antibodies or antigen-binding portions thereof, nucleic acids, vectors, cells, and/or compositions.
  • the combination therapeutic agent includes the single therapeutic agent and an immune checkpoint modulator.
  • the immune checkpoint modulator is an immune checkpoint selected from the group consisting of PD-1, CTLA4, PD-L1, PD-L2, CD47, TIGIT, GITR, TIM3, LAG3, 4-1BB, CD27, and B7H4 Inhibitors of dot proteins.
  • the inhibitor of an immune checkpoint protein is an antibody or antigen-binding fragment thereof.
  • the immune checkpoint protein is PD-1 or CTLA4.
  • the inhibitor of the immune checkpoint protein is selected from anti-PD-1 antibodies or antigen-binding fragments thereof or anti-CTLA-4 antibodies or antigen-binding fragments thereof.
  • the kit of the present disclosure includes the antibody of the present disclosure, its fragments, homologues, derivatives thereof, etc., such as labeled or cytotoxic conjugates, as well as instructions for use of the antibody, and conjugates that kill specific types of cells. etc.
  • the instructions may include instructions for using the antibody, conjugate, etc. in vitro, in vivo, or ex vivo.
  • Antibodies can be in liquid form or solid, usually lyophilized.
  • the kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. Combinations of reagents packaged in predetermined quantities are contemplated together with instructions for their use, for example for therapeutic use or for performing diagnostic assays.
  • the kit may include the substrate and cofactors required for the enzyme (eg, a substrate precursor that provides a detectable chromophore or fluorophore).
  • the substrate and cofactors required for the enzyme eg, a substrate precursor that provides a detectable chromophore or fluorophore.
  • other additives such as stabilizers, buffers (eg blocking buffer or lysis buffer), etc. may also be included.
  • the relative amounts of multiple reagents can be varied to provide a concentration of the reagent solution, which provides user flexibility, space savings, reagent savings, etc.
  • These reagents are also available in dry powder form, usually in lyophilized form, and include excipients which, when dissolved, provide a solution of the reagent at an appropriate concentration.
  • the present disclosure provides the antibodies or antigen-binding portions thereof, nucleic acid molecules, vectors, cells and/or compositions for use in the preparation of medicaments or kits for the diagnosis, treatment or prevention of cancer-related diseases. the use of.
  • antibodies of the present disclosure can also be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
  • the present disclosure also provides a composition comprising an anti-CCR8 antibody of the present disclosure or a fragment thereof, which is conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, which is a common practice in the art. .
  • composition refers to the formulation of various preparations.
  • Formulations containing a therapeutically effective amount of the multivalent antibody are in sterile liquid solutions, liquid suspensions, or lyophilized forms, optionally containing stabilizers or excipients.
  • therapeutic agents according to the described embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • suitable preparations can be found in the pharmacopoeia known to all medicinal chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa. (1975)), especially Chapter 87 of Blaug, Seymour therein.
  • formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (eg Lipofectin TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycols. Any of the foregoing mixtures may be suitable for use in treatment or therapy according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and tolerates the route of administration.
  • the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a suitable propellant such as a gas such as carbon dioxide or a nebulizer.
  • a suitable propellant such as a gas such as carbon dioxide or a nebulizer.
  • Systemic administration can also be done by transmucosal or transdermal means.
  • penetrants suitable for penetrating the barrier are used in the formulation.
  • penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration.
  • Transmucosal administration can be achieved through the use of nasal sprays or suppositories.
  • one or more of the antibodies may be formulated as a paste, ointment, gel, or cream as is generally known in the art.
  • the compounds may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.
  • suppositories eg, with conventional suppository bases such as cocoa butter or other glycerides
  • retention enemas e.g., retention enemas.
  • the antibody can be prepared with a carrier that will protect it from rapid elimination by the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems.
  • a sustained/controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.
  • dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. one or more of said antibodies. Specifications for dosage unit forms of the described embodiments are dictated by and directly dependent upon the unique characteristics of the antibodies and the specific therapeutic effect to be achieved, as well as the limitations inherent in the formulation of such antibodies for the treatment of individuals.
  • the pharmaceutical composition can be placed in a container, package, or dispenser together with instructions for administration.
  • compositions described herein may also contain more than one such antibody depending on the specific condition to be treated, preferably those with complementary activities but without negative effects on each other.
  • the composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • agents that enhances its function such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • Such molecules are appropriately present in combination and in amounts effective for the intended purpose. For example, they may be present together in the kit or during use.
  • one or more of the antibodies may be administered in combination therapy, i.e., with other agents such as therapeutic agents (which may be used to treat pathological conditions or disorders, such as various forms of cancer and inflammatory diseases) joint.
  • agents such as therapeutic agents (which may be used to treat pathological conditions or disorders, such as various forms of cancer and inflammatory diseases) joint.
  • the term "combination” as used herein means that the agents are administered substantially simultaneously, simultaneously or sequentially. If administered sequentially, the first of the two compounds is still preferably present at an effective concentration at the treatment site when administration of the second compound is initiated. degree was detected. In one instance, “combination” may also include both an antibody of the present disclosure and another therapeutic agent in a kit.
  • combination therapy may include one or more antibodies described herein together with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below), are co-formulated and/or co-administered.
  • additional therapeutic agents e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below.
  • Human CCR8 cDNA (Sino Biological, product number: HG11450-M, its amino acid sequence is shown in SEQ ID NO.1)
  • cynomolgus monkey CCR8 cDNA (Suzhou Jinweizhi Biosynthesis, its amino acid sequence is shown in SEQ ID NO.2).
  • human CCR4 cDNA (Sino Biological, Cat. No.: HG13064-UT, whose amino acid sequence is shown in SEQ ID NO.3) were used as templates to construct lentiviral vector plasmids containing the full-length sequences of human and monkey CCR8 and human CCR4.
  • Virus packaging kit instructions (Lenti-Pac HIV Expression Packaging Kit, Gene Copoeia, Catalog No.: HPK-LvTR-20), co-transfect the constructed lentiviral plasmid and packaging plasmid into HEK293T cells (ATCC CRL-11268), and perform lentiviral Virus packaging. Collect the culture medium 48 hours after transfection, centrifuge at 500*g for 10 minutes to remove cell debris, and obtain the culture supernatant containing lentiviral particles. After filtering with a 0.45 ⁇ m PES membrane, dispense it into 1.5 mL EP tubes, and take 10 ⁇ L each.
  • HEK293T or CHO cells used to infect 1 ⁇ 10 6 HEK293T or CHO cells (ATCC CCL-61). After 48 hours, 8 ⁇ g/ml puromycin was added for pressure screening to obtain a monoclonal cell line HEK293-hCCR8 (flow) expressing human CCR8 at a relatively high level.
  • the fluorescence value detected by flow cytometry increased by at least 2 log compared with HEK293 cells), HEK293-hCCR8 low (the fluorescence value detected by flow cytometry increased by ⁇ 1 log compared with HEK293 cells) and the cell line HEK293-hCCR4 that stably expresses human CCR4, as well as CHO that stably expresses human CCR8 -hCCR8 cell line, and CHO-cynoCCR8, a cell line stably expressing cynomolgus monkey CCR8.
  • Figure 1 The results are shown in Figure 1.
  • mice use the full-length sequence of human or cynomolgus CCR8 to construct a eukaryotic expression plasmid containing CMV promoter, and use a gene gun to immunize the mice with DNA (20 ⁇ g plasmid/mouse) After 4-5 times of immunization, the mouse serum titer was detected. After the titer met the requirements, CHO-hCCR8 stably transfected cells (5 ⁇ 10 6 cells/mouse) were used for shock immunization. Three days later, the mice were killed by cervical dissection, and the spleens and peripheral lymph nodes of the mice were collected. After grinding and centrifugation, the total RNA was extracted.
  • PCR amplification was performed using light and heavy chain-specific primers to obtain the variable light chain and heavy chain of the antibody. region cDNA library.
  • Phage antibody library display technology was used to construct a CCR8 immune library for subsequent phage panning.
  • the constructed CCR8 immune library was panned using the classic method.
  • Human CCR8+ cells CHO-hCCR8 or cynomolgus CCR8+ cells CHO-cynoCCR8 (1 ⁇ 10 6 /tube) were used alternately, and 1 ⁇ 10 12 phage was added for incubation and panning. , use 100mM triethylamine to elute phage particles with high binding affinity, neutralize them with 1M Tris-HCl (pH 7.4), and infect them into E. coli TG1 bacteria.
  • the phage obtained after repackaging is used for the next round of panning. After two rounds of panning and enrichment of clones, single clones were picked and inoculated into a 96-well U-shaped well plate, cultured and IPTG induced expression, and the induced supernatant was taken for FACS detection.
  • Example 2 The positive clones obtained in Example 1 were re-expressed and purified to obtain Fab antibodies, and their binding to human CCR8, cynomolgus monkey CCR8 and human CCR4 stably transfected cells were respectively detected. Add 5 ⁇ 10 4 detection cells to each well of a 96-well U-shaped plate, centrifuge and discard the supernatant, add 100 ⁇ L/well of purified Fab antibody, and incubate on ice for 60 minutes.
  • the anti-CCR8 mouse anti-heavy chain/light chain variable region sequences are as follows:
  • amino acid sequence of 29A8 heavy chain VH is shown in SEQ ID NO.4, its coding nucleic acid is shown in SEQ ID NO.5, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.6, 7 and 8 respectively.
  • amino acid sequence of 29A8 light chain VK is shown in SEQ ID NO. 9, its encoding nucleic acid is shown in SEQ ID NO. 10, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 11, 12 and 13 respectively.
  • amino acid sequence of 30A7 heavy chain VH is shown in SEQ ID NO. 14
  • its encoding nucleic acid is shown in SEQ ID NO. 15
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 16, 17 and 8 respectively.
  • amino acid sequence of 30A7 light chain VK is shown in SEQ ID NO. 18, its encoding nucleic acid is shown in SEQ ID NO. 19, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 11, 12 and 13 respectively.
  • amino acid sequence of 87D8 heavy chain VH is shown in SEQ ID NO. 20
  • its encoding nucleic acid is shown in SEQ ID NO. 21
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 16, 22 and 8 respectively.
  • amino acid sequence of 87D8 light chain VK is shown in SEQ ID NO. 23, its coding nucleic acid is shown in SEQ ID NO. 24, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 11, 12 and 25 respectively.
  • amino acid sequence of 102H5 heavy chain VH is shown in SEQ ID NO. 26, its encoding nucleic acid is shown in SEQ ID NO. 27, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 28, 22 and 29 respectively.
  • amino acid sequence of 102H5 light chain VK is shown in SEQ ID NO. 30, its coding nucleic acid is shown in SEQ ID NO. 31, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 32, 33 and 34 respectively.
  • amino acid sequence of 109F6 heavy chain VH is shown in SEQ ID NO. 35
  • its coding nucleic acid is shown in SEQ ID NO. 36
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 28, 22 and 29 respectively.
  • amino acid sequence of 109F6 light chain VK is shown in SEQ ID NO. 37
  • its coding nucleic acid is shown in SEQ ID NO. 38
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 39, 40 and 41 respectively.
  • the amino acid sequence of 110G2 heavy chain VH is shown in SEQ ID NO. 35, its coding nucleic acid is shown in SEQ ID NO. 36, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 28, 22 and 29 respectively.
  • the amino acid sequence of 110G2 light chain VK is shown in SEQ ID NO. 42, its coding nucleic acid is shown in SEQ ID NO. 43, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 44, 45 and 46 respectively.
  • amino acid sequence of 113B3 heavy chain VH is shown in SEQ ID NO. 47
  • its encoding nucleic acid is shown in SEQ ID NO. 48
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 28, 22 and 49 respectively.
  • amino acid sequence of 113B3 light chain VK is shown in SEQ ID NO. 50
  • its coding nucleic acid is shown in SEQ ID NO. 51
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 52, 45 and 53 respectively.
  • amino acid sequence of 116F10 heavy chain VH is shown in SEQ ID NO. 54
  • its coding nucleic acid is shown in SEQ ID NO. 55
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 28, 22 and 49 respectively.
  • amino acid sequence of 116F10 light chain VK is shown in SEQ ID NO. 56
  • its coding nucleic acid is shown in SEQ ID NO. 57
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 44, 58 and 59 respectively.
  • amino acid sequence of 139A9 heavy chain VH is shown in SEQ ID NO. 35
  • its coding nucleic acid is shown in SEQ ID NO. 36
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 28, 22 and 29 respectively.
  • amino acid sequence of 139A9 light chain VK is shown in SEQ ID NO. 60
  • its coding nucleic acid is shown in SEQ ID NO. 61
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 62, 63 and 64 respectively.
  • amino acid sequence of 144D2 heavy chain VH is shown in SEQ ID NO.4, its coding nucleic acid is shown in SEQ ID NO.5, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.6, 7 and 8 respectively.
  • amino acid sequence of 144D2 light chain VK is shown in SEQ ID NO. 65
  • its coding nucleic acid is shown in SEQ ID NO. 66
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 11, 12 and 25 respectively.
  • the heavy chain variable region of the positive clone in Example 2 was cloned into a vector containing the human heavy chain fixed region and regulatory elements to express the complete IgG heavy chain in mammalian cells; similarly, the light chain variable region was cloned into Vector containing a human light chain fixation region and regulatory elements to express intact IgG light chains in mammalian cells. After correct sequencing, it was transfected into CHO-S mammalian cells (Thermo Fisher ExpiCHO). The IgG was expressed and secreted into the culture medium. The supernatant was collected and purified by conventional ProA to obtain the anti-CCR8 chimeric antibody. The OD280 was measured by spectrophotometry. Light absorption determines antibody concentration.
  • Mouse anti-m10A11 (chimera) and humanized antibody h10A11 were synthesized with reference to the document WO2020138489A1 (m10A11: Sequence 27 and Sequence 26 of the reference document, h10A11: Sequence 41 and Sequence 59 of the reference document).
  • Chimeric antibodies such as 87D8, 102H5, 109F6, 110G2, 113B3, 139A9 and 144D2 all bound to human CCR8+ cells CHO-hCCR8 and cynomolgus monkey CCR8+ cells CHO-cynoCCR8 with high affinity.
  • the affinity (EC 50 ) of the anti-CCR8 chimeric antibody to the CCR8 receptor of stably transfected cells is shown in Table 3.
  • NK92 cells Construct a lentiviral vector plasmid of the full-length sequence of human CD16A, package and transfect NK92 cells (purchased from ATCC, Cat. No. CRL-2408) according to the Lenti-Pac HIV Expression Packaging Kit (Gene Copoeia, HPK-LvTR-20), and screen to obtain NK92 cells -CD16A stably transfected cells.
  • Add 5 ⁇ 10 4 HEK293-hCCR8 cells to each well of a 96-well U-shaped plate, add 50 ⁇ L of serially diluted anti-CCR8 chimeric antibody or isotype control, and incubate in the incubator for 30 minutes; add 50 ⁇ L of 2 ⁇ 10 6 to each well.
  • NK92-CD16A cells continue to culture for 4 hours.
  • 30 minutes before detection add 2 ⁇ L cell lysis solution (100 ⁇ ) to the largest well of the target cells, centrifuge at 300*g for 3 minutes, transfer 50 ⁇ L supernatant to a black enzyme plate, and add 50 ⁇ L lactate dehydrogenase (LDH) detection substrate.
  • LDH lactate dehydrogenase
  • Vortex and mix add 25 ⁇ L of stop solution after 10 minutes, vortex for 10 seconds, and select fluorescence (excitation wavelength 560 nm, emission wavelength 590 nm) for plate reading.
  • the target cell killing rate was calculated based on the LDH release level, and the antibody concentration-killing rate (%) curve was obtained through four-parameter model fitting.
  • the regression curve was used to calculate the half effective concentration (EC 50 ) of the test product.
  • Figure 3 shows that the anti-CCR8 chimeric antibody has significant ADCC killing activity on CCR8+ cells in a dose-dependent manner.
  • the half-effective killing concentrations of antibodies such as 29A8, 87D8, 102H5, 109F6, 110G2, 113B3, 116F10, 139A9 and 144D2 all have EC 50 Better than m10A11 (Table 4).
  • Example 5 Anti-CCR8 chimeric antibody inhibits the calcium flux signal of target cells caused by CCL1
  • Clones 87D8 and 144D2 have almost identical light chain sequences.
  • the overlapping PCR site-directed mutagenesis method was used to construct mutants for the potential deamination site N33 and nearby sites as well as the site M56 where oxidative modification may occur. Become Cys, Trp, Phe and other amino acids other than the parent sequence.
  • the OD280 light absorption is measured by spectrophotometry, and the antibody concentration is determined by referring to the theoretical extinction coefficient. .
  • the EC 50 of the binding ability to CHO-hCCR8 was detected.
  • the 144D2 mutants G34Y, G34T, G34R and M56T all retained the same or better affinity than the parent 144D2. The results are shown in Table 5 and Figure 5.
  • Clones 87D8 and 144D2 which have high affinity to human and cynomolgus CCR8 and strong ADCC killing activity, were selected for humanized antibody construction.
  • the light and heavy chain germline genes used in 87D8 and 144D2 are the same, and the CDR region sequences are highly similar.
  • the heavy chain CDR1 and CDR2 have 2 and 1 amino acid differences respectively. It is speculated that they may be generated during the expansion of mouse B cells. Cell mutations.
  • mouse antibody 87D8 and 144D2 sequences were compared with the human antibody germline sequence to identify the highly homologous human germline light chain genes IMGT_hVK2-28 and IGKJ2*01 and the human germline heavy chain genes IMGT_hVH3-7 and IGHJ4. *01, perform mouse antibody CDR transplantation, and combine CDR1 and CDR2 of 87D8 and 144D2 heavy chains at the same time.
  • Computer analysis showed that there is potential deamination modification at the N33 site of the light chain CDR1, and oxidative modification may occur at the CDR2 M52 site.
  • the light and heavy chains were fully sequence synthesized, cloned into vectors containing the antibody kappa chain constant region or the human IgG1 constant region CH1-CH3 respectively, combined and paired, and transfected into CHO cells. After 6 days of expression, the cell culture medium was filtered and collected, and Protein was used to A humanized antibody was purified by affinity chromatography. Determine the antibody concentration by measuring OD280 light absorption spectrophotometrically.
  • the humanized antibody heavy chain/light chain variable region sequences are as follows:
  • amino acid sequence of h87D8 VHv1 is shown in SEQ ID NO. 80, its coding nucleic acid is shown in SEQ ID NO. 81, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 16, 82 and 8 respectively.
  • amino acid sequence of h144D2 VHv1 is shown in SEQ ID NO. 83
  • its coding nucleic acid is shown in SEQ ID NO. 84
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 6, 82 and 8 respectively.
  • amino acid sequence of h87D8 VHv2 is shown in SEQ ID NO. 85
  • its coding nucleic acid is shown in SEQ ID NO. 86
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 16, 7 and 8 respectively.
  • amino acid sequence of h87D8 VKv1 is shown in SEQ ID NO. 87
  • its coding nucleic acid is shown in SEQ ID NO. 88
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 11, 12 and 25 respectively.
  • amino acid sequence of h87D8 VKv4 is shown in SEQ ID NO. 89
  • its coding nucleic acid is shown in SEQ ID NO. 90
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 91, 63 and 25 respectively.
  • Vortex and mix add 25 ⁇ L of stop solution after 10 minutes, vortex for 10 seconds, and select fluorescence (excitation wavelength 560 nm, emission wavelength 590 nm) for plate reading.
  • the target cell killing rate was calculated based on the LDH release level, and the antibody concentration-killing rate (%) curve was obtained through four-parameter model fitting. The regression curve was used to calculate the half effective concentration (EC 50 ) of the test product.
  • h87D8 H1K1 hIgG1 e5 two Fc function-enhanced humanized antibodies, h87D8 H1K1 hIgG1 e5 (hereinafter referred to as h87D8 H1K1 e5) and h87D8 H2K4 hIgG1 e5 (hereinafter referred to as h87D8 H2K4 e5), were obtained (Table 9).
  • the heavy chain amino acid sequence of h87D8 H1K1 hIgG1 e5 is shown in SEQ ID NO. 92, and its encoding nucleic acid is shown in SEQ ID NO. 93.
  • the light chain amino acid sequence of h87D8 H1K1 hIgG1 e5 is shown in SEQ ID NO. 94, and its encoding nucleic acid is shown in SEQ ID NO. 95.
  • the heavy chain amino acid sequence of h87D8 H2K4 hIgG1 e5 is shown in SEQ ID NO. 96, and its encoding nucleic acid is shown in SEQ ID NO. 97.
  • the light chain amino acid sequence of h87D8 H2K4 hIgG1 e5 is shown in SEQ ID NO.98, and its encoding nucleic acid is shown in SEQ ID NO.99.
  • the heavy chain amino acid sequence of h87D8 H2K4 hIgG1 is shown in SEQ ID NO. 100, and its encoding nucleic acid is shown in SEQ ID NO. 101.
  • the light chain amino acid sequence of h87D8 H2K4 hIgG1 is shown in SEQ ID NO. 98, and its encoding nucleic acid is shown in SEQ ID NO. 99.
  • the coupling reagent Amine Coupling Kit (GE Healthcare BR-1000-50) is used to couple Goat Anti-Human IgG F(ab')2 to the CM5 chip surface.
  • h87D8 H2K4 e5 or h87D8 H2K4 Capture then inject serially diluted Fc ⁇ RIIIAV158 or Fc ⁇ RIIIAF158, set the binding time to 120 seconds, the dissociation time to 200 seconds, regenerate with 10mM Glycine-HCl at pH 1.75 for 30 seconds, and set the flow rate to 30 ⁇ l/min.
  • the measured data were fitted to obtain the equilibrium dissociation constant. Determine the affinity of h87D8 H2K4 e5 or h87D8 H2K4 to Fc ⁇ RIIIAV158 and Fc ⁇ RIIIAF158, respectively.
  • h87D8 H2K4 e5 and h87D8 H2K4 both bind Fc ⁇ RIIIAV158 and Fc ⁇ RIIIAF158.
  • the affinity KD of h87D8 H2K4 e5 are 270.0nM and 432.7nM respectively; the affinity KD of h87D8 H2K4 are 861.2nM and 1.3 ⁇ M respectively.
  • the binding of h87D8 H2K4 e5 to Fc ⁇ RIIIAV158 and Fc ⁇ RIIIAF158 is stronger than that of h87D8 H2K4.
  • FIG. 9A shows that the Fc function-enhanced humanized anti-CCR8 antibodies h87D8 H1K1 e5 and h87D8 H2K4 e5 significantly improved the ADCC killing activity against hCCR8+ cells, with EC 50s of 0.016nM and 0.023nM, respectively, and maximum lysis rates of 58% and 58%, respectively. 61%, which is approximately doubled compared with 87D8 and m10A11 (maximum cleavage rates of 33% and 30% respectively).
  • FIG. 9C shows that the anti-CCR8 humanized antibody h87D8 H2K4 e5 also has ADCC activity against HuT78 cells expressing human CCR8, with a half-effective concentration EC 50 of 0.53 nM.
  • the target cell killing rate was calculated based on the LDH release level, and the antibody concentration-killing rate (%) curve was obtained through four-parameter model fitting. The regression curve was used to calculate the half effective concentration (EC 50 ) of the test product.
  • Figure 9D shows that the ADCC activity of the Fc function-enhanced anti-CCR8 humanized antibodies h87D8 H1K1 e5 and h87D8 H2K4 e5 on low-affinity Fc ⁇ RIIIA 158F/F PBMC as effector cells was significantly improved compared to the control group, and the maximum lysis rate was better than that of the wild type.
  • type IgG1 which was comparable to the ADCC activity using high-affinity Fc ⁇ RIIIA 158V/V PBMC as effector cells (Fig. 9E, Table 10).
  • CHO-hCCR8 cells Collect CHO-hCCR8 cells, resuspend them in F12+10% FBS culture medium to 2 ⁇ 10 5 /mL, spread them into a 96-well plate at 100 ⁇ L/well, and culture them in a 37°C, 5% CO 2 incubator overnight. Wash the cells twice with HBSS/probenecid (containing probenecid 2.5mM) solution, add 4 ⁇ M Fluo-4AM working solution, and incubate 100 ⁇ L per well at 37°C for 60 min; wash the cells twice with HBSS/probenecid solution to remove residual Fluo-4AM working solution, add 50ul of HBSS/probenecid solution to each well and incubate at 37°C for 20 minutes.
  • HBSS/probenecid containing probenecid 2.5mM
  • Figure 10 shows that h87D8 H1K1 e5 and h87D8 H2K4 e5 can inhibit the Ca 2+ flow signal caused by CCL1-CCR8, and the half-effective concentrations EC 50 are 5.59nM and 5.20nM respectively.
  • HEK293-rabbitCCR8 cells Use rabbit CCR8 cDNA (Suzhou Jinweizhi Biosynthesis, its amino acid sequence is shown in SEQ ID NO. 112) as a template to construct a lentiviral vector plasmid containing rabbit CCR8, refer to the transfection reagent instructions and use the transfection reagent PEI (Polysciences 9002-98-6)
  • the lentiviral vector plasmid containing rabbit CCR8 was transfected into HEK293 cells (hereinafter referred to as HEK293-rabbitCCR8 cells).
  • Example 13 Effect of humanized CCR8 antibody on the growth of subcutaneous transplanted tumors in MC38 mice
  • the MC38 subcutaneous transplant tumor model was established on the CCR8 humanized transgenic mouse C57BL/6J-Ccr8 em3(hCCR8)/Smoc (purchased from Shanghai Southern Model Biotechnology, Catalog No. NM-HU-2000054).
  • the test is set up: h87D8 H2K4 e5 0.12mg/kg, 0.6mg/kg and 3mg/kg groups, h87D8 H2K4 0.12mg/kg, 0.6mg/kg and 3mg/kg groups, h10A11 0.6mg/kg, 3mg/kg group, IgG1 isotype Control 3mg/kg group.
  • FIG. 12 shows that h87D8 H2K4 e5 can inhibit tumor growth in all dose groups.
  • the tumor inhibition rates of the 3mg/kg, 0.6mg/kg and 0.12mg/kg groups were 113.96%, 110.64% and 95.44% respectively.
  • the tumor inhibition rates of h87D8 H2K4 3mg/kg, 0.6mg/kg and 0.12mg/kg groups were 85.08%, 60.18% and 26.81% respectively.
  • the tumor inhibition rates of h10A11 3mg/kg and 0.6mg/kg groups were 66.10% and 4.07% respectively.
  • T/C (%) (T-T0)/(C-C0) ⁇ 100, where T and T0 are the tumor volumes of the drug group at the end of the experiment and at the beginning of the experiment, respectively, and C and C0 are respectively is the tumor volume of the control group at the end of the experiment and at the beginning of the experiment.
  • TGI tumor inhibition rate
  • TGI tumor inhibition rate
  • Example 14 Effect of humanized CCR8 antibody on the growth of subcutaneous transplanted tumors in B16/F10 mice
  • a B16/F10 subcutaneous transplanted tumor model was established on the CCR8 humanized transgenic mouse C57BL/6J-Ccr8 em3(hCCR8)/Smoc .
  • Experimental settings h87D8 H2K4 e5 10 mg/kg, anti-mouse PD-1 antibody (mIgG1, clone G4C2) 10 mg/kg and h87D8 H2K4 e5 10 mg/kg + anti-mouse PD-1 antibody 10 mg/kg group, IgG1 isotype control 10 mg/kg group, blank control group (i.e. solvent control group).
  • CCR8 humanized transgenic mice were administered intravenously once every 3 days for a total of 3 times.
  • Figure 13 shows that anti-mouse PD-1 antibody cannot inhibit B16/F10 tumor growth, but h87D8 H2K4 e5 can inhibit tumor growth, with a tumor inhibition rate of 48.2%.
  • the combination of h87D8 H2K4 e5 + anti-mouse PD-1 antibody can inhibit tumor growth more effectively than h87D8 H2K4 e5 alone, with a tumor inhibition rate of 75.4%.
  • Example 15 Effect of humanized CCR8 antibody on the growth of subcutaneous transplanted tumors in MDA-MB-231 mice
  • An MDA-MB-231 subcutaneous transplant tumor model was established on B-NGD hIL15 mice (purchased from Biocytogen), and human 1 ⁇ 10 7 PBMC/mouse (purchased from Shanghai Miaoshun Biotechnology) was intravenously administered for immune reconstitution.
  • the experiment set up h87D8 H2K4 e5 0.4 mg/kg, 2 mg/kg and 10 mg/kg groups, and the IgG1 isotype control 10 mg/kg group.
  • CCR8 humanized transgenic mice were administered intravenously once every 3 days for a total of 4 Second-rate.
  • Figure 14 shows that h87D8 H2K4 e5 can inhibit tumor growth in all dose groups, and the tumor inhibition rates in the 10mg/kg, 2mg/kg and 0.4mg/kg groups were 74.4%, 49.3% and 40.4% respectively.

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Abstract

提供了一种新型免疫调节剂的开发和应用，所述免疫调节剂为结合CCR8的抗体或其抗原结合部分。所述抗体具有阻断CCL1/CCR8轴的中和活性，可抑制CCL1诱导的细胞活化，同时针对人群中低亲和力FcγRIIIA158F等位基因携带者，通过Fc优化增强了ADCC效应。

Description

一种新型免疫调节剂的开发和应用 技术领域
本公开涉及一种免疫调节剂及其应用，特别是抗CCR8抗体及其应用。
背景技术
趋化因子受体8(CCR8)是一种C-C趋化因子受体超家族成员，在活化II型Th细胞表达上调，其配体CCL1/I-309可有效募集活化II型Th细胞，CCL1与CCR8的相互作用(也称为CCL1/CCR8轴)与II型Th细胞介导的免疫应答相关(D'Ambrosio,D.,et al.1998.Selective up-regulation of chemokine receptors CCR4 and CCR8 upon activation of polarized human type 2 Th cells.J Immunol 161(10):5111-5；Zingoni,A.,et al.1998.The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells.J Immunol 161(2):547-51.)。CCL1可激活CCR8介导的RAS/MAPK信号通路，促进细胞内钙流释放，并抑制细胞凋亡(Denis,C.,et al.2012.C-terminal clipping of chemokine CCL1/I-309 enhances CCR8-mediated intracellular calcium release and anti-apoptotic activity.PLoS One 7(3):e34199；Louahed,J.,et al.2003.CCR8-dependent activation of the RAS/MAPK pathway mediates anti-apoptotic activity of I-309/CCL1 and vMIP-I.Eur J Immunol 33(2):494-501)。CCR8对维持调节性T细胞(Treg)存活，抑制移植物抗宿主反应(GVHD)也具有重要作用(Coghill,J.M.,et al.2013 CC chemokine receptor 8potentiates donor Treg survival and is critical for the prevention of murine graft-versus-host disease.Blood 122(5):825-36.)。CCR8+Treg被认为是驱动免疫抑制作用的关键细胞，是自身免疫疾病的潜在治疗靶点(Barsheshet,Y.,et al.2017.CCR8(+)FOXp3(+)T(reg)cells as master drivers of immune regulation.Proc Natl Acad Sci U S A 114(23):6086-6091)。
除了维持免疫稳态，Treg细胞也被认为是抑制肿瘤免疫的关键因素，和常规T(Tconv)细胞数量相比，Treg比率偏低的肿瘤患者生存率高，包括卵巢癌、乳腺癌、非小细胞肺癌、肝细胞癌、肾细胞癌、胰腺癌、胃癌、宫颈癌和结肠直肠癌等(Bates,G.J.,et al.2006.Quantification of regulatory T cells enables the identification of high-risk breast cancer patients and those at risk of late relapse.J Clin Oncol 24(34):5373-80；Curiel,et al.2004；Gao,Q.,et al.2007.Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection.J Clin Oncol 25(18):2586-93；Griffiths,R.W.,et al 2007.Frequency of regulatory T cells in renal cell carcinoma patients and investigation of correlation with survival.Cancer Immunol Immunother 56(11):1743-53；Hiraoka,N.,et al.2006.Prevalence of FOXP3+regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions.Clin Cancer Res 12(18):5423-34；Jordanova,E.S.,et al.2008.Human leukocyte antigen class I,MHC class I chain-related molecule A,and CD8+/regulatory T-cell ratio:which variable determines survival of cervical cancer patients？Clin Cancer Res 14(7):2028-35；Perrone,G.,et al.2008.Intratumoural FOXP3-positive regulatory T cells are associated with adverse prognosis in radically resected gastric cancer.Eur J Cancer 44(13):1875-82；Petersen,R.P.,et al.2006Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients.Cancer107(12):2866-72；Sato,et al.2005；Sinicrope,F.A.,et al.2009 Intraepithelial effector(CD3+)/regulatory(FoxP3+)T-cell ratio predicts a clinical outcome of human colon carcinoma.Gastroenterology 137(4):1270-9)。Ruckes T等发现，T细胞淋巴瘤和白血病可通过CCL1自分泌环路方式抵御凋亡(Ruckes,T.,et al.2001 Autocrine antiapoptotic stimulation of cultured adult T-cell leukemia cells by overexpression of the chemokine I-309.Blood 98(4):1150-9)。Hoelzinger D等显示阻断CCL1可消除Treg对肿瘤免疫的抑制作用(Hoelzinger,D.B.,et al.2010 Blockade of CCL1 inhibits T regulatory cell suppressive function enhancing tumor immunity without affecting T effector responses.J Immunol 184(12):6833-42)。Eruslanov E等发现，尿路上皮癌和肾癌患者肿瘤相关巨噬细胞中CCR8表达增加，肿瘤浸润性CD11b+CCR8+细胞可在自体T淋巴细胞中诱导FoxP3的表达，同时还发现原发性肿瘤产生大量CCL1，表明CCL1/CCR8轴是癌症相关炎症的组成部分(Eruslanov,E.,et al.2013 Expansion of CCR8(+)inflammatory myeloid cells in cancer patients with urothelial and renal carcinomas.Clin Cancer Res 19(7):1670-80)。De Simone M等在对人结直肠癌或非小细胞肺癌的转录组分析中发现，肿瘤浸润的Treg细胞特征性上调CCR8等信号分子的表达，具有高度的免疫抑制性，肿瘤组织中CCR8基因高表达与不良预后相关(De Simone,M.,et al.2016 Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating Zingoni,T Regulatory Cells.Immunity 45(5):1135-1147)。Plitas G等在乳腺癌中，同样检测到CCR8在肿瘤浸润Treg细胞上表达升高，提示通过靶向CCR8去除肿瘤部位的Treg细胞，是一种非常有希望的免疫治疗策略(Plitas,G.,et al.2016 Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer.Immunity 45(5):1122-1134)。Magnuson A等比较来自小鼠结直肠癌、黑色素瘤模型和从早期到晚期不同阶段人结直肠癌患者的肿瘤组织，发现肿瘤浸润Treg特征性表达谱具有显著共性，包括CCR8等一系列与Treg分化相关的基因上调，抗CCR8抗体可以使治疗后小鼠的肿瘤消退(Magnuson,A.M.,et al.2018 Identification and validation of a tumor-infiltrating Treg transcriptional signature conserved across species and tumor types.Proc Natl Acad Sci U S A 115(45):E10672-e10681)。Villarreal D等报道，抗CCR8抗体在结直肠癌小鼠模型中可显著抑制肿瘤生长，抗肿瘤活性与肿瘤特异性T细胞的增加、CD4+和CD8+T细胞浸润增强，以及肿瘤内部CD4+CCR8+Tregs显著减少相关(Villarreal,D.O.,et al.2018 Targeting CCR8 Induces Protective Antitumor Immunity and Enhances Vaccine-Induced  Responses in Colon Cancer.Cancer Res 78(18):5340-5348)。
现有疗法中尚无针对肿瘤浸润Treg细胞的有效手段。从肿瘤微环境中，实现特异性清除抑制性Treg细胞一直是非常大的挑战，例如：使用非岩藻糖化抗CCR4抗体mogamulizumab在清除Treg的同时，也显著减少了正常CD4+ T细胞和CD8+T细胞(Kurose,K.,et al.2015 Phase Ia Study of FoxP3+CD4 Treg Depletion by Infusion of a Humanized Anti-CCR4 Antibody,KW-0761,in Cancer Patients.Clin Cancer Res 21(19):4327-36)。CCR8在肿瘤浸润Treg细胞上表达显著升高，有利于靶向CCR8的抗体区分肿瘤浸润Treg细胞与正常T细胞。因此，开发靶向CCR8的单克隆抗体，有效清除肿瘤浸润Treg细胞或同时抑制其活性，是目前临床急需的免疫治疗。最近有报道显示，抗CCR8抗体通过去岩藻糖修饰增强抗体依赖细胞介导的细胞毒作用(ADCC)，在临床前动物模型中显示了抑瘤作用(Andrew Lake,Michael Warren,Sonia Das,Christopher Wells,Maria Scrivens,Ernest Smith,Vito Palombella,Bijan Etemad-Gilbertson,Pamela Holland.2020 SRF114 is a Fully Human,CCR8-Selective IgG1 Antibody that Induces Destruction of Tumor Tregs Through ADCC.J Immunother Cancer 2020；8(Suppl 3):A1–A559；Campbell,J.R.,et al.2021 Fc-Optimized Anti-CCR8 Antibody Depletes Regulatory T Cells in Human Tumor Models.Cancer Res 81(11):2983-2994；Dépis,F.,Hu,C.,Weaver,J.,McGrath,L.,Klebanov,B.,Buggé,J.,&Shaffer,D.R.(2020).Preclinical evaluation of JTX-1811,an anti-CCR8antibody with enhanced ADCC activity,for preferential depletion of tumor-infiltrating regulatory T cells.[abstract].In:Proceedings of the Annual Meeting of the American Association for Cancer Research 2020；2020Apr 27-28and Jun 22-24.Philadelphia(PA):AACR；Cancer Res 2020；80(16Suppl):Abstract nr 4532.)。
发明内容
本公开涉及全新的高亲和力CCR8单克隆抗体及人源化抗体，具有阻断CCL1/CCR8轴的中和活性，可抑制CCL1诱导的细胞活化，同时针对人群中低亲和力FcγRIIIA158F等位基因携带者，通过Fc优化增强了ADCC效应。在体外药效研究中，新的人源化抗体可有效清除CCR8+细胞，显著抑制人源化小鼠皮下移植瘤模型中MC38、B16/F10和MDA-MB-231肿瘤的生长，同时显示了与抗PD-1抗体联用的突出疗效。相对于现有技术，本公开中的CCR8抗体具有全新序列和显著的生物学活性优势。
本公开涉及新型抗CCR8抗体或其抗原结合部分，用于抑制CCL1诱导的钙流，提高效应细胞的ADCC杀伤活性。进一步地，本公开涉及治疗癌症的新型抗CCR8抗体或其抗原结合部分。
在一方面，本公开公开了一种结合CCR8的单克隆抗体或其抗原结合部分。
在一方面，本公开提供了编码前述的结合CCR8的抗体或其抗原结合部分的核酸。
在一方面，本公开提供了包含前述的核酸的载体。
在一方面，本公开提供了包含前述的核酸或前述的载体的细胞。
在一方面，本公开提供了包含前述的抗体或其抗原结合部分、编码核酸、载体、细胞的组合物。
在一方面，本公开提供了包含前述抗体或其抗原结合部分、编码核酸、载体、细胞、组合物的试剂盒。
在一方面，本公开提供了治疗与癌症的相关疾病的方法，其包括下述步骤：向所述受试者施用治疗有效量的前述任一方面的抗体或其抗原结合片段、核酸、载体、细胞和/或组合物。
在一方面，本公开提供了前述任一方面的抗体或其抗原结合部分、核酸、载体、细胞和/或药物组合物在制备用于治疗受试者患有的癌症的药物中的用途。
附图说明
图1示出了稳定表达CCR8或CCR4细胞株的FACS检测结果，其中：
A)人CCR8稳转CHO细胞株CHO-hCCR8；
B)人CCR8稳转HEK293细胞株HEK293-hCCR8；
C)表达水平较低的人CCR8稳转HEK293细胞株HEK293-hCCR8low；
D)食蟹猴CCR8稳转CHO细胞株CHO-cynoCCR8；
E)人CCR4稳转CHO细胞株CHO-hCCR4。
图2示出了抗CCR8嵌合抗体的FACS检测结果，显示抗CCR8嵌合抗体与人或食蟹猴CCR8+细胞株的高亲和力结合。
图3示出了抗CCR8嵌合抗体的ADCC活性。
图4示出了抗CCR8嵌合抗体对CCL1引起靶细胞钙流具有显著抑制活性。
图5示出了抗CCR8嵌合抗体CDR序列优化后与CCR8+细胞的结合，其中：
A)M56位点突变对CCR8+细胞结合的影响；
B)N33、G34位点突变对CCR8+细胞结合的影响。
图6示出了人源化抗CCR8抗体与人CCR8+细胞的高亲和力结合。
图7示出了人源化抗CCR8抗体与食蟹猴CCR8+细胞的高亲和力结合。
图8示出了人源化抗CCR8抗体以NK92细胞为效应细胞检测的ADCC杀伤活性。
图9示出了Fc功能增强型人源化抗CCR8抗体对不同表达水平人CCR8+细胞的ADCC杀伤活性，其中：
A)以NK92-CD16A细胞为效应细胞、以高表达CCR8的HEK293-hCCR8为靶细胞检测的ADCC杀伤活性；
B)以NK92-CD16A细胞为效应细胞、以中低表达人CCR8的HEK293-hCCR8low为靶细胞检测的ADCC杀伤活性；
C)以NK92-CD16A细胞为效应细胞、以内源性表达人CCR8的HuT78细胞为靶细胞检测的ADCC杀伤活性；
D)以分离自基因型为FcγRⅢA158F/F的健康志愿者PBMC为效应细胞、以HEK293-hCCR8为靶细胞检测的ADCC杀伤活性；
E)以分离自基因型为FcγRⅢA158V/V的健康志愿者PBMC为效应细胞、以HEK293-hCCR8为靶细胞检测的ADCC杀伤活性。
图10示出了人源化抗CCR8抗体对CCL1引发Ca2+流信号的抑制活性。
图11示出了人源化抗CCR8抗体与兔CCR8+细胞的结合高亲和力结合。
图12示出了人源化抗CCR8抗体显著抑制人源化小鼠中MC38皮下移植瘤的生长。
图13示出了人源化抗CCR8抗体显著抑制人源化小鼠中B16/10皮下移植瘤的生长，与抗小鼠PD-1抗体联用后，人源化抗CCR8抗体能更有效抑制肿瘤生长。
图14示出了人源化抗CCR8抗体显著抑制人源化小鼠中MDA-MB-231皮下移植瘤的生长。
具体实施方式
I.定义
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
本文提供的是特异性结合CCR8的抗体(例如，单克隆抗体)及其抗原结合片段。在具体的方面，本文提供的是特异性结合CCR8的单克隆抗CCR8抗体，其中所述抗CCR8抗体包括亲本抗体的变体。在具体的方面，本文提供的是特异性结合CCR8(例如，人CCR8)的抗体。在特定的方面，本文提供的是包含一个或更多个氨基酸残基中的修饰的抗CCR8抗体(例如，重链可变区的框架区中1-3个氨基酸取代)，与没有所述修饰的亲本抗体相比，其保持与抗原的亲和力。术语“CCR8”是指本领域技术人员已知的任何CCR8受体。例如所述CCR8可以来自哺乳动物，例如CCR8可以来自人或食蟹猴。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核苷酸序列。
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性(参见，例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Pres,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987；and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。
“取代型”变体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变体。所述取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变体包括取代和插入二者。“插入型”变体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。“缺失型”变体是天然氨基酸序列中一个或多个氨基酸被除去的变体。通常情况下，缺失型变体在其分子的特定区域内有一个或两个氨基酸被缺失。
就抗体的可变结构域而言，术语“可变”系指抗体之间有广泛序列差异的相关分子的某些部分，且被用于针对其特异靶的特定抗体的特异识别和结合。但是，可变性在抗体的整个可变结构域内不是均匀分布的。可变性集中在被称为互补决定区域(CDRs；即CDR1、CDR2和CDR3)或超变区的三个区段，它们均位于轻链和重链的可变结构域内。可变结构域内保守程度更高的部分被称为构架(FR)区或构架序列。天然重链和轻链的每个可变结构域均包括四个FR区，其主要采用β-折叠构型，它们籍三个CDRs连接起来，CDRs形成环，所述环连接β-折叠结构并在某些情形下形成部分的β-折叠结构。每条链的CDRs通常被FR区在邻近连接起来，并且借助于来自其它链的CDR，有助于抗体靶结合位点(表位或决定簇)的形成(参看Kabat等人Sequences of Proteins of Immunological Interest,NationalInstitute of Health,Bethesda,MD(1987))。正如本文所使用，免疫球蛋白氨基酸残基的编号是依据Kabat等人的免疫球蛋白氨基酸残基编号系统而进行的，除非另有说明。一个CDR可具有特异结合关联表位的能力。
如本文所用，抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重 组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab'、F(ab')2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段以及其他片段，包括修饰的片段(参见，例如，Methods in Molecular Biology，Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25,Kipriyanov)。所述片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约107-108M-1的Ka)抗原的抗体。“功能片段”或“抗CCR8抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段″含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab')2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域以非共价结合方式而形成的二聚体(VH-VL二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定VH-VL二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半)，仍可具有识别和结合靶的能力。
如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备(Smith et al.(2004)J.Clin.Pathol.57,912-917；和Nelson et al.,J Clin Pathol(2000),53,111-117)。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
如本文所用，全长抗体是具有两条全长重链(例如VH-CH1-CH2-CH3或VH-CH1-CH2-CH3-CH4)和两条全长轻链(VL-CL)和铰链区的抗体，例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab')2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本公开所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
“保守氨基酸取代”可涉及用非原生残基取代原生氨基酸残基使得对该位置处的氨基酸残基的极性或电荷影响较小或没有影响。保守取代的例子包括将一个非极性(疏水性)氨基酸残基如异亮氨酸、缬氨酸、亮氨酸、正亮氨酸、丙氨酸或甲硫氨酸取代为另一个；将一个极性(亲水性)氨基酸残基取代为另一个，例如精氨酸和赖氨酸之间的取代、谷氨酰胺和天冬酰胺之间的取代、甘氨酸和丝氨酸之间的取代；将一个碱性氨基酸残基如赖氨酸、精氨酸或组氨酸取代为另一个；或将一个酸性残基如天冬氨酸或谷氨酸取代为另一个。短语“保守氨基酸取代”还包括使用化学衍生化残基替换非衍生化残基，前提条件是该多肽展示所需的生物活性。可根据本发明使用的其他示例性氨基酸取代示于下表1中。
表1一些有用的氨基酸取代。

如本文所用，术语“CDR”指互补决定区(complementarity-determining region)，已知抗体分子的每个重链和轻链具有3个CDR。CDR也称作高变区，且存在于抗体的每个重链和轻链的可变区中，在CDR的一级结构中具有非常高的变异性位点。本说明书中，重链的CDR由来自重链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，轻链的CDR由来自轻链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示。这些位点在三级结构中彼此临近，并决定抗体所结合的抗原的特异性。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×107M-1或1×108M-1或更大的亲和常数Ka(或者1×10-7M或1×10-8M或更低的解离常数(Kd))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)(Rich and Myszka(2000)Curr.Opin.Biotechnol 11:54；Englebienne(1998)Analyst.123:1599)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定(参见，例如，Paul,ed.,Fundamental Immunology,2nd ed.,Raven Press,New York,pages 332-336(1989)；还参见描述用于计算抗体的结合亲和力的示例性SPR和ITC方法的美国专利第7，229，619号)。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购(参见，BiaCore 2000,Biacore AB,Upsala,Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27:335)。
如本文所用，术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。如本文所使用，术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链，且可为cDNA。
作为另一选择，在另一实施方案中，可通过例如饱和诱变沿抗CCR8抗体编码序列的全部或一部分随机引入突变，且可针对改良的结合活性筛选所得经修饰抗CCR8抗体。
如本文所用，“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合片段以及本文所提供的组合物的任何药学用途。
如本文所用，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
如本文所用，“预防有效量”或“预防有效剂量”指在施用于对象时会具有预期的预防效果的物质、化合物、材料或包含化合物的组合物的量，例如，防止或延迟疾病或症状的发生或复发，减少疾病或症状发生或复发的可能性。 完全预防有效剂量不必通过施用一个剂量发生，并且可以仅在施用一系列剂量之后发生。因此，预防有效量可以在一次或多次施用中施用。
如本文中所使用的，术语“患者”或“受试者”是指哺乳动物，例如人。
如本文使用的和除非另作说明，术语“包含”，“包括”，“具有”，“含有”，包括其语法上的等同形式，通常应当理解为开放式且非限制性的，例如，不排除其他未列举的要素或步骤。
II.具体实施方式详述
在一方面，本公开提供一种结合CCR8的抗体或其抗原结合部分，其包含重链可变区和轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含重链可变区和轻链可变区，其中：
(i)重链可变区包含：
(a)具有下式的HCDR1序列：
NX1X2AMN(SEQ ID NO.102)，其中：
X1为A或T；
X2为F或Y；和
(b)具有下式的HCDR2序列：
RIRSKSNX3YATX4X5ADSVX6X7(SEQ ID NO.103)，其中：
X3为N或Y；
X4为Y或H；
X5为Y或S；
X6为I或K；
X7为D或G；和
(c)具有下式的HCDR3序列：
GKEAGX8X9X10X11X12X13DY(SEQ ID NO.104)，其中：
X8为A或不存在；
X9为Y、T或A；
X10为Y或N；
X11为A或Y；
X12为M或A；
X13为M或不存在；和
(ii)轻链可变区包含：
(a)具有下式的LCDR1序列：
RSSKSLLHSX14X15NTYLY(SEQ ID NO.105)，其中：
X14为N、A、I、V、Y或R；
X15为G、I、V、Y、T或R；和
(b)具有下式的LCDR2序列：
RX16SNLAS(SEQ ID NO.106)，其中：
X16为M、A、G或T；和
(c)具有下式的LCDR3序列：
MQHLEYPX17(SEQ ID NO.107)，其中：
X17为F或L。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含重链可变区和轻链可变区，其中：
(i)重链可变区包含：
(a)具有下式的HCDR1序列：
NAYAMN(SEQ ID NO.28)；和
(b)具有下式的HCDR2序列：
RIRSKSNNYATYYADSVKD(SEQ ID NO.22)；和
(c)具有下式的HCDR3序列：
QKFGX18RGYYALDF(SEQ ID NO.108)，其中：
X18为A或S；和
(ii)轻链可变区包含：
(a)具有下式的LCDR1序列：
X19ASX20SX21X22X23X24X25X26X27X28X29X30(SEQ ID NO.109)，其中：
X19为R、T或S，X20为E、K或S，X21为V或I，X22为E或S，X23为Y、T或不存在，X24为Y、S或不存在，X25为G或不存在，X26为T、Y或S，X27为S或N，X28为L或Y，X29为L或M，X30为Q或H；和
(b)具有下式的LCDR2序列：
X31X32SX33X34X35S(SEQ ID NO.110)，其中：
X31为R、A、L或S，X32为A或T，X33为N或T，X34为L或V，X35为A或E；和
(c)具有下式的LCDR3序列：
X36X37X38X39X40X41PX42X43(SEQ ID NO.111)，其中：
X36为Q或H，X37为Q或H，X38为S、Y或G，X39为R、S、H或T，X40为E、K、G、R、V或S，X41为Y、V、L、S或I，X42为L或不存在，X43为L、Y、P、R或G。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含选自氨基酸序列SEQ ID NO.6、16、28或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.7、17、22、82或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.8、29、49或其任何变体的重链CDR3；和选自氨基酸序列SEQ ID NO.11、32、39、44、52、62、91或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.12、33、40、45、58、63或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.13、25、34、41、46、53、59、64或其任何变体的轻链CDR3。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.6、7、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、13的轻链CDR1、CDR2及CDR3序列的重链和轻链的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.16、17、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、13的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.16、22、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、25的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.28、22、29的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.32、33、34的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.28、22、29的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.39、40、41的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.28、22、29的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.44、45、46的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.28、22、49的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.52、45、53的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.28、22、49的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.44、58、59的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.28、22、29的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.62、63、64的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.6、7、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、15的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.16、82、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、25的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.6、82、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、25的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含分别包含SEQ ID NO.16、7、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.91、63、25的轻链CDR1、CDR2及CDR3序列的CDR组合。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含选自氨基酸序列SEQ ID NO.4、14、20、26、35、47、54、80、83、85、92、96、100或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.9、18、23、30、37、42、50、56、60、65、67、68、69、70、71、72、73、74、75、76、77、78、79、87、89、94、98或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.9或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.67或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.68或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.69或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.70或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.71或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.72或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可 变区，和氨基酸序列SEQ ID NO.73或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.74或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.75或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.76或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.77或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.78或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.79或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.14或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.18或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.20或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.23或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.26或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.30或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.35或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.37或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.35或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.42或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.47或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.50或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.54或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.56或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.35或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.60或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.65或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.80或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.87或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.83或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.87或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.85或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.89或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.92或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.94或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.96或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.98或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分包含氨基酸序列SEQ ID NO.100或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.98或其任何变体的轻链可变区。
在一些优选地实施方式中，前述抗体或其抗原结合部分是人源化的。
另一方面，本公开还提供编码前述抗体或其抗原结合部分的核酸。
优选地，所述核酸包含选自SEQ ID NO.5、15、21、27、36、48、55、81、84、86、93、97、101或其任何变体的抗体重链可变区核酸序列，和/或选自SEQ ID NO.10、19、24、31、38、43、51、57、61、66、88、90、95、99或其任何变体的抗体轻链可变区核酸序列；
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.5或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.10或其任何变体的轻链可变区核苷酸序列。
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.15或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.19或其任何变体的轻链可变区核苷酸序列；
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.21或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.24或其任何变体的轻链可变区核苷酸序列；
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.27或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.31或其任何变体的轻链可变区核苷酸序列；
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.36或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.38或其任何变体的轻链可变区核苷酸序列；
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.36或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.43或其任何变体的轻链可变区核苷酸序列；
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.48或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.51或其任何变体的轻链可变区核苷酸序列；
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.55或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.57或其任何变体的轻链可变区核苷酸序列；
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.36或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.61或其任何变体的轻链可变区核苷酸序列。
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.5或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.66或其任何变体的轻链可变区核苷酸序列。
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.81或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.88或其任何变体的轻链可变区核苷酸序列。
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.84或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.88或其任何变体的轻链可变区核苷酸序列。
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.86或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.90或其任何变体的轻链可变区核苷酸序列。
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.93或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.95或其任何变体的轻链可变区核苷酸序列。
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.97或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.99或其任何变体的轻链可变区核苷酸序列。
在一些优选地实施方式中，前述核酸包含核苷酸序列SEQ ID NO.101或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.99或其任何变体的轻链可变区核苷酸序列。
另一方面，本公开还提供包含编码前述核酸的载体。
另一方面，本公开还提供包含前述核酸或前述载体的细胞。
另一方面，本公开还提供一种包含前述抗体或其抗原结合部分、核酸、载体和/或细胞的组合物。
另一方面，本公开提供了一种试剂盒，其包含前述的抗体或其抗原结合部分、核酸分子、载体、细胞和/或组合物。
用本公开的CCR8抗体治疗与癌症相关的疾病的方法包括下述步骤：向所述受试者施用治疗有效量的前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物。
与癌症相关的疾病包括乳癌、子宫癌、子宫颈癌、卵巢癌、前列腺癌、肺癌、胃(胃腺)癌、非小细胞肺癌、胰脏癌、头颈部鳞状细胞癌、食道癌、膀胱癌、黑色素瘤、大肠癌、肾癌、非霍奇金氏淋巴瘤、泌尿上皮癌、肉瘤、血球癌(白血病、淋巴瘤等)、胆管癌、胆囊癌、甲状腺癌、睾丸癌、胸腺癌、肝脏癌等癌症，优选为乳癌、子宫癌、卵巢癌、肺癌、大肠癌、肾癌及肉瘤，更优选为乳癌、大肠癌、肾癌及肉瘤。
在本公开一个优选地实施方式中，优选地与癌症相关的疾病是结肠癌或直肠癌。
本公开的一个实施方案中，以单剂量或多剂量施用抗体。
如果根据本公开以多次剂量施用所述抗体，则优选以至少3次剂量、至少4次剂量、至少5次剂量、至少6次剂量、至少7次剂量、至少8次剂量、至少9次剂量或至少10次剂量且优选至多30次剂量、25次剂量、20次剂量、15次剂量或10次剂量施用所述抗体。优选以至少7天、至少10天、至少14天或至少20天的时间间隔施用所述抗体的剂量。优选以7至30天、10至20天且优选为约14天的时间间隔施用所述抗体的剂量。
在一方面，本公开提供了治疗剂在制备用于预防或治疗癌症相关的疾病的药物中的用途，所述治疗剂选自单一治疗剂或联合治疗剂。
在一些实施方案中，所述单一治疗剂选自前述抗体或其抗原结合部分、核酸、载体、细胞和/或组合物。
在一些实施方案中，所述联合治疗剂包括所述单一治疗剂和免疫检查点调节剂。在一些实施方案中，所述免疫检查点调节剂为选自PD-1、CTLA4、PD-L1、PD-L2、CD47、TIGIT、GITR、TIM3、LAG3、4-1BB、CD27和B7H4的免疫检查点蛋白的抑制剂。在一些实施方案中，所述免疫检查点蛋白的抑制剂为抗体或其抗原结合片段。在一些优选的实施方案中，免疫检查点蛋白是PD-1或CTLA4。在一些优选的实施方案中，所述免疫检查点蛋白的抑制剂选自抗PD-1抗体或其抗原结合片段或抗CTLA-4抗体或其抗原结合片段。
在另一方面，本公开提供了治疗受试者与癌症相关的疾病的方法，所述治疗方法使用治疗剂来治疗所述受试者，所述治疗剂选自单一治疗剂或联合治疗剂。
在一些实施方案中，所述单一治疗剂选自前述抗体或其抗原结合部分、核酸、载体、细胞和/或组合物。
在一些实施方案中，所述联合治疗剂包括所述单一治疗剂和免疫检查点调节剂。在一些实施方案中，所述免疫检查点调节剂为选自PD-1、CTLA4、PD-L1、PD-L2、CD47、TIGIT、GITR、TIM3、LAG3、4-1BB、CD27和B7H4的免疫检查点蛋白的抑制剂。在一些实施方案中，所述免疫检查点蛋白的抑制剂为抗体或其抗原结合片段。在一些优选的实施方案中，免疫检查点蛋白是PD-1或CTLA4。在一些优选的实施方案中，所述免疫检查点蛋白的抑制剂 选自抗PD-1抗体或其抗原结合片段或抗CTLA-4抗体或其抗原结合片段。
在另一方面，本公开提供了用于治疗受试者与癌症相关的疾病的治疗剂，所述治疗剂选自单一治疗剂或联合治疗剂。
在一些实施方案中，所述单一治疗剂选自前述抗体或其抗原结合部分、核酸、载体、细胞和/或组合物。
在一些实施方案中，所述联合治疗剂包括所述单一治疗剂和免疫检查点调节剂。在一些实施方案中，所述免疫检查点调节剂为选自PD-1、CTLA4、PD-L1、PD-L2、CD47、TIGIT、GITR、TIM3、LAG3、4-1BB、CD27和B7H4的免疫检查点蛋白的抑制剂。在一些实施方案中，所述免疫检查点蛋白的抑制剂为抗体或其抗原结合片段。在一些优选的实施方案中，免疫检查点蛋白是PD-1或CTLA4。在一些优选的实施方案中，所述免疫检查点蛋白的抑制剂选自抗PD-1抗体或其抗原结合片段或抗CTLA-4抗体或其抗原结合片段。
本公开所述试剂盒包括本公开的抗体、其片段、同源物、其衍生物等，例如带标记或具有细胞毒性的缀合物，以及抗体使用说明书、杀死特定类型细胞的缀合物等等。该说明书可包括在体外、体内或离体使用抗体、缀合物等的指导。抗体可以是液体形式或固体，通常是冻干的。该试剂盒可包含其它适宜的试剂，如缓冲液、重构溶液以及为了预定用途的其它必要成分。考虑了以预定量包装好的试剂组合与用于其用途的说明书，所述用途例如用于治疗用途或用于进行诊断测定。当抗体是带标记的时，例如用酶标记的，那么该试剂盒可包括底物和酶所需的辅因子(例如提供可检测生色团或荧光团的底物前体)。此外，其它添加剂，如稳定剂、缓冲液(例如封闭缓冲液或裂解缓冲液)等也可包括在内。多种试剂的相对量可以改变而提供试剂溶液的浓缩物，这就提供了用户灵活性、节省空间、节省试剂等。这些试剂也可以干粉形式提供，通常是冻干形式，包括赋形剂，它在溶解时可提供具有适当浓度的试剂溶液。
在一方面，本公开提供了所述的抗体或其抗原结合部分、核酸分子、载体、细胞和/或组合物抗在制备用于诊断、治疗或预防与癌症相关的疾病的药物或试剂盒中的用途。
此外，本公开的抗体还可用于免疫测定、纯化方法以及其它用到免疫球蛋白或其片段的方法。此类用途在本领域为人所熟知。
相应地，本公开还提供包含本公开的抗CCR8的抗体或其片段的组合物，所述抗体方便地和药学上可接受的载体、稀释剂或赋形剂组合，这是本领域的常规做法。
本公开所使用的术语“药物组合物”系指多种制备物的制剂。含有治疗有效量的多价抗体的制剂为无菌液体溶液、液体悬浮剂或冻干形式，任选地包含稳定剂或赋形剂。
应当理解，根据所述实施方案的治疗剂将与合适的药学上可接受的载体、赋形剂、以及其它被掺入制剂中以提供改善的转移、递送、耐受性等的试剂一同施用。大量适当的制剂可见于所有药物化学工作者已知的药典中：Remington's Pharmaceutical Sciences(第15版，Mack Publishing Company，Easton，Pa.(1975))，特别是其中Blaug、Seymour的第87章。这些制剂包括例如粉末、糊剂、膏剂、凝胶剂、蜡、油、脂质、含脂质(阳离子或阴离子)载体(例如LipofectinTM)、DNA缀合物、无水吸浆、水包油和油包水乳液、乳液聚乙二醇(各种分子量的聚乙二醇)、半固态凝胶以及含有聚乙二醇的半固态混合物。任何前述混合物均可适用于根据本公开的治疗或疗法，条件是制剂中的活性成分不被制剂灭活并且制剂在生理学上是相容的并耐受给药途径。
对于吸入给药，从包含合适推进剂如二氧化碳等气体的加压容器或分配器或者喷雾器以气溶胶喷雾形式递送化合物。
还可以通过经粘膜或透皮方式全身给药。对于经粘膜或透皮给药，在制剂中使用适于渗透屏障的渗透剂。此类渗透剂通常在本领域是通常所知的，并且包括如用于经粘膜给药的去污剂、胆盐和夫西地酸衍生物。经粘膜给药可以通过使用喷鼻剂或栓剂来实现。对于透皮给药，可将一种或多种所述抗体配制成如本领域通常所知的膏剂、软膏、凝胶、或霜膏。
还可将化合物以栓剂(例如，具有常规栓剂基质，如可可脂或其它甘油酯)或滞留性灌肠剂形式进行制备以用于经直肠递送。
在一个实施方案中，所述抗体可用防止其不被身体迅速消除的载体制备，例如缓释/控释制剂，包括植入体和微胶囊化递送体系。可使用可生物降解、可生物相容的聚合物，例如乙烯-乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。用于制备此类制剂的方法对于本领域技术人员而言是显而易见的。
尤其有利的是以剂量单位形式配制肠胃外组合物以易于施用和剂量的一致性。如本文所用，剂量单位形式是指用于待治疗的受试者，适合作为单位剂量的物理上可分离的单位；每个单位含有经计算与所需药物载体结合产生期望治疗效果的预定量的一种或多种所述抗体。所述实施方案的剂量单位形式的规格由以下指示并直接取决于：抗体的独特特征和待实现的具体治疗效果，和用于治疗个体的此类抗体的调配领域中固有的局限性。
所述药物组合物可与给药说明书一起放于容器、包装、或分配器中。
本文所述制剂还可根据要治疗的具体情况而包含多于一种所述抗体，优选具有互补活性但对彼此无负面影响的那些。另选地或除此之外，组合物可例如包含增强其功能的试剂，诸如细胞毒素试剂、细胞因子、化学治疗剂、或生长抑制剂。此类分子以对预期目的有效的量适当地联合存在。例如，可以在试剂盒中联合存在，也可以在使用中联合存在。
在一个实施方案中，一种或多种所述抗体可在联合治疗中施用，即与其它试剂例如治疗剂(其可用于治疗病理学病症或障碍，例如各种形式的癌症和炎性疾病)联合。术语“联合”在本文中是指将试剂基本上同步地，同时地或顺次地给予。如果顺次给予，则在开始施用第二种化合物时，两种化合物中的第一种仍优选在治疗位点处以有效浓 度被检测到。在一种情况下，“联合”也可以是在试剂盒中同时包含本公开的抗体和其他治疗剂。
例如，联合治疗可包含本文所述一种或多种抗体与一种或多种附加治疗剂(例如一种或多种细胞因子和生长因子抑制剂、免疫抑制剂、抗炎剂、代谢抑制剂、酶抑制剂、和/或细胞毒素或细胞生长抑制剂，如下更详述的)共同配制和/或共同施用。此类联合治疗可有利地利用较低剂量的施用的治疗剂，因而避免了与各种单一疗法相关的可能毒性或并发症。
本公开实验证明，本公开提供的多种可高亲和力结合CCR8的单克隆抗体及其变体具有阻断CCL1/CCR8轴的中和活性，可抑制CCL1诱导的细胞活化，同时针对人群中低亲和力FcγRIIIA158F等位基因携带者，通过对人源化抗体进行Fc优化增强了Fc优化的人源化抗体的ADCC效应。在体外药效研究中，新的人源化抗体可有效清除CCR8+细胞，显著抑制人源化小鼠中MC38、B16/F10和MDA-MB-231肿瘤细胞的皮下移植瘤生长，在PD-1抗体不敏感的B16/F10皮下移植瘤模型中，与抗PD-1抗体联用后，人源化CCR8抗体能更有效地抑制肿瘤生长。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
实施例
实施例1、抗体制备
构建CCR8+或CCR4+稳转细胞株。分别以人CCR8 cDNA(Sino Biological，货号：HG11450-M，其氨基酸序列如SEQ ID NO.1所示)、食蟹猴CCR8cDNA(苏州金唯智生物合成，其氨基酸序列如SEQ ID NO.2所示)和人CCR4 cDNA(Sino Biological，货号：HG13064-UT，其氨基酸序列如SEQ ID NO.3所示)为模板，构建含有人、猴CCR8和人CCR4全长序列的慢病毒载体质粒，按照慢病毒包装试剂盒说明书(Lenti-Pac HIV Expression Packaging Kit，Gene Copoeia，货号：HPK-LvTR-20)，将构建好的慢病毒质粒和包装质粒共转染HEK293T细胞(ATCC CRL-11268)，进行慢病毒包装。转染48小时后收集培养基，以500*g离心10分钟去除细胞碎片，获得含慢病毒颗粒的培养上清，0.45μm的PES膜过滤后，分装至1.5mL EP管中，分别取10μL用于感染1×106的HEK293T或CHO细胞(ATCC CCL-61)，48小时后添加8μg/ml嘌呤霉素加压筛选，得到相对高水平表达人CCR8的单克隆细胞株HEK293-hCCR8(流式检测荧光值相对HEK293细胞，增加至少2log)、HEK293-hCCR8low(流式检测荧光值相对HEK293细胞，增加<1log)和稳定表达人CCR4的细胞株HEK293-hCCR4，以及稳定表达人CCR8的CHO-hCCR8细胞株，和稳定表达食蟹猴CCR8的细胞株CHO-cynoCCR8，结果见图1。
选择4-6周龄雌性Balb/C小鼠，使用人或食蟹猴CCR8全长序列构建含CMV启动子的真核表达质粒，对小鼠使用基因枪进行DNA免疫(20μg质粒/小鼠)，免疫4-5次后检测小鼠血清滴度，待滴度符合要求后以CHO-hCCR8稳转细胞(5×106细胞/只)进行冲击免疫。3天后，断颈处死小鼠，收集小鼠脾脏与外周淋巴结，研磨离心收集后提取总RNA，逆转录后分别利用轻重链特异性引物进行PCR扩增，得到抗体的轻链和重链可变区cDNA文库。利用噬菌体抗体库展示技术，构建CCR8免疫文库，用于后续噬菌体淘选。
采用经典方法对构建的CCR8免疫文库进行淘洗，交替使用人CCR8+细胞CHO-hCCR8或食蟹猴CCR8+细胞CHO-cynoCCR8(1×106/管)，加入1×1012噬菌体进行孵育和淘洗，用100mM三乙胺洗脱结合亲和力高的噬菌体颗粒，以1M Tris-HCl(pH 7.4)中和后，感染入大肠杆菌TG1细菌中，再次包装后得到的噬菌体用于下一轮淘洗。经过2轮淘洗富集得到的克隆，挑取单克隆接种至96孔U形孔板中，进行培养和IPTG诱导表达，取诱导上清进行FACS检测。
实施例2、筛选高特异性CCR8结合抗体
将实施例1中得到的阳性克隆，重新诱导表达后通过纯化得到Fab抗体，分别检测与人CCR8、食蟹猴CCR8和人CCR4稳转细胞的结合。在96孔U型板中每孔加入5×104个检测细胞，离心弃上清，加入100μL/孔纯化的Fab抗体，冰上孵育60分钟。洗去未结合的一抗后，每孔加入50μL二抗溶液(Anti-human IgG，F(ab')2-AF647，Jackson Immuno Research)，冰上孵育30分钟；洗涤后每孔加入50μL的5μg/mL PI/BSA溶液，冰上孵育5分钟。之后用0.5％BSA洗去多余的二抗，加入PBS溶液重悬细胞，使用流式细胞仪(Intellicyte iQue)检测抗体与细胞的结合。选择识别人CCR8+细胞和食蟹猴CCR8+细胞，同时不结合人CCR4+细胞的约200个阳性克隆，通过Sanger测序确定轻、重链可变区序列，排除重复序列后，结合活性较优的前10个克隆序列见下表(表2)。
表2抗CCR8抗体轻重链可变区序列

抗CCR8鼠抗重链/轻链可变区序列如下：
克隆：29A8
29A8重链VH的氨基酸序列如SEQ ID NO.4所示，其编码核酸如SEQ ID NO.5所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.6、7、8所示。
核酸序列
29A8轻链VK的氨基酸序列如SEQ ID NO.9所示，其编码核酸如SEQ ID NO.10所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.11、12、13所示。
核酸序列
克隆：30A7
30A7重链VH的氨基酸序列如SEQ ID NO.14所示，其编码核酸如SEQ ID NO.15所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.16、17、8所示。
核酸序列
30A7轻链VK的氨基酸序列如SEQ ID NO.18所示，其编码核酸如SEQ ID NO.19所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.11、12、13所示。
核酸序列
克隆：87D8
87D8重链VH的氨基酸序列如SEQ ID NO.20所示，其编码核酸如SEQ ID NO.21所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.16、22、8所示。
核酸序列
87D8轻链VK的氨基酸序列如SEQ ID NO.23所示，其编码核酸如SEQ ID NO.24所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.11、12、25所示。
核酸序列
克隆：102H5
102H5重链VH的氨基酸序列如SEQ ID NO.26所示，其编码核酸如SEQ ID NO.27所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.28、22、29所示。
核酸序列
102H5轻链VK的氨基酸序列如SEQ ID NO.30所示，其编码核酸如SEQ ID NO.31所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.32、33、34所示。
核酸序列
克隆：109F6
109F6重链VH的氨基酸序列如SEQ ID NO.35所示，其编码核酸如SEQ ID NO.36所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.28、22、29所示。
核酸序列
109F6轻链VK的氨基酸序列如SEQ ID NO.37所示，其编码核酸如SEQ ID NO.38所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.39、40、41所示。

核酸序列
克隆：110G2
110G2重链VH的氨基酸序列如SEQ ID NO.35所示，其编码核酸如SEQ ID NO.36所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.28、22、29所示。
核酸序列
110G2轻链VK的氨基酸序列如SEQ ID NO.42所示，其编码核酸如SEQ ID NO.43所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.44、45、46所示。
核酸序列
克隆：113B3
113B3重链VH的氨基酸序列如SEQ ID NO.47所示，其编码核酸如SEQ ID NO.48所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.28、22、49所示。
核酸序列
113B3轻链VK的氨基酸序列如SEQ ID NO.50所示，其编码核酸如SEQ ID NO.51所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.52、45、53所示。
核酸序列
克隆：116F10
116F10重链VH的氨基酸序列如SEQ ID NO.54所示，其编码核酸如SEQ ID NO.55所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.28、22、49所示。

核酸序列
116F10轻链VK的氨基酸序列如SEQ ID NO.56所示，其编码核酸如SEQ ID NO.57所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.44、58、59所示。
核酸序列
克隆：139A9
139A9重链VH的氨基酸序列如SEQ ID NO.35所示，其编码核酸如SEQ ID NO.36所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.28、22、29所示。
核酸序列
139A9轻链VK的氨基酸序列如SEQ ID NO.60所示，其编码核酸如SEQ ID NO.61所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.62、63、64所示。
核酸序列
克隆：144D2
144D2重链VH的氨基酸序列如SEQ ID NO.4所示，其编码核酸如SEQ ID NO.5所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.6、7、8所示。
核酸序列
144D2轻链VK的氨基酸序列如SEQ ID NO.65所示，其编码核酸如SEQ ID NO.66所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.11、12、25所示。
核酸序列
实施例3、抗CCR8嵌合抗体的表达和亲和力检测
将实施例2阳性克隆的重链可变区克隆入含有人重链固定区和调节元件的载体，以在哺乳动物细胞中表达完整的IgG重链；类似地，将轻链可变区克隆入含有人轻链固定区和调节元件的载体，以在哺乳动物细胞中表达完整的IgG轻链。经测序正确后转染入CHO-S哺乳动物细胞(Thermo Fisher ExpiCHO)中，IgG经表达分泌入培养基中，收集上清，通过常规ProA纯化得到抗CCR8嵌合抗体，通过分光光度法测定OD280光吸收，确定抗体浓度。鼠抗m10A11(chimera)及人源化抗体h10A11参照文献WO2020138489A1合成(m10A11：该参照文献的序列27和序列26，h10A11：该参照文献的序列41和序列59)。
在96孔U型板中每孔加入5×104个CHO-hCCR8或CHO-cynoCCR8细胞，离心弃上清，每孔加入100μL梯度稀释的抗CCR8嵌合抗体或抗KLH同型对照抗体，冰上孵育60分钟；洗去未结合的一抗后，每孔加入50μL的二抗AlexaFluro647抗人IgG(Jackson Immuno Research#109-606-170)，冰上孵育30分钟。洗涤后每孔加入50μL的5μg/mL PI/BSA溶液，冰上孵育5分钟，之后用0.5％BSA洗去多余的二抗，加入PBS溶液重悬细胞，使用流式细胞仪(Intellicyte iQue)检测抗体与细胞的结合，分析PI阴性细胞群的荧光值。通过Graphpad Prism 7拟合细胞结合曲线(图2)，87D8、102H5、109F6、110G2、113B3、139A9和144D2等嵌合抗体，均高亲和力结合人CCR8+细胞CHO-hCCR8和食蟹猴CCR8+细胞CHO-cynoCCR8。抗CCR8嵌合抗体与稳转细胞CCR8受体的亲和力(EC50)见表3。
表3抗CCR8嵌合抗体与人和食蟹猴CCR8+细胞的结合
实施例4、抗CCR8嵌合抗体的ADCC活性检测
构建人CD16A全长序列的慢病毒载体质粒，按照Lenti-Pac HIV Expression Packaging Kit(Gene Copoeia，HPK-LvTR-20)包装并转染NK92细胞(购自ATCC，货号CRL-2408)，筛选得到NK92-CD16A稳转细胞。在96孔U型板中每孔加入5×104个HEK293-hCCR8细胞，分别加入50μL梯度稀释的抗CCR8嵌合抗体或同型对照，在培养箱孵育30分钟；每孔加入50μL 2×106/mL NK92-CD16A细胞，继续培养4小时。检测前30分钟，向靶细胞最大孔加入2μL细胞裂解液(100×)，300*g离心3分钟，取50μL上清至黑色酶标板，加入50μL乳酸脱氢酶(LDH)检测底物，震荡混匀，10分钟后加入25μL终止液，震荡10秒，选择荧光(激发波长560nm，发射波长590nm)进行读板。根据LDH释放水平计算靶细胞杀伤率，并通过四参数模型拟合得到抗体浓度-杀伤率(％)曲线，采用该回归曲线计算供试品半数有效浓度(EC50)。图3显示，抗CCR8嵌合抗体对CCR8+细胞有显著ADCC杀伤活性，呈剂量依赖关系，29A8、87D8、102H5、109F6、110G2、113B3、116F10、139A9和144D2等抗体的半效杀伤浓度EC50均优于m10A11(表4)。
表4抗CCR8嵌合抗体的ADCC活性

实施例5、抗CCR8嵌合抗体抑制CCL1引起的靶细胞钙流信号
在96孔U型板中每孔加入5×104个CHO-hCCR8细胞，培养箱中培养过夜。用2.5mM丙磺舒/HBSS溶液洗细胞2次，每孔加入100μL 4μM Fluo-4AM工作液，37℃孵育60分钟。用丙磺舒/HBSS溶液洗细胞2次后，每孔加入50μL丙磺舒/HBSS于37℃孵育20分钟，接着加入50μL待测抗体或同型对照(终浓度10μg/mL)，室温孵育30分钟。使用酶标仪(Molecular Device SpectraMax i3x)，加入50μL l80nM CCL1，并读取在激发494nm、发射519nm的波长下的荧光信号。以抗体浓度为0的CCL1处理组的钙流信号为100％，计算各处理组钙流信号百分率。结果显示，抗CCR8嵌合抗体可显著抑制CCL1引起的钙流信号(图4)。
实施例6、抗CCR8抗体突变体与CCR8的结合
克隆87D8和144D2具有几乎完全相同的轻链序列，对潜在的脱氨基位点N33及附近位点及可能发生氧化修饰的位点M56，使用重叠延伸(overlapping)PCR定点突变法构建突变体，突变成Cys、Trp、Phe和母本序列以外的其它氨基酸。在大肠杆菌TG1中表达后，检测诱导上清与CHO-hCCR8细胞的结合。选择结合活性较好的克隆，重新诱导表达，通过抽提周质腔蛋白和Ni-NTA纯化，得到纯化的Fab抗体突变体，通过分光光度法测定OD280光吸收，参照理论消光系数，确定抗体浓度。检测与CHO-hCCR8的结合力EC50，其中144D2突变体G34Y、G34T、G34R和M56T均保留了与母本144D2相当或更优的亲和力，结果见表5及图5。
表5抗CCR8抗体突变体与人CCR8+细胞的结合
实施例7、抗CCR8抗体人源化及序列优化
选择与人和食蟹猴CCR8亲和力高，且ADCC杀伤活性强的克隆87D8和144D2进行人源化抗体构建。87D8和144D2采用的轻、重链胚系基因一样，CDR区序列高度相似，重链CDR1和CDR2分别有2个和1个氨基酸差异，推测可能是在小鼠B细胞扩增过程中产生的体细胞突变。首先将鼠抗体87D8和144D2序列与人抗体胚系序列进行比对，找出同源性高的人胚系轻链基因IMGT_hVK2-28、IGKJ2*01和人胚系重链基因IMGT_hVH3-7、IGHJ4*01，进行鼠抗体CDR移植，同时组合87D8和144D2重链的CDR1和CDR2。计算机分析显示，轻链CDR1 N33位点存在潜在的脱氨基化修饰，CDR2 M52位点可能产生氧化修饰，因此在设计人源化变体过程中，参考实施例6，引入G34Y和M52T突变，设计共得到3个重链变体h87D8 VHv1、h144D2 VHv1、h87D8 VHv2和2个轻链变体h87D8 VKv1、h87D8 VKv4(表6、表7)。
将轻、重链进行全序列合成，分别克隆到含有抗体kappa链恒定区或人IgG1恒定区CH1-CH3的载体，进行组合配对并转染CHO细胞，表达6天后过滤收集细胞培养液，利用Protein A亲和层析纯化得到人源化抗体。通过分光光度法测定OD280光吸收，确定抗体浓度。
表6、CCR8人源化抗体的重链可变区序列
表7、CCR8人源化抗体的轻链可变区序列
人源化抗体重链/轻链可变区序列如下：
h87D8 VHv1的氨基酸序列如SEQ ID NO.80所示，其编码核酸如SEQ ID NO.81所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.16、82、8所示。
核酸序列
h144D2 VHv1的氨基酸序列如SEQ ID NO.83所示，其编码核酸如SEQ ID NO.84所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.6、82、8所示。
核酸序列
h87D8 VHv2的氨基酸序列如SEQ ID NO.85所示，其编码核酸如SEQ ID NO.86所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.16、7、8所示。
核酸序列
h87D8 VKv1的氨基酸序列如SEQ ID NO.87所示，其编码核酸如SEQ ID NO.88所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.11、12、25所示。
核酸序列

h87D8 VKv4的氨基酸序列如SEQ ID NO.89所示，其编码核酸如SEQ ID NO.90所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.91、63、25所示。
核酸序列
实施例8、人源化抗CCR8抗体的亲和力检测
在96孔U型板中每孔加入5×104个CHO-hCCR8或CHO-cynoCCR8细胞，离心弃上清，每孔加入100μL梯度稀释抗CCR8嵌合抗体，冰上孵育60分钟；洗去未结合的一抗后，每孔加入50μL的二抗AlexaFluro647抗人IgG(Jackson Immuno Research#109-606-170)，冰上孵育30分钟。洗涤后每孔加入50μL的5μg/mL PI/BSA溶液，冰上孵育5分钟，之后用0.5％BSA洗去多余的二抗，加入PBS溶液重悬细胞，使用流式细胞仪(BD Celesta)检测抗体与细胞的结合，分析PI阴性细胞群的荧光值。通过Graphpad Prism 7拟合细胞结合曲线，图6和图7显示，人源化抗体h87D8 H1K1(由h87D8 VHv1+h87D8 VKv1组成)、h144D2 H1K1(h144D2 VHv1+h87D8 VKv1)和h87D8 H2K4(h87D8 VHv2+h87D8 VKv4)与母本嵌合抗体保持一致可高亲和力结合CCR8+细胞，与人CCR8+细胞的半效结合浓度EC50＝0.53-0.89nM，与食蟹猴CCR8+细胞的结合EC50＝1.16-2.28nM(表8)。
表8人源化抗体与CCR8+细胞的结合
实施例9、人源化抗CCR8抗体对人CCR8+细胞的ADCC活性
在96孔U型板中每孔加入5×104个HEK293-hCCR8细胞，分别加入50μL梯度稀释的抗CCR8人源化抗体或同型对照，在培养箱孵育30分钟；每孔加入50μL 2×106/mL NK92-CD16A细胞，继续培养4小时。检测前30分钟，向靶细胞最大孔加入2μL细胞裂解液(100×)，300*g离心3分钟，取50μL上清至黑色酶标板，加入50μL乳酸脱氢酶(LDH)检测底物，震荡混匀，10分钟后加入25μL终止液，震荡10秒，选择荧光(激发波长560nm，发射波长590nm)进行读板。根据LDH释放水平计算靶细胞杀伤率，并通过四参数模型拟合得到抗体浓度-杀伤率(％)曲线，采用该回归曲线计算供试品半数有效浓度(EC50)。结果显示，人源化抗体h87D8 H1K1和h144D2 H1K1对CCR8+细胞具有ADCC活性，半效杀伤浓度EC50分别为0.01nM和0.02nM，强于m10A11(EC50＝0.03nM)(图8A、图8B)。
实施例10、人源化抗CCR8抗体的Fc功能优化
1)Fc功能优化
FcγRⅢA基因存在多态性，其中携带高亲和力受体FcγRⅢA158V/V基因型仅占全部人群10％-20％，而对于携带低亲和力受体FcγRⅢA158F/F基因型的患者，野生型IgG1 Fc介导的ADCC活性通常较弱。采用专利申请202011627881.1中所描述的技术方案，在对CCR8嵌合抗体进行人源化改造的同时，针对人群中低亲和力FcγRIIIA158F等位基因携带者，构建具有Fc活性增强的变体R292P Y300L，得到h87D8 H1K1 hIgG1 e5(下文简称h87D8 H1K1 e5)和h87D8 H2K4 hIgG1 e5(下文简称h87D8 H2K4 e5)两个Fc功能增强型人源化抗体(表9)。
表9人源化抗CCR8抗体的序列
h87D8 H1K1 hIgG1 e5的重链氨基酸序列如SEQ ID NO.92所示，其编码核酸如SEQ ID NO.93所示。
核酸序列
h87D8 H1K1 hIgG1 e5的轻链氨基酸序列如SEQ ID NO.94所示，其编码核酸如SEQ ID NO.95所示。
核酸序列
h87D8 H2K4 hIgG1 e5的重链氨基酸序列如SEQ ID NO.96所示，其编码核酸如SEQ ID NO.97所示。
核酸序列

h87D8 H2K4 hIgG1 e5的轻链氨基酸序列如SEQ ID NO.98所示，其编码核酸如SEQ ID NO.99所示。
核酸序列
h87D8 H2K4 hIgG1的重链氨基酸序列如SEQ ID NO.100所示，其编码核酸如SEQ ID NO.101所示。
核酸序列
h87D8 H2K4 hIgG1的轻链氨基酸序列如SEQ ID NO.98所示，其编码核酸如SEQ ID NO.99所示。
核酸序列
2)Fc功能增强型人源化CCR8抗体与FcγRIIIA的亲和力
采用表面等离子共振技术(SPR/Biacore T200)，检测Fc功能增强型人源化CCR8抗体的与FcγRIIIAV158和FcγRIIIAF158的亲和力。采用偶联试剂Amine Coupling Kit(GE Healthcare BR-1000-50)将Goat Anti-Human IgG F(ab’)2偶联到CM5芯片表面，通过与抗体的高亲和力结合，将h87D8 H2K4 e5或h87D8 H2K4捕获，再依次注入梯度稀释的FcγRIIIAV158或FcγRIIIAF158，设置结合时间120秒，解离时间200秒，用pH 1.75的10mM Glycine-HCl再生30秒，流速设为30μl/min。所测数据进行拟合得到平衡解离常数。分别测定h87D8 H2K4 e5或h87D8 H2K4与FcγRIIIAV158和FcγRIIIAF158的亲和力。结果显示，h87D8 H2K4 e5、h87D8 H2K4均结合FcγRIIIAV158和FcγRIIIAF158，h87D8 H2K4 e5的亲和力KD分别为270.0nM和432.7nM；h87D8 H2K4的亲和力KD分别为861.2nM和1.3μM。h87D8 H2K4 e5与FcγRIIIAV158、FcγRIIIAF158的结合强于h87D8 H2K4。
3)Fc功能增强型人源化CCR8抗体的ADCC活性
参照实施例9方法，以NK92-CD16A为效应细胞，以HEK293-hCCR8为靶细胞，比较Fc功能增强型与野生型Fc的抗CCR8人源化抗体活性。图9A显示，Fc功能增强型人源化抗CCR8抗体h87D8 H1K1 e5和h87D8 H2K4 e5显著提高了对hCCR8+细胞的ADCC杀伤活性，EC50分别为0.016nM和0.023nM，最大裂解率分别为58％和61％，较87D8和m10A11(最大裂解率分别为33％和30％)提高约一倍。
参照实施例9的方法，以NK92-CD16A为效应细胞，以中低表达人CCR8的HEK293-hCCR8low为靶细胞，比较Fc功能增强型与野生型Fc的抗CCR8人源化抗体活性。图9B显示，Fc功能增强型h87D8H2K4e5显著提高了对中低表达人CCR8的靶细胞的杀伤。
参考实施例9方法，以NK92-CD16A为效应细胞，以内源性表达人CCR8的HuT78细胞为靶细胞，检测Fc功能增强型抗CCR8人源化抗体活性。图9C显示，抗CCR8人源化抗体h87D8 H2K4 e5对表达人CCR8的HuT78细胞同样具有ADCC活性，半效浓度EC50为0.53nM。
取FcγRⅢA158V/V和FcγRⅢA158F/F健康志愿者外周血，利用Ficoll试剂密度梯度离心法分离PBMC，用RPMI1640+2％FBS缓冲液重悬细胞至1×107细胞/mL，作为ADCC实验的效应细胞。在96孔U型细胞培养板中，每孔加入50μL靶细胞HEK293-hCCR8(1×106细胞/mL)，再分别加入50μL梯度稀释的人源化抗体或母本嵌合体或同型对照抗体，每孔加入50μL效应细胞悬液，于37℃、5％CO2条件下培养4小时。检测前30分钟，向靶细胞最大孔加入2μL细胞裂解液(100×)，300*g离心3分钟，取50μL上清至黑色酶标板，加入50μL乳酸脱氢酶(LDH)检测底物，震荡混匀，10分钟后加入25μL终止液，震荡10秒，选择荧光(激发波长560nm，发射波长590nm)进行读板。根据LDH释放水平计算靶细胞杀伤率，并通过四参数模型拟合得到抗体浓度-杀伤率(％)曲线，采用该回归曲线计算供试品半数有效浓度(EC50)。图9D显示，Fc功能增强型抗CCR8人源化抗体h87D8 H1K1 e5和h87D8 H2K4 e5，对以低亲和力FcγRⅢA158F/F PBMC为效应细胞的ADCC活性相对于对照组显著提高，最大裂解率优于野生型IgG1，与以高亲和力FcγRⅢA158V/V PBMC为效应细胞的ADCC活性相当(图9E，表10)。
表10Fc功能增强型抗CCR8人源化抗体的ADCC活性
实施例11、人源化抗CCR8抗体对CCL1/hCCR8的中和阻断活性
收集CHO-hCCR8细胞，F12+10％FBS培养液重悬至2×105/mL，以100μL/孔铺至96孔板中，37℃、5％CO2培养箱培养过夜。用HBSS/丙磺舒(含丙磺舒2.5mM)溶液洗细胞2次，加入4μM Fluo-4AM工作液，每孔100μL37℃孵育60min；用HBSS/丙磺舒溶液洗细胞2次，去除残留的Fluo-4AM工作液，加入HBSS/丙磺舒溶液每孔50ul于37℃孵育20分钟。加入50μL不同浓度的抗体，室温孵育30分钟。使用酶标仪加入50μL l80nM CCL1或HBSS溶液并读取在激发494nm、发射519nm的波长下的荧光信号。以抗体浓度为0的CCL1处理组的Ca2+流信号为100％，计算各处理组Ca2+流信号百分率。并通过四参数模型拟合得到抗体浓度-Ca2+流信号(％)曲线，采用该回归曲线计算供试品半效浓度(EC50)。图10显示，h87D8 H1K1 e5和h87D8 H2K4 e5可抑制CCL1-CCR8引起的Ca2+流信号，半效浓度EC50分别为5.59nM和5.20nM。
实施例12、人源化抗CCR8抗体与兔CCR8的结合
以兔CCR8 cDNA(苏州金唯智生物合成，其氨基酸序列如SEQ ID NO.112所示)为模板，构建含有兔CCR8的慢病毒载体质粒，参考转染试剂说明书利用转染试剂PEI(Polysciences 9002-98-6)将含有兔CCR8的慢病毒载体质粒转染HEK293细胞(下称HEK293-rabbitCCR8细胞)。转染24小时后在96孔U型板中每孔加入5×104个HEK293-rabbitCCR8细胞，离心弃上清，每孔加入100μL梯度稀释的h87D8 H2K4 e5或抗KLH同型对照抗体，冰上孵育60分钟；洗去未结合的一抗后，每孔加入50μL的二抗AlexaFluro647抗人IgG(Jackson Immuno Research#109-606-170)，冰上孵育30分钟。洗涤后每孔加入50μL的5μg/mL PI/BSA溶液，冰上孵育5分钟，之后用0.5％BSA洗去多余的二抗，加入PBS溶液重悬细胞，使用流式细胞仪(Intellicyte iQue)检测抗体与细胞的结合，分析 PI阴性细胞群的荧光值。通过Graphpad Prism 7拟合细胞结合曲线。图11显示，人源化抗CCR8抗体h87D8 H2K4 e5结合兔CCR8，半效浓度EC50为0.21nM。
实施例13、人源化CCR8抗体对MC38小鼠皮下移植瘤生长的影响
在CCR8人源化转基因小鼠C57BL/6J-Ccr8em3(hCCR8)/Smoc(购自上海南方模式生物，货号NM-HU-2000054)上建立MC38皮下移植瘤模型。试验设h87D8 H2K4 e5 0.12mg/kg、0.6mg/kg和3mg/kg组，h87D8 H2K4 0.12mg/kg、0.6mg/kg和3mg/kg组，h10A11 0.6mg/kg、3mg/kg组，IgG1同型对照3mg/kg组。通过对CCR8人源化转基因小鼠静脉给药，每3天1次，共给药4次。图12显示，h87D8 H2K4 e5各剂量组均能抑制肿瘤生长，3mg/kg、0.6mg/kg和0.12mg/kg组抑瘤率分别为113.96％、110.64％、95.44％。h87D8 H2K4 3mg/kg、0.6mg/kg和0.12mg/kg组抑瘤率分别为85.08％、60.18％、26.81％。h10A11 3mg/kg和0.6mg/kg组抑瘤率分别为66.10％、4.07％。h87D8 H2K4 e5抑瘤效果优于h87D8 H2K4和h10A11。(肿瘤相对生长率T/C(％)＝(T-T0)/(C-C0)×100，其中T和T0分别为实验结束时和实验开始时给药组的肿瘤体积，C和C0分别为实验结束时和实验开始时对照组的肿瘤体积。当肿瘤结束体积大于起始体积时，抑瘤率(TGI)(％)＝100-T/C(％)。当肿瘤结束体积小于起始体积时，抑瘤率(TGI)(％)＝100-(T-T0)/T0×100)。
实施例14、人源化CCR8抗体对B16/F10小鼠皮下移植瘤生长的影响
在CCR8人源化转基因小鼠C57BL/6J-Ccr8em3(hCCR8)/Smoc上建立B16/F10皮下移植瘤模型。试验设h87D8 H2K4 e5 10mg/kg、抗小鼠PD-1抗体(mIgG1，克隆G4C2)10mg/kg和h87D8 H2K4 e5 10mg/kg+抗小鼠PD-1抗体10mg/kg组，IgG1同型对照10mg/kg组，空白对照组(即溶剂对照组)。通过对CCR8人源化转基因小鼠静脉给药，每3天1次，共给药3次。图13显示，抗小鼠PD-1抗体不能抑制B16/F10肿瘤生长，h87D8 H2K4 e5能抑制肿瘤生长，抑瘤率为48.2％。h87D8 H2K4 e5+抗小鼠PD-1抗体联用较h87D8 H2K4 e5单用能更有效抑制肿瘤生长，抑瘤率为75.4％。
实施例15、人源化CCR8抗体对MDA-MB-231小鼠皮下移植瘤生长的影响
在B-NGD hIL15小鼠(购自百奥赛图)上建立MDA-MB-231皮下移植瘤模型，并静脉给予人1×107PBMC/只(购自上海妙顺生物)进行免疫重建。试验设h87D8 H2K4 e5 0.4mg/kg、2mg/kg和10mg/kg组，IgG1同型对照10mg/kg组，通过对CCR8人源化转基因小鼠静脉给药，每3天1次，共给药4次。图14显示，h87D8 H2K4 e5各剂量组均能抑制肿瘤生长，10mg/kg、2mg/kg和0.4mg/kg组抑瘤率分别为74.4％、49.3％和40.4％。

Claims (10)

  1. 一种结合CCR8的抗体或其抗原结合部分，其包含：
    (1)重链可变区和轻链可变区，其中：
    (i)所述重链可变区包含：
    (a)具有下式的HCDR1序列：
    NX1X2AMN(SEQ ID NO.102)，其中：
    X1为A或T；
    X2为F或Y；和
    (b)具有下式的HCDR2序列：
    RIRSKSNX3YATX4X5ADSVX6X7(SEQ ID NO.103)，其中：
    X3为N或Y；
    X4为Y或H；
    X5为Y或S；
    X6为I或K；
    X7为D或G；和
    (c)具有下式的HCDR3序列：
    GKEAGX8X9X10X11X12X13DY(SEQ ID NO.104)，其中：
    X8为A或不存在；
    X9为Y、T或A；
    X10为Y或N；
    X11为A或Y；
    X12为M或A；
    X13为M或不存在；和
    (ii)所述轻链可变区包含：
    (a)具有下式的LCDR1序列：
    RSSKSLLHSX14X15NTYLY(SEQ ID NO.105)，其中：
    X14为N、A、I、V、Y或R；
    X15为G、I、V、Y、T或R；和
    (b)具有下式的LCDR2序列：
    RX16SNLAS(SEQ ID NO.106)，其中：
    X16为M、A、G或T；和
    (c)具有下式的LCDR3序列：
    MQHLEYPX17(SEQ ID NO.107)，其中：
    X17为F或L；或
    (2)重链可变区和轻链可变区，其中：
    (i)所述重链可变区包含：
    (a)具有下式的HCDR1序列：
    NAYAMN(SEQ ID NO.28)；和
    (b)具有下式的HCDR2序列：
    RIRSKSNNYATYYADSVKD(SEQ ID NO.22)；和
    (c)具有下式的HCDR3序列：
    QKFGX18RGYYALDF(SEQ ID NO.108)，其中：
    X18为A或S；和
    (ii)所述轻链可变区包含：
    (a)具有下式的LCDR1序列：
    X19ASX20SX21X22X23X24X25X26X27X28X29X30(SEQ ID NO.109)，其中：
    X19为R、T或S，X20为E、K或S，X21为V或I，X22为E或S，X23为Y、T或不存在，X24为Y、S或不存在，X25为G或不存在，X26为T、Y或S，X27为S或N，X28为L或Y，X29为L或M，X30为Q或H；和
    (b)具有下式的LCDR2序列：
    X31X32SX33X34X35S(SEQ ID NO.110)，其中：
    X31为R、A、L或S，X32为A或T，X33为N或T，X34为L或V，X35为A或E；和
    (c)具有下式的LCDR3序列：
    X36X37X38X39X40X41PX42X43(SEQ ID NO.111)，其中：
    X36为Q或H，X37为Q或H，X38为S、Y或G，X39为R、S、H或T，X40为E、K、G、R、V或S，X41为Y、V、L、S或I，X42为L或不存在，X43为L、Y、P、R或G。
  2. 根据权利要求1的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO.6、16、28或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.7、17、22、82或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.8、29、49或其任何变体的重链CDR3；和选自氨基酸序列SEQ ID NO.11、32、39、44、52、62、91或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.12、33、40、45、58、63或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.13、25、34、41、46、53、59、64或其任何变体的轻链CDR3。
  3. 根据权利要求1或2的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
    (1)分别包含SEQ ID NO.6、7、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、13的轻链CDR1、CDR2及CDR3序列；
    (2)分别包含SEQ ID NO.16、17、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、13的轻链CDR1、CDR2及CDR3序列；
    (3)分别包含SEQ ID NO.16、22、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、25的轻链CDR1、CDR2及CDR3序列；
    (4)分别包含SEQ ID NO.28、22、29的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.32、33、34的轻链CDR1、CDR2及CDR3序列；
    (5)分别包含SEQ ID NO.28、22、29的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.39、40、41的轻链CDR1、CDR2及CDR3序列；
    (6)分别包含SEQ ID NO.28、22、29的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.44、45、46的轻链CDR1、CDR2及CDR3序列；
    (7)分别包含SEQ ID NO.28、22、49的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.52、45、53的轻链CDR1、CDR2及CDR3序列；
    (8)分别包含SEQ ID NO.28、22、49的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.44、58、59的轻链CDR1、CDR2及CDR3序列；
    (9)分别包含SEQ ID NO.28、22、29的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.62、63、64的轻链CDR1、CDR2及CDR3序列；
    (10)分别包含SEQ ID NO.6、7、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、15的轻链CDR1、CDR2及CDR3序列；
    (11)分别包含SEQ ID NO.16、82、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、25的轻链CDR1、CDR2及CDR3序列；
    (12)分别包含SEQ ID NO.6、82、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.11、12、25的轻链CDR1、CDR2及CDR3序列；
    (13)分别包含SEQ ID NO.16、7、8的重链CDR1、CDR2及CDR3序列，和分别包含SEQ ID NO.91、63、25的轻链CDR1、CDR2及CDR3序列。
  4. 根据权利要求1-3任一项所述的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO.4、14、20、26、35、47、54、80、83、85、92、96、100或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.9、18、23、30、37、42、50、56、60、65、67、68、69、70、71、72、73、74、75、76、77、78、79、87、89、94、98或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.9或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.67或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.68或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.69或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.70或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.71或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.72或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.73或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.74或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.75或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.76或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.77或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.78或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.79或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.14或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.18或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.20或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.23或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.26或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.30或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.35或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.37或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.35或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.42或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.47或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.50或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.54或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.56或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.35或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.60或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.4或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.65或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.80或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.87或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.83或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.87或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.85或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.89或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.92或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.94或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.96或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.98或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO.100或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.98或其任何变体的轻链可变区；
    优选地，所述抗体或其抗原结合部分是人源化的。
  5. 编码根据权利要求1-4任一项所述的抗体或其抗原结合部分的核酸；
    优选地，所述核酸包含选自SEQ ID NO.5、15、21、27、36、48、55、81、84、86、93、97、101或其任何变体的抗体重链可变区核酸序列，和/或选自SEQ ID NO.10、19、24、31、38、43、51、57、61、66、88、90、95、99或其任何变体的抗体轻链可变区核酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.5或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.10或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.15或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.19或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.21或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.24或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.27或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.31或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.36或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.38或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.36或其任何变体的重链可变区核苷酸序列，和核苷酸序列 SEQ ID NO.43或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.48或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.51或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.55或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.57或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.36或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.61或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.5或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.66或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.81或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.88或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.84或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.88或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.86或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.90或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.93或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.95或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.97或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.99或其任何变体的轻链可变区核苷酸序列；
    更优选地，所述核酸包含核苷酸序列SEQ ID NO.101或其任何变体的重链可变区核苷酸序列，和核苷酸序列SEQ ID NO.99或其任何变体的轻链可变区核苷酸序列。
  6. 包含权利要求5所述核酸的载体。
  7. 包含权利要求5所述的核酸或权利要求6所述载体的细胞。
  8. 一种组合物，其包含权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的核酸、权利要求6所述的载体和/或权利要求7所述的细胞。
  9. 一种试剂盒，其包含权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的核酸、权利要求6所述的载体、权利要求7所述的细胞和/或权利要求8所述的组合物。
  10. 治疗剂在制备用于诊断、治疗或预防癌症相关疾病的药物或试剂盒中的用途，所述治疗剂选自单一治疗剂或联合治疗剂；
    所述单一治疗剂选自权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的核酸、权利要求6所述的载体、权利要求7所述的细胞和/或权利要求8所述的组合物；
    所述联合治疗剂包括所述单一治疗剂和免疫检查点调节剂，优选地，所述免疫检查点调节剂为选自PD-1、CTLA4、PD-L1、PD-L2、CD47、TIGIT、GITR、TIM3、LAG3、4-1BB、CD27和B7H4的免疫检查点蛋白的抑制剂；优选地，所述免疫检查点蛋白的抑制剂为抗体或其抗原结合片段；优选地，免疫检查点蛋白是PD-1或CTLA4；更优选地，所述免疫检查点蛋白的抑制剂选自抗PD-1抗体或其抗原结合片段或抗CTLA-4抗体或其抗原结合片段；
    优选地，与癌症相关的疾病选自乳癌、子宫癌、子宫颈癌、卵巢癌、前列腺癌、肺癌、胃(胃腺)癌、非小细胞肺癌、胰脏癌、头颈部鳞状细胞癌、食道癌、膀胱癌、黑色素瘤、大肠癌、肾癌、非霍奇金氏淋巴瘤、泌尿上皮癌、肉瘤、血球癌(白血病、淋巴瘤等)、胆管癌、胆囊癌、甲状腺癌、睾丸癌、胸腺癌、肝脏癌等癌症，优选为乳癌、子宫癌、卵巢癌、肺癌、大肠癌、肾癌及肉瘤，更优选为乳癌、大肠癌、肾癌及肉瘤。
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