Anti-CCR8 antibody and anti-CCR8 / CTLA4 bispecific antibody

Abstract

This disclosure relates to an anti-CCR8 antibody or its antigen-binding fragment, or an anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment, a nucleic acid encoding the antibody or its antigen-binding fragment, a pharmaceutical composition comprising the antibody or its antigen-binding fragment, a method for producing the antibody or its antigen-binding fragment, and the use thereof.

Description

[Technical Field]

[0001] This disclosure relates to the biomedical field, and more particularly to an anti-CCR8 antibody or its antigen-binding fragment that specifically binds to CCR8, an anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment that specifically binds to CCR8 and CTLA4, a method for producing the antibody or its antigen-binding fragment, and the use thereof. [Background technology]

[0002] While tumor immunotherapy has achieved revolutionary success, tumor tissue is infiltrated by a large number of immunosuppressive cells, resulting in lower response rates to tumor immunotherapy in patients with different types of tumors. The most crucial aspect of treating solid tumors is eliminating the suppression of the tumor microenvironment (TME) against immune effector cells in order to improve treatment outcomes.

[0003] Regulatory T cells (Tregs) in the tumor microenvironment are associated with tumor progression and can be a major obstacle to tumor immunotherapy. Therefore, removal of tumor-infiltrating Tregs (TI-Tregs) is a promising approach to overcome resistance to immunotherapy while maintaining peripheral homeostasis. Regulatory T cells (Tregs) are an essential suppressive CD4+ T cell subset for preventing autoimmunity, but they also suppress effective tumor immunity. [1、2] Tregs infiltrate tumor tissue in large numbers, which is often associated with a poor prognosis in cancer patients. [3] It has been reported that removing Tregs may also trigger a strong antitumor immune response in tumor-bearing animals. [4-6] However, removing Tregs may also trigger autoimmunity. [7] A key challenge in developing Treg-targeted cancer immunotherapies is finding a way to specifically deplete Tregs that have infiltrated tumor tissue without affecting tumor-responsive effector T cells (Teffs) and without suppressing autoimmune suppression.

[0004] CTLA-4 is a well-known target for Tregs, and anti-CTLA-4 antibodies have shown clinically potential and durable anti-tumor activity either as monotherapy or in combination with PD-1 targeted therapy. However, severe immune therapy-related side effects (irAEs) due to systemic activation of T cells may limit its application. CTLA-4 is constitutively expressed on regulatory T cells (Tregs) and upregulated on other T cells after activation. [8、9] CTLA-4 is highly expressed in the TME, especially on Tregs, and is considered an important molecule in the regulation of Treg function and anti-tumor immunity. [10、11] To date, several mechanisms of action of therapeutic CTLA-4 antibodies have been proposed, including activation of effector T cells by blocking the B7-CTLA-4 pathway and depletion of Tregs by antibody-dependent cell cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP). [12、13] However, the relative clinical importance of these mechanisms remains controversial.

[13]

[0005] Ipilimumab (Yervoy®) is currently the most well-known anti-CTLA-4 antibody and was approved for the treatment of advanced melanoma in 2011. Ipilimumab has shown clinically potent and broad cancer immunotherapy effects (CITE) either as monotherapy

[14] or as part of combination therapy with nivolumab.

[15] However, CTLA-4 therapy shows severe immune therapy-related side effects (irAEs),

[16] and particularly when combined with nivolumab, the tolerable dose is limited in cancer patients due to systemic activation of T cells by blocking the B7-CTLA-4 pathway. [17、18]

[0006] One approach to improving the therapeutic window of anti-CTLA-4 antibodies and reducing their irAEs is to direct the action to the TME by bispecific antibodies (bsAbs).

[19] ​​CCR8 is a chemokine receptor and has recently been identified as a potential specific marker for tumor infiltrating Tregs (TI-Tregs). [20、21] This is because CCR8 is selectively upregulated in these Tregs in multiple cancers, including breast cancer, colorectal cancer, and lung cancer, but is rarely expressed in Tregs and effector T cells (Teffs) in peripheral blood or other tissues.

[0007] Therefore, there is an unmet need for bispecific antibodies that reduce antibody exposure in the periphery by simultaneously targeting two cell surface antigens (CCR8 and CTLA-4) that overlap on TI-Tregs. By targeting two receptors that overlap on TI-Tregs, localization in the tumor microenvironment (TME) may be increased compared to single specific antibodies, resulting in reduced activation of systemic T cells and improved response rates.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Outdoor Tools 15

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Outdoor Tools 19

[0009] In one embodiment, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human CC motif chemokine receptor 8 (CCR8), comprising a heavy chain complementarity-determining region (HCDR) 1 having an amino acid sequence having at least 80% identity with SEQ ID NO:1, an HCDR2 having an amino acid sequence having at least 80% identity with SEQ ID NO:2, an HCDR3 having an amino acid sequence having at least 80% identity with SEQ ID NO:3, a light chain complementarity-determining region (LCDR) 1 having an amino acid sequence having at least 80% identity with SEQ ID NO:4, an LCDR2 having an amino acid sequence having at least 80% identity with SEQ ID NO:5, and an LCDR3 having an amino acid sequence having at least 80% identity with SEQ ID NO:6.

[0010] In some embodiments, the antibody or its antigen-binding fragment includes or comprises the amino acid sequence described in SEQ ID NO:1, or HCDR1 thereof; the amino acid sequence described in SEQ ID NO:2, or HCDR2 thereof; the amino acid sequence described in SEQ ID NO:3, or HCDR3 thereof; the amino acid sequence described in SEQ ID NO:4, or LCDR1 thereof; the amino acid sequence described in SEQ ID NO:5, or LCDR2 thereof; and the amino acid sequence described in SEQ ID NO:6, or LCDR3 thereof.

[0011] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region having an amino acid sequence that is at least 80% identical to SEQ ID NO:7 or SEQ ID NO:13, and a light chain variable region having an amino acid sequence that is at least 80% identical to SEQ ID NO:8 or SEQ ID NO:14.

[0012] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region comprising or consisting thereof of the amino acid sequence described in SEQ ID NO:7 or SEQ ID NO:13, and a light chain variable region comprising or consisting thereof of the amino acid sequence described in SEQ ID NO:8 or SEQ ID NO:14.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:15, and a light chain having the amino acid sequence of SEQ ID NO:10.

[0014] In some embodiments, the antibody or its antigen-binding fragment is a human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, diabody, triabody, tetrabody, Fab fragment, F(ab')2 fragment, scFv fragment, Fv fragment, Fab' fragment, or domain antibody.

[0015] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment that competes for binding to CCR8 with the antibody or antigen-binding fragment described above.

[0016] In one embodiment, the present disclosure provides a bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment comprising the antibody or antigen-binding fragment thereof, comprising a first domain that specifically binds to CCR8 and a second domain that specifically binds to CTLA4.

[0017] In some embodiments, the first domain is an antibody or an antigen-binding fragment thereof as described above.

[0018] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment is a) To strongly induce the ADCC reaction and kill human Treg cells or cells that overexpress CHOK1-human CCR8 / CTLA-4, b) Compared to anti-CCR8 antibodies, it reduces tumor growth and significantly extends survival time. c) Partially block CD80 or CD86-CTLA4 interactions on cells overexpressing CHOK1-human CTLA-4, but significantly block the binding of CD80 or CD86 to CTLA-4 on cells overexpressing CHOK1-human CCR8 / CTLA-4. d) Compared to ipilimumab, better resistance and longer survival, and e) The in vivo antitumor effect is dose-dependently superior to that of anti-CCR8 antibodies and ipilimumab. It has at least one of the properties selected from the following.

[0019] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment is defucosylated.

[0020] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment is a human antibody or a humanized antibody.

[0021] In some embodiments, the second domain includes an anti-CTLA-4 nanobody.

[0022] In some embodiments, the second domain includes an amino acid sequence having at least 80% identity with SEQ ID NO:22 or SEQ ID NO:23.

[0023] In some embodiments, the second domain comprises or consists of the amino acid sequence described in SEQ ID NO:22 or SEQ ID NO:23.

[0024] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody further comprises the Fc region of IgG. In one embodiment, the Fc region is the Fc region of IgG1.

[0025] In some embodiments, the first domain that specifically binds to CCR8 and the second domain that specifically binds to CTLA-4 are connected to each other directly or via one or more linkers. In one embodiment, the linkers may be the same or different. In one embodiment, the linkers are flexible connections. In one embodiment, the linkers are peptide linkers.

[0026] Therefore, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment further comprises one or more linkers, each optionally containing one or more amino acids selected from the group consisting of glycine and serine.

[0027] In some embodiments, the one or more linkers are inserted into the C-end, N-end, or both ends of the second domain.

[0028] In some embodiments, the linker includes one or more amino acid sequences selected from GGGGSGGGGS (SEQ ID NO: 24) and GS.

[0029] In some embodiments, the linker includes GGGGSGGGGS and GS.

[0030] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20, and a light chain having the amino acid sequence of SEQ ID NO:10.

[0031] In one embodiment, the present disclosure provides an antibody or its antigen-binding fragment as described above, or a nucleic acid encoding a bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment.

[0032] In some embodiments, the nucleic acid includes a nucleic acid encoding a heavy chain having a nucleotide sequence of SEQ ID NO: 11 or 16, and a nucleic acid encoding a light chain having a nucleotide sequence of SEQ ID NO: 12 or 17.

[0033] In some embodiments, the nucleic acid includes a nucleic acid encoding a heavy chain having a nucleotide sequence with SEQ ID NO: 19 or 21, and a nucleic acid encoding a light chain having a nucleotide sequence with SEQ ID NO: 17.

[0034] In one embodiment, the present disclosure provides an expression vector containing the nucleic acid described above.

[0035] In one embodiment, the present disclosure provides a host cell containing the expression vector described above.

[0036] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or its antigen-binding fragment, a bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment, a nucleic acid or expression vector, and a pharmaceutically acceptable carrier.

[0037] In one embodiment, the present disclosure provides a method for producing an antibody or its antigen-binding fragment as described above, or a bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment as described above, comprising culturing host cells as described above under conditions that enable the expression of the antibody or its antigen-binding fragment.

[0038] In one embodiment, the present disclosure provides a method for treating a disease or illness associated with abnormal expression of CCR8 and / or CTLA-4 in a subject, comprising administering to the subject a therapeutically effective amount of the above-described antibody or its antigen-binding fragment, a bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment, a nucleic acid, an expression vector, host cells, or a pharmaceutical composition.

[0039] In one embodiment, the present disclosure provides the use of such antibodies or their antigen-binding fragments, or bispecific anti-CCR8 / CTLA-4 antibodies or their antigen-binding fragments, in the manufacture of pharmaceuticals for the treatment of diseases associated with CCR8 and / or CTLA-4 abnormal expression in subjects.

[0040] In some embodiments, the disease is cancer.

[0041] In some embodiments, the cancer is squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, colon carcinoma, anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, paranasal sinus natural killer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium, fallopian tube cancer, ovarian cancer, carcinoma of the The following are selected from the group consisting of cervix, vaginal cancer, vulvar cancer, testicular cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, penile cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epithelioid carcinoma, squamous cell carcinoma, pediatric solid tumors, environment-induced cancer, virus-related cancer, virus-derived cancer, advanced cancer, unresectable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination thereof. Selected solid tumors; or hematological malignancies selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), T-cell lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal immunoglobulinemia of unknown meaning (MGUS), progressive, metastatic, refractory and / or recurrent hematological malignancies, and any combination of these hematological malignancies.

[0042] In some embodiments, the cancer is bladder cancer.

[0043] These and other embodiments will be described in more detail herein. Each embodiment provided may include various embodiments provided herein. Thus, it is expected that each embodiment described may include each embodiment relating to one element or combination of elements, and that all such embodiments and combinations of embodiments are clearly considered. [Brief explanation of the drawing]

[0044] [Figure 1] This involves the binding of an anti-CCR8 antibody to the human CCR8 protein on the cell surface. [Figure 2] This involves the inhibition of the binding of CCR8 to its ligand CCL1 by an anti-CCR8 antibody. [Figure 3] This involves the binding of an anti-CCR8 antibody to in vitro-induced human Treg cells. [Figure 4] This involves the suppression of the binding of CCR8 to its ligand CCL1 in in vitro-induced human Treg cells by an anti-CCR8 antibody. [Figure 5] This involves the binding of an anti-CCR8 antibody to the cyno-CCR8 protein on the cell surface. [Figure 6] This is an ADCC (antibody-dependent cytotoxicity) bioassay using PBMCs as effector cells, incubated with human Treg target cells induced in vitro with a defucosylated (AF) anti-CCR8 antibody (E:T=20:1). [Figure 7] This shows the changes in tumor growth in the MC38 model after administration of an anti-CCR8 antibody. [Figure 8] This is the survival rate of MC38 models treated with anti-CCR8 antibodies. [Figure 9] This is a schematic diagram of the tetravalent structure of bsAb-38-64-05-3. [Figure 10A]The binding of bsAb-38-64-05-2 or bsAb-38-64-05-3, parental CCR8 mAb (HC 64-23-AF), or parental CTLA-4 nanobody (GBD008-hS005-3-2) to CCR8 single-positive cells (A), CTLA-4 single-positive cells (B), and CCR8 / CTLA-4 double-positive cells (C). [Figure 10B] The binding of bsAb-38-64-05-2 or bsAb-38-64-05-3, parental CCR8 mAb (HC 64-23-AF), or parental CTLA-4 nanobody (GBD008-hS005-3-2) to CCR8 single-positive cells (A), CTLA-4 single-positive cells (B), and CCR8 / CTLA-4 double-positive cells (C). [Figure 10C] The binding of bsAb-38-64-05-2 or bsAb-38-64-05-3, parental CCR8 mAb (HC 64-23-AF), or parental CTLA-4 nanobody (GBD008-hS005-3-2) to CCR8 single-positive cells (A), CTLA-4 single-positive cells (B), and CCR8 / CTLA-4 double-positive cells (C). [Figure 11A] The inhibition of CCR8 binding to its ligand CCL1 in CCR8 monopositive cells (A) and CCR8 / CTLA-4 bipositive cells (B) by bsAb-38-64-05-2 or the parent CCR8 mAb (HC64-23-AF). [Figure 11B] The inhibition of CCR8 binding to its ligand CCL1 in CCR8 monopositive cells (A) and CCR8 / CTLA-4 bipositive cells (B) by bsAb-38-64-05-2 or the parent CCR8 mAb (HC64-23-AF). [Figure 12A] The inhibitory effect is the inhibition of bsAb-38-64-05-2, bsAb-38-64-05-3, or parental CTLA-4 nanobody (GBD008-hS005-3-2) on the binding of CTLA-4 to its ligand CD80 in CTLA-4 mono-positive cells (A) and CCR8 / CTLA-4 bi-positive cells (B). [Figure 12B]The inhibitory effect is the inhibition of bsAb-38-64-05-2, bsAb-38-64-05-3, or parental CTLA-4 nanobody (GBD008-hS005-3-2) on the binding of CTLA-4 to its ligand CD80 in CTLA-4 mono-positive cells (A) and CCR8 / CTLA-4 bi-positive cells (B). [Figure 13A] This involves the inhibition of bsAb-38-64-05-2 or the parental CTLA-4 nanobody (GBD008-hS005-3-2) of the binding of CTLA-4 to its ligand CD86 in CTLA-4 single-positive cells (A) and CCR8 / CTLA-4 double-positive cells (B). [Figure 13B] This involves the inhibition of bsAb-38-64-05-2 or the parental CTLA-4 nanobody (GBD008-hS005-3-2) of the binding of CTLA-4 to its ligand CD86 in CTLA-4 single-positive cells (A) and CCR8 / CTLA-4 double-positive cells (B). [Figure 14A] Binding of bsAb-38-64-05-2, bsAb-38-64-05-3, or parent nanobody to cyno-CCR8 protein (A) or coated cyno-CTLA-4 protein (B) on the cell surface. [Figure 14B] Binding of bsAb-38-64-05-2, bsAb-38-64-05-3, or parent nanobody to cyno-CCR8 protein (A) or coated cyno-CTLA-4 protein (B) on the cell surface. [Figure 15] This is an ADCC (antibody-dependent cytotoxicity) bioassay using PBMCs as effector cells, incubated with bsAb-38-64-05-2, parental CCR8 mAb(AF)(HC64-23-AF) or parental CTLA-4 nanobody(GBD008-hS005-3-2), and CHOK1-hCCR8 / hCTLA-4 target cells (E:T=20:1). [Figure 16] This shows the change in body weight gain in 10-day-old C57BL / 6CCR8 / CTLA-4 double knock-in mice after co-administration of bsAb-38-64-05-2 or ipilimumab with anti-mPD1. [Figure 17] This is the survival rate of 10-day-old C57BL / 6 CCR8 / CTLA-4 double knock-in mice administered in combination with bsAb-38-64-05-2 or ipilimumab and anti-mPD1. [Figure 18] This shows the changes in tumor growth in the MB49 model after administration of bsAb-38-64-05-2 or bsAb-38-64-05-3 antibodies in C57BL / 6 CCR8 / CTLA-4 double knock-in mice. [Modes for carrying out the invention]

[0045] All publications cited herein, including but not limited to patents and patent applications, are incorporated herein by reference, as they are enumerated in their entirety.

[0046] Unless otherwise defined herein, scientific and technical terms used herein should have meanings generally understood by those skilled in the art. Furthermore, unless otherwise indicated in the context, singular terms are intended to include plural terms, and vice versa. For the sake of readability, some terms are defined below.

[0047] definition

[0048] As used herein, the indefinite articles “a” or “an” should be understood to refer to “one or more” of any listed or enumerated components.

[0049] As used herein, the term “approximately” means, when applied to a numerical value, a value that is reasonably close to the stated value and within an acceptable margin of error determined by those skilled in the art, the margin of error which depends in part on the method of measuring or determining the value, i.e., the limits of the measuring system. For example, “approximately” may mean within ±50%, preferably ±25%, and more preferably ±10% of the given reference value. Where a particular value is provided in this application, unless otherwise specified, the meaning of “approximately” should be understood, in accordance with the conventions of the art, to be within an acceptable margin of error for that particular value.

[0050] An "antibody" (Ab) should include, but not limited to, a glycoprotein immunoglobulin (Ig) or an antigen-binding fragment thereof, which specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each H chain has a heavy chain variable region (V in this specification). H It includes a heavy chain constant region (abbreviated as CH1, CH2, and CH3). The heavy chain constant region of IgG Ab includes three constant domains, namely CH1, CH2, and CH3. Each light chain has a light chain variable region (V as specified herein). L It includes a constant domain (abbreviated as C) and a constant light chain region. The constant light chain region of IgG Ab is one constant domain, namely C L Includes. V H and V L The domain can be further subdivided into hyper-variable domains called complementarity-determining domains (CDRs), with more conservative domains called framework domains (FRs) scattered between them. H and V LEach antibody contains three CDRs (LCDRs including LCDR1, LCDR2, and LCDR3, and heavy chain CDRs including HCDR1, HCDR2, and HCDR3) and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Both the heavy chain and light chain variable regions contain binding domains that interact with the antigen. Various methods have been used to define the CDR domains in Ab, including Kabat, Chothia, AbM, Contact, and IMGT definitions. Unless otherwise indicated, Kabat numbering is used as the default in this disclosure. The constant region of Ab can mediate the binding of Ig to various cells of the immune system (e.g., effector cells) and host tissues or factors including the first component (Clq) of the classical complement system. Antibodies are designated based on the amino acid sequence of their heavy chain constant region. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM. Several major antibody classes are classified into subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0051] As used herein, the term “antibody” includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. Intact antibodies or antibody fragments containing the antigen-binding moiety of an antibody may be used. As used herein, the term “antigen-binding fragment” refers to an antibody fragment formed from a portion of an intact antibody containing one or more CDRs, or any other antibody fragment capable of binding to an antigen but not containing an intact native antibody structure. As used herein, the term “antibody or its antigen-binding fragment” refers to an intact antibody or an antibody fragment containing the antigen-binding moiety. The antigen-binding moiety can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antibodies or their antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), fragments containing complementarity-determining regions (CDRs), single-chain variable fragments (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides containing at least a portion of immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0052] "Fab fragment" is V L , V H , C L The "F(ab')2 fragment" is a monovalent fragment having a CH1 domain. The "Fv fragment" is a bivalent fragment having two Fab fragments connected at a hinge region by a disulfide bond. The "Fv fragment" is a V fragment derived from a single arm of the antibody. L and V H It has a domain. "Domain antibody (dAb)" is V H or V L It consists of domains. A "single-chain variable fragment (scFv)" is V L and V HA region is an antibody in which protein chains are linked by a linker (e.g., a synthetic sequence of amino acid residues) of sufficient length to form a monovalent antigen-binding site, thereby forming a continuous protein chain. The term "diabody" refers to a bivalent antibody containing two polypeptide chains, where each polypeptide chain is linked by a linker. H and V L The linker contains domains and is too short to pair two domains on the same chain, thereby pairing each domain with a complementary domain on another polypeptide chain. If the two polypeptide chains of a diabody are identical, the resulting diabody will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to produce diabodies or bispecific antibodies having two different antigen-binding sites. A diabody or bispecific antibody also refers to an artificial antibody or antigen-binding fragment having fragments derived from two different monoclonal antibodies and capable of binding to two different epitopes. These two epitopes may be present on the same antigen or on two different antigens. Similarly, a triabody, tetrabody, or other multispecific antibody is an antibody containing three, four, or more polypeptide chains, which may be identical or different, and thus form three, four, or more antigen-binding sites, each of which may be identical or different.

[0053] As used herein, the term “human” antibody refers to an antibody having a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody also contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence.

[0054] As used herein, the term “humanized” antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulin. In one embodiment of a humanized antibody, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids derived from human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not eliminate the antibody’s ability to bind to a particular antigen. “Humanized” antibodies retain antigen specificity similar to that of the original antibody.

[0055] As used herein, the term “monoclonal antibody” (mAb) refers to an antibody molecule with a single molecular composition, i.e., a naturally occurring formulation of an antibody molecule whose primary sequence is substantially identical and which exhibits single-binding specificity and affinity for a particular epitope. mAbs can be manufactured by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0056] As used herein, the term “chimeric” antibody refers to an antibody in which the variable region originates from one species and the constant region originates from another species, for example, an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody. In exemplary cases, a chimeric antibody may include a constant region of human origin and a variable region of a non-human animal (e.g., mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0057] As used herein, the terms “specific binding” or “specific binds” refer to non-random binding reactions between two molecules, such as between an antibody and an antigen. The binding affinity of the antibody and antigen-binding fragments provided herein is the ratio (k) of the dissociation rate to the binding rate when the binding between the antigen and the antigen-binding molecule (e.g., antibody and antigen-binding fragment) reaches equilibrium. off / k on ) represents KD It can be expressed as a value. Antigen binding affinity (e.g., K D ) can be appropriately measured using appropriate methods known in the art, such as Biacore technology, Kinexa technology, and flow cytometry.

[0058] As used herein, the term “competing for binding” refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human CCR8 and an anti-CCR8 antibody) to any detectable degree (e.g., at least 85%, at least 90%, or at least 95%). Those skilled in the art will acknowledge that it is possible to determine, without excessive experimentation, whether a given antibody competes with the antibody of this disclosure for binding to CCR8.

[0059] As used herein, the term “epitope” refers to a specific group of atoms or amino acids in an antigen to which an antibody or antigen-binding moiety binds. The minimum size of an epitope may be approximately 3, 4, 5, 6, or 7 amino acids; however, these amino acids do not need to be a continuous linear sequence in the antigen's primary structure, as the epitope may depend on the antigen's three-dimensional conformation based on its secondary and tertiary structures. CDRs are important for identifying antigen epitopes.

[0060] "Multispecific" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes in an antigen, for example, three, four, or five different antigens or epitopes.

[0061] "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific antibodies may cross-react with other related antigens, such as those of humans or other species (e.g., cynomolgus monkeys (Macaca fascicularis), chimpanzees (Pan troglodytes), or common marmosets (Callithrix jacchus)), or they may bind to epitopes shared between two or more different antigens.

[0062] A "bispecific anti-CCR8 / CTLA-4 antibody," "CCR8 / CTLA-4 antibody," "anti-CCR8 / CTLA-4 antibody," or "antibody that specifically binds to CCR8 and CTLA-4" refers to a molecule containing at least one domain that specifically binds to CCR8 and at least one domain that specifically binds to CTLA-4. The domains that specifically bind to CCR8 and CTLA-4 are typically VH / VL pairs. With respect to binding to CCR8 or CTLA-4, a bispecific anti-CCR8 / CTLA-4 antibody may be monovalent.

[0063] As used herein, “percent (%) identity” of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment. Sequence identity refers to a perfect match between nucleotides or amino acids in two sequences being compared. Those skilled in the art can determine sequence identity by conventional means such as the BLAST algorithm.

[0064] As used herein, “administering” (“administered” or “administration”) means the physical introduction of a composition containing a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. The preferred route of administration for therapeutic antibodies is intravenous (IV) administration. Other routes of administration include subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), spinal, or other parenteral routes (e.g., by injection or infusion). As used herein, the phrase “parenteral administration” means a mode of administration other than enteral and topical administration, which is usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions, as well as internal electroperforation. Alternatively, the antibodies or antigen-binding fragments according to this disclosure may be administered via parenteral routes, such as topical, cutaneous, or mucosal administration routes (e.g., intranasal, oral, transvaginal, rectal, sublingual, or topical). Administration may be, for example, one dose, multiple doses, and / or over one or more extended periods.

[0065] As used herein, the term “CC motif chemokine receptor 8” (“CCR8”; also known as CY6, TER1, CCR-8, CKRL1, CDw198, CMKBR8, GPRCY6, CMKBRL2, or CC-KR-8) refers to FOXP3 in tumors. hi This is a 7-transmembrane GPCR that has been shown to be primarily expressed in Tregs.

[0066] As used herein, the term “CCR8” includes human CCR8 (hCCR8), variants, isoforms, species homologs of hCCR8 such as mouse CCR8 (mCCR8), and analogs having at least one common epitope with hCCR8. The complete amino acid sequences of hCCR8 and mCCR8 can be found at GENBANK® accession numbers AAI07160.1 and NP_031746.1, respectively.

[0067] As used herein, the term “CTLA4 protein” includes full-length CTLA4 protein, CTLA4 protein fragments, CTLA4 protein variants, and CTLA4 fusion proteins (e.g., CTLA4 / Fc fusion proteins) to which an anti-CTLA4 antibody (e.g., MDX-010) can bind.

[0068] As used herein, the term “cancer” refers to a broad group of various diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division and growth lead to the formation of malignant tumors, which can invade neighboring tissues and metastasize to distant parts of the body via the lymphatic system and bloodstream. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignant-transforming cells, tissues or organs, regardless of histopathological type or stage of invasion. As used herein, the term “tumor” refers to cancer cells, e.g., a mass of cancer cells. Cancers that can be treated or diagnosed by the methods described herein include, for example, malignant tumors of various organ systems affecting the lungs, breasts, thyroid, lymph nodes, gastrointestinal tract and urogenital tract, and adenocarcinomas, including most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, small intestine cancer, esophageal cancer, and other malignant tumors. In some embodiments, antibodies or antigen-binding fragments provided herein are designed to treat or diagnose cancer in a subject. The term "cancer" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissue, including respiratory cancers, gastrointestinal cancers, urogenital cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. In some embodiments, the cancer is renal cancer or melanoma. Exemplary cancers include cancers that form from cervical, lung, prostate, breast, head and neck, colon, and ovarian tissues. The term also includes carcinosarcomas, which include malignant tumors consisting of cancerous and sarcomatous tissues. "Adenocarcinoma" refers to cancers of glandular origin, or cancers in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and refers to malignant tumors of mesenchymal origin.

[0069] As used herein, “subject” includes any human or non-human animal. The term “non-human animal” includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, monkeys, chimpanzees, gorillas, and rodents (such as mice, rats, and guinea pigs). In preferred embodiments, the subject is human. The terms “subject” and “patient” can be used interchangeably herein.

[0070] As used herein, “vector” refers to a polynucleotide molecule that enables the replication / cloning of a desired nucleic acid fragment contained therein, or enables the expression of a protein encoded by the desired nucleic acid fragment when introduced into a suitable cell host. Examples of vectors include both cloning vectors and expression vectors. As used herein, the term “expression vector” refers to a vehicle into which a protein-encoding polynucleotide can be operably inserted to enable protein expression. Expression vectors may contain various elements to control expression, including promoter sequences, transcription start sequences, enhancer sequences, selectable elements, and reporter genes. Vectors may also contain origins of replication. Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0071] As used herein, “host cell” means a cell that is transformed or can be transformed by a nucleic acid sequence and is therefore capable of expressing a target gene. The host cell may be a prokaryotic cell (e.g., Escherichia coli), a eukaryotic cell (e.g., a plant including yeast, tobacco and tomato, or an animal including human, monkey, hamster, rat, mouse, or insect), or a hybridoma cell.

[0072] As used herein, “therapeutic effective dose” or “therapeutic effective amount” of a drug or therapeutic agent is any amount of the drug or agent that, when used alone or in combination with other therapeutic agents, protects a subject from the onset of the disease, or promotes disease regression demonstrated by a reduction in the severity of disease symptoms in a subject, an increase in the frequency and duration of disease-free periods, the prevention or mitigation of damage or impairment caused by the suffering of the disease, or otherwise improves disease symptoms. Furthermore, the terms “effective” and “effectiveness” in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote disease regression in a patient (e.g., cancer regression). Physiological safety refers to an acceptable level of toxicity or other adverse physiological effects (side effects) at the cellular, organ and / or biological level caused by drug administration. The effectiveness of a therapeutic agent can be evaluated using a variety of methods known to the employee, such as evaluating it in human subjects during a clinical trial, evaluating it in animal model systems to predict human efficacy, or measuring the activity of the drug in in vitro measurements.

[0073] As used herein, the terms “treating” or “treatment” of a disease or illness include preventing or mitigating a disease, slowing the onset or progression of a disease, reducing the risk of developing a disease, preventing or delaying the development of symptoms associated with a disease, reducing or ending symptoms associated with a disease, completely or partially regressing a disease, curing a disease, or a combination thereof.

[0074] As used herein, the term “pharmaceutically acceptable carrier” means that a designated carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other components of the formulation and physiologically compatible with its recipient.

[0075] Antibodies and antigen-binding fragments This disclosure provides novel anti-CCR8 antibodies (e.g., HC64, HC64-23) or antigen-binding fragments thereof that specifically bind to CCR8, such as human CCR8, expressed on the cell surface.

[0076] This disclosure also provides novel anti-CCR8 / CTLA4 bispecific antibodies (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) or antigen-binding fragments thereof that specifically bind to CCR8 and CTLA4, such as human CCR8 and CTLA4 expressed on the cell surface.

[0077] In certain embodiments, bsAb-38-64-05-2 / bsAb-38-64-05-3 is designed by inserting an anti-CTLA-4 nanobody (e.g., GBD008-hS005-3-2) between the anti-CCR8 Fab and the hinge region, in the same form of defucosylated IgG1. The bispecific antibodies bsAb-38-64-05-2 / bsAb-38-64-05-3 reduce peripheral antibody exposure by simultaneously targeting these two cell surface antigens (CCR8 and CTLA-4) that overlap with TI-Tregs. By targeting two receptors that overlap with TI-Tregs, localization in the TME may be increased compared to single-specific antibodies, resulting in reduced systemic T cell activation and improved response rates.

[0078] Experiments have shown that the binding of the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) to cells is increased when both target antigens are conjugated compared to single-antigen conjugation. Experiments have also demonstrated that the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) mediate TI-Treg depletion, enabling a potent antitumor response in various mouse tumor models. Furthermore, the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) did not cause toxicity in mouse irAEs models compared to ipilimumab in combination with an anti-PD-1 antibody. These data demonstrate the potential and safe clinical utility of the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) for the selective depletion of tumor-resident T regulatory cells.

[0079] Our preclinical mouse tumor modeling demonstrated that the Fc-optimized defucosylated (AF) anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) showed significant tumor suppression by specifically depleting TI-Tregs rather than Teffs. Furthermore, in irAEs models, the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) showed better resistance compared to the combination of ipilimumab and an anti-PD-1 antibody. These research findings indicate that the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) can achieve clinically optimal TI-Treg depletion, provide antitumor immunity, and simultaneously limit peripheral toxicity.

[0080] The antibodies or antigen-binding fragments provided herein have an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with SEQ ID NO:1, or a heavy chain complementarity-determining region (HCDR) 1 having up to one amino acid addition, substitution, and / or deletion compared to SEQ ID NO:1, an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with SEQ ID NO:2, or an HCDR2 having up to three (e.g., one, two, or three) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:2, and SEQ ID HCDR3 has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to NO:3, or has up to one amino acid addition, substitution, and / or deletion compared to SEQ ID NO:3, HCDR3 has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:4, or has up to two (e.g., one or two) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:4, LCDR1 has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:5, or SEQ ID The present invention includes LCDR2 having up to one amino acid addition, substitution, and / or deletion compared to NO:5, and LCDR3 having an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:6, or having up to one amino acid addition, substitution, and / or deletion compared to SEQ ID NO:6.

[0081] In a preferred embodiment, the antibody or its antigen-binding fragment includes HCDR1 having the amino acid sequence of SEQ ID NO:1, HCDR2 having the amino acid sequence of SEQ ID NO:2, and HCDR3 having the amino acid sequence of SEQ ID NO:3, LCDR1 having the amino acid sequence of SEQ ID NO:4, LCDR2 having the amino acid sequence of SEQ ID NO:5, and LCDR3 having the amino acid sequence of SEQ ID NO:6.

[0082] In a preferred embodiment, the antibody or its antigen-binding fragment contains the CDR sequence of HC64 (see Table 1).

[0083] [Table 1]

[0084] In a preferred embodiment, the antibody or its antigen-binding fragment includes HCDR1 consisting of the amino acid sequence described in SEQ ID NO:1, HCDR2 consisting of the amino acid sequence described in SEQ ID NO:2, HCDR3 consisting of the amino acid sequence described in SEQ ID NO:3, LCDR1 consisting of the amino acid sequence described in SEQ ID NO:4, LCDR2 consisting of the amino acid sequence described in SEQ ID NO:5, and LCDR3 consisting of the amino acid sequence described in SEQ ID NO:6.

[0085] While CDRs are known to be responsible for antigen binding, it has been found that not all six CDRs are essential or immutable. In other words, substituting, altering, or modifying one or more CDRs from HC64 or HC64-23 can substantially maintain specific binding affinity to CCR8.

[0086] In some embodiments, the antibodies or antigen-binding fragments provided herein may include one or more modifications or substitutions of one or more CDR regions provided in Table 1. Such variants retain the specific binding affinity of their parent antibody to CCR8 but may have improvements to one or more properties, such as higher antigen-binding affinity or reduced glycosylation potential.

[0087] In some embodiments, the antibodies or antigen-binding fragments provided herein may be modified to remove one or more Asn or Asp hotspots within the CDR region (or variable region). Such Asn and Asp hotspots can cause antibody degradation, resulting in reduced antibody stability.

[0088] In some embodiments, the one or more modifications or substitutions are conservative substitutions.

[0089] In some embodiments, the antibody or its antigen-binding fragment has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:7 or SEQ ID NO:13, or a heavy chain variable region having up to 20 (e.g., 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 20, or 20) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:7 or SEQ ID NO:13 (i.e., the heavy chain variable region of HC64 or HC64-23), and SEQ ID NO:8 or SEQ ID The amino acid sequence has at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with NO:14, or includes a light chain variable region having up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 19, or 20) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:8 or SEQ ID NO:14 (i.e., the light chain variable region of HC64 or HC64-23). ​​In preferred embodiments, the substitutions are conservative substitutions.

[0090] In a preferred embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO:7 or SEQ ID NO:13, and a light chain variable region consisting of the amino acid sequence described in SEQ ID NO:8 or SEQ ID NO:14.

[0091] The antibodies or antigen-binding fragments provided herein further comprise an immunoglobulin constant region, optionally a human immunoglobulin constant region, and optionally a human IgG constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or light chain constant region. The heavy chain constant region comprises a CH1 region, a hinge region, and / or a CH2-CH3 region. In some embodiments, the heavy chain constant region comprises an Fc region. In some embodiments, the light chain constant region comprises a C κ or C λThis includes. In a preferred embodiment, the steady region is derived from human IgG1 (hIgG1). In a preferred embodiment, the steady region is the steady region of human IgG1.

[0092] In some embodiments, the antibodies or antigen-binding fragments provided herein include at least one heavy chain and / or at least one light chain. In one embodiment, the heavy chain has an amino acid sequence that is at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:9 or SEQ ID NO:15, or has up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 35, 40, 45, or 50) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:9 or SEQ ID NO:15 (i.e., the full-length heavy chain sequence of HC64 or HC64-23). In one embodiment, the light chain has an amino acid sequence that is at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:10, or has up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:10 (i.e., the full-length light chain sequence of HC64 or HC64-23). ​​In a preferred embodiment, the substitutions are conservative substitutions.

[0093] In a preferred embodiment, the antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:15, and a light chain having the amino acid sequence of SEQ ID NO:10.

[0094] Table 2 shows the amino acid sequence and nucleotide sequence of HC64.

[0095] [Table 2-1] [Table 2-2]

[0096] Table 3 shows the amino acid and nucleotide sequences of HC64-23.

[0097] [Table 3-1] [Table 3-2]

[0098] This disclosure provides a bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment thereof, comprising a first domain that specifically binds to CCR8 and a second domain that specifically binds to CTLA4, comprising the antibody or antigen-binding fragment described above.

[0099] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment is isolated.

[0100] In some embodiments, the first domain is an antibody or its antigen-binding fragment as described above.

[0101] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment is a) To strongly induce the ADCC reaction and kill human Treg cells or cells that overexpress CHOK1-human CCR8 / CTLA-4, b) Compared to anti-CCR8 antibodies, it reduces tumor growth and significantly extends survival time. c) Partially block CD80 or CD86-CTLA4 interactions on cells overexpressing CHOK1-human CTLA-4, but significantly block the binding of CD80 or CD86 to CTLA-4 on cells overexpressing CHOK1-human CCR8 / CTLA-4. d) Compared to ipilimumab, better resistance and longer survival, and e) The in vivo antitumor effect is dose-dependently superior to that of anti-CCR8 antibodies and ipilimumab. It has at least one of the properties selected from the following.

[0102] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment is defucosylated.

[0103] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment is a human antibody or a humanized antibody.

[0104] In some embodiments, the second domain includes an anti-CTLA-4 nanobody such as GBD008-hS005-3-2.

[0105] In some embodiments, the second domain includes an amino acid sequence having at least 80% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with SEQ ID NO:22 or SEQ ID NO:23.

[0106] In some embodiments, the second domain comprises or consists of the amino acid sequence described in SEQ ID NO:22 or SEQ ID NO:23.

[0107] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment can be generated by covalently linking the first domain and the second domain of the present invention directly or via a linker.

[0108] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment further comprises one or more linkers, each optionally containing one or more amino acids selected from the group consisting of glycine and serine. Those skilled in the art can determine the linkers by conventional experiments.

[0109] In some embodiments, the one or more linkers are inserted into the C-end, N-end, or both ends of the second domain.

[0110] In some embodiments, the linker includes one or more amino acid sequences selected from GGGGSGGGGS (SEQ ID NO: 24) and GS.

[0111] In some embodiments, the linker includes GGGGSGGGGS and GS.

[0112] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment has an amino acid sequence that is at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:18 or SEQ ID NO:20, or a heavy chain having up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:18 or SEQ ID NO:20 (i.e., the full-length heavy chain sequence of bsAb-38-64-05-2 or bsAb-38-64-05-3), and SEQ ID The light chain has an amino acid sequence that is at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to NO:10, or has up to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) amino acid additions, substitutions, and / or deletions compared to SEQ ID NO:10 (i.e., the full-length light chain sequence of bsAb-38-64-05-2 or bsAb-38-64-05-3). In preferred embodiments, the substitutions are conservative substitutions.

[0113] In a preferred embodiment, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20, and a light chain having the amino acid sequence of SEQ ID NO:10.

[0114] Tables 4 and 5 show the amino acid sequences and nucleotide sequences of bsAb-38-64-05-2 and bsAb-38-64-05-3, respectively.

[0115] [Table 4-1] [Table 4-2]

[0116] [Table 5-1] [Table 5-2]

[0117] In some embodiments, the anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody, or its antigen-binding fragment provided herein may contain one or more modifications or substitutions of the one or more sequences provided herein, while maintaining specific binding affinity to CCR8. To achieve this objective, various methods known in the art can be used. For example, computer software can be used to virtually simulate the binding of the antibody to CCR8 and to identify amino acid residues that form a binding interface with the antibody. Such residues can be avoided in substitution to prevent a decrease in binding affinity, or they can be targeted for substitution to provide stronger binding.

[0118] As used herein, “conservative modified variant” or “conservative substitution” refers to the substitution of an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain stereochemistry, rigidity, etc.) so that such substitutions can be made frequently without generally altering the protein’s biological activity. Those skilled in the art will recognize that single amino acid substitutions in non-essential regions of polypeptides generally do not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). Furthermore, substitutions of structurally and / or functionally similar amino acids are less likely to disrupt biological activity. Various embodiments of the anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment according to this disclosure include polypeptide chains having sequences containing up to 0 (unchanged), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more conserved amino acid substitutions compared to specific amino acid sequences disclosed herein, such as SEQ ID NO: 7, 8, 9, or 10. The phrase “up to X” conserved amino acid substitutions as used herein includes 0 substitutions and any number of substitutions up to X substitutions (including X substitutions). Such exemplary substitutions are preferably made as shown in the following table.

[0119] [Table 6]

[0120] This disclosure also considers functionally conserved variants of the anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment. A “functionally conserved variant” is a variant that alters one or more amino acid residues in a protein without altering the conformation and function of the entire polypeptide, and includes, but is not limited to, substituting an amino acid with an amino acid having similar properties.

[0121] The anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody, or its antigen-binding fragment provided herein further comprises a constant region capable of inducing effector function. In some embodiments, the constant region includes one or more modifications to enhance antibody-dependent cytotoxicity (ADCC). In some embodiments, the anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody, or its antigen-binding fragment is defucosylated.

[0122] In some embodiments, a defucosylated antibody can enhance the effector function (e.g., ADCC) of the antibody or its antigen-binding fragment by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1x, 2x, 5x, 10x, 20x, 50x, or 100x compared to a wild-type antibody or its antigen-binding fragment.

[0123] As stated herein, “antibody or antigen-binding fragment” refers to an intact antibody or an antibody fragment having an antigen-binding moiety. Various types of antibodies or antigen-binding fragments are known in the art and can be developed based on the antigen-binding moiety of the anti-CCR8 antibody (e.g., HC64) provided herein.

[0124] In some embodiments, the anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment of the Disclosure is a human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, diabody, triabody, tetrabody, Fab fragment, F(Fab')2 fragment, scFv fragment, Fv fragment, Fab' fragment, or domain antibody.

[0125] Antibody properties This disclosure provides novel anti-CCR8 antibodies (e.g., HC64, HC64-23) or antigen-binding fragments thereof that specifically bind to CCR8, such as human CCR8, expressed on the cell surface.

[0126] This disclosure also provides novel anti-CCR8 / CTLA4 bispecific antibodies (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) or antigen-binding fragments thereof that specifically bind to CCR8 and CTLA4, such as human CCR8 and CTLA4 expressed on the cell surface.

[0127] The bispecific antibodies bsAb-38-64-05-2 / bsAb-38-64-05-3 reduce peripheral antibody exposure by simultaneously targeting these two cell surface antigens (CCR8 and CTLA-4) that overlap with TI-Tregs. By targeting two receptors that overlap with TI-Tregs, they increase localization in the TME compared to single-specific antibodies, resulting in reduced systemic T cell activation and improved response rates.

[0128] Experiments have shown that the binding of the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) to cells is increased when both target antigens are conjugated compared to single-antigen conjugation. Experiments have also demonstrated that the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) mediate TI-Treg depletion, enabling a potent antitumor response in various mouse tumor models. Furthermore, the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) did not cause toxicity in mouse irAEs models compared to ipilimumab in combination with an anti-PD-1 antibody. These data demonstrate the potential and safe clinical utility of the anti-CCR8 / CTLA4 bispecific antibodies of this disclosure (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) for the selective depletion of tumor-resident T regulatory cells.

[0129] The bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment (e.g., bsAb-38-64-05-2, bsAb-38-64-05-3) disclosed in this invention is, a) To strongly induce the ADCC reaction and kill human Treg cells or cells that overexpress CHOK1-human CCR8 / CTLA-4, b) Compared to anti-CCR8 antibodies, it reduces tumor growth and significantly extends survival time. c) Partially block CD80 or CD86-CTLA4 interactions on cells overexpressing CHOK1-human CTLA-4, but significantly block the binding of CD80 or CD86 to CTLA-4 on cells overexpressing CHOK1-human CCR8 / CTLA-4. d) Compared to ipilimumab, better resistance and longer survival, and e) The in vivo antitumor effect is dose-dependently superior to that of anti-CCR8 antibodies and ipilimumab. It has at least one of the properties selected from the following.

[0130] In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment provided herein specifically binds CCR8 and has a binding affinity (K D ) is 1 × 10 -8 Less than M, 1 x 10 -9 Less than M, or 1 × 10 -10 It is less than M. In some embodiments, K D The methyl group is 50nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or less than 1nM. In some embodiments, the bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment provided herein specifically binds to CTLA-4 and has a binding affinity (K D ) is 1 × 10 -8 Less than M, 1 x 10 -9 Less than M, or 1 × 10 -10 It is less than M. In some embodiments, K D These values ​​are 50nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or less than 1nM.

[0131] Common techniques for measuring the affinity of antibodies to antigens include, for example, ELISA, RIA, and surface plasmon resonance (SPR).

[0132] Polynucleotides and methods for their production This disclosure also provides nucleic acids encoding anti-CCR8 antibodies, anti-CCR8 / CTLA4 bispecific antibodies, or antigen-binding fragments thereof, as provided herein.

[0133] As used herein, the terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, having single-stranded or double-stranded forms. Unless otherwise specified, the term encompasses polynucleotides including known analogs of native nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, specific polynucleotide sequences also implicitly encompass their conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitution can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (see Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985), and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0134] In some embodiments, the nucleic acid encoding the anti-CCR8 antibody or its antigen-binding fragment comprises a nucleic acid encoding a heavy chain having a nucleotide sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 11 or 16, and a nucleic acid encoding a light chain having a nucleotide sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12 or 17.

[0135] In some embodiments, the nucleic acid encoding the anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment comprises a nucleic acid encoding a heavy chain having a nucleotide sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 19 or 21, and / or a nucleic acid encoding a light chain having a nucleotide sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 17.

[0136] DNA encoding anti-CCR8 antibodies or anti-CCR8 / CTLA4 bispecific antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the antibody heavy and light chains). Encoding DNA can also be obtained by synthetic methods.

[0137] The anti-CCR8 antibodies, anti-CCR8 / CTLA4 bispecific antibodies, or their antigen-binding fragments provided herein can be produced by any protein (e.g., antibody) synthesis method known in the art, particularly by chemical synthesis or preferably by recombinant expression techniques.

[0138] Recombinant expression of an antibody requires the construction of an expression vector containing the nucleic acid encoding the antibody. Once the nucleic acid encoding the antibody is obtained, a vector for generating the antibody can be produced using recombinant DNA technology. This disclosure describes the construction of an expression vector containing an antibody-coding sequence and appropriate transcription and translation regulatory elements. These methods include, but are not limited to, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination.

[0139] An expression vector is transferred to host cells using conventional techniques, and then the transfected cells are cultured using conventional techniques to produce the antibody or its antigen-binding fragment according to this disclosure.

[0140] In one embodiment, a method for producing an anti-CCR8 antibody, an anti-CCR8 / CTLA4 bispecific antibody, or its antigen-binding fragment according to the Disclosure comprises culturing host cells according to the Disclosure under conditions that enable the expression of the anti-CCR8 antibody, an anti-CCR8 / CTLA4 bispecific antibody, or its antigen-binding fragment. In a preferred embodiment, the method further comprises recovering and / or purifying the anti-CCR8 antibody, an anti-CCR8 / CTLA4 bispecific antibody, or its antigen-binding fragment obtained from the host cells and / or culture medium.

[0141] Pharmaceutical composition This disclosure provides pharmaceutical compositions comprising an anti-CCR8 antibody, an anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment provided herein, a nucleic acid provided herein, an expression vector provided herein, or an antibody-drug conjugate provided herein, and one or more pharmaceutically acceptable carriers. In preferred embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an anti-CCR8 antibody, an anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment provided herein, and one or more additional components such as a pharmaceutically acceptable carrier, vehicle, or culture medium. In some embodiments, the pharmaceutical composition may include a pharmaceutically acceptable carrier (e.g., a pharmaceutically acceptable liquid, gel, or solid carrier), an aqueous vehicle, a non-aqueous vehicle, an antimicrobial agent, an isotonic agent, a buffer, an antioxidant, an anesthetic, a suspension / dispersant, a metal ion sequestering agent or chelating agent, a diluent, an adjuvant, an excipient or non-toxic auxiliary, other components known in the art, or various combinations thereof.

[0142] In some embodiments, the composition may include a sterile diluent (e.g., sterile water or saline solution), a non-volatile oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent, an antimicrobial or antifungal agent (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thiomersal, etc.), an antioxidant (e.g., ascorbic acid or sodium bisulfite), a chelating agent (e.g., ethylenediaminetetraacetic acid), a buffer (e.g., acetate, citrate, or phosphate), and an isotonic agent (e.g., sugars (e.g., dextrose), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride), or any combination thereof. The liposome suspension may be pharmaceutically acceptable. It can also be used as a carrier (see, for example, U.S. Patent No. 4,522,811). Formulations of the composition may be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. Appropriate fluidity can be maintained as needed (e.g., in injectable formulations) by coating with, for example, lecithin or by using a surfactant. The absorption of anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody, or its antigen-binding fragment can be prolonged by including absorption-delaying agents (e.g., aluminum monostearate and gelatin). Alternatively, release control can be achieved by implants and microencapsulation delivery systems that may include biodegradable biocompatible polymers (e.g., ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoester, polylactic acid; Alza Corporation, and Nova Pharmaceutical, Inc.).

[0143] The pharmaceutical composition may be administered by any suitable method known to those skilled in the art, for example, by the parenteral and parenteral routes described above. In preferred embodiments, the pharmaceutical composition may be administered by intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, or intrasternal injection.

[0144] This disclosure also provides kits comprising an anti-CCR8 antibody, an anti-CCR8 / CTLA4 bispecific antibody, or an antigen-binding fragment thereof, as provided herein. In preferred embodiments, the kits according to this disclosure further include instructions for the use of the anti-CCR8 antibody, the anti-CCR8 / CTLA4 bispecific antibody, or an antigen-binding fragment thereof, for example, for the treatment or prevention of a disease (e.g., cancer) associated with abnormal CCR8 expression in a subject.

[0145] In some embodiments, the anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment, the nucleic acid provided herein, the expression vector provided herein, or the antibody-drug conjugate provided herein, and at least one additional therapeutic agent are administered in the same composition. In some embodiments, the anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment, the nucleic acid provided herein, the expression vector provided herein, or the antibody-drug conjugate provided herein, and at least one additional therapeutic agent are administered in two different compositions.

[0146] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) inhibitors and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent may be an indoleamine-2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0147] In some embodiments, additional therapeutic agents may include one or more inhibitors selected from HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, activated Hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

[0148] In some embodiments, the additional therapeutic agents are trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reorizin, alimta, zykadia, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, AGS-003, and The treatment may include one or more therapeutic agents selected from the following: cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0149] In some embodiments, additional therapeutic agents may include one or more therapeutic agents selected from adjuvants, TLR agonists, tumor necrosis factor (TNF)α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0150] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, or an anti-GITR antibody.

[0151] Treatment methods and use The anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment, nucleic acid, expression vector, host cell, and pharmaceutical composition according to this disclosure can be used to treat or prevent diseases associated with abnormal expression of CCR8 and / or CTLA-4, such as cancer, in a subject. In one embodiment, a method for treating or preventing a disease associated with abnormal expression of CCR8 and / or CTLA-4 in a subject comprises administering to a subject a therapeutically effective amount of the anti-CCR8 antibody, anti-CCR8 / CTLA-4 bispecific antibody or its antigen-binding fragment, nucleic acid, expression vector, host cell, or pharmaceutical composition according to this disclosure.

[0152] Furthermore, the anti-CCR8 antibody, anti-CCR8 / CTLA4 bispecific antibody or its antigen-binding fragment, nucleic acid, expression vector, host cell, and pharmaceutical composition relating to this disclosure can be used, for example, in the manufacture of pharmaceuticals and / or kits for the treatment or prevention of diseases associated with abnormal expression of CCR8 and / or CTLA-4 in subjects.

[0153] In one embodiment, the disease is cancer. In preferred embodiments, the cancers include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, colon carcinoma, anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinus natural killer cancer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium, fallopian tube cancer, ovarian cancer, and carcinoma of the Solid tumors are selected from the group consisting of cervix, vaginal cancer, vulvar cancer, testicular cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, penile cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epithelioid carcinoma, squamous cell carcinoma, pediatric solid tumors, environment-induced cancers, virus-associated cancers, virus-derived cancers, advanced cancers, unresectable cancers, metastatic cancers, refractory cancers, recurrent cancers, and any combination thereof. In another preferred embodiment, the cancer is a hematological malignancy selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), T-cell lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal immunoglobulinemia of unknown meaning (MGUS), progressive, metastatic, refractory and / or recurrent hematological malignancies, and any combination of the hematological malignancies. [Examples]

[0154] The present invention has been described with reference to the following embodiments, but these embodiments should not be construed as limiting.

[0155] Example 1: Production of anti-human CCR8 antibody Anti-human CCR8 antibodies were generated by immunizing inbred SJL mice with one or more antigens from the following: 1) HEK293F cells overexpressing recombinant human CCR8, and 2) a full-length human CCR8 expression vector in pCP (pCP-hCCR8).

[0156] Plasmid DNA immunization was performed by intraperitoneal injection (ip). Cellular immunization was performed by intraperitoneal injection (ip) of HEK293F cells transfected with human CCR8. One group of animals was first immunized with HEK293F cells overexpressing human CCR8, followed by DNA-enhanced immunization as follows: Prepared antigens were injected into the peritoneal cavity of mice every two weeks. Animals that produced anti-CCR8 titers were given 1 × 10⁶ 7 HEK293F cells overexpressing 10¹ human CCR8 cells, or pCP-hCCR8+1×10¹ cells. 7 Human CCR8-overexpressing HEK293F cells were injected intraperitoneally (ip). The spleen and / or lymph nodes were collected, and splenic and / or lymph node cells were used to prepare hybridomas.

[0157] To select animals that produced CCR8-binding antibodies, serum from immunized animals was examined by ELISA to determine binding to cells overexpressing CHOK1-human CCR8 and to CHOK1-blank cells. In short, polyclonal serum binding was evaluated by incubating cells overexpressing CHOK1-human CCR8 with diluted serum samples. The cells were then washed, and binding was detected using Aleax Fluor® 488 donkey anti-mouse IgG (H+L) antibody. Flow cytometry analysis was performed using fluorescence microplate cytometry (Acumen Explorer microplate cytometer). Mice with the highest titer anti-CCR8 antibody were used for fusion. Fusion was performed as follows. The hybridoma supernatant was examined for anti-CCR8 activity by flow cytometry.

[0158] Mouse-derived spleen cells and / or lymphocytes were fused to the Sp2 / 0-Ag14 cell line by electrical fusion. The supernatant, which was evaluated as positive for mouse IgG antibodies, was then screened for anti-CCR8 IgG antibodies by flow cytometry. Subcloning was then performed on hybridomas secreting anti-CCR8 antibodies using the limiting dilution method. Stable subclones were cultured in vitro, and binding to human CCR8 was measured by flow cytometry. Clones confirmed to be positive were scaled up, and small amounts of antibody were produced in tissue medium for further characterization. Antibodies from the hybridoma supernatant were purified by protein A column chromatography.

[0159] Several anti-CCR8 antibodies, including HC64, were obtained.

[0160] Example 2: Humanization of anti-CCR8 antibody The heavy and light chain variable regions (VH and VL) sequences of mouse anti-human CCR8 antibodies were used to search the IMGT database for the corresponding variable region sequences of human CCR8 antibodies. The mouse antibody heavy and light chain variable regions were compared with those of human antibodies using MOE software. Germline genes with high homology to the mouse antibody were selected as templates, and the CDR of the mouse antibody was transplanted into the corresponding human template to form the variable region sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. To ensure affinity to the original mouse antibody, key amino acids in the skeleton sequences were reverted and mutated to the corresponding amino acids of the mouse antibody according to structural analysis of the heavy and light chain variable regions of the selected human antibody and the mouse antibody (i.e., humanized anti-CCR8 antibody). The amino acid residues of the CDR region were determined and annotated using the Kabat numbering system.

[0161] The humanized antibody for antibody HC64 was designated HC64-23.

[0162] Example 3: Production of bsAb-38-64-05-2, bsAb-38-64-05-3, and defucosylated (AF) anti-CCR8 monoclonal antibodies. To generate defucosylated (AF) bsAb-38-64-05-2, bsAb-38-64-05-3, or anti-CCR8 monoclonal antibodies, expression plasmids pCDNA 3.4 provided by Biometas (Shanghai) Limited were transformed into E. coli for growth at appropriate scales. The NucleoBond Xtra Maxi Plus EF kit was used for large-scale plasmid generation. Constructs containing the heavy and light chains of each antibody were transfected into FUT8-KO CHO cells with PEI. Acclimatization medium was collected 9–11 days post-transfection. Acclimatization medium expressing the target antibody was collected by centrifugation and filtration and then loaded onto a protein A affinity column. Purified antibodies were analyzed by SDS-PAGE, SEC-HPLC, and endotoxin measurement.

[0163] Example 4: Binding of anti-CCR8 or anti-CTLA-4 antibody to cell surface antigens Regarding CCR8 binding, the binding of the antibody of this disclosure was evaluated by incubating cells overexpressing Raji-human CCR8 with serially diluted (1:5) antibodies. After washing the cells, binding was detected by flow cytometry (BD LRFortessa) using APC anti-human IgG Fc antibody (Jackson Lab, catalog no. 309-605-008). Compared to a reference (i.e., 4A19 (anti-CCR8, BMS)), the antibody of this disclosure showed equivalent or superior binding ability to cell lines overexpressing Raji-human CCR8 (Figure 1 and Figure 10A).

[0164] For CTLA-4 binding, the binding of the antibody disclosed herein was evaluated by incubating cells overexpressing CHOK1-human CTLA-4 with serially diluted (1:5) antibodies. Similar to the CCR8 binding procedure, the cells were ultimately analyzed by flow cytometry. Compared to a baseline (i.e., ipilimumab), the antibody disclosed herein showed weak binding to cell lines overexpressing CHOK1-human CTLA-4 (Figure 10B).

[0165] For CCR8 / CTLA-4 binding, the binding of the antibody disclosed herein was evaluated by incubating cells overexpressing CHOK1-human CCR8 / CTLA-4 with serially diluted (1:5) antibodies. Similar to the CCR8 binding procedure, the cells were ultimately analyzed by flow cytometry. Compared to the parental anti-CCR8 antibody (GBD008-hS 005-3-2), the antibody disclosed herein showed equivalent or superior binding ability to cell lines overexpressing CHOK1-human CCR8 / CTLA-4 (Figure 10C).

[0166] Example 5: Blocking of CCR8-CCL1 binding The blocking effect of the antibody of this disclosure against the CCL1 ligand was evaluated by incubating cells overexpressing Raji-human CCR8 or Raji-human CCR8 / CTLA-4 with serially diluted (1:5) antibodies. The cells were washed twice, 0.4 μg / mL of human I-309-AF 647 was added to the designated row of the measurement plate, mixed well, and incubated at 4°C for 30 minutes. After incubation, the cells were washed once and resuspended in 120 μL of FACS buffer (BioLegend, catalog no. 420201). The measurement plates were measured by flow cytometry. The results are shown in Figures 2 and 11. The antibody of this disclosure significantly blocked the binding of CCL1 to CCR8.

[0167] Example 6: Binding and blockade of anti-CCR8 antibody in human Treg cells For the isolation of human Treg cells, see EasySep. TM Treg cells (CD4+CD127lowCD25+) were isolated from human peripheral blood mononuclear cells (PBMCs) (Milestone® Biotechnologies) using the Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (Stem Cell, catalog number 18063).

[0168] Regarding Treg cell induction, StemSep TM Human CD14-positive selection kit (StemCell, catalog number 14758) was used to isolate human monocytes from PBMCs, and human T cell transacting was performed. TM Treg cells were mixed in a 1:1 ratio with hIL-2 (10 ng / mL, Miltenyi Biotec, catalog no. 130-111-160) and hIL-2 (10 ng / mL, PeproTech, catalog no. 200-02). After incubation for 3 days, monocytes were removed, and the Treg cells were continued to culture for a further 10–13 days for expansion.

[0169] Regarding the binding and blocking of anti-CCR8 antibodies, the antibody of this disclosure showed superior binding ability to human Treg cells compared to the reference (i.e., 4A19 (anti-CCR8, BMS)) (Figure 3) and significantly blocked the binding of CCL1 to CCR8 (Figure 4).

[0170] Example 7: Cross-binding of anti-CCR8 antibody with cyno CCR8 Cross-binding between anti-CCR8 antibody and cyno-CCR8 was evaluated by incubating 293T cells overexpressing cyno-CCR8 with serially diluted (1:10) antibody. After washing the cells, binding was detected by flow cytometry using APC anti-human IgG Fc antibody. The results are shown in Figures 5 and 14A. The anti-CCR8 antibody described herein showed remarkable binding ability to cyno-CCR8, which is advantageous for subsequent toxicological evaluation.

[0171] Example 8: Cross-binding of anti-CTLA-4 antibody with cyno-CTLA-4 Cross-binding of anti-CTLA-4 antibody to cyno-CTLA-4 was measured using an ELISA assay. Each step of the assay was performed by incubating at room temperature for 1 hour with appropriate reagents, except that the initial plate coating step was performed overnight at 4°C. Between steps, the plates were washed three times with PBS containing 0.05% Tween 20.

[0172] Plates were coated with 1 μg / mL cyno CTLA-4 (Sino Biological, catalog number 90213-C08H) and subsequently blocked with 2% BSA. Next, the plates were incubated with serially diluted (1:5) anti-CTLA-4 mAbs, and antibodies conjugated with HRP-hFc (Sigma, catalog number A0170) and TMB substrate (Cell Signaling, catalog number 7004P6) were detected. The results are shown in Figure 14B. The anti-CTLA-4 antibodies described herein showed remarkable binding affinity to cyno CTLA-4, which is advantageous for subsequent toxicological evaluation.

[0173] Example 9: ADCC of bsAb-38-64-05-2 or anti-CCR8 antibody Cytotoxic activity was evaluated using FACS. Effector cells, human PBMCs, were obtained from individual human donors and cultured overnight in 10 ng / mL hIL-2. Target human Treg cells or cells overexpressing CHOK1-human CCR8 / CTLA-4 were labeled with Celltrace Far red (Thermo, catalog no. C34564) at 37°C for 10 minutes, washed twice with RPMI 1640 medium (Gibco, catalog no. A10491-01) containing 10% FBS (Gibco, catalog no. 10099-141), and plated in a 96-well round-bottom plate in an effector cell to target cell ratio (20:1). Serially diluted antibodies (1:5) were added to the designated rows of the analysis plate. After incubation at 37°C for 2 hours, 2 μl of propidium iodide staining solution (BD Biosciences, catalog no. 556547) was added to each well, and the dead cells were stained at room temperature for 10 minutes. Cells were directly analyzed by flow cytometry. The results are shown in Figures 6 and 15, and the antibodies of this disclosure, including HC64-23-AF and bsAb-38-64-05-2, induced a strong ADCC reaction to kill human Treg cells or cells overexpressing CHOK1-human CCR8 / CTLA-4.

[0174] Example 10: Effect study in the MC38 model The antitumor activity of the anti-CCR8 antibody (defucosylated hIgG 1) was measured in the MC38 mouse colon adenocarcinoma model. The study involved 6-8 week old female C57BL / 6-CCR8 mice. em3(hCCR8) / Smoc Each mouse (Shanghai Model Organisms Center, Inc.) has 1 x 10 6Individual MC38 tumor cells were subcutaneously transplanted, and six days after transplantation, mice were randomized into treatment groups of six. Antibodies (anti-hCCR8-hIgG1-AF or control hIgG1-AF antibody) were administered intraperitoneally via injection at a rate of 3 mpk / mouse in 200 μL volume on days 0, 4, 7, and 11 post-transplant. Tumor measurements were recorded twice weekly until up to 16 days post-transplant, after which the mice were euthanized. The results are shown in Figure 7. Compared to the negative control (human IgG1-AF), both 4A19 (anti-CCR8, BMS)-AF and HC64-23-AF reduced tumor growth and significantly extended survival (Figure 8), with HC64-23-AF showing superior efficacy compared to 4A19 (anti-CCR8, BMS)-AF.

[0175] Example 11: Kinetic parameters of antibodies detected by Octet Kinetic assays were performed by first capturing mAbs with an anti-human Fc(AHC) Octet(Sartorius) biosensor, followed by at least two baseline steps of 30 seconds each in HBS-EBT buffer. The biosensor containing the captured mAbs was then immersed in wells containing different concentrations of antigen for 4–6 minutes, followed by dissociation in HBS-EBT buffer for 10–15 minutes. To compensate for the spontaneous dissociation of the captured mAbs, the sensor was also immersed in a well containing HBS-EBT buffer to enable single-reference subtraction. Binding sensograms were collected using the high-sensitivity 16-channel detection mode on the Octet HTX biosensor. Unless otherwise specified, new AHC biosensors were used without a regeneration step. The kinetic parameters of the antibodies disclosed are shown in Table 7.

[0176] [Table 7]

[0177] Example 12: Blocking of CTLA4-CD80 / 86 binding Blockade of CD80 or CD86 ligands by the antibodies of this disclosure was evaluated by incubating cells overexpressing CHOK1-human CTLA-4 or CHOK1-human CCR8 / CTLA-4 with serially diluted (1:5) antibodies in the presence of CD80 Fc-AF647 (Kactus, catalog no. B71-HM280) or CD86 Fc-AF647 (Kactus, catalog no. B72-HM286). After incubation at 4°C for 30 minutes, the cells were washed once and resuspended in 120 μL of FACS buffer. The measurement plates were measured by flow cytometry. The antibodies described herein partially blocked CD80 or CD86-CTLA4 interactions on cells overexpressing CHOK1-human CTLA-4 (Figures 12A and 13A), but significantly blocked the binding of CD80 or CD86 to CTLA-4 on cells overexpressing CHOK1-human CCR8 / CTLA-4 (Figures 12B and 13B).

[0178] Example 13: irAE model in 10-day-old C57BL / 6 CCR8 / CTLA-4 double knock-in mice Young CCR8 / CTLA-4 double knock-in mice were co-treated with a specified antibody at a dose of 100 or 300 μg / mouse / injection, respectively, and an anti-mPD1 antibody (BioXcell, catalog number BE0146) at a dose of 100 μg / mouse / injection, for a total of 7 injections, once every 3 days starting from postnatal day 10. To avoid cage variability, mice in the same cage were tagged separately and treated with different antibodies. To avoid sex and weight variability, female mice with similar weights (4.5–5.3g) were used in all studies, although a similar trend was observed in male mice. For Kaplan-Meier survival analysis, the endpoint was considered reached when the mouse became mortal or died. All experiments were performed double-blind. As shown in Figures 16 and 17, the antibodies of this disclosure, including bsAb-38-64-05-2, showed superior resistance and longer survival than ipilimumab.

[0179] Example 14: Effect study in the MB49 model The antitumor activity of bsAb-38-64-05-3 was measured in a bladder cancer model of MB49 mice. The mice used were 6-8 week old male C57BL / 6-CCR8. em3(hCCR8) / Smoc CTLA-4 em1(hCTLA4)Smoc A mouse (Shanghai Model Organisms Center, Inc.) has 1 x 10 6 Each individual MB49 tumor cell was subcutaneously transplanted, and six days after transplantation, mice were randomized into treatment groups of six. Antibodies (bsAb-38-64-05-3 or control hIgG1-AF antibody) were administered intraperitoneally via 200 μL volume at a dose of 10 mpk / mouse on days 0, 4, 7, and 11 post-transplant. Tumor measurements were recorded twice weekly until a maximum of 32 days post-transplant, after which the mice were euthanized. Compared to the negative control (human IgG1-AF), bsAb-38-64-05-3 reduced tumor growth (Figure 18). Furthermore, bsAb-38-64-05-3 demonstrated dose-dependent superiority of in vivo antitumor efficacy compared to 4A19 (anti-CCR8)-AF and ipilimumab (Figure 18).

[0180] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to illustrate, and not limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0181] References 1. Sakaguchi, S., et al., Regulatory T cells and immune tolerance. Cell, 2008. 133(5): p. 775-87. 2. Lee, HM, JL Bautista, and CS Hsieh, Thymic and peripheral differentiation of regulatory T cells. Adv Immunol, 2011. 112: p. 25-71. 3. Tanaka, A. and S. Sakaguchi, Targeting Treg cells in cancer immunotherapy. Eur J Immunol, 2019. 49(8): p. 1140-1146. 4. Shimizu, J., S. Yamazaki, and S. Sakaguchi, Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J Immunol, 1999. 163(10): p. 5211-8. 5. Onizuka, S., et al., Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res, 1999. 59(13): p. 3128-33. 6. Yamaguchi, T. and S. Sakaguchi, Regulatory T cells in immune surveillance and treatment of cancer. Semin Cancer Biol, 2006. 16(2): p. 115-23. 7. Chen, B.J., et al., Immunotherapy of Cancer by Targeting Regulatory T cells. Int Immunopharmacol, 2022. 104: p. 108469. 8. Sledzinska, A., et al., Negative immune checkpoints on T lymphocytes and their relevance to cancer immunotherapy. Mol Oncol, 2015. 9(10): p. 1936-65. 9. Jago, C.B., et al., Differential expression of CTLA-4 among T cell subsets. Clin Exp Immunol, 2004. 136(3): p. 463-71. 10. Montler, R., et al., OX40, PD-1 and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and neck cancer. Clin Transl Immunology, 2016. 5(4): p. e70. 11. Sutmuller, R.P., et al., Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med, 2001. 194(6): p. 823-32. 12. Arce Vargas, F., et al., Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies. Cancer Cell, 2018. 33(4): p. 649-663.e4. 13. Du, X., et al., A reappraisal of CTLA-4 checkpoint blockade in cancer immunotherapy. Cell Res, 2018. 28(4): p. 416-432. 14. Hodi, F.S., et al., Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med, 2010. 363(8): p. 711-23. 15. Valsecchi, M.E., Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med, 2015. 373(13): p. 1270. 16. Calabrese, L.H., C. Calabrese, and L.C. Cappelli, Rheumatic immune-related adverse events from cancer immunotherapy. Nat Rev Rheumatol, 2018. 14(10): p. 569-579. 17. Hodi, F.S., Overcoming immunological tolerance to melanoma: Targeting CTLA-4. Asia Pac J Clin Oncol, 2010. 6 Suppl 1: p. S16-23. 18. Bertrand, A., et al., Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis. BMC Med, 2015. 13: p. 211. 19. Ellmark, P., et al., Tumor-directed immunotherapy can generate tumor-specific T cell responses through localized co-stimulation. Cancer Immunol Immunother, 2017. 66(1): p. 1-7. 20. Plitas, G., et al., Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity, 2016. 45(5): p. 1122-1134. 21. De Simone, M., et al., Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells. Immunity, 2016. 45(5): p. 1135-1147. 22. Whiteside, S.K., et al., CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function. Immunology, 2021. 163(4): p. 512-520.

Claims

1. SEQ ID NO: Heavy chain complementarity-determining region (HCDR) 1 having an amino acid sequence with at least 80% identity with 1, SEQ ID NO: HCDR2 having an amino acid sequence that is at least 80% identical to 2. HCDR3 having an amino acid sequence that is at least 80% identical to SEQ ID NO: 3, SEQ ID NO: Light chain complementarity-determining region (LCDR) 1 having an amino acid sequence with at least 80% identity with 4, LCDR2 having an amino acid sequence that is at least 80% identical to SEQ ID NO: 5, and LCDR3 contains an amino acid sequence having at least 80% identity with SEQ ID NO:

6. An antibody or its antigen-binding fragment that specifically binds to human CC motif chemokine receptor 8 (CCR8).

2. HCDR1 containing or consisting of the amino acid sequence described in SEQ ID NO: 1, HCDR2 containing or consisting of the amino acid sequence described in SEQ ID NO: 2, HCDR3 containing or consisting of the amino acid sequence described in SEQ ID NO: 3, LCDR1 containing or consisting of the amino acid sequence described in SEQ ID NO: 4, LCDR2, which contains or consists of the amino acid sequence described in SEQ ID NO: 5, and Contains or comprises LCDR3, which includes the amino acid sequence described in SEQ ID NO:

6. The antibody or antigen-binding fragment according to claim 1.

3. A heavy chain variable region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 7 or SEQ ID NO: 13, and It includes a light chain variable region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 8 or SEQ ID NO:

14. The antibody or antigen-binding fragment according to claim 1.

4. A heavy chain variable region comprising or consisting of the amino acid sequence described in SEQ ID NO: 7 or SEQ ID NO: 13, and A light chain variable region comprising or consisting of the amino acid sequence described in SEQ ID NO: 8 or SEQ ID NO: 14, The antibody or antigen-binding fragment according to claim 3.

5. Heavy chains having amino acid sequences of SEQ ID NO: 9 or SEQ ID NO: 15, and SEQ ID NO: Contains a light chain having an amino acid sequence of 10. The antibody or antigen-binding fragment according to any one of claims 1 to 4.

6. Human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F(ab') 2 An antibody or its antigen-binding fragment according to any one of claims 1 to 5, which is a fragment, scFv fragment, Fv fragment, Fab' fragment, or domain antibody.

7. An antibody or its antigen-binding fragment that competes for binding to CCR8 with the antibody or its antigen-binding fragment according to any one of claims 1 to 6.

8. A bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment comprising the antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a first domain that specifically binds to CCR8 and a second domain that specifically binds to CTLA-4.

9. a) To strongly induce the ADCC reaction and kill human Treg cells or cells that overexpress CHOK1-human CCR8 / CTLA-4, b) Compared to anti-CCR8 antibodies, it reduces tumor growth and significantly extends survival, and / or c) Partially block CD80 or CD86-CTLA4 interaction on cells overexpressing CHOK1-human CTLA-4, but significantly block CD80 or CD86 binding to CTLA-4 on cells overexpressing CHOK1-human CCR8 / CTLA-4. A bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment according to claim 8, having at least one of the properties selected from the above.

10. A defucosylated, bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment according to claim 8.

11. A bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment according to any one of claims 8 to 10, which is a human antibody or a humanized antibody.

12. The bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment according to any one of claims 8 to 11, wherein the second domain comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 22 or SEQ ID NO:

23.

13. The bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment according to any one of claims 8 to 12, wherein the second domain comprises or consists of the amino acid sequence described in SEQ ID NO: 22 or SEQ ID NO:

23.

14. A bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment according to any one of claims 8 to 13, further comprising one or more linkers optionally containing one or more amino acids selected from the group consisting of glycine and serine.

15. The bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment according to claim 14, wherein the linker comprises one or more amino acid sequences selected from GGGGSGGGGS (SEQ ID NO: 24) and GS.

16. The bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment according to claim 15, wherein the linker comprises GGGGSGGGGS and GS.

17. Heavy chains having amino acid sequences of SEQ ID NO: 18 or SEQ ID NO: 20, A bispecific anti-CCR8 / CTLA-4 antibody or its antigen-binding fragment according to any one of claims 8 to 16, comprising a light chain having the amino acid sequence SEQ ID NO:

10.

18. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or a bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment thereof according to any one of claims 8 to 17.

19. SEQ ID NO: Nucleic acid encoding a heavy chain having a nucleotide sequence of 11 or 16, SEQ ID NO: Containing nucleic acids encoding a light chain having a nucleotide sequence of 12 or 17, The nucleic acid according to claim 18.

20. SEQ ID NO: Nucleic acid encoding a heavy chain having a nucleotide sequence of 19 or 21, and SEQ ID NO: Contains nucleic acids encoding a light chain having a nucleotide sequence of 17, The nucleic acid according to claim 18.

21. An expression vector comprising the nucleic acid according to any one of claims 18 to 20.

22. A host cell comprising the expression vector described in claim 21.

23. An antibody or antigen-binding fragment according to any one of claims 1 to 7, a bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment according to any one of claims 8 to 17, a nucleic acid according to any one of claims 18 to 20, or an expression vector according to claim 21, and A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

24. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or a bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment thereof according to any one of claims 8 to 17, comprising the step of culturing a host cell according to claim 22 under conditions that enable the expression of the antibody or antigen-binding fragment thereof.

25. A method for treating a disease or illness associated with abnormal expression of CCR8 and / or CTLA-4 in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 7, a bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment according to any one of claims 8 to 17, a nucleic acid according to any one of claims 18 to 20, an expression vector according to claim 21, a host cell according to claim 22, or a pharmaceutical composition according to claim 23.

26. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 7, or a bispecific anti-CCR8 / CTLA-4 antibody or antigen-binding fragment according to any one of claims 8 to 17, in the manufacture of a pharmaceutical product for the treatment of a disease associated with abnormal expression of CCR8 and / or CTLA-4 in a subject.

27. The method or use according to claim 25 or 26, wherein the disease is cancer.

28. The aforementioned cancer, Squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, esophageal cancer, stomach cancer, gastrointestinal cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, colon carcinoma, anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinus natural killer cancer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium, fallopian tube cancer, ovarian cancer, carcinoma of the Solid tumors selected from the group consisting of cervix, vaginal cancer, vulvar cancer, testicular cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, penile cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epithelioid carcinoma, squamous cell carcinoma, pediatric solid tumors, environment-induced cancers, virus-associated cancers, virus-derived cancers, advanced cancers, unresectable cancers, metastatic cancers, refractory cancers, recurrent cancers, and any combination thereof, or The method or use of claim 27, wherein the hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), T-cell lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal immunoglobulinemia of unknown meaning (MGUS), progressive, metastatic, refractory and / or recurrent hematological malignancies, and any combination thereof.

29. The method or use of claim 27, wherein the cancer is bladder cancer.

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