Development and Use of Novel Immunomodulators

Abstract

The present disclosure relates to the development and use of novel immunomodulatory agents, which are antibodies or antigen-binding portions thereof that bind to CCR8. The antibodies of the present disclosure have neutralizing activity that blocks the CCL1 / CCR8 axis and inhibit CCL1-induced cell activation while suppressing the low affinity FcγRIIIA in the population. 158F Fc optimization enhances ADCC efficacy against allele carriers.

Description

[Technical field]

[0001] The present disclosure relates to immunomodulatory agents and uses thereof, in particular anti-CCR8 antibodies and uses thereof. [Background technology]

[0002] Chemokine receptor 8 (CCR8), a member of the CC chemokine receptor superfamily, is upregulated in activated type II Th cells, its ligand CCL1 / I-309 efficiently recruits activated type II Th cells, and the interaction between CCL1 and CCR8 (also known as the CCL1 / CCR8 axis) correlates with type II Th cell-mediated immune responses (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 activates the CCR8-mediated RAS / MAPK signaling pathway, promotes intracellular calcium release, and inhibits apoptosis (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 also plays an important role in maintaining the survival of regulatory T cells (Treg) and suppressing graft-versus-host reaction (GVHD) (Coghill, JM, et al. 2013 CC chemokine receptor 8 potentiates donor Treg survival and is critical for the prevention of murine graft-versus-host disease. Blood 122(5):825-36.). CCR8. + Tregs are important cells that promote immune suppression and are thought to be 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 USA 114(23):6086-6091).

[0003] Treg cells are considered to be important factors that inhibit tumor immunity in addition to maintaining immune homeostasis. Compared to conventional T (Tconv) cell counts, patients with tumors with low Treg ratios have a higher survival rate in ovarian cancer, breast cancer, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, cervical cancer, colorectal cancer, etc. (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.Clin Cancer Res 12(18):5423-34;Jordanova,ES,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,RP,et al.2006 Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients. Cancer 107(12):2866-72;Sato,et al.2005;Sinicrope,FA,et al.2009Intraepithelial effector (CD3+) / regulatory (FoxP3+) T-cell ratio predicts a clinical outcome of human colon carcinoma.Gastroenterology 137(4):1270-9). Ruckes T et al. found that T-cell lymphoma and leukemia can resist apoptosis by CCL1 autocrine loop (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 et al. showed that inhibition of CCL1 can abolish the inhibitory effect of Treg on tumor immunity (Hoelzinger,DB,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 et al. found that CCR8 expression was increased in tumor-associated macrophages from patients with urothelial and renal carcinoma, that tumor-infiltrating CD11b+CCR8+ cells induced FoxP3 expression in autologous T lymphocytes, and that primary tumors produced high levels of CCL1, suggesting that the CCL1 / CCR8 axis is a component of cancer-associated inflammation (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). In a transcriptome analysis of human colon cancer or non-small cell lung cancer, De Simone M et al. found that tumor-infiltrating Treg cells characteristically upregulate the expression of signal molecules such as CCR8 and are highly immunosuppressive, and that high expression of the CCR8 gene in tumor tissue correlates with poor prognosis (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). In breast cancer, Pritas G et al. also detected increased CCR8 expression in tumor-infiltrating Treg cells, suggesting that depleting Treg cells from tumor sites by targeting CCR8 is a very promising immunotherapeutic strategy (Plitas,G.,et al.2016 Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer.Immunity 45(5):1122-1134). Magnuson A et al. compared tumor tissue from mouse colorectal cancer, melanoma models, and human colorectal cancer patients at different stages of disease, from early to advanced, and found that the expression profiles of tumor-infiltrating Treg signatures shared important commonalities, including upregulation of a set of genes associated with Treg differentiation, such as CCR8, and that anti-CCR8 antibodies led to tumor regression in treated mice (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 USA 115(45):E10672-e10681). Villarreal D et al. reported that anti-CCR8 antibodies significantly inhibited tumor growth in a mouse model of colorectal cancer, and that antitumor activity was associated with an increase in tumor-specific T cells, enhanced infiltration of CD4+ and CD8+ T cells, and a significant decrease in CD4+CCR8+ Tregs in the tumor (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).

[0004] Existing therapies have no effective means of targeting tumor-infiltrating Treg cells. It is very difficult to achieve specific removal of suppressive Treg cells from the tumor microenvironment. For example, the use of mogamulizumab, a non-fucosylated anti-CCR4 antibody, significantly reduced normal CD4+ and CD8+ T cells while depleting Tregs (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 expression is significantly elevated in tumor-infiltrating Treg cells, so antibodies targeting CCR8 can easily distinguish tumor-infiltrating Treg cells from normal T cells. Therefore, the development of monoclonal antibodies targeting CCR8 that can effectively remove tumor-infiltrating Treg cells or simultaneously inhibit their activity is eagerly awaited as a clinical immunotherapy.Recently, it has been reported that anti-CCR8 antibodies, when defucosylated, enhance antibody-dependent cellular cytotoxicity (ADCC) and exhibit tumor inhibition effects in preclinical animal models (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, JR, et al.2021 Fc-Optimized Anti-CCR8 Antibody Depletes Regulatory T Cells in Human Tumor Models. Cancer Res 81(11):2983-2994;Depis,F.,Hu,C.,Weaver,J.,McGrath,L.,Klebanov,B.,Bugge,J.,&Shaffer,DR(2020).Preclinical evaluation of JTX-1811, an anti-CCR8 antibody 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;2020 Apr 27-28 and Jun 22-24.Philadelphia(PA):AACR;Cancer Res 2020;80(16 Suppl):Abstract nr 4532.). Summary of the Invention

[0005] The present disclosure relates to novel high affinity CCR8 monoclonal and humanized antibodies with neutralizing activity that block the CCL1 / CCR8 axis, inhibit CCL1-induced cell activation, and inhibit low affinity FcγRIIIA in the human population. 158F For allele carriers, the ADCC effect is enhanced by Fc optimization. In vitro efficacy studies have shown that this novel humanized antibody effectively eliminates CCR8+ cells, significantly inhibits the growth of MC38, B16 / F10, and MDA-MB-231 tumors in humanized mouse subcutaneous tumor models, and shows excellent therapeutic effects in combination with anti-PD-1 antibodies. Compared with the prior art, the CCR8 antibody of the present disclosure has the advantages of a novel sequence and remarkable biological activity.

[0006] The present disclosure relates to a novel anti-CCR8 antibody or antigen-binding portion thereof for inhibiting CCL1-induced calcium flux and enhancing the ADCC killing activity of effector cells.Furthermore, the present disclosure relates to a novel anti-CCR8 antibody or antigen-binding portion thereof for the treatment of cancer.

[0007] In one embodiment, the present disclosure discloses a monoclonal antibody or antigen-binding portion thereof that binds to CCR8.

[0008] In one aspect, the disclosure provides a nucleic acid encoding the aforementioned CCR8-binding antibody or antigen-binding portion thereof.

[0009] In one aspect, the disclosure provides a vector comprising the aforementioned nucleic acid.

[0010] In one aspect, the disclosure provides a cell comprising the aforementioned nucleic acid or the aforementioned vector.

[0011] In one aspect, the disclosure provides compositions comprising the aforementioned antibodies or antigen-binding portions thereof, encoding nucleic acids, vectors, and cells.

[0012] In one aspect, the disclosure provides kits comprising the aforementioned antibodies or antigen-binding portions thereof, encoding nucleic acids, vectors, cells, and compositions.

[0013] In one aspect, the disclosure provides a method of treating a cancer-related disease comprising administering to said subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, and / or composition of any one of the preceding aspects.

[0014] In one aspect, the disclosure provides for the use of an antibody, or antigen-binding portion thereof, nucleic acid, vector, cell, and / or pharmaceutical composition of any one of the preceding aspects in the preparation of a medicament for use in treating a cancer afflicted by a subject. [Brief description of the drawings]

[0015] [Figure 1] The results of FACS assay of CCR8 or CCR4 stably expressing cell lines are shown. (A) CHO cell line stably transfected with human CCR8, CHO-hCCR8; (B) HEK293 cell line stably transfected with human CCR8, HEK293-hCCR8; (C) HEK293 cell line stably transfected with low expression level of human CCR8, HEK293-hCCR8low; (D) CHO cell line stably transfected with cynomolgus monkey CCR8, CHO-cynoCCR8; (E) CHO cell line stably transfected with human CCR4, CHO-hCCR4. [Diagram 2] The results of FACS assay of the anti-CCR8 chimeric antibodies are shown, which demonstrated that the anti-CCR8 chimeric antibodies bound to human or cynomolgus monkey CCR8+ cell lines with high affinity. [Diagram 3] 1 shows the ADCC activity of anti-CCR8 chimeric antibodies. [Figure 4] These results show that anti-CCR8 chimeric antibodies have significant inhibitory activity against CCL1-induced calcium flux in target cells. [Diagram 5]Binding of anti-CCR8 chimeric antibodies with optimized CDR sequences to CCR8+ cells is shown. (A) Effect of M56 mutation on CCR8+ cell binding, (B) Effect of N33 and G34 mutations on CCR8+ cell binding. [Figure 6] 1 shows high affinity binding of humanized anti-CCR8 antibodies to human CCR8+ cells. [Figure 7] 1 shows high affinity binding of humanized anti-CCR8 antibodies to cynomolgus monkey CCR8+ cells. [Figure 8] 1 shows the ADCC killing activity of humanized anti-CCR8 antibodies detecting NK92 cells as effector cells. [Figure 9] The ADCC killing activity of Fc function-enhanced humanized anti-CCR8 antibodies against human CCR8+ cells with different expression levels is shown. (A) ADCC killing activity detected using NK92-CD16A cells as effector cells and HEK293-hCCR8 with high CCR8 expression as target cells, (B) ADCC killing activity detected using NK92-CD16A cells as effector cells and HEK293-hCCR8low with low to medium expression of human CCR8 as target cells, (C) ADCC killing activity detected using NK92-CD16A cells as effector cells and HuT78 cells that endogenously express human CCR8 as target cells. (D) ADCC killing activity detected using PBMCs isolated from peripheral blood of a healthy volunteer with the genotype FcγRIIIA158F / F as effector cells and HEK293-hCCR8 as target cells. (E) ADCC killing activity detected using PBMCs isolated from peripheral blood of a healthy volunteer with the genotype FcγRIIIA158V / V as effector cells and HEK293-hCCR8 as target cells. [Figure 10] 1 shows the inhibitory activity of humanized anti-CCR8 antibodies against CCL1-mediated Ca2+ flux signaling. [Figure 11] 1 shows high affinity binding of humanized anti-CCR8 antibodies to rabbit CCR8+ cells. [Figure 12] 1 shows that humanized anti-CCR8 antibodies significantly inhibit the growth of subcutaneously implanted MC38 tumors in humanized mice. [Figure 13]This indicates that humanized anti-CCR8 antibody significantly inhibits the growth of B16 / 10 subcutaneously implanted tumors in humanized mice, and that when combined with anti-mouse PD-1 antibody, humanized anti-CCR8 antibody can more effectively inhibit tumor growth. [Figure 14] 1 shows that humanized anti-CCR8 antibodies significantly inhibit the growth of subcutaneously implanted MDA-MB-231 tumors in humanized mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] I. Definition In this disclosure, scientific and technical terms used herein have the meanings that are generally understood by those skilled in the art, unless otherwise specified.In addition, the terms and experimental procedures used herein for protein chemistry and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and experimental procedures are terms and routine procedures that are widely used in the respective fields.In addition, in order to better understand this disclosure, the definitions and explanations of related terms are provided below.

[0017] Provided herein are antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to CCR8. In a specific embodiment, provided herein are monoclonal anti-CCR8 antibodies that specifically bind to CCR8, the anti-CCR8 antibodies including variants of a parent antibody. In a specific embodiment, provided herein are antibodies that specifically bind to CCR8 (e.g., human CCR8). In a particular embodiment, provided herein are anti-CCR8 antibodies that include modifications at one or more amino acid residues (e.g., 1-3 amino acid substitutions in the framework region of the heavy chain variable region), which retain affinity for the antigen compared to a parent antibody that does not include the modifications. The term "CCR8" refers to any CCR8 receptor known to those of skill in the art. For example, the CCR8 may be from a mammal, for example, the CCR8 may be from a human or a cynomolgus monkey.

[0018] As used herein, unless otherwise specified, 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 within plus or minus 10% of the given value or range, rounded up or down to the nearest integer.

[0019] In the context of antibody chain polypeptide sequences, the phrase "substantially identical" is understood to mean antibody chains that exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. In the context of nucleic acid sequences, the term is understood to mean a nucleotide sequence that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

[0020] Sequence "identity" or "matching" has its art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed 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, AM, ed., Oxford University Pres, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, 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). Although there are many methods for determining identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0021] A "substitutional" variant is one in which at least one amino acid residue in a native sequence has been removed and a different amino acid has been inserted at the same position. The substitution may be a single substitution, where only one amino acid has been replaced in the molecule, or a multiple substitution, where two or more amino acids have been replaced in the molecule. Multiple substitutions may be located at contiguous sites. Similarly, an amino acid may be replaced by multiple residues, and such variants include both substitutions and insertions. An "insertion" variant is one in which one or more amino acids have been inserted immediately adjacent to a particular position in a native sequence. Immediately adjacent to an amino acid means that it is attached to the α-carboxy or α-amino functional group of that amino acid. A "deletion" variant is one in which one or more amino acids have been removed from a native amino acid sequence. In general, a deletion variant is missing one or two amino acids in a particular region of the molecule.

[0022] In the case of the variable domain of an antibody, the term "variable" refers to a portion of the associated molecule that exhibits extensive sequence differences among antibodies and is used for the specific recognition and binding of a particular antibody to its specific target. However, variability is not uniformly distributed throughout the variable domain of an antibody. The variability is concentrated in three regions known as complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are located in both the light and heavy chain variable domains. The more conserved portions of the variable domains are called framework (FR) regions or framework sequences. Natural heavy and light chain variable domains each contain four FR regions, which are primarily β-folded, connected by three CDRs to form loops that connect the β-folds and, in some cases, form partial β-folds. The CDRs of each chain are usually closely linked by the FR region and contribute to the formation of the antibody target binding site (epitope or determinant) with the help of the CDRs of the other chain (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). In this specification, the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system by Kabat et al., unless otherwise specified. A CDR may have the ability to specifically bind to an associated epitope.

[0023] As used herein, 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 of said antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites), and thus retains at least a portion of the binding specificity and specific binding ability of said full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Antibody includes antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, as well as other fragments, including modified fragments (see, e.g., 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, for example, multiple chains linked together by disulfide bonds and / or peptide linkages. Antibody fragments generally contain at least about 50 amino acids, 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 substitution of the corresponding region), immunospecifically binds to an antigen (i.e., binds at least or at least about 10 amino acids). 7 -10 8The antibody has a Ka of M-1. A "functional fragment" or "anti-CCR8 antibody analog" is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind to a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as an "antibody fragment" and, in the case of antibodies, can refer to a fragment that prevents or substantially reduces the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. An "Fv" fragment is a dimer (V) formed by non-covalent bonding of a heavy chain variable domain and a light chain variable domain. H -V L In this configuration, the three CDRs of each variable domain are arranged in a V H -V L interact to define a target-binding site on the surface of the dimer. Collectively, the six CDRs confer target-binding specificity to the intact antibody. However, even a single variable domain (or even just half of an Fv containing three target-specific CDRs) can be capable of recognizing and binding to a target.

[0024] As used herein, "monoclonal antibody" refers to a population of identical antibodies, indicating that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to the property of a polyclonal population of antibodies, which contains antibodies with many different sequences. Monoclonal antibodies can be prepared by many known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al. (2004) J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, for example by fusing 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 a clonal population of host cells by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0025] As used herein, a full-length antibody is an antibody having 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, e.g., antibodies naturally produced by antibody-secreting B cells and synthetically produced antibodies having the same structural domains.

[0026] The term "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, e.g., the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0027] A "humanized" antibody is a non-human (e.g., murine) form of an antibody that is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from the non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.

[0028] Furthermore, in humanization, amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL can be mutated, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, by PCR-mediated mutagenesis, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests, as described herein. Usually, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. Also, there are usually no more than one or two mutations in the CDRs. Thus, the humanized antibodies described in this disclosure also include antibodies that contain one or two double amino acid mutations in the CDRs.

[0029] "Conservative amino acid substitution" may include the substitution of a unique amino acid residue with a non-unique residue, with little or no effect on the polarity or charge of the amino acid residue at that position. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, n-leucine, alanine or methionine, for another; the substitution of a polar (hydrophilic) amino acid residue, such as, for example, the substitution between arginine and lysine, the substitution between glutamine and asparagine, the substitution between glycine and serine; the substitution of a basic amino acid residue, such as lysine, arginine or histidine, for another; or the substitution of an acidic residue, such as aspartic acid or glutamic acid, for another. The phrase "conservative amino acid substitution" also encompasses the substitution 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 in accordance with the present invention are shown in Table 1 below.

[0030] Table 1. Some useful amino acid substitutions. [Table 1]

[0031] As used herein, the term "CDR" refers to a complementarity-determining region, and it is known that three of each are present in each heavy and light chain of an antibody molecule. CDRs are also called hypervariable regions, and are present in the variable regions of each heavy and light chain of an antibody, and have highly variable sites in the primary structure of the CDR. In this specification, the CDR of the heavy chain is represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino terminal sequence of the heavy chain, and the CDR of the light chain is represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino terminal sequence of the light chain. These sites are close to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0032] As used herein, the term "epitope" refers to any antigenic determinant on an antigen that binds to a complementary site on an antibody. Epitope determinants usually include chemically active surface types of molecules, such as amino acid or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics.

[0033] As used herein, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through non-covalent interactions between the antibody and the antigen-binding site. The antigen may be an isolated antigen or may be present in a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen has a binding affinity of about 1× or 1×10 8 M -1 or higher affinity constant Ka (or 1×10 -7 M or 1×10 -8The affinity constant is one that binds the antigen with a dissociation constant (Kd) of M or less. Affinity constants can be determined by standard kinetic methods of antibody reactions, such as 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 (e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Devices and methods for real-time detection and monitoring of binding rates are known and commercially available (BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0034] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include RNA and DNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0035] Alternatively, in another embodiment, mutations can be introduced randomly, for example by saturation mutagenesis along all or part of the anti-CCR8 antibody coding sequence, and the resulting modified anti-CCR8 antibodies can be screened for improved binding activity.

[0036] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, such as, for example, a method for determining the amount of a polypeptide produced from a host cell. Such methods include, but are not limited to, quantification of polypeptide in cell lysates by ELISA, Comas Blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0037] As used herein, a "host cell" refers to a cell that is used to receive, maintain, replicate, and amplify a vector. A host cell may also be used to express a polypeptide encoded by a vector. When the host cell divides, the nucleic acid contained in the vector is replicated, thereby amplifying the nucleic acid. A host cell may be a eukaryotic or 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, and the like.

[0038] As used herein, a "vector" is a replicable nucleic acid that can express one or more heterologous proteins when the vector is transformed into a suitable host cell. Vectors include those into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced, usually by restriction digestion and ligation. Vectors also include vectors that contain a nucleic acid encoding a polypeptide. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express / display the polypeptide encoded by the nucleic acid. Vectors are usually left free, but can be designed to allow a gene or a part thereof to be integrated into a chromosome of a genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles are well known to those skilled in the art.

[0039] As used herein, vector also includes "viral vectors" or "viral vectors." Viral vectors are man-made viruses that are operably linked to foreign genes to introduce the foreign genes into cells (as a vehicle or shuttle).

[0040] As used herein, an "expression vector" includes a vector capable of expressing DNA, said DNA being operably linked to a regulatory sequence, such as a promoter region, capable of affecting the expression of such a DNA fragment. Such additional fragments may include promoter and terminator sequences, and may optionally 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. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as, for example, a plasmid, phage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of the 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, expression vectors that remain free, or that are integrated into the host cell genome.

[0041] As used herein, "treating" an individual with a disease or disease state indicates that the individual's symptoms are partially or completely alleviated or remain unchanged following treatment. Thus, treatment includes prevention, therapy and / or cure. Prevention refers to the prevention of underlying disease and / or the prevention of worsening of symptoms or progression of disease. Treatment also includes any pharmaceutical use of any of the provided antibodies or antigen-binding fragments thereof and compositions provided herein.

[0042] As used herein, "therapeutic effect" refers to the effect that results from treating an individual and that alters, usually improves or ameliorates the symptoms of a disease or disease state, or cures the disease or disease state.

[0043] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition containing a compound, at least in an amount sufficient to produce a therapeutic effect after administration to a subject, and thus is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or condition.

[0044] As used herein, "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition containing a compound that has the desired prophylactic effect when applied to a subject, such as preventing or delaying the onset or recurrence of a disease or condition, reducing the likelihood of the onset or recurrence of a disease or condition, etc. A complete prophylactically effective amount does not necessarily occur by a single administration, but may occur only after a series of administrations. Thus, a prophylactically effective amount can be administered in one or multiple applications.

[0045] As used herein, the term "patient" or "subject" refers to a mammal, such as a human.

[0046] As used herein, unless otherwise stated, the terms "comprise," "include," "have," and "contain," including their grammatical equivalents, are generally to be understood as open-ended and non-limiting, e.g., not excluding other unrecited elements or steps.

[0047] II. MODE FOR CARRYING OUT THE DISCLOSURE In one aspect, the disclosure provides an antibody or antigen-binding portion thereof that binds to CCR8 comprising a heavy chain variable region and a light chain variable region.

[0048] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region: (i) the heavy chain variable region comprises (a), (b), and (c); (a) an HCDR1 sequence having the formula: NX1X2AMN (SEQ ID NO. 102), where X1 is A or T; X2 is F or Y; (b) an HCDR2 sequence having the formula: RIRSKSNX3YATX4X5ADSVX6X7 (SEQ ID NO. 103), where X3 is N or Y; X4 is Y or H; X5 is Y or S; X6 is I or K; X7 is D or G, and (c) an HCDR3 sequence having the following formula: GKEAGX8X9X 10 X 11 X 12 X 13 DY (SEQ ID NO. 104), where X8 is A or absent; X9 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 absent, (ii) the light chain variable region comprises: (a) an LCDR1 sequence having the following formula: 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, (b) an LCDR2 sequence having the formula: RX 16 SNLAS (SEQ ID NO. 106), where X 16 is M, A, G, or T, (c) an LCDR3 sequence having the following formula: MQHLEYPX 17 (SEQ ID NO. 107), where X 17 is F or L.

[0049] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region: (i) the heavy chain variable region comprises: (a), (b), and (c) (a) an HCDR1 sequence having the formula: NAYAMN (SEQ ID NO. 28), (b) an HCDR2 sequence having the formula: RIRSKSNNYATYYADSVKD(SEQ ID NO.22), (c) an HCDR3 sequence having the following formula: QKFGX 18 RGYYALDF (SEQ ID NO. 108), where: X 18 is A or S, (ii) the light chain variable region comprises: (a) an LCDR1 sequence having the following formula: X 19 ASX 20 SX 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 (SEQ ID NO. 109), where X19 is R, T or S, and X 20 is E, K or S, and X 21 is V or I, and X 22 is E or S, and X 23 is Y, T or not present, X 24 is Y, S or not present, X 25 G or not present, X 26 is T, Y or S, and X 27 is S or N, and X 28 is L or Y, and X 29 is L or M, and X 30 is Q or H, (b) an LCDR2 sequence having the formula: X 31 X 32 SX 33 X 34 X 35 S (SEQ ID NO. 110), where X 31 is R, A, L or S, and X 32 is A or T, and X 33 is N or T, and X 34 is L or V, and X 35 is A or E, (c) an LCDR3 sequence having the following formula: X 36 X 37 X 38 X 39 X 40 X 41 PX 42 X 43 (SEQ ID NO. 111), where X 36 is Q or H, and X 37 is Q or H, and X 38 is S, Y or G, and X 39 is R, S, H or T, and X 40 is E, K, G, R, V or S, and X 41 is Y, V, L, S or I, and X 42 is L or absent, X 43 is L, Y, P, R or G.

[0050] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.6, 16, 28, or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.7, 17, 22, 82, or any variant thereof, a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.8, 29, 49, or any variant thereof, and a light chain CDR1 selected from the amino acid sequence SEQ ID NO.11, 32, 39, 44, 52, 62, 91, or any variant thereof, a light chain CDR2 selected from the amino acid sequence SEQ ID NO.12, 33, 40, 45, 58, 63, or any variant thereof, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.13, 25, 34, 41, 46, 53, 59, 64, or any variant thereof.

[0051] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a combination of heavy and light chain CDRs with heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 6, 7, 8, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 11, 12, 13, respectively.

[0052] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 16, 17, and 8, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 11, 12, and 13, respectively.

[0053] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 16, 22, and 8, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 11, 12, and 25, respectively.

[0054] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 28, 22, and 29, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 32, 33, and 34, respectively.

[0055] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 28, 22, and 29, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 39, 40, and 41, respectively.

[0056] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 28, 22, and 29, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 44, 45, and 46, respectively.

[0057] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 28, 22, 49, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 52, 45, 53, respectively.

[0058] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 28, 22, and 49, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 44, 58, and 59, respectively.

[0059] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 28, 22, and 29, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 62, 63, and 64, respectively.

[0060] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 6, 7, 8, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 11, 12, 15, respectively.

[0061] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 16, 82, 8, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 11, 12, 25, respectively.

[0062] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 6, 82, 8, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 11, 12, 25, respectively.

[0063] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise CDR combinations of heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 16, 7, 8, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 91, 63, 25, respectively.

[0064] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region selected from the amino acid sequence of SEQ ID NOs. 4, 14, 20, 26, 35, 47, 54, 80, 83, 85, 92, 96, 100, or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NOs. 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 any variant thereof.

[0065] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.9 or any variant thereof.

[0066] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.67 or any variant thereof.

[0067] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.68 or any variant thereof.

[0068] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.69 or any variant thereof.

[0069] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.70 or any variant thereof.

[0070] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.71 or any variant thereof.

[0071] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.72 or any variant thereof.

[0072] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.73 or any variant thereof.

[0073] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.74 or any variant thereof.

[0074] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.75 or any variant thereof.

[0075] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.76 or any variant thereof.

[0076] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.77 or any variant thereof.

[0077] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.78 or any variant thereof.

[0078] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.79 or any variant thereof.

[0079] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.14 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.18 or any variant thereof.

[0080] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.20 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.23 or any variant thereof.

[0081] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.26 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.30 or any variant thereof.

[0082] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.35 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.37 or any variant thereof.

[0083] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.35 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.42 or any variant thereof.

[0084] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.47 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.50 or any variant thereof.

[0085] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.54 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.56 or any variant thereof.

[0086] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.35 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.60 or any variant thereof.

[0087] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.4 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.65 or any variant thereof.

[0088] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.80 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.87 or any variant thereof.

[0089] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.83 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.87 or any variant thereof.

[0090] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.85 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.89 or any variant thereof.

[0091] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.92 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.94 or any variant thereof.

[0092] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.96 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.98 or any variant thereof.

[0093] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof comprise a heavy chain variable region of amino acid sequence SEQ ID NO.100 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO.98 or any variant thereof.

[0094] In some preferred embodiments, the aforementioned antibodies or antigen-binding portions thereof are humanized.

[0095] In another aspect, the present disclosure also provides a nucleic acid encoding the aforementioned antibody or antigen-binding portion thereof.

[0096] Preferably, the nucleic acid comprises an antibody heavy chain variable region nucleic acid sequence selected from SEQ ID NOs.5, 15, 21, 27, 36, 48, 55, 81, 84, 86, 93, 97, 101 or any variant thereof, and / or an antibody light chain variable region nucleic acid sequence selected from SEQ ID NOs.10, 19, 24, 31, 38, 43, 51, 57, 61, 66, 88, 90, 95, 99 or any variant thereof.

[0097] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.5, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.10, or any variant thereof.

[0098] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.15, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.19, or any variant thereof.

[0099] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.21, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.24, or any variant thereof.

[0100] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.27, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.31, or any variant thereof.

[0101] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.36, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.38, or any variant thereof.

[0102] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.36, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.43, or any variant thereof.

[0103] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.48, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.51, or any variant thereof.

[0104] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.55, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.57, or any variant thereof.

[0105] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.36, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.61, or any variant thereof.

[0106] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.5, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.66, or any variant thereof.

[0107] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.81, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.88, or any variant thereof.

[0108] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.84, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.88, or any variant thereof.

[0109] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.86, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.90, or any variant thereof.

[0110] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.93, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.95, or any variant thereof.

[0111] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.97, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.99, or any variant thereof.

[0112] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.101, or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO.99, or any variant thereof.

[0113] In another aspect, the present disclosure also provides a vector comprising the aforementioned nucleic acid.

[0114] In another aspect, the disclosure also provides a cell comprising the aforementioned nucleic acid or the aforementioned vector.

[0115] In another embodiment, the present disclosure also provides compositions comprising the aforementioned antibodies or antigen-binding portions thereof, nucleic acids, vectors, and / or cells.

[0116] In another aspect, the present disclosure provides kits comprising the aforementioned antibodies or antigen-binding portions thereof, nucleic acid molecules, vectors, cells, and / or compositions.

[0117] A method of treating a cancer-related disease using a CCR8 antibody of the present disclosure, comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, or a nucleic acid molecule, vector, cell, or pharmaceutical composition of any one of the preceding aspects.

[0118] Cancer-related diseases include breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach (gastric adenocarcinoma), non-small cell lung cancer, pancreatic cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colon cancer, kidney cancer, non-Hodgkin's lymphoma, urothelial cancer, sarcoma, blood cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, liver cancer, and other cancers, preferably breast cancer, uterine cancer, ovarian cancer, lung cancer, colon cancer, kidney cancer, and sarcoma, and more preferably breast cancer, colon cancer, kidney cancer, and sarcoma.

[0119] In one preferred embodiment of the present disclosure, preferably the cancer related disease is colon cancer or rectal cancer.

[0120] In one embodiment of the disclosure, the antibody is administered in a single dose or in multiple doses.

[0121] When the antibody is administered in multiple doses according to the present disclosure, preferably the antibody is administered in 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, or at least 10 doses, preferably up to 30 doses, 25 doses, 20 doses, 15 doses, or 10 doses. Preferably, the 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. Preferably, the doses of the antibody are administered at intervals of 7-30 days, 10-20 days, preferably about 14 days.

[0122] In one aspect, the disclosure provides the use of a therapeutic agent in the preparation of a medicament for the prevention or treatment of a cancer-related disease, wherein said therapeutic agent is selected from a monotherapy or a combination therapy.

[0123] In some embodiments, the single agent therapeutic is selected from the aforementioned antibodies or antigen-binding portions thereof, nucleic acids, vectors, cells, and / or compositions.

[0124] In some embodiments, the combination therapy comprises the monotherapy and an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an inhibitor of an immune checkpoint protein selected from PD-1, CTLA4, PD-L1, PD-L2, CD47, TIGIT, GITR, TIM3, LAG3, 4-1BB, CD27, and B7H4. In some embodiments, the inhibitor of an immune checkpoint protein is an antibody or an antigen-binding fragment thereof. In some preferred embodiments, the immune checkpoint protein is PD-1 or CTLA4. In some preferred embodiments, the inhibitor of an immune checkpoint protein is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

[0125] In another aspect, the disclosure provides a method of treating a cancer-related disease in a subject, the method of treatment using a therapeutic agent to treat the subject, the therapeutic agent being selected from a single therapeutic agent or a combination therapeutic agent.

[0126] In some embodiments, the single agent therapeutic is selected from the aforementioned antibodies or antigen-binding portions thereof, nucleic acids, vectors, cells, and / or compositions.

[0127] In some embodiments, the combination therapy comprises the monotherapy and an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an inhibitor of an immune checkpoint protein selected from PD-1, CTLA4, PD-L1, PD-L2, CD47, TIGIT, GITR, TIM3, LAG3, 4-1BB, CD27, and B7H4. In some embodiments, the inhibitor of an immune checkpoint protein is an antibody or an antigen-binding fragment thereof. In some preferred embodiments, the immune checkpoint protein is PD-1 or CTLA4. In some preferred embodiments, the inhibitor of an immune checkpoint protein is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

[0128] In another aspect, the present disclosure provides a therapeutic agent for treating a cancer-related disease in a subject, wherein the therapeutic agent is selected from a single therapeutic agent or a combination therapeutic agent.

[0129] In some embodiments, the single agent therapeutic is selected from the aforementioned antibodies or antigen-binding portions thereof, nucleic acids, vectors, cells, and / or compositions.

[0130] In some embodiments, the combination therapy comprises the monotherapy and an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an inhibitor of an immune checkpoint protein selected from PD-1, CTLA4, PD-L1, PD-L2, CD47, TIGIT, GITR, TIM3, LAG3, 4-1BB, CD27, and B7H4. In some embodiments, the inhibitor of an immune checkpoint protein is an antibody or an antigen-binding fragment thereof. In some preferred embodiments, the immune checkpoint protein is PD-1 or CTLA4. In some preferred embodiments, the inhibitor of an immune checkpoint protein is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

[0131] The kits described herein include the antibodies, fragments, homologs, derivatives, etc. of the present disclosure, such as labeled or cytotoxic conjugates, instructions for use of the antibodies, conjugates that kill specific types of cells, etc. The instructions may include directions for using the antibodies, conjugates, etc. in vitro, in vivo, or ex vivo. The antibodies may be in liquid or solid form, typically lyophilized. The kits may include other suitable reagents, such as buffers, reconstitution solutions, and other components required for the intended use. Combinations of reagents packaged in predetermined amounts and instructions for their use are contemplated, such as for therapeutic use or for performing diagnostic assays. If the antibody is labeled, such as with an enzyme, the kit may include a substrate and cofactors required for the enzyme (e.g., to provide a substrate precursor for detecting a chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffers, or lysis buffers), may be included. The relative amounts of the various reagents may be varied to provide a concentrate of the reagent solution, which provides the user with flexibility, space savings, reagent savings, etc. These reagents may also be supplied in the form of a dry powder, typically lyophilized and containing excipients that upon dissolution provide a reagent solution of the appropriate concentration.

[0132] In one aspect, the disclosure provides the use of said antibody or antigen-binding portion thereof, nucleic acid molecule, vector, cell, and / or composition in the preparation of a medicament or kit for use in the diagnosis, treatment, or prevention of a cancer-related disease.

[0133] Furthermore, the antibodies of the present disclosure can be used in immunoassays, purification methods, and other methods which employ immunoglobulins or fragments thereof, such uses being well known in the art.

[0134] Thus, the present disclosure also provides compositions comprising an anti-CCR8 antibody or fragment thereof of the present disclosure, conveniently combined with a pharma- ceutically acceptable carrier, diluent, or excipient, as is conventional in the art.

[0135] As used in this disclosure, the term "pharmaceutical composition" refers to a formulation of any of a variety of preparations. The formulations containing a therapeutically effective amount of a multivalent antibody are in sterile liquid solution, liquid suspension, or lyophilized form, and optionally contain stabilizers or excipients.

[0136] It will be understood that the therapeutic agents according to the above embodiments are administered with suitable pharma- ceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to improve migration, delivery, tolerance, etc. Many suitable formulations are described in the pharmacopoeia known to every pharmaceutical chemist: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa. (1975)), in particular Chapter 87 by Blaug, Seymour. These formulations include, for example, powders, pastes, salves, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsions of polyglycols (polyglycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyglycols. Any of the foregoing mixtures are suitable for use in treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable to the route of administration.

[0137] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container, dispenser, or nebulizer, which contains a suitable propellant, such as a gas such as carbon dioxide.

[0138] It can also be administered systemically by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for penetrating the barrier is used in the formulation.Such penetrants are generally known in the art, and include detergents, bile salts, and fusidic acid derivatives used for transmucosal administration.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, one or more of the above-mentioned antibodies can be prepared as ointments, ointments, gels or creams, as generally known in the art.

[0139] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

[0140] In one embodiment, the antibody can be prepared in a carrier that prevents rapid elimination from the body, such as a sustained / controlled release formulation, including implants and microencapsulated delivery systems. For example, biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyglycogenates, and polylactic acid. Methods for preparing such formulations are clear to those skilled in the art.

[0141] In particular, it is advantageous to prepare parenteral compositions in dosage units for ease of administration and consistency of dosage.As used herein, dosage unit form refers to a physically separable unit suitable for use as a unitary dosage to a subject to be treated.Each unit contains a predetermined amount of one or more of the antibodies calculated to combine with a desired drug carrier to produce a desired therapeutic effect.The specifications of the dosage unit form of the above embodiment are dictated by and directly depend on the inherent characteristics of the antibody and the specific therapeutic effect to be achieved, as well as the limitations inherent in the field of formulating such antibodies for use in treating individuals.

[0142] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0143] The formulations described herein may also contain one or more of the above antibodies, preferably antibodies with complementary activities but that do not adversely affect each other, depending on the specific condition being treated. Alternatively or in addition, the compositions may contain agents that enhance their function, such as, for example, cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitory agents. Such molecules may be suitably co-present in amounts effective for the intended purpose. For example, they may be co-present in a kit or during use.

[0144] In one embodiment, one or more of the antibodies can be administered in combination therapy, i.e., in combination with other reagents, such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer and inflammatory diseases). As used herein, the term "combined" refers to substantially simultaneous, parallel, or sequential administration of reagents. When administered sequentially, the first of the two compounds is preferably detected at an effective concentration at the treatment site at the time the administration of the second compound is initiated. In one case, "combined" can also refer to the inclusion of both the antibody of the present disclosure and the other therapeutic agent in a kit.

[0145] For example, combination therapy may involve the co-preparation and / or co-application of one or more antibodies described herein with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immune inhibitors, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cell growth inhibitors, described in more detail below). Such combination therapy may advantageously allow lower dosages of the therapeutic agents to be administered, thereby avoiding possible toxicities or complications associated with various monotherapies.

[0146] The present disclosure provides a variety of monoclonal antibodies and variants thereof that bind to CCR8 with high affinity, have neutralizing activity to block the CCL1 / CCR8 axis, inhibit CCL1-induced cell activation, while inhibiting the low affinity FcγRIIIA in the population. 158FWe experimentally demonstrated that Fc-optimization of humanized antibodies against allele carriers also enhanced the ADCC effect of Fc-optimized humanized antibodies.In in vitro efficacy studies, the novel humanized antibodies effectively eliminated CCR8+ cells and significantly inhibited the subcutaneous tumor growth of MC38, B16 / F10 and MDA-MB-231 tumor cells in humanized mice, and in the PD-1 antibody-insensitive B16 / F10 subcutaneous tumor model, the humanized CCR8 antibody could more effectively inhibit tumor growth in combination with anti-PD-1 antibody.

[0147] Although for clarity and conciseness of description, features are described herein as part of the same or separate embodiments, it will be understood that the scope of the disclosure may encompass multiple embodiments having all or a partial combination of the described features.

[0148] Working Example Example 1: Preparation of antibodies Construction of CCR8+ or CCR4+ stable transfected cell lines Using human CCR8 cDNA (manufactured by Sino Biological Co., Ltd., product code: HG11450-M, the amino acid sequence of which is shown in SEQ ID NO.1), cynomolgus monkey CCR8 cDNA (synthesized by Suzhou Jinweizhi Biological Co., Ltd., the amino acid sequence of which is shown in SEQ ID NO.2), and human CCR4 cDNA (manufactured by Sino Biological Co., Ltd., product code: HG13064-UT, the amino acid sequence of which is shown in SEQ ID NO.3), respectively, as templates, lentivirus vector plasmids containing the full-length sequences of human, monkey CCR8, and human CCR4 were constructed, and the constructed lentivirus plasmids and packaging plasmids were co-transfected into HEK293T cells (ATCC CRL-11268) according to the instructions of the lentivirus packaging kit (Lenti-Pac HIV Expression Packaging Kit, Gene Copoeia, product code: HPK-LvTR-20), and lentivirus packaging was performed. 48 hours after transfection, the medium was collected and centrifuged at 500*g for 10 minutes to remove cell debris, and the culture supernatant containing lentivirus particles was obtained. This was filtered through a 0.45 μm PES membrane and then dispensed into 1.5 mL EP tubes. 10 μL of each tube was used to obtain 1 × 10 6 HEK293T cells or CHO cells (ATCC CCL-61) were infected with 1000 cells, and after 48 hours, 8 μg / ml puromycin was added and pressure was applied to screen the monoclonal cell line HEK293-hCCR8, which expresses human CCR8 at a relatively high level (fluorescence value by flow assay is at least 2 log higher than that of HEK293 cells), HEK293-hCCR8 low (Fluorescence values ​​by flow assay increased by <1 log compared to HEK293 cells.) We also obtained cell lines stably expressing human CCR4, HEK293-hCCR4, human CCR8, and cynomolgus monkey CCR8, and the results are shown in Figure 1.

[0149] Select 4-6 week old female Balb / C mice, construct eukaryotic expression plasmids containing CMV promoters using the full-length sequence of human or cynomolgus monkey CCR8, immunize the mice with DNA using a gene gun (20 μg of plasmid / mouse), measure serum titers after 4-5 immunizations, and transfect CHO-hCCR8 stable cells (5 × 10 6 Shock immunization was performed using 1000 cells / mouse. After 3 days, the mice were sacrificed by cervical dislocation, and the spleens and peripheral lymph nodes of the mice were collected, and total RNA was extracted by grinding and centrifugation. After reverse transcription, PCR amplification was performed using light- and heavy-chain specific primers, respectively, to obtain antibody light- and heavy-chain variable region cDNA libraries. A CCR8 immune library was constructed using phage antibody library display technology for subsequent phage elution.

[0150] The constructed CCR8 immune library was washed by a conventional method and then incubated with human CCR8+ cells CHO-hCCR8 or cynomolgus monkey CCR8+ cells CHO-cynoCCR8 (1 × 10 6 / tube) alternately, 1×10 12 phages were added, incubated and washed, and the phage particles with high binding affinity were eluted with 100 mM triethylamine, neutralized with 1 M Tris-HCl (pH 7.4), infected with E. coli TG1, and the repackaged phages were used for the next round of washing. A single clone was selected from the clones obtained by the two rounds of washing and enrichment, inoculated into a 96-well U-shaped well plate for culture and IPTG-induced expression, and the induced supernatant was collected for FACS detection.

[0151] Example 2 Screening for highly specific CCR8-binding antibodies The positive clone obtained in Example 1 was re-induced and expressed, and purified to obtain Fab antibodies, and their binding to cells stably transfected with human CCR8, cynomolgus monkey CCR8, and human CCR4 was detected. 410 detection cells were added, centrifuged, the supernatant was discarded, purified Fab antibody was added at 100μL / well, and incubated on ice for 60 minutes. After removing unbound primary antibody by washing, 50μL of secondary antibody solution (anti-human IgG, F(ab')2-AF647, Jackson Immuno Research) was added to each well and incubated on ice for 30 minutes. After washing, 50μL of 5μg / mL PI / BSA solution was added to each well and incubated on ice for 5 minutes. Then, excess secondary antibody was washed with 0.5% BSA, PBS solution was added to resuspend the cells, and the binding of the antibody to the cells was detected using a flow cytometer (Intellicyte iQue). Approximately 200 positive clones that recognized human CCR8+ cells and cynomolgus monkey CCR8+ cells but did not bind to human CCR4+ cells were selected, and the sequences of the light and heavy chain variable regions were determined by Sanger sequencing. After excluding duplicated sequences, the sequences of the first 10 clones with superior binding activity are shown in the table below (Table 2).

[0152] Table 2. Anti-CCR8 antibody light and heavy chain variable region sequences [Table 2]

[0153] The anti-CCR8 mouse antibody heavy / light chain variable region sequences are as follows:

[0154] Clone:29A8 The amino acid sequence of 29A8 heavy chain VH is shown in SEQ ID NO.4, its encoding nucleic acid in SEQ ID NO.5, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.6, 7 and 8, respectively. [Table 3]

[0155] The amino acid sequence of 29A8 light chain VK is shown in SEQ ID NO.9, its encoding nucleic acid in SEQ ID NO.10, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.11, 12 and 13, respectively. [Table 4]

[0156] Clone:30A7 The amino acid sequence of 30A7 heavy chain VH is shown in SEQ ID NO.14, its encoding nucleic acid in SEQ ID NO.15, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.16, 17 and 8, respectively. [Table 5]

[0157] The amino acid sequence of 30A7 light chain VK is shown in SEQ ID NO.18, its encoding nucleic acid in SEQ ID NO.19, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.11, 12 and 13, respectively. [Table 6]

[0158] Clone:87D8 The amino acid sequence of 87D8 heavy chain VH is shown in SEQ ID NO.20, its encoding nucleic acid in SEQ ID NO.21, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.16, 22 and 8, respectively. [Table 7]

[0159] The amino acid sequence of 87D8 light chain VK is shown in SEQ ID NO.23, its encoding nucleic acid in SEQ ID NO.24, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.11, 12 and 25, respectively. [Table 8]

[0160] Clone: ​​102H5 The amino acid sequence of 102H5 heavy chain VH is shown in SEQ ID NO.26, its encoding nucleic acid in SEQ ID NO.27, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.28, 22 and 29, respectively. [Table 9]

[0161] The amino acid sequence of 102H5 light chain VK is shown in SEQ ID NO.30, its encoding nucleic acid in SEQ ID NO.31, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.32, 33 and 34, respectively. [Table 10]

[0162] Clone: ​​109F6 The amino acid sequence of 109F6 heavy chain VH is shown in SEQ ID NO.35, its encoding nucleic acid in SEQ ID NO.36, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.28, 22 and 29, respectively. [Table 11]

[0163] The amino acid sequence of 109F6 light chain VK is shown in SEQ ID NO.37, its encoding nucleic acid in SEQ ID NO.38, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.39, 40 and 41, respectively. [Table 12]

[0164] Clone: ​​110G2 The amino acid sequence of 110G2 heavy chain VH is shown in SEQ ID NO.35, its encoding nucleic acid in SEQ ID NO.36, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.28, 22 and 29, respectively. [Table 13]

[0165] The amino acid sequence of 110G2 light chain VK is shown in SEQ ID NO.42, its encoding nucleic acid in SEQ ID NO.43, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.44, 45 and 46, respectively. [Table 14]

[0166] Clone:113B3 The amino acid sequence of 113B3 heavy chain VH is shown in SEQ ID NO.47, its encoding nucleic acid in SEQ ID NO.48, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.28, 22 and 49, respectively. [Table 15]

[0167] The amino acid sequence of 113B3 light chain VK is shown in SEQ ID NO.50, its encoding nucleic acid in SEQ ID NO.51, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.52, 45 and 53, respectively. [Table 16]

[0168] Clone: ​​116F10 The amino acid sequence of 116F10 heavy chain VH is shown in SEQ ID NO.54, its encoding nucleic acid in SEQ ID NO.55, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.28, 22 and 49, respectively. [Table 17]

[0169] The amino acid sequence of 116F10 light chain VK is shown in SEQ ID NO.56, its encoding nucleic acid in SEQ ID NO.57, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.44, 58 and 59, respectively. [Table 18]

[0170] Clone:139A9 The amino acid sequence of 139A9 heavy chain VH is shown in SEQ ID NO.35, its encoding nucleic acid in SEQ ID NO.36, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.28, 22 and 29, respectively. [Table 19]

[0171] The amino acid sequence of 139A9 light chain VK is shown in SEQ ID NO.60, its encoding nucleic acid in SEQ ID NO.61, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.62, 63 and 64, respectively. [Table 20]

[0172] Clone:144D2 The amino acid sequence of 144D2 heavy chain VH is shown in SEQ ID NO.4, its encoding nucleic acid in SEQ ID NO.5, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.6, 7 and 8, respectively. [Table 21]

[0173] The amino acid sequence of 144D2 light chain VK is shown in SEQ ID NO.65, its encoding nucleic acid in SEQ ID NO.66, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.11, 12 and 25, respectively. [Table 22]

[0174] Example 3 Expression of anti-CCR8 chimeric antibody and detection of affinity The heavy chain variable region of the positive clone of Example 2 was cloned into a vector containing a human heavy chain fixing region and a regulatory element, and a complete IgG heavy chain was expressed in a mammalian cell. Similarly, the light chain variable region was cloned into a vector containing a human light chain fixing region and a regulatory element, and a complete IgG light chain was expressed in a mammalian cell. The correct sequence was transfected into CHO-S mammalian cells (Thermo Fisher ExpiCHO), and IgG was secreted into the culture medium by expression. The supernatant was collected, and anti-CCR8 chimeric antibody was obtained by conventional ProA purification, and the antibody concentration was determined by spectrophotometric measurement of OD280 absorbance. Mouse anti-m10A11 (chimeric) and humanized antibody h10A11 were synthesized with reference to the literature WO2020138489A1 (m10A11: sequences 27 and 26 of the literature, h10A11: sequences 41 and 59 of the literature).

[0175] Place 5 × 10 CHO-hCCR8 or CHO-cynoCCR8 cells in each well of a 96-well U-shaped plate. 4The wells were then centrifuged to discard the supernatant, and 100 μL of gradient-diluted anti-CCR8 chimeric antibody or anti-KLH isotype control antibody was added to each well and incubated on ice for 60 minutes. After washing to remove unbound primary antibody, 50 μL of secondary AlexaFluro647 anti-human IgG (Jackson Immuno Research #109-606-170) was added to each well and incubated on ice for 30 minutes. After washing, 50 μL of 5 μg / mL PI / BSA solution was added to each well and incubated on ice for 5 minutes, after which excess secondary antibody was washed off with 0.5% BSA, PBS solution was added to resuspend the cells, and the fluorescence value of the PI-negative cell population was analyzed by detecting the binding of the antibody to the cells using flow cytometry (Intellicyte iQue). The cell binding curves were fitted using Graphpad Prism 7 (Figure 2), and the chimeric antibodies 87D8, 102H5, 109F6, 110G2, 113B3, 139A9, and 144D2 all bound with high affinity to human CCR8+ cells CHO-hCCR8 and cynomolgus monkey CCR8+ cells CHO-cynoCCR8. The affinity (EC 50 ) are shown in Table 3.

[0176] Table 3. Binding of anti-CCR8 chimeric antibodies to human and cynomolgus monkey CCR8+ cells [Table 23]

[0177] Example 4 Detection of ADCC activity of anti-CCR8 chimeric antibody A lentiviral vector plasmid of the full-length human CD16A sequence was constructed, packaged according to the Lenti-Pac HIV Expression Packaging Kit (Gene Copoeia, HPK-LvTR-20), transfected into NK92 cells (purchased from ATCC, product code: CRL-2408), and screened to obtain NK92-CD16A stable transfected cells. 5 × 10 per well in a 96-well U-shaped plate. 4 HEK293-hCCR8 cells were added, and 50 μL of gradient-diluted anti-CCR8 chimeric antibodies or isotype controls were added, and incubated in an incubator for 30 minutes. 2 × 10 6 50μL of NK92-CD16A cells at 100x / mL were added to each well and incubated for 4 hours. 30 minutes before detection, 2μL of cell lysis solution (100x) was added to the largest well of the target cells, centrifuged at 300*g for 3 minutes, 50μL of the supernatant was transferred to a black enzyme plate, 50μL of lactate dehydrogenase (LDH) detection substrate was added, and the well was mixed by shaking. After 10 minutes, 25μL of stop solution was added, the well was shaken for 10 seconds, and the plate was read by selecting fluorescence (excitation wavelength 560nm, emission wavelength 590nm). The killing rate of the target cells was calculated based on the LDH release level, and the antibody concentration-killing rate (%) curve was obtained by fitting to a 4-parameter model, and the median effective concentration (EC 50 ) was calculated. As shown in FIG. 3, the anti-CCR8 chimeric antibodies showed significant ADCC killing activity against CCR8+ cells in a dose-dependent manner, and the EC50 values ​​of the median effective killing concentrations of the antibodies 29A8, 87D8, 102H5, 109F6, 110G2, 113B3, 116F10, 139A9, and 144D2 were 50 was superior to that of m10A11 (Table 4).

[0178] Table 4 ADCC activity of anti-CCR8 chimeric antibodies [Table 24]

[0179] Example 5 Anti-CCR8 chimeric antibody inhibits CCL1-induced calcium flux signaling in target cells 5 x 10 per well in a 96-well U-shaped plate 4 CHO-hCCR8 cells were added and cultured overnight in an incubator. After washing twice with 2.5 mM probenecid / HBSS solution, 100 μL of 4 μM Fluo-4 AM working solution was added to each well and incubated at 37 °C for 60 min. After washing the cells twice with probenecid / HBSS solution, 50 μL of probenecid / HBSS was added to each well and incubated at 37 °C for 20 min, after which 50 μL of test antibody or isotype control (final concentration 10 μg / mL) was added and incubated at room temperature for 30 min. Using a microplate reader (Molecular Device SpectraMax i3x), 50 μL of 180 nM CCL1 was added and the fluorescence signal was read at an excitation wavelength of 494 nm and an emission wavelength of 519 nm. The calcium flux signal of the CCL1 treatment group with antibody concentration 0 was taken as 100%, and the percentage of calcium flux signal of each treatment group was calculated. As a result, the anti-CCR8 chimeric antibody significantly inhibited the calcium flux signal induced by CCL1 (FIG. 4).

[0180] Example 6 Binding of anti-CCR8 antibody mutants to CCR8 Clones 87D8 and 144D2 have almost identical light chain sequences, and mutants were constructed using overlapping PCR site-directed mutagenesis for the potential deamidation site N33 and its neighboring sites, and the site M56, where oxidative modification may occur, to mutate to Cys, Trp, Phe and other amino acids other than the parent sequence. After expression in E. coli TG1, the binding of the induced supernatant to CHO-hCCR8 cells was detected. A clone with better binding activity was selected, re-induced expression, and purified by extraction of periplasmic lumen proteins and Ni-NTA to obtain purified Fab antibody mutants, and the absorbance at OD280 was measured spectrophotometrically, and the antibody concentration was determined by referring to the theoretical extinction coefficient. Binding EC with CHO-hCCR8 50The 144D2 mutants G34Y, G34T, G34R, and M56T all retained affinity equal to or greater than that of the parent 144D2, and the results are shown in Table 5 and FIG.

[0181] Table 5. Binding of anti-CCR8 antibody mutants to human CCR8+ cells [Table 25]

[0182] Example 7 Humanization and sequence optimization of anti-CCR8 antibodies Clones 87D8 and 144D2, which have high affinity for human and cynomolgus CCR8 and strong ADCC killing activity, were selected to construct humanized antibodies. The light and heavy chain germline genes used in 87D8 and 144D2 are the same, and the sequences of the CDR regions are very similar. The heavy chain CDR1 and CDR2 have two and one amino acid differences, respectively, which may be due to somatic mutations that occurred during the mouse B cell expansion process. First, the sequences of mouse antibodies 87D8 and 144D2 were compared with the sequences of human antibody germline, and the highly homologous human germline light chain genes IMGT_hVK2-28 and IGKJ2*01, and human germline heavy chain genes IMGT_hVH3-7 and IGHJ4*01 were identified. Then, CDR grafting of mouse antibodies was performed to combine the CDR1 and CDR2 of the 87D8 heavy chain and the 144D2 heavy chain. Computer analysis indicated the possibility of deamidation modification at the N33 site of light chain CDR1 and the possibility of oxidative modification at the M52 site of CDR2. Therefore, in the process of designing humanized variants, G34Y and M52T mutations were introduced with reference to Example 6, and a total of three heavy chain variants, h87D8 VHv1, h144D2 VHv1, h87D8 VHv2, and two light chain variants, h87D8 VKv1 and h87D8 VKv4, were designed (Tables 6 and 7).

[0183] The light and heavy chains were synthesized in their entirety and cloned into vectors containing the constant region of the antibody κ chain or the constant regions of human IgG1 CH1-CH3, respectively, and transfected in pairs into CHO cells. After 6 days of expression, the cell culture medium was collected by filtration and subjected to affinity chromatography using Protein A to obtain humanized antibodies. The antibody concentration was determined by spectrophotometric measurement of OD280 absorbance.

[0184] Table 6. Sequences of the heavy chain variable region of the CCR8 humanized antibody [Table 26]

[0185] Table 7. Sequence of the light chain variable region of the CCR8 humanized antibody [Table 27]

[0186] The sequences of the heavy and light chain variable regions of the humanized antibody are shown below.

[0187] The amino acid sequence of h87D8 VHv1 is shown in SEQ ID NO.80, its encoding nucleic acid is shown in SEQ ID NO.81, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.16, 82 and 8, respectively. [Table 28]

[0188] The amino acid sequence of h144D2 VHv1 is shown in SEQ ID NO.83, its encoding nucleic acid is shown in SEQ ID NO.84, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.6, 82 and 8, respectively. [Table 29]

[0189] The amino acid sequence of h87D8 VHv2 is shown in SEQ ID NO.85, its encoding nucleic acid is shown in SEQ ID NO.86, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.16, 7 and 8, respectively. [Table 30]

[0190] The amino acid sequence of h87D8 VKv1 is shown in SEQ ID NO.87, its encoding nucleic acid in SEQ ID NO.88, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.11, 12 and 25, respectively. [Table 31]

[0191] The amino acid sequence of h87D8 VKv4 is shown in SEQ ID NO.89, its encoding nucleic acid in SEQ ID NO.90, and its CDR1, CDR2 and CDR3 in SEQ ID NOs.91, 63 and 25, respectively. [Table 32]

[0192] Example 8. Detection of affinity of humanized anti-CCR8 antibodies Place 5 × 10 CHO-hCCR8 or CHO-cynoCCR8 cells in each well of a 96-well U-shaped plate. 4The cells were centrifuged and the supernatant was discarded, and 100 μL of gradient-diluted anti-CCR8 chimeric antibody was added to each well and incubated on ice for 60 minutes. After washing to remove unbound primary antibody, 50 μL of secondary AlexaFluro647 anti-human IgG (Jackson Immuno Research #109-606-170) was added to each well and incubated on ice for 30 minutes. After washing, 50 μL of 5 μg / mL PI / BSA solution was added to each well and incubated on ice for 5 minutes, after which excess secondary antibody was washed off with 0.5% BSA, PBS solution was added to resuspend the cells, and the fluorescence value of the PI-negative cell population was analyzed by detecting the binding of the antibody to the cells using flow cytometry (BD Celesta). The cell binding curves were fitted using Graphpad Prism 7. From Figures 6 and 7, it was found that the humanized antibodies h87D8 H1K1 (consisting of h87D8 VHv1+h87D8 VKv1), h144D2 H1K1 (h144D2 VHv1+h87D8 VKv1) and h87D8 H2K4 (h87D8 VHv2+h87D8VKv4) were consistent with the parent chimeric antibodies and could bind to CCR8+ cells with high affinity, and the half effective binding concentration with human CCR8+ cells was EC 50 = 0.53-0.89 nM, and the half-effective binding concentration with cynomolgus monkey CCR8+ cells was EC 50 =1.16-2.28 nM (Table 8).

[0193] Table 8. Binding of humanized antibodies to CCR8+ cells [Table 33]

[0194] Example 9 ADCC activity of humanized anti-CCR8 antibodies against human CCR8+ cells 5 x 10 per well in a 96-well U-shaped plate 4 HEK293-hCCR8 cells were added, and 50 μL of gradient-diluted anti-CCR8 humanized antibodies or isotype controls were added, and incubated in an incubator for 30 minutes. 2 × 10 650μL of NK92-CD16A cells at 100x / mL were added to each well and incubated for 4 hours. 30 minutes before detection, 2μL of cell lysis solution (100x) was added to the largest well of the target cells, centrifuged at 300*g for 3 minutes, 50μL of the supernatant was transferred to a black enzyme plate, 50μL of lactate dehydrogenase (LDH) detection substrate was added, and the well was mixed by shaking. After 10 minutes, 25μL of stop solution was added, the well was shaken for 10 seconds, and the plate was read by selecting fluorescence (excitation wavelength 560nm, emission wavelength 590nm). The killing rate of the target cells was calculated based on the LDH release level, and the antibody concentration-killing rate (%) curve was obtained by fitting to a 4-parameter model, and the median effective concentration (EC 50 The results showed that the humanized antibodies h87D8 H1K1 and h144D2 H1K1 had ADCC activity against CCR8+ cells, and the median effective killing concentration (EC 50 were 0.01 nM and 0.02 nM, respectively, and m10A11 (EC 50 =0.03 nM) (Figures 8A and 8B).

[0195] Example 10 Optimization of Fc function of humanized anti-CCR8 antibodies 1) Optimization of Fc function There is a polymorphism in the FcγRIIIA gene, among which the high affinity receptor FcγRIIIA 158V / V Only 10-20% of people have the genotype, which is the low-affinity receptor FcγRIIIA 158F / F In patients with the genotype, wild-type IgG1 Fc-mediated ADCC activity is usually weak. Using the technical solution described in patent application 202011627881.1, the CCR8 chimeric antibody is humanized while the low affinity FcγRIIIA in the population is eliminated. 158F Variant R with enhanced Fc activity in carriers of the allele 292 PY 300 L was constructed to obtain two Fc function-enhanced humanized antibodies, h87D8 H1K1 hIgG1 e5 (hereinafter abbreviated as h87D8 H1K1 e5) and h87D8 H2K4 hIgG1 e5 (hereinafter abbreviated as h87D8 H2K4 hIgG1 e5) (Table 9).

[0196] Table 9. Sequences of humanized anti-CCR8 antibodies [Table 34]

[0197] The heavy chain amino acid sequence of h87D8 H1K1 hIgG1 e5 is shown in SEQ ID NO 92 and its encoding nucleic acid in SEQ ID NO 93. [Table 35]

[0198] The light chain amino acid sequence of h87D8 H1K1 hIgG1 e5 is shown in SEQ ID NO 94 and its encoding nucleic acid in SEQ ID NO 95. [Table 36]

[0199] The heavy chain amino acid sequence of h87D8 H2K4 hIgG1 e5 is shown in SEQ ID NO 96 and its encoding nucleic acid in SEQ ID NO 97. [Table 37]

[0200] The light chain amino acid sequence of h87D8 H2K4 hIgG1 e5 is shown in SEQ ID NO 98 and its encoding nucleic acid in SEQ ID NO 99. [Table 38]

[0201] The heavy chain amino acid sequence of h87D8 H2K4 hIgG1 is shown in SEQ ID NO:100 and its encoding nucleic acid in SEQ ID NO:101. [Table 39]

[0202] The light chain amino acid sequence of h87D8 H2K4 hIgG1 is shown in SEQ ID NO 98 and its encoding nucleic acid in SEQ ID NO 99. [Table 40]

[0203] 2) Affinity of Fc-function-enhanced humanized CCR8 antibodies to FcγRIIIA Surface plasmon resonance (SPR / Biacore T200) was used to detect the affinity of Fc-functionalized humanized CCR8 antibodies to FcγRIIIAV158 and FcγRIIIAF158. Goat Anti-Human IgG F(ab')2 was coupled to the CM5 chip surface using the coupling reagent Amine Coupling Kit (GE Healthcare BR-1000-50), and h87D8 H2K4 e5 or h87D8 H2K4 was captured by high affinity binding with the antibody. Gradient dilutions of FcγRIIIAV158 or FcγRIIIAF158 were then sequentially injected, with the binding time set to 120 s, the dissociation time set to 200 s, and the regeneration with 10 mM Glycine-HCl at pH 1.75 for 30 s, and the flow rate set to 30 μl / min. The measured data was fitted to obtain the equilibrium dissociation constant. The affinity of h87D8 H2K4 e5 or h87D8 H2K4 to FcγRIIIAV158 and FcγRIIIAF158 was measured. As a result, both h87D8 H2K4 e5 and h87D8 H2K4 bound to FcγRIIIAV158 and FcγRIIIAF158, and the affinity KD of h87D8 H2K4 e5 was 270.0 nM and 432.7 nM, respectively, and the affinity KD of h87D8 H2K4 was 861.2 nM and 1.3 μM, respectively. The binding of h87D8 H2K4 e5 to FcγRIIIAV158 and FcγRIIIAF158 was stronger than that of h87D8 H2K4.

[0204] 3) ADCC activity of Fc-enhanced humanized CCR8 antibodies Referring to the method of Example 9, the anti-CCR8 antibody activity of Fc-function-enhanced humanized antibodies and wild-type Fc was compared using NK92-CD16A as effector cells and HEK293-hCCR8 as target cells. As shown in Figure 9A, the Fc-function-enhanced humanized anti-CCR8 antibodies h87D8 H1K1 e5 and h87D8 H2K4 e5 significantly enhanced the ADCC killing activity against hCCR8+ cells and increased the EC 50 were 0.016 nM and 0.023 nM, respectively, and the maximum lysis rates were 58% and 61%, respectively, which were approximately 1-fold higher than those of 87D8 and m10A11 (maximum lysis rates were 33% and 30%, respectively).

[0205] Referring to the method of Example 9, NK92-CD16A was used as an effector cell, and HEK293-hCCR8 expressing human CCR8 at low to moderate levels was used. low We compared the activity of anti-CCR8 humanized antibodies with Fc function-enhanced and wild-type Fc using target cells expressing human CCR8 at low to moderate levels. As shown in Figure 9B, the Fc function-enhanced h87D8H2K4e5 significantly enhanced the killing ability of target cells expressing human CCR8 at low to moderate levels.

[0206] Referring to the method of Example 9, the activity of Fc function-enhanced anti-CCR8 humanized antibodies was detected using NK92-CD16A as effector cells and HuT78 cells that endogenously express human CCR8 as target cells. As shown in Figure 9C, the anti-CCR8 humanized antibody h87D8 H2K4 e5 showed similar ADCC activity against HuT78 cells that express human CCR8, and its half effective concentration EC 50 was 0.53 nM.

[0207] FcγRIIIA 158V / V and FcγRIIIA 158F / F Peripheral blood was collected from healthy volunteers, and PBMCs were separated by density gradient centrifugation using Ficoll reagent. 7 The cells were resuspended at 1 × 10 cells / mL and used as effector cells in the ADCC experiment. Target cells HEK293-hCCR8 (1 × 10 650μL of 100x10 cells / mL was added to each well, 50μL of gradient diluted humanized antibody, parent chimeric antibody, and isotype control antibody were added to each well, and 50μL of effector cell suspension was added to each well, and the wells were incubated at 37℃ and 5% CO2 for 4 hours. 30 minutes before detection, 2μL of cell lysis solution (100x) was added to the largest well of target cells, centrifuged at 300*g for 3 minutes, 50μL of the supernatant was transferred to a black enzyme plate, 50μL of lactate dehydrogenase (LDH) detection substrate was added, and the wells were mixed by shaking. After 10 minutes, 25μL of stop solution was added, the wells were shaken for 10 seconds, and the plate was read by selecting fluorescence (excitation wavelength 560nm, emission wavelength 590nm). The killing rate of target cells was calculated based on the LDH release level, and the antibody concentration-killing rate (%) curve was obtained by fitting to a four-parameter model, and the median effective concentration (EC 50 As shown in FIG. 9D, the Fc-functionalized anti-CCR8 humanized antibodies h87D8 H1K1 e5 and h87D8 H2K4 e5 exhibited low affinity FcγRIIIA 158F / F When PBMCs were used as effector cells, ADCC activity was significantly enhanced compared to the control group, and the maximum lysis rate exceeded that of wild-type IgG1. 158V / V The ADCC activity was comparable to that observed when PBMCs were used as effector cells (FIG. 9E, Table 10).

[0208] Table 10 ADCC activity of Fc-function-enhanced anti-CCR8 humanized antibodies [Table 41]

[0209] Example 11 Neutralizing and blocking activity of humanized anti-CCR8 antibodies against CCL1 / hCCR8 CHO-hCCR8 cells were harvested and cultured at 2 × 10 5The cells were resuspended in 100 μL / mL and plated in a 96-well plate at 100 μL / well and incubated overnight at 37°C in a 5% CO2 incubator. The cells were washed twice with HBSS / probenecid (containing 2.5 mM probenecid) solution, 100 μL of 4 μM Fluo-4 AM working solution was added per well, and incubated at 37°C for 60 min. The cells were washed twice with HBSS / probenecid solution, and after removing the remaining Fluo-4 AM working solution, 50 μL of HBSS / probenecid solution was added per well and incubated at 37°C for 20 min. 50 μL of antibodies at different concentrations were added and incubated at room temperature for 30 min. Fluorescence signals were read using a microplate reader at excitation wavelength of 494 nm and emission wavelength of 519 nm after adding 50 μL of 180 nM CCL1 or HBSS solution. Ca in the CCL1-treated group at antibody concentration 0 was measured. 2+ The Ca2+ flux signal percentage for each treatment group was calculated, taking the flux signal as 100%. Then, the antibody concentration-Ca 2+ Flux signal (%) curves were obtained and the median effective concentration (EC 50 As shown in Figure 10, h87D8 H1K1 e5 and h87D8 H2K4 e5 significantly suppressed the Ca2+-induced increase in IL-1 expression by CCL1-CCR8. 2+ Inhibits flux signaling and has a half effective concentration EC 50 were 5.59 nM and 5.20 nM, respectively.

[0210] Example 12 Binding of humanized anti-CCR8 antibodies to rabbit CCR8 A lentiviral vector plasmid containing rabbit CCR8 was constructed using rabbit CCR8 cDNA (synthesized by Suzhou Jinweizhi Biological Co., Ltd., and its amino acid sequence is shown in SEQ ID NO. 112) as a template, and the lentiviral vector plasmid containing rabbit CCR8 was transfected into HEK293 cells (hereinafter abbreviated as HEK293-rabbitCCR8 cells) using a transfection reagent PEI MAX (registered trademark) (Polysciences 9002-98-6) according to the instructions of the transfection reagent. 24 hours after transfection, 5 × 10 cells were transfected into each well of a 96-well U-shaped plate. 4 HEK293-rabbitCCR8 cells were added, centrifuged, and the supernatant was discarded. 100 μL of gradient dilutions of h87D8 H2K4 e5 antibody or anti-KLH isotype control antibody were added to each well, and the plate was incubated on ice for 60 minutes. After washing to remove unbound primary antibody, 50 μL of secondary antibody AlexaFluro647 anti-human IgG (Jackson Immuno Research #109-606-170) was added to each well and incubated on ice for 30 minutes. After washing, 50 μL of 5 μg / mL PI / BSA solution was added to each well and incubated on ice for 5 minutes, after which excess secondary antibody was washed off with 0.5% BSA, PBS solution was added to resuspend the cells, and the fluorescence value of the PI negative cell population was analyzed by detecting the binding of the antibody to the cells using flow cytometry (Intellicyte iQue). The cell binding curve was fitted using Graphpad Prism 7. As shown in Figure 11, the humanized anti-CCR8 antibody h87D8 H2K4 e5 binds to rabbit CCR8 and has a median effective concentration (EC 50 was 0.21 nM.

[0211] Example 13 Effect of humanized CCR8 antibody on the growth of subcutaneously transplanted tumors in MC38 mice CCR8 humanized transgenic mouse C57BL / 6J-Ccr8 em3(hCCR8) / Smoc(purchased from Shanghai Nanfang Model Biology Co., Ltd., product number: NM-HU-2000054) was used to prepare MC38 subcutaneously transplanted tumor models. The study included 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 and 3mg / kg groups, and IgG1 isotype control 3mg / kg group. CCR8 humanized transgenic mice were intravenously administered once every three days for a total of four times. As shown in Figure 12, h87D8 H2K4 e5 inhibited tumor growth in each dosage group, and the tumor inhibition rates were 113.96%, 110.64% and 95.44% in the 3mg / kg, 0.6mg / kg and 0.12mg / kg groups, 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. The tumor inhibition effect of h87D8 H2K4 e5 was better than that of h87D8 H2K4 and h10A11. Relative tumor growth rate T / C(%)=(T-T0) / (C-C0)×100, where T and T0 are the tumor volumes of the treated group at the end and start of the experiment, respectively, and C and C0 are the tumor volumes of the control group at the end and start of the experiment, respectively. When the tumor end volume is greater than the start volume, the tumor inhibition rate (TGI)(%)=100-T / C(%). When the tumor end volume is smaller than the start volume, the tumor inhibition rate (TGI) (%) = 100-(T-T0) / T0 x 100).

[0212] Example 14 Effect of humanized CCR8 antibody on the growth of subcutaneously transplanted tumors in B16 / F10 mice CCR8 humanized transgenic mouse C57BL / 6J-Ccr8 em3(hCCR8) / SmocA B16 / F10 subcutaneously transplanted tumor model was established in the study. The study included h87D8 H2K4 e5 10mg / kg, anti-mouse PD-1 antibody (mIgG1, clone G4C2) 10mg / kg group, and h87D8 H2K4 e5 10mg / kg + anti-mouse PD-1 antibody 10mg / kg group, IgG1 isotype control 10mg / kg group, and blank control group (i.e., solvent control group). The CCR8 humanized transgenic mice were intravenously administered once every three days for a total of three times. As shown in Figure 13, the anti-mouse PD-1 antibody could not inhibit the growth of B16 / F10 tumors, while h87D8 H2K4 e5 could inhibit the growth of tumors, with a tumor inhibition rate of 48.2%. The combination of h87D8 H2K4 e5 + anti-mouse PD-1 antibody could inhibit tumor growth more effectively than h87D8 H2K4 e5 alone, with a tumor inhibition rate of 75.4%.

[0213] Example 15 Effect of humanized CCR8 antibody on the growth of subcutaneously transplanted tumors in MDA-MB-231 mice MDA-MB-231 subcutaneous tumor model was established in B-NGD hIL15 mice (purchased from Bioseto), and 1 × 10 human IgG was added for immune reconstitution. 7 PBMC / mouse (purchased from Shanghai Miaoshun Biological) was administered intravenously. The study included h87D8 H2K4 e5 0.4mg / kg, 2mg / kg and 10mg / kg groups, as well as an IgG1 isotype control 10mg / kg group, which were administered intravenously once every 3 days, a total of 4 times, to CCR8 humanized transgenic mice. As shown in Figure 14, h87D8 H2K4 e5 inhibited tumor growth in each dose group, with tumor inhibition rates of 74.4%, 49.3% and 40.4% in the 10mg / kg group, 2mg / kg group and 0.4mg / kg group, respectively.

Claims

1. An antibody or antigen-binding portion thereof that binds to CCR8, said antibody or antigen-binding portion thereof comprising: (1) a heavy chain variable region and a light chain variable region, (i) the heavy chain variable region comprises the following (a), (b), and (c): (a) a HCDR1 sequence having the following formula: NX １ X ２ AMN (SEQ ID NO. 102), where X １ is A or T, X ２ is F or Y, (b) a HCDR2 sequence having the formula: RIRSKSNX ３ YAT ４ X ５ ADS VX ６ X ７ (SEQ ID NO. 103), wherein 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, and (c) a HCDR3 sequence having the following formula: GKEAGX ８ X ９ X １０ X １１ X １２ X １３ DY (SEQ ID NO. 104), where X ８ is A or absent, 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 absent, (ii) the light chain variable region comprises the following (a), (b), and (c): (a) a LCDR1 sequence having the following formula: 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; (b) an LCDR2 sequence having the following formula: R.X. １６ SNLAS (SEQ ID NO. 106), where X １６ is M, A, G, or T; (c) a LCDR3 sequence having the following formula: MQHLEYPX １７ (SEQ ID NO. 107), wherein X １７ is F or L, or (2) a heavy chain variable region and a light chain variable region, (i) the heavy chain variable region comprises the following (a), (b), and (c): (a) a HCDR1 sequence having the following formula: NAYAMN (SEQ ID NO.28), (b) a HCDR2 sequence having the formula: RIRSKSNNYATYYADSVKD (SEQ ID NO. 22), (c) a HCDR3 sequence having the following formula: QKFGX １８ RGYYALDF (SEQ ID NO. 108), where X １８ is A or S, and (ii) the light chain variable region comprises the following (a), (b), and (c): (a) a LCDR1 sequence having the following formula: X １９ ASX ２０ S.X. ２１ X ２２ X ２３ X ２４ X ２５ X ２６ X ２７ X ２８ X ２９ X ３０ (SEQ ID NO. 109), wherein X １９ is R, T or S; X ２０ is E, K or S, and X ２１ is V or I, X ２２ is E or S, and X ２３ is Y, T or absent, 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, and X ２８ is L or Y, and X ２９ is L or M, and X ３０ is Q or H, (b) an LCDR2 sequence having the following formula: X ３１ X ３２ S.X. ３３ X ３４ X ３５ S (SEQ ID NO. 110), where X ３１ is R, A, L or S; X ３２ is A or T, and X ３３ is N or T, and X ３４ is L or V, and X ３５ is A or E, (c) a LCDR3 sequence having the following formula: X ３６ X ３７ X ３８ X ３９ X ４０ X ４１ P.X. ４２ X ４３ (SEQ ID NO. 111), wherein X ３６ is Q or H, and X ３７ is Q or H, and X ３８ is S, Y or G, and X ３９ is R, S, H or T; X ４０ is E, K, G, R, V or S, and X ４１ is Y, V, L, S or I; X ４２ is L or absent, and X ４３ is L, Y, P, R or G An antibody or antigen-binding portion thereof comprising:

2. 2. The antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 6, 16, 28, or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO. 7, 17, 22, 82, or any variant thereof, a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO. 8, 29, 49, or any variant thereof, and a light chain CDR1 selected from the amino acid sequence SEQ ID NO. 11, 32, 39, 44, 52, 62, 91, or any variant thereof, a light chain CDR2 selected from the amino acid sequence SEQ ID NO. 12, 33, 40, 45, 58, 63, or any variant thereof, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO. 13, 25, 34, 41, 46, 53, 59, 64, or any variant thereof.

3. (1) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 6, 7 and 8, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 11, 12 and 13, respectively; (2) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 16, 17 and 8, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 11, 12 and 13, respectively; (3) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 16, 22 and 8, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 11, 12 and 25, respectively; (4) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 28, 22 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 32, 33 and 34, respectively; (5) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 28, 22 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 39, 40 and 41, respectively; (6) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 28, 22 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 44, 45 and 46, respectively; (7) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 28, 22 and 49, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 52, 45 and 53, respectively; (8) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 28, 22 and 49, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 44, 58 and 59, respectively; (9) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 28, 22 and 29, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 62, 63 and 64, respectively; (10) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 6, 7 and 8, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 11, 12 and 15, respectively; (11) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 16, 82 and 8, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 11, 12 and 25, respectively; (12) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 6, 82 and 8, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 11, 12 and 25, respectively; (13) Heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 16, 7 and 8, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 91, 63 and 25, respectively.

3. The antibody or antigen-binding portion thereof of claim 1 or 2, comprising a combination of heavy chain CDRs and light chain CDRs selected from:

4. a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 4, 14, 20, 26, 35, 47, 54, 80, 83, 85, 92, 96, 100, or any variant thereof, and / or a light chain variable region 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 any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 9 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 67 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 68 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 69 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 70 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 71 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 72 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 73 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 74 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 75 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 76 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 77 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 78 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 79 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 14 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 18 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 20 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 23 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 26 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 30 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 35 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 37 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 35 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 42 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 47 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 50 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 54 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 56 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 35 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 60 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 4 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 65 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 80 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 87 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 83 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 87 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 85 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 89 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 92 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 94 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 96 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 98 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO. 100 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO. 98 or any variant thereof; The antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding portion thereof is humanized.

5. A nucleic acid encoding the antibody or antigen-binding portion thereof according to any one of claims 1 to 4, Preferably, the nucleic acid comprises an antibody heavy chain variable region nucleic acid sequence selected from SEQ ID NO. 5, 15, 21, 27, 36, 48, 55, 81, 84, 86, 93, 97, 101 or any variant thereof, and / or an antibody light chain variable region nucleic acid sequence selected from SEQ ID NO. 10, 19, 24, 31, 38, 43, 51, 57, 61, 66, 88, 90, 95, 99 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 5 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 10 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 15 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 19 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 21 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 24 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 27 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 31 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 36 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 38 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 36 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 43 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 48 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 51 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 55 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 57 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 36 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 61 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 5 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 66 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 81 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 88 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 84 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 88 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 86 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 90 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 93 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 95 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 97 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 99 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 101 or any variant thereof, and a light chain variable region nucleotide sequence of the nucleotide sequence SEQ ID NO. 99 or any variant thereof.

6. A vector comprising the nucleic acid of claim 5.

7. A cell comprising the nucleic acid of claim 5 or the vector of claim 6.

8. A composition comprising an antibody or an antigen-binding portion thereof according to any one of claims 1 to 4, a nucleic acid according to claim 5, a vector according to claim 6 and / or a cell according to claim 7.

9. A kit comprising an antibody or an antigen-binding portion thereof according to any one of claims 1 to 4, a nucleic acid according to claim 5, a vector according to claim 6, a cell according to claim 7 and / or a composition according to claim 8.

10. Use of a therapeutic agent in the preparation of a medicament or kit for the diagnosis, treatment or prevention of a cancer-related disease, said therapeutic agent being selected from a single therapeutic agent or a combination therapeutic agent; The single therapeutic agent is selected from an antibody or antigen-binding portion thereof according to any one of claims 1 to 4, a nucleic acid according to claim 5, a vector according to claim 6, a cell according to claim 7 and / or a composition according to claim 8, said combination therapy comprises said single therapy and an immune checkpoint modulator, preferably said immune checkpoint modulator is an inhibitor of an immune checkpoint protein selected from PD-1, CTLA4, PD-L1, PD-L2, CD47, TIGIT, GITR, TIM3, LAG3, 4-1BB, CD27 and B7H4, preferably said immune checkpoint protein inhibitor is an antibody or antigen-binding fragment thereof, preferably the immune checkpoint protein is PD-1 or CTLA4, more preferably said immune checkpoint protein inhibitor is selected from an anti-PD-1 antibody, an antigen-binding fragment thereof, an anti-CTLA-4 antibody, or an antigen-binding fragment thereof; Preferably, the cancer-related disease is selected from breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach (gastric adenocarcinoma), non-small cell lung cancer, pancreatic cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colon cancer, renal cancer, non-Hodgkin's lymphoma, urothelial cancer, sarcoma, blood cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, liver cancer, and other cancers, preferably breast cancer, uterine cancer, ovarian cancer, lung cancer, colon cancer, renal cancer, and sarcoma, and more preferably breast cancer, colon cancer, renal cancer, and sarcoma.

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