Anti-CCR8 antibodies and uses thereof

Abstract

The present invention relates to anti-CCR8 antibodies and methods of use thereof. The antibodies described herein can be used in the diagnosis and treatment of diseases mediated by CCR8 and / or CCL1, such as various cancers and neuropathic pain associated with an abnormal CCL1 / CCR8 axis.

Description

[Technical field]

[0001] The present invention relates to novel anti-CCR8 antibodies or antigen-binding fragments thereof, nucleic acids encoding said antibodies or antigen-binding fragments thereof, vectors and host cells containing said nucleic acids, methods for producing said antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising said antibodies or antigen-binding fragments thereof as an active ingredient, and use of said antibodies for the treatment of diseases mediated by CCR8. [Background technology]

[0002] Chemokines are a family of low molecular weight chemotactic cytokines involved in inflammatory cell recruitment and activation. Chemokines regulate and exert their effects on a wide range of cell functions by binding to chemokine receptors (G protein-coupled receptors), causing chemotaxis and activation of various cell subsets of the immune system. Depending on the location of the N-terminal cysteine ​​residue of the protein, chemokines are classified into various classes, including CC, CXC, CX3C, and XC. CC chemokines contain a CC motif, where the first two cysteines are not separated by any amino acid, and CXC chemokines contain a CXC motif, where the first two cysteines are separated by random amino acids. The activity of chemokines is mainly mediated by tight binding to leukocyte surface receptors.

[0003] Under normal physiological conditions, the expression of chemokines and chemokine receptors is delicately and tightly controlled, but dysregulated expression and activation of either chemokines or chemokine receptors often leads to diseases such as autoimmune diseases and cancer.

[0004] The human body has many built-in mechanisms designed to prevent the development of cancer. In this regard, the immune system is thought to play a key role in eradicating cells with genetic mutations. Thus, cancer cells often evolve to persist without being recognized by cells of the normal immune system. In particular, studies show that elevated levels of regulatory T lymphocytes (sometimes referred to herein as "Treg cells") in the peripheral circulation and in the tumor microenvironment underlie immune inhibition in cancer patients. Increased numbers of Treg cells are also identified as a barrier to the successful implementation of cancer immunotherapy.

[0005] CCR8 (CC motif chemokine receptor 8) is mainly expressed by Treg cells and Th2 cells, but not by Th1 cells. A subset of CD4+Foxp3+Treg cells expressing CCR8 (CCR8+Treg cells) has been shown to be a major driver of immune inhibition and is important for Treg function and inhibition. In addition, CCR8 is a specific marker that is selectively upregulated by tumor-resident Treg cells in various tumor types. Numerous reports indicate that an increase in CCR8+Treg cells is favorable for tumor escape mechanisms. Clinically, an increase in Treg cells in the tumor microenvironment of various cancer types, such as breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, liver cancer, and colon cancer, is associated with poor prognosis. In the mechanism, Treg cells not only inhibit various anti-tumor immune responses, but also promote angiogenesis in the tumor microenvironment. Cancer cells and immune cells in the tumor microenvironment secrete CCL1, a specific ligand for CCR8, to recruit CCR8+ Treg cells to the tumor microenvironment. CCR8 is also involved in the proliferation and expansion of Tregs in the tumor microenvironment.

[0006] CCR8 inhibitors have been shown to prevent tumor growth by decreasing tumor-infiltrating Treg cells. Thus, CCR8 is considered as a potential therapeutic target for cancer. In addition, CCR8 is expressed in spinal cord neurons and is also the major source of spinal cord CCL1. CCL1 (also called SCYA1, I-309, TCA3, P500 or SISe) is a well-characterized chemokine of the CC subfamily. It attracts immune cells by interacting with the cell surface chemokine receptor CCR8. CCL1 and CCR8 neuronal signaling pathways also play important roles in neuropathic pain caused by diabetes and spinal cord injury. Thus, the CCL1 / CCR8 axis may be a promising new target for drug development in the treatment of diabetic neuropathy.

[0007] CCL1-CCR8 interactions have also been reported to play an important role in IgG4-related diseases (IgG4-RD), such as IgG4-related sclerosing cholangitis (ISC).

[0008] Another recently identified CCR8 ligand is CCL18, a chemokine of the β-chemokine subfamily. Compared with primary Sjögren's syndrome and control subjects, the CCL18-CCR8 axis is specifically upregulated in the labial salivary glands (LSG) and lacrimal glands of IgG4-RD patients. Based on its important pathogenetic role in chemotaxis of various cells, induction of fibrosis, and enhancement of IgG4 production, this axis may be a novel therapeutic target for IgG4-RD.

[0009] Considering the role of CCR8 in the pathogenic mechanisms of various diseases, there is a need to prepare antibodies that inhibit the activity of CCR8, which can be used to treat diseases mediated by CCR8, such as cancer, neuropathic pain, and IgG4-related disease (IgG4-RD), including, but not limited to, autoimmune pancreatitis, eosinophilic angiocentric fibrosis, fibrosing mediastinitis, hypertrophic pachymeningitis, idiopathic hypocomplementemic tubulointerstitial nephritis with extensive tubulointerstitial deposits, inflammatory aortic aneurysm, inflammatory pseudotumor, Kuttner's tumor (chronic sclerosing sialadenitis), mediastinal fibrosis, Mikulicz syndrome, multifocal fibrosclerosis, periaortitis and periarteritis, retroperitoneal fibrosis (Ormond's disease), Riedel's thyroiditis, sclerosing mesenteritis, sclerosing pancreatitis, and sclerosing cholangitis.

[0010] In summary, there is a need in the art for more effective therapeutics, including anti-CCR8 antibodies that effectively inhibit CCR8 signaling while causing minimal side effects in humans. Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide an anti-CCR8 antibody or an antigen-binding fragment thereof that specifically binds to CCR8 and activates immunity by suppressing immunosuppression mediated by Treg cells or the like that specifically express CCR8. Furthermore, the anti-CCR8 antibody or an antigen-binding fragment thereof disclosed herein can alleviate neuropathic pain by controlling the CCL1 / CCR8 axis. Such an antibody or antigen-binding fragment can be used to target cells expressing CCR8 for therapeutic and diagnostic purposes.

[0012] The inventors have also found that the humanized antibodies disclosed herein not only specifically bind to CCR8, but also exhibit potent CCL1-CCR8 signaling blockade and improved species cross-reactivity. Thus, the main advantages of the present invention are as follows: (a) the antibody of the present invention has excellent biological activity and specificity, has good binding affinity to cell surface CCR8, and can be used as a CCR8-targeting antibody; (b) the fully human antibodies of the present invention not only have activity comparable to that of immune antibodies, but also have low immunogenicity; (c) The antibody of the present invention not only has a remarkable effect of inhibiting CCL1-induced chemotaxis, but is also applicable to other abnormal CCL1 / CCR8 axis-related diseases. [Means for solving the problem]

[0013] In a first aspect, an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: a) SEQ ID NOs: 2, 3 and 4; or b) SEQ ID NOs: 2, 3 and 10; or c) SEQ ID NOs: 2, 16 and 17; or d) SEQ ID NOs: 22, 23 and 24; or e) SEQ ID NOs: 32, 33 and 34; or f) SEQ ID NOs: 40, 41 and 42; or g) SEQ ID NOs: 47, 48 and 49; or h) SEQ ID NOs: 40, 3 and 55; or i) SEQ ID NOs: 58, 59 and 60; or j) SEQ ID NOs: 58, 3 and 63; or k) SEQ ID NOs: 2, 3 and 67; or l) SEQ ID NOs: 72, 73 and 74; or m) SEQ ID NOs: 58, 79 and 80; or n) SEQ ID NOs: 58, 83 and 84; or o) SEQ ID NOs: 58, 83 and 88; or at least one heavy chain variable region comprising an HCDR1, HCDR2 and HCDR3 selected from the group consisting of: a) SEQ ID NOs: 6, 7 and 8; or b) SEQ ID NOs: 12, 7 and 14; or c) SEQ ID NOs: 19, 7 and 14; or d) SEQ ID NOs: 26, 20 and 27; or e) SEQ ID NOs: 30, 20 and 27; or f) SEQ ID NOs: 36, 37 and 38; or g) SEQ ID NOs: 44, 37 and 45; or h) SEQ ID NOs: 51, 52 and 53; or i) SEQ ID NOs: 6, 7 and 8; or j) SEQ ID NOs: 6, 7 and 14; or k) SEQ ID NOs: 65, 7 and 14; or l) SEQ ID NOs: 69, 70 and 13; or m) SEQ ID NOs: 76, 37 and 77; or n) SEQ ID NOs: 86, 7 and 14; wherein the antibody or antigen-binding fragment thereof specifically binds to CCR8; and any of the amino acid sequences further comprises a derivative sequence, optionally formed by addition, deletion, modification and / or substitution of 1 to 5 (or 1, 2, 3) amino acids, capable of retaining binding affinity.

[0014] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises: a) SEQ ID NOs: 2, 3, 4, 6, 7 and 8; or b) SEQ ID NOs: 2, 3, 10, 12, 7 and 14; or c) SEQ ID NOs: 2, 16, 17, 19, 7 and 14; or d) SEQ ID NOs: 22, 23, 24, 26, 20 and 27; or e) SEQ ID NOs: 22, 23, 24, 30, 20 and 27; or f) SEQ ID NOs: 32, 33, 34, 36, 37 and 38; or g) SEQ ID NOs: 40, 41, 42, 44, 37 and 45; or h) SEQ ID NOs: 47, 48, 49, 51, 52 and 53; or i) SEQ ID NOs: 40, 3, 55, 6, 7 and 8; or j) SEQ ID NOs: 58, 59, 60, 6, 7 and 14; or k) SEQ ID NOs: 58, 3, 63, 65, 7 and 14; or l) SEQ ID NOs: 2, 3, 67, 69, 70 and 13; or m) SEQ ID NOs: 72, 73, 74, 76, 37 and 77; or n) SEQ ID NOs: 58, 79, 80, 6, 7 and 14; or o) SEQ ID NOs: 58, 83, 84, 86, 7 and 14; or p) SEQ ID NOs: 58, 83, 88, 86, 7 and 14. and wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the group consisting of:

[0015] In a preferred embodiment, said heavy chain further comprises a heavy chain constant region and / or said light chain further comprises a light chain constant region.

[0016] In a preferred embodiment, the number of amino acids added, deleted, modified and / or substituted in the three HCDRs and three LCDRs of the antibody is 1-5 (e.g., 1-3, preferably 1-2, more preferably 1).

[0017] In a preferred embodiment, the heavy chain variable region of said antibody further comprises a human or humanized framework region, and / or the light chain variable region of said antibody further comprises a human or humanized framework region.

[0018] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1 and a light chain variable region having the polypeptide sequence of SEQ ID NO:5; or b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9 and a light chain variable region having the polypeptide sequence of SEQ ID NO:11; or c) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18; or d) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a light chain variable region having the polypeptide sequence of SEQ ID NO:25; or e) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:28 and a light chain variable region having the polypeptide sequence of SEQ ID NO:29; or f) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 31 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 35; or g) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 39 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 43; or h) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 46 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 50; or i) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:54 and a light chain variable region having the polypeptide sequence of SEQ ID NO:56; or j) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:57 and a light chain variable region having the polypeptide sequence of SEQ ID NO:61; or k) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 62 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 64; or l) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:66 and a light chain variable region having the polypeptide sequence of SEQ ID NO:68; or m) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 75; or n) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 78 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 81; or o) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 82 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 85; or p) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 87 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 89; or q) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 90 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 91; or r) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 92 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 93; or s) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 94 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 95; or t) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 96 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 97; or u) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 92 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 98; or v) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 92 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 99; or w) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 100 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 101; or x) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 102 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 103; or y) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 104 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 105; or z) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 106 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 107; or aa) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 108 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 109; or bb) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 110 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 111. The invention comprises a heavy chain variable region and a light chain variable region selected from: In a preferred embodiment, the antibody is a monoclonal antibody.

[0019] In a preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0020] In a preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0021] In a preferred embodiment, the antibody is a recombinant antibody.

[0022] In a preferred embodiment, the antibody or antigen-binding fragment thereof is chimeric.

[0023] In a preferred embodiment, the antibody is a bispecific or multispecific antibody.

[0024] In another preferred embodiment, the CCR8-specific antibody is selected from: (i) a single chain antibody, a single chain variable fragment (scFv), a monovalent antibody lacking the hinge region or a minibody; (ii) a Fab, Fab' or F(ab')2 fragment; (iii) a full-length antibody; and (iv) an antibody comprising a human IgG Fc domain.

[0025] In a preferred embodiment, the antibody or antigen-binding fragment thereof is human or humanized.

[0026] In a preferred embodiment, the antibody is a human anti-CCR8 antibody.

[0027] In a preferred embodiment, the antibody or antigen-binding fragment thereof has a polypeptide sequence that is at least (≧) 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1, 9, 15, 21, 28, 31, 39, 46, 54, 57, 62, 66, 71, 78, 82, 87, 90, 92, 94, 96, 100, 102, 104, 106, 108 or 110. or a light chain variable region having a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:5, 11, 18, 25, 29, 35, 43, 50, 56, 61, 64, 68, 75, 81, 85, 89, 91, 93, 95, 97, 98, 99, 101, 103, 105, 107, 109 or 111.

[0028] In a particularly preferred embodiment, the CCR8-specific antibody VH and VL chains have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the amino acid sequence of each VH and VL chain, respectively, selected from: SEQ ID NOs: 1 and 5; or SEQ ID NOs: 9 and 11; or SEQ ID NOs: 15 and 18; or SEQ ID NOs: 21 and 25; or SEQ ID NOs: 28 and 29; or SEQ ID NOs: 31 and 35; or SEQ ID NOs: 39 and 43; or SEQ ID NOs: 46 and 50; or SEQ ID NOs: 54 and 56; or SEQ ID NOs: 57 and 61; or SEQ ID NOs: Sequence numbers 62 and 64; or SEQ ID NOs: 66 and 68; or SEQ ID NOs: 71 and 75; or SEQ ID NOs: 78 and 81; or SEQ ID NOs: 82 and 85; or SEQ ID NOs: 87 and 89; or SEQ ID NOs: 90 and 91; or SEQ ID NOs: 92 and 93; or SEQ ID NOs: 94 and 95; or SEQ ID NOs: 96 and 97; or SEQ ID NOs: 92 and 98; or SEQ ID NOs: 92 and 99; or SEQ ID NOs: 100 and 101; or SEQ ID NOs: 102 and 103; or SEQ ID NOs: 104 and 105; or SEQ ID NOs: 106 and 107; or SEQ ID NOs: 108 and 109; or SEQ ID NOs: 110 and 111.

[0029] In some embodiments, disclosed herein are antibodies or antigen-binding fragments that bind to human CCR8 at an epitope comprising one or more amino acid residues selected from Y94 to K107 of human CCR8.

[0030] In some embodiments, disclosed herein are antibodies or antigen-binding fragments that bind to human CCR8 at an epitope that includes one or more amino acid residues selected from Y94, L95, L96, D97, Q98, V100, T103, V104, M105 and K107 of human CCR8.

[0031] In some embodiments, disclosed herein is an antibody or antigen-binding fragment that binds to human CCR8 at an epitope that includes one amino acid residue selected from Y94, L95, L96, Q98, V100, T103, V104, M105, and K107 of human CCR8.

[0032] In another particularly preferred embodiment, said CCR8-specific antibody is an IgA, IgD, IgE, IgG or IgM antibody.

[0033] In another preferred embodiment, said CCR8-specific antibody is an IgG selected from IgG1, IgG2, IgG3, IgG4 and synthetic IgG.

[0034] In a preferred embodiment, the antibody is in the form of a drug conjugate.

[0035] In a second aspect, (i) an antibody or antigen-binding fragment thereof according to the first aspect of the present application; and (ii) Providing a recombinant protein (or polypeptide), optionally including a tag sequence to aid in expression and / or purification.

[0036] In a preferred embodiment, the tag sequence comprises a 6His tag.

[0037] In a preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0038] In a preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

[0039] In a third aspect, there is provided an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment of the first aspect of the invention, or the recombinant protein of the second aspect.

[0040] In a third aspect, there is provided an isolated nucleic acid encoding the antibody or antigen-binding fragment of the first aspect or the recombinant protein of the second aspect of the invention.

[0041] In a fourth aspect, there is provided a vector comprising the isolated nucleic acid encoding the antibody or antigen-binding fragment of the first aspect of the invention, or the recombinant protein of the second aspect.

[0042] In preferred embodiments, the vector comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a lentivirus, a retrovirus, or other vector.

[0043] In a fifth aspect, a method of producing a composition comprising the steps of: (i) an antibody portion selected from the group consisting of an antibody or antigen-binding fragment of the first aspect of the invention, or a recombinant protein of the second aspect, or a combination thereof; and (ii) a coupling moiety that is coupled to the antibody moiety, the coupling moiety being selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof; The present invention provides an antibody conjugate comprising:

[0044] In a preferred embodiment, the antibody moiety and the coupling moiety are coupled via a chemical bond or linker.

[0045] In a sixth aspect, there is provided a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an isolated nucleic acid (particularly DNA or RNA) according to the third aspect of the invention, a vector according to the fourth aspect of the invention, an antibody conjugate according to the fifth aspect of the invention or a combination thereof, and (ii) a pharma- ceutically acceptable carrier.

[0046] In a preferred embodiment, the antibody has the effect of depleting tumor-infiltrating Treg cells.

[0047] In a seventh aspect, there is provided a method for treating a disease mediated by CCR8 and / or CCL1 comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the first aspect, a recombinant protein of the second aspect, an isolated nucleic acid (particularly DNA or RNA) of the third aspect, a vector of the fourth aspect, an antibody conjugate of the fifth aspect or a pharmaceutical composition of the seventh aspect or a combination thereof; or a method for treating a disease mediated by CCR8 and / or CCL1 comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the first aspect, a recombinant protein of the second aspect, an isolated nucleic acid (particularly DNA or RNA) of the third aspect, a vector of the fourth aspect, an antibody conjugate of the fifth aspect or a pharmaceutical composition of the seventh aspect or a combination thereof. or use of the vector of the first aspect, the antibody conjugate of the fifth aspect or the pharmaceutical composition of the seventh aspect or a combination thereof for a medicament for treating a disease mediated by CCR8 and / or CCL1; or an antibody or antigen-binding fragment thereof of the first aspect, a recombinant protein of the second aspect, an isolated nucleic acid (in particular DNA or RNA) of the third aspect, a vector of the fourth aspect, the antibody conjugate of the fifth aspect or the pharmaceutical composition of the seventh aspect or a combination thereof for use in treating a disease mediated by CCR8 and / or CCL1.

[0048] In a preferred embodiment, the CCR8 and / or CCL1 mediated disease is cancer.

[0049] In particularly preferred embodiments, the cancer is breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer or pancreatic cancer.

[0050] In a preferred embodiment, the disease is associated with an abnormal CCL1 / CCR8 axis.

[0051] In a particularly preferred embodiment, said disease associated with an abnormal CCL1 / CCR8 axis is neuropathic pain.

[0052] In particularly preferred embodiments, said neuropathic pain is caused by diabetes or spinal cord injury.

[0053] In a particularly preferred embodiment, the disease is an IgG4-related disease.

[0054] In particularly preferred embodiments, said IgG4-related disease is sclerosing cholangitis, autoimmune pancreatitis, eosinophilic angiocentric fibrosis, fibrosing mediastinitis, hypertrophic pachymeningitis, idiopathic hypocomplementemic tubulointerstitial nephritis with widespread tubulointerstitial deposits, inflammatory aortic aneurysm, inflammatory pseudotumor, Kuttner's tumor (chronic sclerosing sialadenitis), mediastinal fibrosis, Mikulicz syndrome, multifocal fibrosclerosis, periaortitis and periarteritis, retroperitoneal fibrosis (Ormond's disease), Riedel's thyroiditis, sclerosing mesenteritis, or sclerosing pancreatitis.

[0055] In an eighth aspect, a method is provided for determining the level of CCR8 in a subject, comprising: (a) obtaining a sample from a subject; (b) contacting the sample with an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention; and (c) determining the level of CCR8 in the subject.

[0056] Preferably, the sample is a tissue sample or a blood sample, and the tissue sample may be a cancer tissue sample.

[0057] In a ninth aspect, there is provided the use of an active ingredient (a) for the manufacture of a diagnostic reagent or kit; and / or (b) for the manufacture of a medicament for the prevention and / or treatment of a disease associated with CCR8, wherein said active ingredient is selected from an antibody or antigen-binding fragment thereof of the first aspect, a recombinant protein of the second aspect, an isolated nucleic acid (in particular DNA or RNA) of the third aspect, a vector of the fourth aspect, an antibody conjugate of the fifth aspect and combinations thereof.

[0058] In a preferred embodiment, the diagnostic reagent is a detection sheet or a detection plate.

[0059] In a preferred embodiment, the CCR8-associated disease comprises cancer.

[0060] In particularly preferred embodiments, the cancer is breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer or pancreatic cancer.

[0061] In a preferred embodiment, the diagnostic reagent or kit comprises: (i) detection of CCR8 protein in a sample; and / or (ii) detection of endogenous CCR8 protein in spinal cord neurons; and / or (iii) It is used to detect regulatory T lymphocytes that express the CCR8 protein.

[0062] In a preferred embodiment, the antibody is in the form of an antibody-drug conjugate (ADC).

[0063] In a tenth aspect, 1. A method for detecting CCR8 protein in a sample in vitro (including diagnostic or non-diagnostic), comprising: (i) contacting said sample in vitro with an antibody according to the first aspect of the invention; and (ii) detecting whether an antigen-antibody complex has been formed, where the formation of the complex indicates the presence of CCR8 protein in the sample.

[0064] In an eleventh aspect, (1) a first container containing an antibody of the present invention as a primary antibody; and (2) a second container containing a secondary antibody against the primary antibody of the present invention; Provide a kit.

[0065] In a thirteenth aspect, there is provided a method for producing a recombinant polypeptide, comprising the steps of: (a) culturing an engineered host cell of the fifth aspect of the invention under conditions suitable for expression; (b) isolating a recombinant polypeptide from the culture, which is the antibody or antigen-binding fragment thereof of the first aspect of the invention or the recombinant protein of the second aspect of the invention. The present invention provides a method comprising: [Brief description of the drawings]

[0066] [Figure 1A] FIG. 1A shows that FACS binding analysis of clones 103G4, 118H1, 101E4, 84B4, 170G6, 172E7, 2P15, 3C11, 3O20, 4M13, 2L15, 4D24, 1G17, 3F11, 3F6 and 4G19 shows that the antibodies have the ability to bind to CCR8-overexpressing 293F cells (293F-CCR8). [Figure 1B] FIG. 1B shows that FACS binding analysis of clones 103G4, 118H1, 101E4, 84B4, 170G6, 172E7, 2P15, 3C11, 3O20, 4M13, 2L15, 4D24, 1G17, 3F11, 3F6 and 4G19 shows that the antibodies have the ability to bind to CCR8-overexpressing 293F cells (293F-CCR8). [Figure 1C] FIG. 1C shows that in FACS binding analysis of several clones, the antibody did not bind to the parental 293F cells. [Figure 1D] FIG. 1D shows that in FACS binding analysis of several clones, the antibodies did not bind to the parental 293F cells. [Figure 2A] FIG. 2A shows the results of the anti-tumor effect of an anti-CCR8 antibody (i.e., antibody clone 84B4) in MDA-MB-231 xenografts. [Figure 2B] FIG. 2B shows the results of the anti-tumor effect of an anti-CCR8 antibody (i.e., antibody clone 101E4) in MDA-MB-231 xenografts. [Figure 2C] FIG. 2C shows the results of the anti-tumor effect of an anti-CCR8 antibody (i.e., antibody clone 170G6) in MDA-MB-231 xenografts. [Figure 2D] FIG. 2D shows the results of the anti-tumor effect of anti-CCR8 antibody (i.e., antibody clone 3C11) in MDA-MB-231 xenografts. [Figure 2E] FIG. 2E shows the results of the anti-tumor effect of anti-CCR8 antibody (i.e., antibody clone 2P15) in MDA-MB-231 xenografts. [Diagram 3]FIG. 3 shows the results of the binding affinity of 3F11hz4 and 3F11 to mouse CCR8 CHOK1. [Figure 4A] FIG. 4A shows that antibody 3F11hz4 has the binding ability to human CCR8-overexpressing 293T cells (human CCR8-293T) in a FACS binding analysis using the 293T cell line. [Figure 4B] FIG. 4B shows that antibody 3F11hz4 has the binding ability to rat CCR8-overexpressing 293T cells (rat CCR8-293T) in FACS binding analysis using the 293T cell line. [Figure 4C] FIG. 4C shows that antibody 3F11hz4 has the binding ability to canine CCR8-overexpressing 293T cells (canine CCR8-293T) in a FACS binding analysis using the 293T cell line. [Figure 4D] FIG. 4D shows that antibody 3F11hz4 has the binding ability to mouse CCR8-overexpressing CHOK1 cells (mouse CCR8-CHOK1) in a FACS binding analysis using the CHOK1 cell line. [Figure 4E] FIG. 4E shows that antibody 3F11hz4 has the binding ability to cynomolgus CCR8-overexpressing 293T cells (cynomolgus CCR8-293T) in a FACS binding analysis using the 293T cell line. [Figure 5A] FIG. 5A shows the binding affinity of 3F11hz4 to mutant CCR8 293T. [Figure 5B] FIG. 5B shows the binding affinity of 3F11hz4 to mutant CCR8 293T. [Figure 6A] FIG. 6A shows the results of the binding affinity of 3F11hz4 to hCCR4 293T. [Figure 6B] FIG. 6B shows the results of the binding affinity of 3F11hz4 to hCX3CR1 293T. [Figure 7] FIG. 7 shows the antitumor effect of the 3F11hz4 antibody against HCC827 lung cancer subcutaneous tumor (CD34 humanized model). [Figure 8]FIG. 8 shows the antitumor effect of the 3F11hz4 antibody against H22 liver cancer subcutaneous tumor (syngenic model). [Figure 9] FIG. 9 shows the antitumor effect of the 3F11hz4 antibody against LLC lung cancer subcutaneous tumor (syngenic model). [Figure 10] FIG. 10 shows a Treg cell migration assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The present inventors have conducted extensive and in-depth research and have unexpectedly obtained a fully human CCR8 antibody having a series of novel amino acid sequences. The CCR8 antibody of the present invention has excellent high affinity for CCR8 protein and is therefore useful for treating CCR8-related diseases such as alloimmune diseases, autoimmune diseases, allergies, inflammatory diseases, tumors, neuropathic pain, or IgG4-related diseases. Based on this, the present invention has been completed.

[0068] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings defined herein.

[0069] It must be noted that as used in this specification and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0070] Unless otherwise indicated, any numerical values, such as concentrations or concentration ranges described herein, should be understood in all cases to be modified by the term "about." Thus, numerical values ​​generally include ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% (w / v) to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within that range, and includes integers and fractions of values ​​within such ranges unless the context clearly dictates otherwise.

[0071] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0072] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variations thereof, are understood to mean the inclusion of a recited integer or group of integers, but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of components is not necessarily limited to only those components, but may include other components not expressly listed or inherent in such composition, mixture, process, method, article, or device. Additionally, unless expressly stated to the contrary, "or" means inclusive "or," not exclusive "or." For example, condition A or B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0073] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within the meaning of, and thus satisfy the requirements of, the term "and / or" as used herein. The simultaneous applicability of two or more options is further understood to be within the meaning of, and thus satisfy the requirements of, the term "and / or."

[0074] As used herein, the term "consists of," or variations thereof such as "consist of" or "consisting of," as used throughout this specification and claims, means inclusive of any recited integer or group of integers, but no additional integer or group of integers can be added to that particular method, structure, or composition.

[0075] As used herein, the term "consists essentially of," or variations thereof such as "consist essentially of" or "consisting essentially of," as used throughout this specification and claims, means inclusive of any recited integer or group of integers, and optionally inclusive of any recited integer or group of integers that do not materially change the basic or novel characteristics of that particular method, structure, or composition. See MPEP section 2111.03.

[0076] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. The term "mammal" as used herein includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like, more preferably humans.

[0077] The words "right", "left", "lower" and "top" designate directions in the drawings to which reference is made.

[0078] In addition, terms such as "about," "approximately," "generally," and "substantially," used herein when referring to dimensions or characteristics of preferred inventive components, are to be understood to indicate that the described dimensions / characteristics are not precise boundaries or parameters, but rather are functionally the same or similar, as would be understood by one of ordinary skill in the art, and do not exclude minor variations therefrom. At the very least, references involving such numerical parameters will include variations that do not alter the lowest order numbers using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0079] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-CCR8 antibodies and the polynucleotides encoding them, and CCR8 polypeptides and the polynucleotides encoding them) refers to two or more sequences or subsequences that have the same or a certain percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence as determined using one of the following sequence comparison algorithms or by visual inspection.

[0080] For sequence comparison, one sequence usually serves as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0081] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally as described in Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.). The invention may be carried out through a joint venture between Ausubel and Sons, Inc. (see 1995 Supplement) (Ausubel).

[0082] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short strings of length W in the query sequence that match or meet some positive threshold score T when aligned with strings of the same length in a database sequence. T is referred to as the contiguous string score threshold (Altschul et al, supra). These initial contiguous string hits act as seeds to initiate searches to find strings containing longer HSPs. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0083] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the accumulation of one or more negatively scoring residue alignments causes the cumulative score to fall below 0; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSEIM62 scoring matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0084] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin & Altschul, Proc. NatT. Acad. Sci. ETSA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0085] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is usually substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0086] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Moreover, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, and synthetic means.

[0087] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, to name a few. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0088] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and / or light chain that binds to an antigen of interest, which in a particularly preferred embodiment described herein binds to the CC motif chemokine receptor 8 (CCR8). In this regard, the antigen-binding fragment of the antibody described herein may contain one, two, three, four, five, or all six CDRs of the VH and / or VL sequences shown herein derived from an antibody that binds to CCR8. The antigen-binding fragment of the CCR8-specific antibody described herein is capable of binding to CCR8. In other embodiments, the binding of the antigen-binding fragment prevents or inhibits the binding of a CCR8 ligand to the CCR8 receptor or otherwise blocks a biological response resulting from the binding of the ligand to the receptor. In certain embodiments, the antigen-binding fragment specifically binds to CCR8 and / or inhibits or modulates its biological activity.

[0089] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and which can be used to produce, in an animal, antibodies capable of binding to an epitope of that antigen. An antigen can have one or more epitopes.

[0090] The term "epitope" encompasses any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen to which an antibody binds. In certain embodiments, epitopic determinants include groupings of chemically active surfaces of molecules, such as amino acids, sugar side chains, phosphoryls or sulfonyls, and in certain embodiments may have specific three-dimensional structural features and / or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. An antibody can be said to specifically bind an antigen when, according to certain embodiments, the equilibrium dissociation constant for antibody-antigen binding is less than or equal to 10-6 M, or less than or equal to 10-7 M, or less than or equal to 10-8 M. In some embodiments, the equilibrium dissociation constant may be less than or equal to 10-9 M, or less than or equal to 10-10 M.

[0091] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to introduce coding information into a host cell. The term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that induce and / or control the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and, if introns are present, RNA splicing.

[0092] CC motif chemokine receptor 8 (CCR8) CCR8, previously known as Cy6, CKR-L1 or TER1, is a G protein-coupled seven-transmembrane CC chemokine receptor protein expressed in the thymus, spleen, etc. The gene encoding this protein is located on human chromosome 3p21. Human CCR8 consists of 355 amino acids. CCL1 is known as the endogenous ligand of CCR8. Human CCR8 cDNA is composed of the nucleotide sequence represented by GenBank ACC number M_005201.3, and mouse CCR8 cDNA is composed of the nucleotide sequence represented by GenBank ACC number NM_007720.2.

[0093] The CCR8 of the present invention includes those derived from mice, rats, hamsters, guinea pigs, dogs, pigs, and monkeys and primate mammals, including humans. Human CCR8 is preferred.

[0094] antibody The present invention generally relates to isolated anti-CCR8 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. Methods of making the antibodies and methods of using the antibodies to treat diseases, including cancer, are also provided. The antibodies of the present invention have one or more desirable functional properties, including, but not limited to, high affinity binding to CCR8, high specificity for CCR8, and the ability to inhibit tumor growth in subjects and animal models in need thereof when administered alone or in combination with other anti-cancer therapies.

[0095] In a general aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CCR8.

[0096] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules, including human, humanized, composite, and chimeric antibodies, whether monoclonal or polyclonal, as well as antibody fragments. In general, an antibody is a protein or peptide chain that exhibits binding specificity to a specific antigen. The structure of an antibody is known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, an antibody of the invention can be of any of the five major classes or corresponding subclasses. Preferably, the antibody of the invention is IgG1, IgG2, IgG3, or IgG4. The antibody light chain of a vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequence of its constant domain. Thus, an antibody of the invention can contain a kappa or lambda light chain constant domain. According to certain embodiments, the antibodies of the invention comprise heavy and / or light chain constant regions derived from a rat or human antibody. In addition to the heavy and light constant domains, the antibodies contain an antigen-binding region consisting of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0097] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds CD33 is substantially free of antibodies that do not bind CCR8). Moreover, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0098] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the invention can be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas that contain B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse or rat, and have a genome that includes a human heavy chain transgene and a light chain transgene.

[0099] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as, for example, a diabody, Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), single domain antibody (sdab) scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. An antigen-binding fragment can bind to the same antigen that the parent antibody or parent antibody fragment binds. According to certain embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd fragment of a heavy chain. According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab').

[0100] As used herein, the term "single chain antibody" refers to a conventional single chain antibody in the art that comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region, or comprises only a heavy chain variable region.

[0101] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies that comprise at least one human heavy and / or light chain polypeptide.

[0102] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to have increased sequence homology to that of a human antibody such that the antigen-binding properties of the antibody are retained but the antigenicity of the antibody in the human body is reduced.

[0103] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0104] As used herein, the term "multispecific antibody" refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or substantially do not overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or subunits of different multimeric proteins). In an embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody branch, or a tetraspecific antibody molecule.

[0105] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or no more than two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap or substantially overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or subunits of different multimeric proteins). In some embodiments, a bispecific antibody comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In some embodiments, a bispecific antibody comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In some embodiments, a bispecific antibody comprises an scFv or fragment thereof having binding specificity for a first epitope and an scFv or fragment thereof having binding specificity for a second epitope. In some embodiments, the first epitope is located on CCR8 and the second epitope is located on PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD47, CD73, apelin, DLL3, claudinl8.2, TIP-l, CD3, and / or other tumor-associated immunosuppressive factors or surface antigens.

[0106] By the term "specifically binds," as used herein with respect to an antibody, it is meant an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody as specific. In some examples, the terms "specifically bind" or "specifically bind" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, but means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies recognize and bind to a particular protein structure rather than a protein in general. If an antibody is specific for epitope "A," then the presence of a molecule that contains epitope A (or free unlabeled A) reduces the amount of labeled A bound to the antibody in a reaction involving labeled "A" and an antibody.

[0107] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs, respectively, interposed between a set of heavy and light chain framework regions (FRs), which provide support for the CDRs and define the spatial relationship of the CDRs to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the V region of a heavy or light chain. Starting from the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2" and "CDR3", respectively. Thus, an antigen-binding site comprises six CDR sets, including the CDRs from each of the V regions of the heavy and light chains. A polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analysis of several antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contacts being with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of the antigen-binding site.

[0108] As used herein, the term "FR set" refers to four adjacent amino acid sequences surrounding the CDRs of the CDR set of a heavy or light chain V region. Some FR residues may contact the bound antigen. However, FRs are primarily involved in folding the V region into the antigen-binding site, especially the FR residues directly adjacent to the CDRs. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form an antigen-binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there are conserved structural regions of FRs that affect the shape of the CDR loops when folded into certain "canonical" structures. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction of antibody heavy and light chains.

[0109] The structure and location of immunoglobulin variable regions are described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, 1987, and revised versions thereof, now available on the Internet (immuno.bme.nwu.edu), Chothia, AbM and IMGT (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme)).Definitions of antigen-binding sites are also found in: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In IMGT, the CDR amino acid residues of the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues of the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (Kabat numbering). In IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align. Unless otherwise specified, the positions of the CDRs and framework regions disclosed herein are determined according to the IMGT numbering scheme.

[0110] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Heavy chains of vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also called α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0111] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, with κ and λ light chains referring to the two major antibody light chain isotypes.

[0112] By the term "synthetic antibody," as used herein, is meant an antibody made using recombinant DNA techniques, such as the antibodies expressed by bacteriophage described herein. The term should also be taken to mean an antibody made by synthesis of a DNA molecule encoding an antibody and which expresses an antibody protein, or an amino acid sequence specifying that antibody, where the DNA or amino acid sequence has been obtained using well-known synthetic DNA or amino acid sequence techniques available in the art.

[0113] Furthermore, the antibody of the present invention may have another protein fused to its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). The protein to be fused can be appropriately selected by those skilled in the art.

[0114] In a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. The polypeptide sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are: 1. SEQ ID NOs: 2, 3, 4, 6, 7 and 8; or 2. SEQ ID NOs: 2, 3, 10, 12, 7 and 14; or 3. SEQ ID NOs: 2, 16, 17, 19, 7 and 14; or 4. SEQ ID NOs: 22, 23, 24, 26, 20 and 27; or 5. SEQ ID NOs: 22, 23, 24, 30, 20 and 27; or 6. SEQ ID NOs: 32, 33, 34, 36, 37 and 38; or 7. SEQ ID NOs: 40, 41, 42, 44, 37 and 45; or 8. SEQ ID NOs: 47, 48, 49, 51, 52 and 53; or 9. SEQ ID NOs: 40, 3, 55, 6, 7 and 8; or 10. SEQ ID NOs: 58, 59, 60, 6, 7, and 14; or 11. SEQ ID NOs: 58, 3, 63, 65, 7, and 14; or 12. SEQ ID NOs: 2, 3, 67, 69, 70, and 13; or 13. SEQ ID NOs: 72, 73, 74, 76, 37, and 77; or 14. SEQ ID NOs: 58, 79, 80, 6, 7, and 14; or 15. SEQ ID NOs: 58, 83, 84, 86, 7, and 14; or 16. SEQ ID NOs: 58, 83, 88, 86, 7, and 14; the antibody or antigen-binding fragment thereof specifically binds to CCR8, preferably human CCR8; Here, the positions of the CDRs are determined according to the IMGT numbering scheme.

[0115] In another particular embodiment, the present invention provides a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, e.g., 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NOs: 1, 9, 15, 21, 28, 31, 39, 46, 54, 57, 62, 66, 71, 78, 82, 87, 90, 92, 94, 96, 100, 102, 104, 106, 108 or 110, or SEQ ID NOs: 5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 66, 71, 78, 82, 87, 90, 92, 94, 96, 100, 102, 104, 106, 108 or 110, 8, 25, 29, 35, 43, 50, 56, 61, 64, 68, 75, 81, 85, 89, 91, 93, 95, 97, 98, 99, 101, 103, 105, 107, 109 or 111, or an antigen-binding fragment thereof, comprising a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, e.g., 95%, 96%, 97%, 98% or 99% identical to any of the above.

[0116] In the present invention, the antibody of the present invention further includes its conservative variants, and refers to a polypeptide in which, compared with the amino acid sequence of the antibody of the present invention, 10 or less, preferably 8 or less, more preferably 5 or less, and most preferably 3 or less amino acids are substituted with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by substituting amino acids as shown in Table A.

[0117] JPEG2024527288000001.jpg118169

[0118] The present invention relates to an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0119] Polynucleotides, vectors, host cells and preparation methods The present invention also provides a polynucleotide encoding any of the antibodies disclosed herein. In some embodiments, disclosed herein is an isolated polynucleotide encoding an antibody or antibody fragment that binds to CCR8, comprising three light chain CDRs having the amino acid sequences shown in SEQ ID NOs: 58, 79, and 80; and / or three heavy chain CDRs having the amino acid sequences shown in SEQ ID NOs: 6, 7, and 14. In some embodiments, disclosed herein is an isolated polynucleotide encoding an antibody or antibody fragment that binds to CCR8, comprising three light chain CDRs having the amino acid sequences shown in SEQ ID NOs: 58, 79, and 80; and three heavy chain CDRs having the amino acid sequences shown in SEQ ID NOs: 6, 7, and 14. In some embodiments, disclosed herein is an isolated polynucleotide encoding an antibody or antibody fragment that binds to CCR8, comprising a heavy chain variable region and a light chain variable region of a polypeptide sequence selected from the following: SEQ ID NOs: 96 and 97; SEQ ID NOs: 78 and 81; SEQ ID NOs: 90 and 91; SEQ ID NOs: 92 and 93; SEQ ID NOs: 94 and 95; SEQ ID NOs: 92 and 98; or SEQ ID NOs: 92 and 99.

[0120] The present invention also provides a vector comprising an isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. Any vector known to one of skill in the art in view of this disclosure can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any elements for establishing the conventional functions of an expression vector, for example, a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or reconfigurable promoter. Numerous expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce antibodies or antigen-binding fragments thereof in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present invention. Such techniques are well known to one of skill in the art in view of this disclosure.

[0121] The present invention also provides a host cell comprising an isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. In view of the present disclosure, any host cell known to one of skill in the art can be used for recombinant expression of the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is an E. coli TG1 or BL21 cell (e.g., for expression of scFv or Fab antibodies), a CHO-DG44 cell, a CHO-K1 cell, or a HEK293 cell (e.g., for expression of full-length IgG antibodies). According to certain embodiments, the recombinant expression vector is transformed into the host cell by conventional methods, such as chemical transfection, heat shock, or electroporation, and the recombinant expression vector is stably integrated into the genome of the host cell such that the recombinant nucleic acid is effectively expressed.

[0122] The invention also provides a method of producing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions to produce the monoclonal antibody or antigen-binding fragment thereof of the invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cells and purified according to conventional techniques known in the art and described herein.

[0123] As will be appreciated by those of skill in the art, polynucleotides can include genomic sequences, extragenomic and plasmid-encoded sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides, etc. Such segments may be isolated in nature or may be synthetically modified by one of skill in the art.

[0124] As will also be appreciated by those skilled in the art, polynucleotides may be single-stranded (coding or antisense) or double-stranded, DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond one-to-one to DNA molecules, and mRNA molecules, which do not contain introns. Polynucleotides according to the present disclosure may or may not have additional coding or non-coding sequences present, and polynucleotides may or may not be linked to other molecules and / or supporting materials. Polynucleotides may include naturally occurring sequences or may include sequences that code for variants or derivatives of such sequences.

[0125] Polynucleotide variants typically contain one or more substitutions, additions, deletions, and / or insertions, preferably such that the binding affinity of the antibody encoded by the variant polynucleotide is not substantially diminished compared to the antibody encoded by the polynucleotide sequences specifically set forth herein.

[0126] Regardless of the length of the coding sequence itself, the polynucleotides described herein or fragments thereof can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., so that their overall length can vary significantly. It is therefore envisioned that nucleic acid fragments of almost any length can be used, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, exemplary polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, etc. (including all intermediate lengths) are envisioned to be useful.

[0127] Site-directed mutagenesis allows for the production of mutants through the use of specific oligonucleotide sequences that encode a DNA sequence having a desired mutation, as well as a sufficient number of flanking nucleotides to provide primer sequences of sufficient size and sequence complexity to form stable duplexes on both sides of the deletion junction to be traversed. Mutations in a selected polynucleotide sequence can also be used to improve, change, reduce, modify, or otherwise alter the properties of the polynucleotide itself and / or to change the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0128] Antibody-dependent cell-mediated cytotoxicity (ADCC) Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, followed by lysis of the target cells. In a preferred embodiment, such cells are human cells. Without wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcR). The primary cells that mediate ADCC, NK cells, express FcγRIII, whereas monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To evaluate the ADCC activity of a molecule, in vitro ADCC assays such as those described in U.S. Patent No. 5,500,362 or 5,821,337 can be carried out.Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of a molecule of interest can be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al., PNAS (USA), 95:652-656 (1998).

[0129] An "effector cell" is a leukocyte that expresses one or more FcRs and performs an effector function. Preferably, the cell expresses at least FcγRI, FcγRII, FcγRIII and / or FcγRIV and carries out ADCC effector function. Examples of human leukocytes that mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells and neutrophils, with PBMCs and NK cells being preferred. In a preferred embodiment, the effector cell is a human cell.

[0130] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In a preferred embodiment, the FcR is a native sequence human FcR. Moreover, in a preferred embodiment, the FcR is one that binds an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also encompassed by the term "FcR" herein. This term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

[0131] Complement-dependent cytotoxicity (CDC) Complement-dependent cytotoxicity (CDC) refers to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement.The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., antibody) complexed with a cognate antigen.To evaluate complement activation, for example, CDC assays can be performed, such as those described in Gazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996).

[0132] Antibody-drug conjugates (ADCs) The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0133] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, and the antibody is coupled to the effector molecule, preferably by chemical coupling. Wherein, the effector molecule is preferably a drug having therapeutic activity. Moreover, the effector molecule can be one or more of a toxic protein, a chemotherapy drug, a small molecule drug or a radionuclide.

[0134] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. The example of the coupling agent can be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent refers to a compound that allows the antibody to form a covalent bond with the effector molecule, such as glutaraldehyde. The coupling agent using a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0135] Certain residues of antibodies (e.g., Cys or Lys) are used to connect various functional groups, such as imaging reagents (e.g., chromophores and fluorescent groups), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., glycol polymers), and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. The functional agent (e.g., drug, detection agent, stabilizer) is coupled (covalently bound) to the antibody. The functional agent can be linked to the antibody directly or indirectly via a linker.

[0136] An antibody can be coupled to a drug to form an antibody-drug conjugate (ADC). Typically, an ADC includes a linker located between the drug and the antibody. The linker can be a degradable linker or a non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, e.g., the linker is degraded at the target site, resulting in the release of the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that are hydrolyzed at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide bond linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0137] The linker has a reactive group that can react with a specific amino acid residue before being attached to the antibody, and is connected via an active reactive group. Sulfhydryl-specific reactive groups are preferred, including, for example, maleimide compounds, halogenated amides (e.g., iodine, bromine, or chlorine); halogenated esters (e.g., iodine, bromine, or chlorine); halogenated methyl ketones (e.g., iodine, bromine, chlorine); benzyl halides (e.g., iodine, bromine, chlorine); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-3,6-bis-(mercurymethyl)dioxane, and counterions such as acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may, for example, include a maleimide linked to the antibody via thiosuccinimide.

[0138] The drug can be any cytotoxic, cell growth inhibitory or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group capable of forming a bond with the linker. When the drug is directly attached to the linker, the drug has a reactive active group before being attached to the antibody.

[0139] Particularly useful classes of drugs include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drug classes include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors; typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids.

[0140] In the present invention, the drug-linker can be used to form the ADC in one simple step. In other embodiments, bifunctional linker compounds can be used to form the ADC in a two-step or multi-step method. For example, the cysteine ​​residue reacts with the reactive moiety of the linker in the first step, and the functional group of the linker reacts with the drug in the subsequent step to form the ADC.

[0141] Typically, the functional group on the linker is selected to promote a specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be based on the azide-based moiety to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via 1,3-dipolar cycloaddition between the azide group and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines); and activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other ligation strategies, as described in "Bioconjugation Technology", 2nd edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that when a complementary pair of reactive functional groups is selected for selective reaction of the drug moiety with the linker, each member of the complementary pair can be used on both the linker and the drug.

[0142] The present invention also provides a method for preparing an ADC, which may further comprise combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0143] In certain embodiments, the methods of the invention comprise linking an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate, in these embodiments, the methods of the invention further comprise coupling the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0144] In some embodiments, the antibody drug conjugate ADC is represented by the following molecular formula: Ab-(LU-D)p where: Ab is antibody; LU is the linker; D is a drug; and the subscript p is a value selected from 1 to 8.

[0145] Drug Compositions and Other Uses CCL1 / CCR8 signaling is an important pathway in the pathogenesis of several diseases, including cancer, inflammatory diseases, and diabetic neuropathy. In particular, the antibodies described herein specifically bind to CCR8 with unexpectedly high affinity, and in certain embodiments, have the ability to block CCR8 signaling and inhibit CCL1-induced chemotaxis. CCR8+Treg cells are known to contribute to tumor escape mechanisms. In some aspects, provided herein are methods for reducing the number or activity of tumor-infiltrating T regulatory cells (TITR) in tumors present in a subject by inhibiting immune suppression mediated by CCR8+Treg cells and the like, and pharmaceutical compositions for treating cancer via this mechanism. Such cancers include, but are not limited to, breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer, pancreatic cancer, and many other cancer types, and are associated with poor prognosis. In some aspects, provided herein are methods for increasing the amount of T effector cells in a tumor of a subject by administering an anti-CCR8 antibody to the subject. The cytotoxicity may be antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The agent may be an antibody, such as a peptide, a small molecule, a protein-drug conjugate, or an interfering nucleic acid. Furthermore, the present invention may also modulate the CCL1 / CCR8 axis, which may treat diseases such as diabetic neuropathy, spinal cord injury, and IgG4-related diseases such as sclerosing cholangitis (ISC). Exemplary antibodies, or antigen-binding fragments thereof, or amino acid sequences of their complementarity determining regions (CDRs).

[0146] The present invention also provides a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a product comprising an active ingredient of the present invention and a pharma- ceutically acceptable carrier, wherein the active ingredient is selected from the isolated monoclonal antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the isolated nucleic acid (particularly DNA or RNA) of the third aspect, the vector of the fourth aspect, the target antibody complex of the sixth aspect, the immune cell of the seventh aspect, or a combination thereof. The active ingredients of the present invention and compositions comprising them can also be used for the manufacture of drugs for the treatments referred to herein.

[0147] As used herein, "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0148] The dosage will depend on variables such as the type and severity of the disease or indication being treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, the serum half-life of the antibody, and the route of administration.

[0149] The frequency of administration can vary depending on factors such as the route of administration, the dosage, the serum half-life of the antibody or fusion protein, and the disease being treated.

[0150] In some embodiments, the antibodies of the invention are used for non-therapeutic purposes, such as in diagnostic tests and assays, for example, to determine CCR8 levels in a sample from a subject by contacting the sample with a CCR8-specific antibody of the invention and detecting immunoreactivity between the antibody and CCR8 in the sample.

[0151] Detection Applications and Kits The antibodies of the invention or ADCs thereof can be used in detection applications, such as for example, detecting a sample, thereby providing diagnostic information.

[0152] In the present invention, the sample used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used in the present invention is intended to include any type of biopsy known to those skilled in the art. Thus, the biopsy as used in the present invention can include a sample of a resected tumor, and a tissue sample prepared by endoscopy or puncture or needle biopsy of an organ.

[0153] Samples for use in the present invention include fixed or preserved cell or tissue samples.

[0154] The present invention further provides a kit comprising the antibody (or a fragment thereof) of the present invention, and in a preferred embodiment of the present invention, the kit further comprises a container, an instruction manual, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0155] According to a further embodiment of the invention, a CCR8-mediated disease is diagnosed in a subject by detecting the presence or amount of CCR8 protein in a sample.

[0156] The present invention provides a kit for predicting or diagnosing the prognosis of cancer, comprising an anti-CCR8 antibody. The kit of the present invention may further comprise tools and / or reagents known in the art for use in ELISA. The kit of the present invention may further comprise, as necessary, a tube for mixing each component, a well plate, an instruction manual for use, etc. EXAMPLES

[0157] The present invention will be further described with reference to the following examples. It should be understood that the following examples are merely used to illustrate the present invention, but do not limit the scope of the present invention. Experimental methods whose specific conditions are not shown in the following examples are usually carried out according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by the manufacturer. Unless otherwise stated, percentages and parts refer to weight percentages and parts by weight. The cell lines are conventional products that are commercially available or purchased from ATCC, and all plasmids are commercially available products.

[0158] Cell line: The 293F cell line was obtained from Thermo Fisher (R79007) and cultured in Expi293 TM expression medium.

[0159] Example 1. Generation of anti-CCR8 monoclonal antibodies Anti-CCR8 monoclonal antibodies were developed by immunizing SJL mice with 293F cells overexpressing CCR8. Briefly, 293F cells were transfected with a lentiviral vector encoding CCR8 by polybrene (8 μg / mL), selected in medium containing puromycin (2 μg / mL), and tested for CCR8 expression by FACS. Individual clones with the highest MFI against CCR8 were selected for further studies.

[0160] Spleen and lymph node cells from these mice were fused to myeloma cells (SP20) using standard methods to generate hybridomas producing unique antibodies. Antibody-containing supernatants produced by pools of these cells were tested by cell-based ELISA for reactivity with CCR8-overexpressing cells. Cell-based ELISA was generally performed as follows: Approximately 3 × 104 293F-CCR8 cells per well were seeded in 96-well plates and cultured overnight, after which the cells were washed with 1 × PBS-T, followed by the addition of 100 μL of 4% paraformaldehyde solution to fix and crosslink the cells to the microplate. The cells were washed twice with 1 × PBS-T. Neat supernatants from hybridoma cultures, including blanks and positive controls, were then incubated with the cells for 60 min at 37 °C. The cells were then washed four times with 1 × PBS-T. Then, the plates were incubated with goat anti-mouse IgG secondary antibody at 1:10000 dilution in 100 μL PBS for 60 min at 37 °C and washed four times with 1 × PBS-T. 100 μL of TMB was added to the 96-well plate. After incubation for 10–12 min, the plate was scanned with detection at a wavelength of 450 nm.

[0161] Supernatants from these positive clones were confirmed by fluorescence-activated cell sorting (FACS). FACS analysis was generally performed as follows: Approximately 5 × 105 293F-CCR8 cells per sample were prepared and blocked with Mouse BD Fc Block. Cells were distributed into 96-well round-bottom polystyrene plates and incubated with neat supernatants from hybridoma cultures for 20-30 min on ice. Cells were then washed with PBS / 0.5% BSA and centrifuged. Pelleted cell samples were incubated with a secondary antibody of FITC-labeled anti-mouse IgG at a dilution of 1:300 in 100 μL PBS / 0.5% BSA for 30 min on ice, followed by washing with PBS / 0.5% BSA and spinning down. Cell pellets were resuspended in PBS / 0.5% BSA and samples were analyzed on a CYTOFLEX (Beckman). Generally, the same supernatants were tested on non-transfected parental cells to confirm that the reactive antibodies specifically recognized CCR8.

[0162] Positive pools were identified and subcloned by limiting dilution. After three fusions, nine clones were obtained that produced unique antibodies that specifically recognized CCR8-overexpressing cells by FACS: 101E4, 172E7, 103G4, 118H1, 170G6, 84B4, 2P15, 3C11, 3O20, 4M13, 2L15, 4D24, 1G17, 3F11, 3F6 and 4G19. Hybridomas were designated by the same name as the antibodies they produced (e.g., hybridoma 84B4 produces antibody 84B4). All anti-CCR8 antibody clones had high affinity antibodies, and their VH and VL amino acid sequences were shown in Table 1.

[0163] [Table 1A]

[0164] [Table 1B]

[0165] [Table 1C]

[0166] [Table 1D]

[0167] [Table 1E]

[0168] [Table 1F]

[0169] [Table 1G]

[0170] [Table 1H]

[0171] [Table 1I]

[0172] [Table 2]

[0173] Example 2. Binding affinity of anti-CCR8 antibodies to CCR8-expressing cells The affinity of anti-CCR8 antibodies was evaluated by FACS. 293F-CCR8, which expresses high levels of CCR8 on the cell surface, was used for the binding study. The affinity analysis was generally performed as follows. Approximately 5 × 105 293F-CCR8 cells per sample were prepared and blocked with Human BD Fc Block. Test antibodies were diluted in PBS / 0.5% BSA (1:3 serial dilutions from 225 μg / mL to 0.00381 μg / mL). Transfected cells and non-transfected parental cells were distributed into 96-well round-bottom polystyrene plates and incubated with diluted antibodies for 20–30 min on ice. Samples were then washed with PBS / 0.5% BSA, and cells were centrifuged. Pelleted cell samples were incubated with anti-mouse IgG-FITC-secondary antibody at a dilution of 1:300 in 100 μL of PBS / 0.5% BSA for 30 min on ice, washed with PBS / 0.5% BSA, and the cells were pelleted. The cell pellet was resuspended in PBS / 0.5% BSA, and the samples were analyzed using a CYTOFLEX (Beckman).

[0174] As a result, as shown in Figure 1, these antibodies bound to human CCR8-overexpressing 293F cells, but did not bind to cells that did not overexpress CCR8. Figures 1A and 1B show that these antibodies have the ability to bind to CCR8-overexpressing 293F cells (293F-CCR8), but do not bind to the parental 293F cells (Figures 1C and 1D). The EC50 of the clones that showed binding ability is summarized in Table 3:

[0175] [Table 3]

[0176] Example 3. Blocking CCL1-CCR8 signaling by anti-CCR8 antibodies To determine whether any of the above antibodies can block CCL1-CCR8 signaling, Tango-CCR8-Gal4-CHO-K1 cells were constructed. Cells were pelleted and resuspended at 1x104cells / 70μL / well, cells were distributed into 96-well plates, and cultured in starvation medium (F12K, 1% FBS, 1% penicillin-streptomycin) at 37℃, 5% CO2 for 6 hours, test antibodies were added and cultured for 1 hour, and then CCL1 (R&D, Cat. No.: 272-I) was added and cultured at 37℃, 5% CO2 for 24 hours.

[0177] After overnight incubation, the cells were incubated with ONE-Glo working reagent for 10 min at room temperature in the dark, and the relative luminescence units (RLU) of each sample were then recorded using a microplate reader at 560 nm wavelength for fluorescence measurements.

[0178] Antibodies from clones such as 101E4, 170G6, 3O20, 4M13, 4D24, 3F11, and 3F6 showed strong CCL1-CCR8 signal blocking. The signal blocking ability of the antibodies from each clone is shown in Table 4.

[0179] [Table 4]

[0180] Example 4. Antitumor effect test of CCR8 antibody The in vivo antitumor effect was evaluated in humanized mice reconstituted with human peripheral blood mononuclear cells (hu-PBMCs, Milestone Biotechnologies). To facilitate the study, an MDA-MB-231 mouse xenograft model was established. Human breast cancer MDA-MB-231 cells (5 × 106 / mouse) were inoculated subcutaneously into the front and right dorsal skin of female NCG mice. When the average size of the transplanted tumors reached 80-100 mm3, tumor-bearing mice with similar tumor sizes were selected and randomly divided into groups to be injected with 2 × 106 hu-PBMCs from healthy donors via the tail vein. One hour later, the mice were intraperitoneally injected with CCR8 antibody once a week. The tumor size was measured twice a week with a caliper, and tumor size was expressed as volume (mm3), with the formula: V = 0.5a × b2 (a and b represent the long and short diameters of the tumor, respectively). As shown in FIG. 2 (AE), antibodies from clones 84B4, 101E4, 170G6, 3C11 and 2P15 showed significant antitumor effects with P≦0.001.

[0181] The results are shown in Figure 2. Tumor growth curves showed that tumor growth was significantly suppressed in the anti-CCR8 antibody-injected mice compared to the vehicle-treated group (P<0.001).

[0182] Example 5. Binding ability of CCR8 antibodies to cynomolgus monkey CCR8 293F cells were transfected with a lentiviral vector encoding cynomolgus CCR8 by polybrene (8 μg / mL), selected in medium containing puromycin (2 μg / mL), and tested for expression of cynomolgus CCR8 by FACS.

[0183] Specific binding of antibodies to cynomolgus CCR8 was examined by flow cytometry using cynomolgus CCR8-overexpressing cell lines and the parental 293F cell line, and the results showed that antibodies 84B4, 170G6, 172E7, 3C11, 3O20, 1G17, 3F11, and 4G19 bound to cynomolgus CCR8-expressing 293F cells, but not to 293F cells.

[0184] Example 6. Humanization of antibodies 3F11, 3O20 and 1G17 Antibodies 3F11, 3O20 and 1G17 were humanized by grafting the CDRs of the lead antibodies into selected human IgG germline frameworks. Human germline IGHV3-73*01, IGKV2-28*01, IGHV1-46*01 and IGKV2-30*02 were selected based on sequence similarity within both frameworks (FR). To maintain canonical loop structures and chain interfaces, specific residues in the human germline frameworks were backmutated to the corresponding murine residues (Table 5).

[0185] In silico predictions implied high-risk sequence liability in the CDRs of 3F11, such as the NG motif in the CDR-L1 region of 3F11. One liability mutation at position N33 of the light chain was evaluated to see if it could remove a potential deamidation site in the VL without affecting activity.

[0186] Humanization of 3F11, 3O20 and 1G17 resulted in monoclonal antibodies 3F11hz0, 3F11hz1, 3F11hz2, 3F11hz3, 3F11hz4, 3F11hz5 and 3F11hz6; 3O20hz0, 3O20hz1, 3O20hz2, and 3O20hz3; and 1G17hz0, 1G17hz1, 1G17hz2, and 1G17hz3.

[0187] [Table 5A]

[0188] [Table 5B]

[0189] [Table 5C]

[0190] All optimized antibodies were confirmed to bind to human CCR8 expressing 293F. The affinity constants of the humanized versions of antibodies 3F11, 3O20 and 1G17 are shown in Table 6. The affinity of 3F11hz4, 3O20hz2, and 1G17hz3 is equivalent to or higher than that of chimeric antibodies such as 3F11hz0, 3O20hz0, and 1G17hz0.

[0191] [Table 6]

[0192] Next, whether 3F11hz4, 3O20hz2, and 1G17hz3 have cross-reactivity with mouse CCR8 and cynomolgus monkey CCR8 was tested by FACS in the same manner as in Example 5. The affinity of humanized antibodies 3F11hz4 and 3F11 for mouse CCR8 is shown in Figure 3. After humanization, the affinity of 3F11hz4 for mouse CCR8 increased from 681.9nM to 1.926nM. After humanization, the affinity of 3O20hz2 and 1G17hz3 for cynomolgus monkey CCR8 was 6418nM and 3142nM, respectively.

[0193] Example 7. Calcium mobilization assay of humanized antibodies The calcium ion mobilization assay is a cell-based second messenger assay to measure calcium flux associated with activation or inhibition of G protein-coupled receptors. The change in fluorescence intensity is directly related to the content of intracellular calcium released into the cytoplasm in response to ligand activation of the target receptor. This assay is used to determine which of the above humanized antibodies can block CCL1-CCR8 signaling.

[0194] hCCR8-Gqi5-293T cells (constructed by Genomeditech) passaged in complete medium (DMEM medium, 10% FBS, 1% penicillin-streptomycin, 0.75 μg / mL puromycin, 400 μg / mL G418) in an incubator (37 °C, 5% CO2) were used for calcium mobilization assay.

[0195] A fluorescent membrane-permeable calcium-binding dye (FLIPR Calcium 6 Assay Kit) was dissolved in assay buffer (20 mM HEPES buffer, pH 7.4, containing 1* Hank's Balanced Salt Solution (HBSS)). A loading buffer was prepared with the dye solution containing 5 mM probenecid. Probenecid was prepared as a 500 mM stock solution in 1N NaOH and diluted to 250 mM in HBSS buffer before use.

[0196] Approximately 1.5*104 hCCR8-Gqi5-293T cells were seeded in a 384-well plate and incubated in 25 μL starvation medium (DMEM, 1% FBS, 1% penicillin-streptomycin) at 37°C with 5% CO2 for 16 hours. The starvation medium was then completely replaced with 25 μL of assay buffer and 25 μL of loading buffer was added to the desired wells. After dye addition, the cell plate was incubated at 37°C with 5% CO2 for 2 hours and then stored at room temperature until use. Compounds in 12.5 μL of assay buffer were added to each well at the desired concentration (5×) and incubated with the cells for 30 minutes at room temperature. After incubation, the microplate was transferred to the FLIPR instrument and the calcium assay was started as described in the instrument's user guide. 12.5 μL of assay buffer with or without CCL1 was added during the assay. The MAX ratio values ​​were plotted versus antibody concentration and analyzed with GraphPad Prism 7.0 to generate concentration curves.

[0197] All humanized antibodies showed CCL1-CCR8 signal blocking, with 3F11hz4 showing the strongest CCL1-CCR8 signal blocking. The signal blocking ability of representative antibodies disclosed herein is shown in Table 7.

[0198] [Table 7]

[0199] Example 8. Stability verification of humanized antibodies Monoclonal antibodies are proteins and therefore suffer from instability problems. Stability testing of monoclonal antibodies is an important regulatory issue in their development and commercialization as therapeutic biological molecules. The stability and activity of humanized antibodies such as 3F11hz4, 3O20hz2, and 1G17hz3 were tested under stress conditions as shown in Table 8.

[0200] [Table 8]

[0201] The binding affinity and calcium mobilization of the humanized antibodies at different time points were performed in the same manner as in Examples 2 and 7. The results are shown in Table 9. 3F11hz4 showed better stability than other humanized antibodies.

[0202] [Table 9]

[0203] Example 9. Cross-reactivity of F11hz4 with 293F / CHOK1 rat, dog, mouse and cynomolgus monkey CCR8 293F cells were transfected with lentiviral vectors encoding rat and canine CCR8 by polybrene (8 μg / mL), selected in medium containing puromycin (2 μg / mL), and tested for rat and canine CCR8 expression by FACS.CHOK1 cells were transfected with lentiviral vectors encoding mouse CCR8 by polybrene (8 μg / mL), selected in medium containing puromycin (6 μg / mL), and tested for mouse CCR8 expression by FACS.

[0204] Specific binding of humanized antibodies to human, rat, dog, mouse and cynomolgus CCR8 was studied by flow cytometry using human, rat, dog, mouse and cynomolgus CCR8 overexpressing and parental 293F or CHOK1 cell lines.

[0205] The results are shown in Figures 4A-4E. Based on binding affinity, 3F11hz4 bound to HEK293 / CHOK1 cells expressing human (4A), rat (4B), dog (4C), mouse (4D) and cynomolgus CCR8 (4E), but not to the parental cells.

[0206] Example 10. Epitope analysis of humanized monoclonal antibodies The sequences of human, rat, dog, mouse and cynomolgus monkey CCR8 were analyzed. The results showed that the ECD2 regions of human, rat, dog, mouse and cynomolgus monkey CCR8 are similar (Table 10). Assuming that the 3F11hz4 humanized antibody binds to ECD2, the binding was evaluated by transiently expressing each ECD2 mutant in 293F cells and reacting the mutant with an antibody solution of humanized 3F11hz4 antibody prepared by serially diluting the mutant 14 times at 3-fold increments from 25 μg / mL. After reacting at 4°C for 1 hour, the mixture was reacted with Alexa Fluor 488 affinipure goat anti-human Ig(H+L) (Jackson, 109-545-003) and analyzed by flow cytometry. The highest MFI of the serial dilutions was taken as 100%, and the inhibition rate was calculated using the following formula.

[0207] % Inhibition = (best MFI-MFI) / best MFI * 100% The results are shown in Figures 5A-B and Table 11. Compared to human CCR8, the 97 amino acid mutant of CCR8 can increase the EC50 from 0.2335 nM to 23.96 nM.

[0208] [Table 10]

[0209] [Table 11]

[0210] Example 11. Study of the binding of 3F11hz4 humanized antibody to human CCR4 and human CX3CR1 The 3F11hz4 humanized antibody was tested for binding to CCR4 and CX3CR1 overexpressing cells by flow cytometry. Briefly, 293F cells were transfected with lentiviral vectors encoding human CCR4 and CX3CR1 by polybrene (8 μg / mL), selected in medium containing puromycin (2 μg / mL), and tested for expression of human CCR4 and CX3CR1 by FACS using CCR4 antibody (Biolegend, 359408) and CX3CR1 antibody (Biolegend, 341610). Binding evaluation was performed by expressing human CCR4 and CXCR1 in 293F cells and reacting them with antibody solutions of humanized 3F11hz4 antibody, which were serially diluted 3-fold from 25 μg / mL. After reacting at 4°C for 1 hour, the mixture was reacted with Alexa Fluor 488 affinipure goat anti-human Ig(H+L) (Jackson, 109-545-003) and analyzed by flow cytometry.

[0211] As shown in Figures 6A-6B, the humanized 3F11hz4 antibody bound to 293F cells expressing human CCR8 (4A), but did not bind to 293F cells expressing human CCR4 (6A), human CX3CR1 (6B), or 293F cells expressing human CCR4 (6B).

[0212] Example 12. Comparison of 3F11hz4 humanized antibody with other CCR8 antibodies The binding affinity, species cross-reactivity, and signal transduction block of the 3F11hz4 humanized antibody disclosed herein and other prior art CCR8 antibodies were studied. Prior art CCR8 antibodies were produced and characterized according to relevant literature. Specifically, the CCR8 antibody sequences of Gilead (WO2021163064), Shionogi (EP3903817A1), Surface (SRF-114) and Bayer (TPP-23411, WO2021152186) were expressed through CHO cells. Various antibodies were compared in terms of affinity (EC50), species cross-reactivity, and calcium mobilization (IC50). The results are shown in Table 12.

[0213] [Table 12]

[0214] As shown in Table 12, 3F11hz4 demonstrated superiority in binding affinity, species cross-reactivity and signal blocking Ca2+.

[0215] Example 13. Antitumor effect of 3F11hz4 antibody in CD34-humanized mice Preparation and detection of hu-HSC-NPG human immune system mouse model: 4-week-old female NPG mice were irradiated with an X-ray biological irradiation device for 4-24 hours and then injected with CD34+ hematopoietic stem cells derived from umbilical cord blood through the tail vein. 16 weeks after transplantation, blood samples were taken from the orbit and collected in EDTA-Na2 anticoagulant tubes. Flow cytometry was used to analyze the content of human CD45, CD3, CD4 and CD8 positive cells in the peripheral blood of the animals, and the proportion of transplanted human T cells was determined. To establish a tumor-bearing model, Hu-HSC-NPG with a high T cell engraftment ratio was selected.

[0216] Tumor inoculation: Lung cancer (HCC827) was cultured and grown to sufficient numbers, suspended in PBS solution, and injected subcutaneously on the dorsal side at a volume of 5×106 cells / 0.2 ml. A total of 45 mice were inoculated in each round, and the volume was observed when the subcutaneous tumor was clearly visible. After 7-14 days, the subcutaneous tumor grew to 50-100 mm3. Mice with subcutaneous tumors that were too large or too small were excluded, and randomly divided into 4 groups with 10 mice per group. The classification was as follows: Group 1: IgG1 10 mpk, iv, biw; Group 2: CCR8-Ab-IgG1 1 mpk, iv, biw; Group 3: CCR8-Ab-IgG1 3 mpk, iv, biw; Group 4: CCR8-Ab-IgG1 10 mpk, iv, biw. The animals were intravenously injected with CCR8 antibody. The antibody was administered twice a week for a total of 5 to 6 times depending on the growth rate of the subcutaneous tumor. The volume of the subcutaneous tumor and the body weight of the mouse were measured twice a week. The tumor volume was calculated using the following formula: volume = major axis × minor axis × minor axis / 2.

[0217] Statistical methods: Data analysis GraphPad Prism 8.0 statistical analysis software was used to analyze whether there was a statistical difference in subcutaneous tumor volume between the CCR8 antibody-treated group and the isotype control group. First, the data were tested for normal distribution and homogeneity of variance. The results showed that the data were normally distributed (p>0.20) and had homogeneity of variance (p>0.10): Comparisons between multiple groups were tested using one-way ANOVA, with P<0.05 considered statistically significant. When the data did not show a normal distribution or the variances were not equal, the nonparametric Kruskal-Wallis H method was used for analysis. Tumor volume inhibition (TGI) was calculated as follows: TGI=100%×[1-RTV(experimental group) / RTV(control group)].

[0218] Conclusion: In a CD34 humanized animal model, the 3F11hz4 antibody exhibited a dose-dependent antitumor effect against subcutaneous lung cancer (HCC827 cell line) (Figure 7).

[0219] Example 14. Antitumor effect of 3F11hz4 antibody in a syngenic model The dose-effect relationship of 3F11hz4 was investigated in solid cancer models, including lung cancer and liver cancer, to explore the minimum and maximum effective doses.

[0220] In vivo efficacy validation in syngenic transplanted tumor model mice of lung and liver cancer: 50 8-week-old female mice were prepared for each round of experiments. 1 × 106 lung cancer (LLC) and liver cancer (H22) cells in 200 μL of PBS were subcutaneously inoculated into the right flank of the mice, and the day of inoculation was recorded as day 0. After 4-7 days, the subcutaneous tumors grew to 50-100 mm3. According to the volume of the subcutaneous tumor, the mice were randomly divided into 4 groups with 10 mice per group, excluding some mice whose subcutaneous tumors were too large or too small. The classification was as follows: Group 1: mIgG2a 10mpk, ip, biw; Group 2: CCR8-Ab-mIgG2a 1mpk, ip, biw; Group 3: CCR8-Ab-mIgG2a 3mpk, ip, biw; Group 4: CCR8-Ab-mIgG2a 10mpk, ip, biw. Animals were injected intraperitoneally (ip) with CCR8 antibody. Depending on the growth rate of the subcutaneous tumor, the antibody was administered twice a week for a total of 3-6 times. The volume of the subcutaneous tumor and the weight of the mice were measured twice a week. The formula for calculating the tumor volume was: volume = major axis × minor axis × minor axis / 2. Using GraphPad software, we analyzed whether there was a statistical difference in the subcutaneous tumor volume between the CCR8 antibody-administered group and the homozygous control group.

[0221] Conclusion: The dose-dependent antitumor effect of 3F11hz4 antibody against subcutaneous lung cancer (HCC827 cell line) was confirmed in a CD34 humanized animal model (Figure 7), a mouse liver tumor (H22 cell line) and a mouse lung cancer (LLC cell line) synthetic model (Figures 8 and 9).

[0222] Example 15. Treg cell migration assay Treg cell migration test: CD4+CD25+Treg cells in mouse H22 subcutaneous tumors were enriched by magnetic bead sorting, and the CCR8 expression rate of enriched Treg cells was detected by CCR8 antibody flow cytometry. When the CCR8 expression rate reached 80% or more, a migration test was performed. CD4+CD25+Treg cells were treated with starvation medium 1640+1%FBS(Ce)+1%P / S for 3 hours, and the cells were collected, centrifuged, counted, and suspended in 1640+0.5%BSA medium to a concentration of 2.66x106 / mL. Dilution of CCR8 antibody with 1640+0.5%BSA+1%P / S medium: First, 2 μL of 10 mg / mL CCR8 antibody was added to 998 μL of 1640+0.5%BSA+1%P / S medium and diluted to 50 μg / mL; a positive control group without CCR8 antibody was set up by 5-fold 6-point serial dilution. 75 μL of diluted CCR8 antibody was mixed with 75 μL of cell suspension and left in a 37°C incubator and incubated for 30 minutes. CCL1 was diluted in migration medium 1640+0.5%BSA+1%P / S (1 mL per 8 wells). 100 μL of 10 ng / mL CCL1 diluted solution was pre-added to the lower layer of the transwell plate. 75 μL of cells co-cultured with CCR8 antibody were added to the upper layer of the transwell plate. Cultured for 3 hours in a 37°C incubator. The number of cells in the lower layer was counted by flow cytometry, with the high speed set at 60ul / min for 1 min. The data was processed with GraphPad Prism, and the inhibitory effect of CCR8 antibody on Treg cell migration was analyzed.

[0223] Conclusion: Using Treg cells selected from subcutaneous tumor tissue of H22 liver cancer, we verified that CCR8 antibody can inhibit Treg cell migration (Figure 10).

[0224] All documents mentioned in this application are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Furthermore, after reading the teachings of this invention, various modifications and changes may be made to the present invention by those skilled in the art, and it should be understood that these equivalents are also included in the scope defined by the claims.

Claims

**Claim 1** A monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment comprises a heavy-chain variable region comprising HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 58, 79 and 80, respectively, and a light-chain variable region comprising LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 6, 7 and 14, respectively, wherein the antibody or its antigen-binding fragment specifically binds to CCR8. An antibody or an antigen-binding fragment thereof. **Claim 2** The antibody according to claim 1, wherein the heavy-chain variable region of the antibody further comprises a human or humanized framework region and / or the light-chain variable region of the antibody further comprises a human or humanized framework region. **Claim 3** The antibody according to claim 2, wherein the antibody or its antigen-binding fragment comprises a heavy-chain variable region having the polypeptide sequence of SEQ ID NO: 96 and a light-chain variable region having the polypeptide sequence of SEQ ID NO:

97. **Claim 4** The antibody according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy-chain variable region having a polypeptide sequence that is at least (≧) 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 96, or a light-chain variable region having a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:

97. **Claim 5** The antibody according to claim 1, wherein the antibody is (i) a single-chain antibody, a single-chain variable fragment (scFv), a monovalent antibody lacking a hinge region, or a minibody; (ii) a Fab, Fab', or F(ab') 2 fragment; (iii) a full-length antibody; and (iv) an antibody comprising a human IgG Fc domain. **Claim 6** The antibody according to claim 1, wherein the antibody is human or humanized. **Claim 7** The antibody according to claim 1, wherein the antibody or its antigen-binding fragment is a CCR8-specific antibody that binds to human CCR8 and can block CCL1-CCR8 signal transduction. **Claim 8** The antibody according to claim 1, wherein the antibody or its antigen-binding fragment specifically binds to human CCR8 with an epitope comprising one or more amino acid residues selected from Y94 to K107 of human CCR8. **Claim 9** The antibody according to claim 8, wherein the antibody or its antigen-binding fragment specifically binds to human CCR8 with an epitope comprising one or more amino acid residues selected from Y94, L95, L96, D97, Q98, V100, T103, V104, M105 and K107 of human CCR8. **Claim 10** (i) An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; and (ii) An optionally selected tag sequence that aids in expression and / or purification A recombinant protein comprising. **Claim 11** An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment according to any one of claims 1 to 9. **Claim 12** A vector comprising the isolated nucleic acid molecule according to claim 11. **Claim 13** (i) An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or a combination thereof, and (ii) A pharmaceutical composition comprising a pharmaceutically acceptable carrier. **Claim 14** The pharmaceutical composition according to claim 13 for treating a disease mediated by CCR8 and / or CCL1, wherein the disease is cancer, neuropathic pain, or an IgG4-related disease. **Claim 15** The pharmaceutical composition according to claim 14, wherein the disease is breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer or pancreatic cancer, neuropathic pain caused by diabetes or spinal cord injury, or IgG4-related sclerosing cholangitis.

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