Antibodies that bind to human CCR8

Abstract

The present disclosure provides an isolated antibody that specifically binds to C-C motif chemokine receptor 8 (CCR8) expressed on the surface of cells and exhibits various functional properties including desirable properties for diagnostic antibodies. These properties include binding with high affinity and specificity to CCR8-expressing cells, such as tumor-infiltrating activated CD4 + FOXP3 high Tregs, etc., and binding to a human CCR8 (hCCR8) epitope that is outside the N-terminal domain of hCCR8 to which most therapeutic anti-CCR8 antibodies bind.

Description

Technical Field

[0001] Throughout this application, various publications are cited in parentheses by author name and date, or by patent number or patent publication number. All citation information regarding these publications can be found at the end of this specification, near the end of the claims. The disclosures of these publications are incorporated herein by reference in their entirety as of the date of the present invention described and claimed herein, to more fully describe the state of the art known to those skilled in the art. However, these disclosures are incorporated into this application only to the extent that there is no conflict between the information incorporated by reference and the information provided by the explicit disclosure in this application. In particular, even though a reference is cited herein, such reference should not be construed as an admission that the reference is prior art to the present invention.

[0002] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 365,255, filed May 24, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing This application includes a sequence listing submitted electronically via the Patent Center in ST.26 compliant XML format, which is incorporated herein by reference in its entirety. A copy of ST.26 was created on May 18, 2023, named 20230518_SEQL_13429WOPCT.xml, and its size is 48,452 bytes.

[0004] The present invention mainly relates to monoclonal antibodies (mAbs) that specifically bind to human C-C motif chemokine receptor 8 (hCCR8), as well as methods related to using such antibodies (Abs) including detecting and quantifying the expression of hCCR8 on the surface of cells, estimating receptor engagement by therapeutic anti-CCR8 mAbs, and measuring the depletion of CCR8-expressing regulatory T cells (Tregs) mediated by therapeutic anti-CCR8 mAbs. The mAbs according to the present invention, first disclosed herein (the Abs of the present invention), bind to an epitope distinct from the epitope located in the N-terminal region of CCR8 that has been shown to bind to a particular therapeutic anti-CCR8 mAb; thus, the binding of any of the Abs of the present invention to CCR8 is not affected by the presence of an Ab that binds to the N-terminal epitope.

Background Art

[0005] Immune checkpoint molecules, such as PD-1, PD-L1, CTLA-4, or LAG-3, etc., are known to suppress host anti-tumor immunity within the tumor microenvironment, but by blocking them, immunotherapy that stimulates the activity of cytotoxic T cells has achieved great success in the treatment of various solid tumors and hematological malignancies (Pardoll, 2012; Lesokhin et al., 2015; Baumeister et al., 2016; Pianko et al., 2017). However, even in cancers where this treatment is effective, generally less than about 15% of patients obtain long-term benefits from treatment with checkpoint inhibitors (Haslam and Prasad, 2019), and checkpoint inhibitors have proven to be less effective in certain cancers including breast cancer and prostate cancer. Therefore, there is an urgent need for biomarkers that can be reliably used for both predicting which cancers or patients are suitable for treatment with which immunotherapeutic agents and monitoring the mechanistic course of treatment at the molecular level.

[0006] When immunosuppressive mechanisms, particularly those mediated by regulatory T cells (Tregs), are prolonged, resistance to treatment with checkpoint inhibitors in certain cancers or in certain patients may be observed (Fares et al., 2019; Han et al., 2019). Therefore, reducing the activity or number of tumor-infiltrating Tregs has been recognized as an attractive approach for reversing immunosuppression and enhancing antitumor immunity (Finotello and Trajanoski, 2017; Han et al., 2019). It has been demonstrated in recent years that the expression of CCR8 is selectively upregulated in tumor-resident Tregs within multiple cancers (De Simone et al., 2016; Plitas et al., 2016), and CCR8 is an attractive target for depleting tumor-resident Tregs for the purpose of enhancing antitumor immunity.

[0007] PCT Publication No. WO2021 / 194942 specifically binds with high affinity to hCCR8 expressed on the cell surface in various mouse tumor models when administered to mice as a monotherapy or in combination therapy with checkpoint blockers, and CCR8 + mediates the depletion of tumor-infiltrating Tregs and discloses several human or humanized mAbs that potently inhibit tumor growth. One of these mAbs, A419, is currently in Phase I / II clinical trials (NCT04895709; https: / / clinicaltrials.gov / ct2 / show / NCT04895709) and has been shown to bind to an epitope within the N-terminal region of hCCR8.

[0008] As disclosed herein, the majority of mAbs generated targeting cell surface-expressed hCCR8 immunogens bind to the N-terminal epitope. Several recent publications describing the generation of therapeutic anti-CCR8 Abs, such as PCT Publication Nos. WO2020 / 138489, WO2021 / 142002, WO2021 / 152186, WO2021 / 163064, WO2021 / 194942, WO2021 / 260209, and WO2022 / 136649, also describe Abs that bind to epitopes within the N-terminal domain. In contrast, the present invention relates to several mAbs that bind to epitopes other than the N-terminal epitope of mAb4A19 described in WO2021 / 194942 and do not compete with mAb4A19 for binding to CCR8. This mAb, herein referred to as a "non-competing" mAb because it does not have the ability to compete with therapeutic mAb4A19 for binding to hCCR8, can be used in many pharmaceutical applications, including detection of hCCR8 on the surface of cells, measurement of its expression, measurement of its receptor occupancy (RO), and detection of depletion of CCR8-expressing cells, even in the presence of a therapeutic anti-hCCR8 Ab that binds to the N-terminal epitope. These properties of the mAbs disclosed herein make the mAbs useful for several purposes, including measurement of depletion of CCR8-expressing Tregs mediated by anti-CCR8 therapeutic Abs, as well as various diagnostic and biomarker applications.

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present disclosure provides an isolated Ab, preferably an mAb, of the invention that exhibits various functional properties including desirable properties for diagnostic Abs that specifically bind to CCR8 expressed on the surface of cells, such as human CCR8 (hCCR8), and can be used to measure CCR8 expression in patients treated with therapeutic anti-CCR8 Abs. These properties include CCR8-expressing cells, such as tumor-infiltrating activated CD4 + FOXP3 highBinding to Tregs and other cells with high affinity and specificity, and binding to an hCCR8 epitope distinct from the epitope within the N-terminal domain of hCCR8 to which therapeutic anti-CCR8 Abs, such as mAb4A19, bind.

Means for Solving the Problems

[0010] Specifically, the present disclosure provides an mAb or an antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of a cell and binds to an epitope other than the epitope within the N-terminal domain of hCCR8. mAb4A19 (WO2021 / 194942) binds to at least one amino acid within the peptide having the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73), and has been shown to bind to the N-terminal epitope containing, in a preferred embodiment, all 11 amino acids. In certain embodiments, the N-terminal epitope to which the anti-hCCR8 mAb of the present disclosure does not bind contains at least one amino acid within the peptide having the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73), and includes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the amino acids within the peptide having the sequence represented by SEQ ID NO: 73. In certain preferred embodiments, the N-terminal epitope to which the anti-hCCR8 mAb of the present disclosure does not bind is the sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21It contains a peptide having (Sequence Number 2). In other preferred embodiments, the N-terminal epitope to which the anti-hCCR8 mAb of the present disclosure does not bind is the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It contains a peptide having (Sequence Number 73). In other preferred embodiments, the N-terminal epitope to which the anti-hCCR8 mAb of the present disclosure does not bind is the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It is composed of a peptide having (Sequence Number 73).

[0011] In certain preferred embodiments, the binding of the Ab of the invention or its antigen-binding portion to hCCR8 is not affected by the presence of an Ab that binds to an epitope, such as an N-terminal epitope, for example, the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It contains at least one amino acid within the peptide having (Sequence Number 73), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of such amino acids, etc. In certain embodiments, the amino acid Y 15 or Y 17 , preferably Y 15 and Y 17All of them are sulfated. In a further embodiment, the Ab that binds to the N-terminal epitope is a mAb comprising the six CDRs (SEQ ID NOs: 53-58), the heavy chain and / or the heavy chain variable region (SEQ ID NOs: 9 and / or 16), or the heavy chain and / or the light chain (SEQ ID NOs: 65 and / or 72) of mAb4A19 as described in WO2021 / 194942. In a further embodiment, the Ab that binds to the N-terminal epitope is mAb4A19 comprising the heavy chain and / or the light chain (SEQ ID NOs: 65 and / or 72) of mAb4A19.

[0012] In certain preferred embodiments, the mAb or antigen-binding portion thereof that binds to an epitope other than the N-terminal epitope is a mAb or antigen-binding portion thereof comprising the six CDRs, the heavy chain and / or the light chain variable regions, or the heavy chain and / or the light chain of the mAb named herein as 25T40, 21C17, 28P3, 22B13, 33H18, or 23A14.

[0013] The disclosed invention also provides a labeled Ab or antigen-binding portion thereof comprising the mAb of the invention and a detectable label. In different embodiments, the detectable label is a fluorophore, a chromophore, an enzyme, a radioisotope, a micropolymer, or a metal.

[0014] The present disclosure further provides a method for generating a first Ab (e.g., an Ab of the present invention) that does not bind to a defined epitope on an antigen (e.g., the N-terminal epitope of hCCR8) or does not cross-compete with a second Ab for binding (e.g., Clone L263G8 against hCCR8 commercialized by BioLegend, mAb433H marketed by BD Bioscience, or any of the mAbs 4A19, 18Y12, 8D55, 10R3, 14S15, and 14S15h described in WO2021 / 194942). The method includes immunizing a vertebrate with an immunogen that expresses the antigen and also expresses a second Ab or an antigen-binding portion thereof that specifically binds to the epitope, or a component of the cell line, wherein binding of the second Ab or its antigen-binding portion to the epitope shields the epitope from the vertebrate's immune system and reduces the production of Abs that bind to the epitope, thereby causing the production of a first Ab that does not bind to the epitope or does not cross-compete with the second Ab for binding to the epitope.

[0015] The present disclosure also provides a method for measuring depletion of the number of Tregs in a subject, comprising: (a) determining a baseline percentage (%) of T cells that are CCR8-expressing Tregs in a first test tissue within or taken from the subject, the first test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering a treatment to the subject; and (c) determining the percentage (%) of T cells that are CCR8-expressing Tregs in a second test tissue within or taken from the subject during or after the treatment, wherein a decrease in the percentage (%) of T cells that are CCR8-expressing Tregs in the second test tissue indicates depletion of the number of Tregs in the test tissue. In certain preferred embodiments, the treatment administered to the subject is a treatment for cancer. In a further preferred embodiment, the treatment for cancer comprises administering to the subject, as a monotherapy or in combination therapy with another anti-cancer therapy, an anti-CCR8 Ab or an antigen-binding portion thereof for Treg depletion therapy.

[0016] A method for predicting the efficacy of a therapeutic Treg-depleting anti-CCR8 Ab or an antigen-binding portion thereof when treating cancer in a subject, comprising: (a) determining the percentage of T cells that are CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) comparing the percentage of T cells that are CCR8-expressing Tregs with a pre-determined threshold; and (c) predicting the efficacy of the therapeutic anti-CCR8 Ab, wherein a percentage of T cells that are CCR8-expressing Tregs above the threshold indicates that the therapeutic Ab or an antigen-binding portion thereof is effective when treating the subject, and a percentage of T cells that are CCR8-expressing Tregs below the threshold indicates that the therapeutic Ab or an antigen-binding portion thereof is not effective when treating the subject. The method is also disclosed by the present disclosure.

[0017] The present invention is also related to a method for treating cancer in a patient. Accordingly, the present disclosure provides a method for treating cancer in a subject, comprising: (a) (i) determining the percentage of T cells that are CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) comparing the percentage of T cells that are CCR8-expressing Tregs with a pre-determined threshold; (iii) selecting the subject as a suitable candidate for immunotherapy using a therapeutic anti-CCR8 Ab or an antigen-binding portion thereof based on an evaluation that the percentage of T cells that are CCR8-expressing Tregs in the cells of the test tissue exceeds the pre-determined threshold; and (b) administering to the selected subject a composition comprising a therapeutically effective amount of the therapeutic anti-CCR8 Ab or an antigen-binding portion thereof.

[0018] The present disclosure is a method for treating cancer in a subject, comprising: (a) (i) determining the percentage of T cells that are CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) comparing the percentage of T cells that are CCR8-expressing Tregs with a pre-determined threshold; and (iii) selecting the subject as not suitable for immunotherapy using a therapeutic anti-CCR8 Ab or an antigen-binding portion thereof based on an assessment that the percentage of T cells that are CCR8-expressing Tregs in the cells of the test tissue is less than the pre-determined threshold, thereby selecting a subject who is not a suitable candidate for immunotherapy using a therapeutic anti-CCR8 Ab or an antigen-binding portion thereof for Treg depletion therapy; and (b) administering a standard therapeutic agent other than the therapeutic anti-CCR8 Ab or an antigen-binding portion thereof to the selected subject.

[0019] The present disclosure further provides a method for treating cancer in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a therapeutic anti-CCR8 Ab or an antigen-binding portion thereof for Treg depletion therapy, wherein the subject is selected based on the determination that the percentage of T cells that are CCR8-expressing Tregs in the cells of a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs, exceeds a pre-determined threshold level.

[0020] The present disclosure still further provides a method for treating cancer in a subject, comprising administering to the subject a standard therapeutic treatment other than a therapeutic anti-CCR8 Ab or an antigen-binding portion thereof for Treg depletion therapy, wherein the subject is selected based on the determination that the percentage of T cells that are CCR8-expressing Tregs in the cells of a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs, is less than a pre-determined threshold level.

[0021] In certain embodiments related to any of such treatment methods for treating cancer, the anti-CCR8 Ab for Treg depletion therapy or an antigen-binding portion thereof specifically binds with high affinity to CCR8 expressed on the cell surface and mediates cell depletion, and comprises the six CDRs, heavy and / or light chain variable regions, or heavy and / or light chains, or an antigen-binding portion thereof, of mAb4A19 described in WO2021 / 194942. In further embodiments, the anti-CCR8 Ab for Treg depletion therapy or an antigen-binding portion thereof is an mAb or an antigen-binding portion thereof named 4A19, 18Y12, 10R3, 8D55, 14S15, or 14S15h. These Abs disclosed in WO2021 / 194942 bind to the N-terminal epitope of hCCR8.

[0022] In still other embodiments, the treatment method further comprises administering, to a subject in need thereof, a therapeutically effective amount of an additional therapy for treating cancer. Such additional anti-cancer therapies can be small molecule agents, polypeptides, antibodies, immunomodulatory agents, chemotherapy, targeted therapy, radiation therapy, surgery, or any combination thereof. In certain embodiments, the immunotherapy comprises an agent that reduces the inhibition of or increases the stimulation of the immune system. In certain preferred embodiments, the immunomodulatory agent that reduces the inhibition of the immune system is an immune checkpoint inhibitor. In a more preferred embodiment, the immunomodulatory agent that reduces the inhibition of the immune system is an antagonist of an immune checkpoint inhibitor, such as PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, and / or TIM-3. In certain embodiments, the chemotherapy comprises an alkylating agent, such as dacarbazine, ifosfamide, cyclophosphamide, etc., or platinum-based chemotherapeutic agents such as cisplatin, bendamustine, carboplatin, and oxaliplatin; a mitotic inhibitor, such as vinca alkaloids vincristine and vinblastine, or taxanes such as docetaxel, paclitaxel, and cabazitaxel; a topoisomerase inhibitor, such as etoposide or irinotecan; an antimetabolite, such as 5-fluorouracil, azacitidine, or gemcitabine; or an antitumor antibiotic, such as bleomycin, mitomycin-C, or anthracyclines such as daunorubicin, doxorubicin, and mitoxantrone. In certain preferred embodiments, the additional anti-cancer therapy is an anti-PD-1 antibody or docetaxel.

[0023] The present disclosure measures the receptor (e.g., CCR8) occupancy by an Ab for Treg depletion (e.g., anti-CCR8, Ab for Treg depletion); measures the depletion of the number of Tregs in a subject, predicts the efficacy of a therapeutic anti-CCR8 Ab, selects a subject suffering from cancer as a suitable candidate for immunotherapy using the therapeutic anti-CCR8 Ab, and also provides various kits for implementing the methods described herein for use in a method of treating cancer in a subject. By way of example, the present disclosure provides a kit for use in measuring the depletion of the number of Tregs in a subject, the kit comprising: (a) a mAb or an antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of a cell and that binds to an epitope other than an epitope within the N-terminal domain of hCCR8, and (b) instructions for using the mAb or a portion thereof in any one of the methods for measuring Treg depletion disclosed herein.

[0024] As another example, the present disclosure provides a kit for use in treating cancer in a subject, the kit comprising: (a) a mAb or an antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of a cell and that binds to an epitope other than an epitope within the N-terminal domain of hCCR8, (b) a therapeutic anti-CCR8 Ab for Treg depletion or an antigen-binding portion thereof, and (c) instructions for using the mAb or a portion thereof and the therapeutic anti-CCR8 Ab or an antigen-binding portion thereof in any one of the methods for treating cancer in a subject disclosed herein.

[0025] Other characteristics and advantages of the present invention are apparent from the "Modes for Carrying Out the Invention" and "Examples" which should not be construed as limiting. The content of all cited references, including scientific literature, GenBank entries, patents, and patent applications cited throughout the present application, are hereby expressly incorporated by reference herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

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Mode for Carrying Out the Invention

[0027] The present invention relates to an mAb that does not specifically bind to an epitope within the N-terminus of hCCR8, and to the use of such an mAb. That is, the present invention relates to an mAb that specifically binds to an epitope of hCCR8 that is different from the epitope within the N-terminal domain to which a Treg depletion therapy Ab, such as mAb4A19, etc., binds, and to a method for detecting and / or measuring the level of expression of hCCR8 on the surface of cells using, for example, such an mAb. The mAb that binds to this epitope of hCCR8 that is outside the N-terminal domain does not compete with the mAb that binds to the N-terminal epitope, and thus the binding of the aforementioned mAb to CCR8 is not affected thereby even in the presence of an Ab that binds to the N-terminal domain. Therefore, the mAb of the present invention can be used to detect and / or measure the level of expression of hCCR8 on the surface of cells even in the presence of a therapeutic mAb that binds to the N-terminal epitope. This property is very useful when using the mAb of the present invention for diagnostic or biomarker applications.

[0028] Term To make the present disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly stated herein, each of the following terms shall have the meaning set forth below. Additional definitions are provided throughout this application.

[0029] "Administering", "administer", or "administration" refers to physically introducing a composition, including a drug, e.g., a therapeutic or diagnostic agent, to a subject using various methods and delivery systems known to those skilled in the art. A preferred route for administering a therapeutic or diagnostic Ab, e.g., an anti-CCR8 Ab, etc., is intravenous (IV) administration. Other routes of administration include, for example, subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intraspinal, or other parenteral administration routes by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, and includes intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intra-articular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation, but is not limited thereto. Alternatively, the Abs of the present invention can also be administered via routes other than parenteral, e.g., topical, epidermal, or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration routes. Administration can be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0030] "Antibody (Ab)" includes, but is not limited to, a glycoprotein immunoglobulin (Ig) that specifically binds to an antigen and contains at least two heavy chains (H chains) and two light chains (L chains) interconnected by disulfide bonds or an antigen-binding portion thereof. Each H chain includes a heavy chain variable region (abbreviated as V H herein) and a heavy chain constant region. The heavy chain constant region of an IgG Ab includes three constant domains C H1 , C H2 , and C H3 . Each light chain includes a light chain variable region (abbreviated as V L herein) and a light chain constant region. The light chain constant region of an IgG Ab includes one constant domain C L . V H and VL The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs) that are interspersed with those regions. V H and V L Each contains three CDRs and four FRs arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus toward the carboxy terminus. The variable regions of the heavy and light chains comprise binding domains that interact with an antigen. Various methods, including the definitions of Kabat, Chothia, AbM, contact, and IMGT, have been used to define the CDR domains within an Ab. The constant regions of an Ab can mediate the binding of the Ig to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0031] As used herein and based on conventional usage, an Ab described as including "one" heavy chain and / or "one" light chain refers to an Ab that includes "at least one" of the recited heavy chain and / or light chain, and thus also includes an Ab having two or more heavy chains and / or light chains. In particular, an Ab so described is intended to include conventional Abs having two substantially identical heavy chains and two substantially identical light chains. The Ab chains can be substantially identical but are not completely identical if they differ, for example, due to C-terminal truncation of lysine residues or post-translational modifications including alternative glycosylation patterns.

[0032] Ig can be derived from any generally known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the class or subclass of Ab encoded by the heavy chain constant region gene (e.g., IgM, IgG1, IgG4). The term "antibody" includes, by way of example, both naturally occurring Abs and non-naturally occurring Abs, monoclonal and polyclonal Abs, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs, and single-chain Abs. Non-human Abs can be partially or fully humanized by recombinant methods to reduce their immunogenicity in humans. Unless specified otherwise and unless the context indicates otherwise, the term "antibody" also includes any antigen-binding fragment or antigen-binding portion of any of the above Igs, as well as monovalent and bivalent fragments or portions, and single-chain Abs.

[0033] "Isolated" Ab refers to an Ab that is substantially free of other Abs having different antigen specificities (e.g., an isolated Ab that specifically binds to CCR8 is substantially free of Abs that specifically bind to antigens other than CCR8, such as Abs that bind to CCR4). However, an isolated Ab that specifically binds to human CCR8 (hCCR8) may cross-react with other antigens, such as CCR8 polypeptides, from different species, such as mice or cynomolgus monkeys. Further, in certain contexts, "isolated" Ab may refer to an Ab that has been purified so as to be substantially free of other cellular materials and / or chemical substances. By comparison, "isolated" nucleic acid clearly differs from nucleic acid as it exists in nature and refers to a nucleic acid composition having a unique chemical identity, properties, and use. For example, isolated DNA differs from natural DNA in that it is an independent portion of natural DNA and is not an essential part of a larger structural complex (chromosome) found in nature. Further, isolated DNA, unlike natural DNA, can be used, inter alia, as a PCR primer or hybridization probe to measure gene expression, detect biomarker genes or mutations for diagnosing diseases, or predict the effect of therapeutic agents. In addition, in certain contexts, "isolated" nucleic acid may mean a nucleic acid that has been purified using standard techniques well known in the art so as to be substantially free of other cellular components or other contaminants, such as other cellular nucleic acids or proteins.

[0034] The term "monoclonal" Ab (mAb) refers to a non-naturally occurring preparation of Ab molecules consisting of a single molecular composition, i.e., Ab molecules having essentially the same primary sequence and exhibiting a single binding specificity and affinity for a particular epitope. mAbs are an example of isolated Abs. mAbs can be produced by the hybridoma method, recombinant methods, transgenic methods, or other techniques known to those of skill in the art.

[0035] The "chimeric" Ab refers to an Ab in which the variable region is derived from one species and the constant region is derived from another species. For example, an Ab in which the variable region is derived from a mouse Ab and the constant region is derived from a human Ab.

[0036] The "human" mAb (HumAb) refers to an mAb having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Further, when the Ab includes a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human Abs of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutations in vivo). However, the term "human" Ab, as used herein, is not intended to include Abs in which CDR sequences derived from the germline of another mammalian species, such as a mouse, etc., are transplanted onto a human framework sequence. The terms "human" Ab and "fully human" Ab are used synonymously.

[0037] The "humanized" mAb refers to an mAb in which some, most, or all of the amino acids outside the CDR domains of a non-human mAb are replaced with the corresponding amino acids derived from human immunoglobulins. In one embodiment regarding the humanized form of an Ab, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids derived from human immunoglobulins, 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 tolerated as long as the ability of the Ab to bind to a specific antigen is not impaired. The "humanized" Ab retains an antigen specificity similar to that of the original Ab.

[0038] The "anti-antigen" Ab refers to an Ab that specifically binds to an antigen. For example, the anti-CCR8 Ab is an Ab that specifically binds to CCR8.

[0039] The "antigen-binding portion" or "antigen-binding fragment" of Ab refers to one or more fragments of an mAb that retain the ability to specifically bind to an antigen to which the whole Ab binds. For example, an anti-CCR8 antigen-binding portion or fragment that mediates depletion of CCR8-expressing cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) necessarily includes the Fc region portion of the Ab required to mediate these effector functions via interaction with Fc receptors or C1q on immune cells.

[0040] "Antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to in vitro or in vivo cell-mediated cytotoxic activity in which non-specific effector cells [e.g., natural killer (NK) cells, macrophages, neutrophils, and eosinophils] that express Fc receptors (FcRs) on their surface recognize the Fc region of an Ab bound to a surface antigen of a target cell and actively lyse the target cell. In principle, any effector cell with an activated FcR can be induced to mediate ADCC.

[0041] Antibody-dependent cell-mediated phagocytosis ("ADCP") refers to an immunological mechanism for removing cells, whereby phagocytic immune cells that express Fc receptors (FcRs) on their cell surface, such as monocytes, macrophages, neutrophils, etc., recognize the Fc region of an Ab bound to a surface antigen on a target cell, thereby inducing phagocytosis, which results in internalization and degradation of the target cell through acidification of the phagosome.

[0042] "Cancer" refers to a broad group of various diseases characterized by uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth cause the formation of malignant tumors that can invade adjacent tissues and may also metastasize to distant sites in the body through the lymphatic system or bloodstream.

[0043] "C-C motif chemokine receptor 8" ("CCR8"; also known as, for example, CY6, TER1, CCR-8, CKRL1, CDw198, CMKBR8, GPRCY6, CMKBRL2, or CC-CKR-8) is a G protein-coupled seven-transmembrane chemokine receptor (GPCR) that is mainly expressed on FOXP3 hi Tregs. As used herein, "CCR8" includes human CCR8 (hCCR8), variants of hCCR8, isoforms, interspecies homologs, such as mouse CCR8 (mCCR8), etc., and analogs having at least one epitope common to hCCR8. The amino acid sequences of full-length hCCR8 and mCCR8 can be found in GENBANK® accession numbers AAI07160.1 and NP_031746.1, respectively.

[0044] A "cell surface receptor" refers to a molecule or complex of molecules expressed on the surface of a cell that has the ability to receive signals and transmit those signals across the cell's plasma membrane.

[0045] Complement-dependent cytotoxicity ("CDC") is an immune response in which target cells are lysed through the activation of the complement cascade and the recruitment of the complement cascade to the surface of the target cells. This is an effector function of IgG (mainly IgG1 and IgG3) and IgM Abs. When bound to surface antigens on target cells, C1q binds to this Ab, triggering the activation of the classical complement pathway, resulting in the formation of the membrane attack complex (MAC) and lysis of the target cells.

[0046] The term "immune response" refers to the biological response to foreign substances that occurs within vertebrates, and through this response, the living body is protected from these substances and the diseases caused by them. The immune response is mediated by the action of one or more types of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils), and soluble macromolecules (including Abs, cytokines, and complement) produced by either these cells or the liver. As a result, invading pathogens, cells or tissues infected with pathogens, cancer cells or other abnormal cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues are selectively targeted, bound to, damaged, destroyed, and / or eliminated from the body of the vertebrate.

[0047] The term "immunotherapy" refers to treating a disease in a subject, or treating a subject at risk of developing or relapsing from a disease, by a method that includes inducing, enhancing, suppressing, or otherwise modifying the immune response. "Cancer immunotherapy" refers to applying immunotherapy for the treatment or prevention of cancer, and is usually performed by inducing or enhancing the immune response, for example, by blocking immunosuppressive pathways or mechanisms within the subject.

[0048] The term "positron emission tomography" or "PET" refers to a non-invasive imaging technique that uses radioactive substances to visualize molecular targets within the body of a subject and measure metabolic processes. This technique detects pairs of gamma rays indirectly emitted by positron-emitting radionuclides (tracers) introduced into the body on bioactive molecules, and generates a three-dimensional image regarding the tracer position within the body. Representative uses of PET imaging tools in drug development include direct visualization of changes in the quantity of targets, monitoring of drug uptake and metabolism in different tissues for the purpose of predicting toxicity or inter-patient differences, quantification of diseased tissues, evaluation of tumor metastasis, and long-term monitoring of the efficacy or resistance of drugs.

[0049] "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, rodents such as mice, rats, guinea pigs, etc. In a preferred embodiment, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0050] The "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug or therapeutic agent that, when used alone or in combination with another therapeutic agent, protects the subject from the onset of a disease, or proves to promote the regression of the disease as evidenced by a decrease in the severity of the disease symptoms, an increase in the frequency and duration of the disease symptom-free period, the prevention or alleviation of functional or capacity impairment caused by the pain of the disease, or other improvement of the disease symptoms in the subject.

[0051] "Treatment" or "therapy" of a subject refers to any type of intervention or process carried out on the subject, including the administration of an active agent to the subject, with the aim of reversing, alleviating, improving, inhibiting, delaying, or preventing the expression, progression, onset, exacerbation, or recurrence of symptoms, complications, or conditions, or biochemical signs associated with a disease.

[0052] As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any of the components described or recited.

[0053] The term "about", when applied to a numerical value, refers to a value that is reasonably close to the recited numerical value and falls within the tolerance range determined by those skilled in the art, which in part depends on the method by which the numerical value is measured or determined, such as the limitations of the measurement system. By way of example, "about" can mean within a range of plus or minus 50% of the recited reference value, preferably within a range of plus or minus 25%, or more preferably within a range of plus or minus 10%. According to the practice in the art, this range will typically fall within the tolerance range for such a particular numerical value.

[0054] The terms "substantially identical" or "essentially identical" refer to a sufficiently high degree of similarity recognized among two or more numerical values, substances, compositions of matter, or features such that a person skilled in the art would consider the differences among such numerical values, substances, compositions of matter, or features to be biologically and / or statistically hardly or not at all significant in the context of the property being measured. The difference in the measured numerical values can be, for example, less than about 50%, preferably less than about 25%, and more preferably less than about 10%.

[0055] As described herein, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the recited range and, where applicable, its decimal values (e.g., 1 / 10, 1 / 100, etc. of an integer), unless otherwise indicated.

[0056] The various aspects of the present invention are further detailed in the following subsections.

[0057] Targeting of CCR8 due to its specific expression on tumor-infiltrating Tregs The expression of CCR8 has been shown to be selectively upregulated in tumor-resident Tregs in multiple cancers (De Simone et al., 2016; Plitas et al., 2016), and FOXP3, which is associated with poor survival hi It is expressed on the most activated and suppressive subset of tumor Tregs (Plitas et al., 2016; Wang et al., 2019, WO2021 / 194942). From the correlation between the genes of human CCR8 and FOXP3 in The Cancer Genome Atlas (TCGA), the expression of CCR8 has the highest correlation with FOXP3 (the master transcriptional regulator of Tregs) in most cancer types, and CCR8 is tumor FOXP3 hi While it is expressed on Tregs, it has been further clarified that it is hardly observed on Tregs and Teffs in peripheral blood (WO2021 / 194942). Also, CCR8 is FOXP3 in hepatocellular carcinoma tumor sampleshi Lymphocytes (FOXP3 in the patient's tumor mid and FOXP3 neg are not expressed in CD8 and CD4 effector T cells), most tumor-resident Tregs (tumor-infiltrating CD4 + T cells and CD8 + T cells have a much lower proportion), and a small part of peripheral Tregs, but selectively expressed in the majority of tumor-infiltrating Tregs (WO2021 / 194942).

[0058] From such a CCR8 expression pattern, CCR8 is a highly desirable target for mediating the depletion of such highly immunosuppressive Tregs through ADCC and ADCP using anti-CCR8 Ab. Also, since CCR8 is hardly expressed on Tregs and Teffs in peripheral blood or other tissues, targeting Tregs results in minimal toxic risk. WO2021 / 194942 describes the generation and characterization of multiple anti-hCCR8 mAbs that exhibit desirable properties for therapeutic Abs for treating cancer, including the high efficiency in mediating the depletion of CCR8-expressing tumor-associated Tregs. Since this therapeutic Ab binds to epitopes within the extracellular N-terminal domain of hCCR8, it gives rise to the need for Abs that bind to epitopes outside the N-terminal domain. Such Abs are useful for diagnostic applications related to the measurement or monitoring of CCR8 expression on Tregs and the number of CCR8-expressing Tregs, without affecting or being affected by the binding of the therapeutic Ab to the N-terminal domain of CCR8, even in the presence of a therapeutic Ab bound to the N-terminal of CCR8.

[0059] Generation of anti-hCCR8 mAbs that do not bind to the N-terminal domain Mice were immunized with an immunogen containing a plasma membrane material derived from hCCR8-overexpressing cells to generate mAbs. Since many anti-hCCR8 Abs have been found to bind to one or more epitopes within the N-terminal domain of hCCR8, a strategy has been developed to preferentially generate Abs that bind to epitopes different from these N-terminal epitopes. This strategy (see Example 1) involved using proteoliposomes derived from cells that overexpressed hCCR8 and also expressed an anti-hCCR8 Ab, Clone L263G8 (BioLegend), that binds to the N-terminal epitope, as the immunogen. The expressed Ab was expected to bind to the N-terminal epitope of CCR8. Thus, by using the proteoliposomes prepared from this cell as the immunogen, the bound Ab(s) shielded the N-terminal epitope(s) from the mouse immune system, and the generated Abs preferentially targeted epitopes other than the N-terminal epitope.

[0060] This strategy was successful in demonstrating the generation of mouse Abs that bind to epitopes on hCCR8 different from the N-terminal epitope. Specifically, as described in Example 1, using this method, mice were immunized with an immunogen containing proteoliposome material derived from cells that overexpressed both an hCCR8 / hCCR5 chimeric protein and an anti-hCCR8 Ab (Clone L263G8) that binds to an epitope within the N-terminal domain of hCCR8, thereby generating Abs that bind to epitopes outside the N-terminal domain of hCCR8. However, this approach is also widely applicable when generating Abs that do not bind to pre-defined epitopes of the antigen.

[0061] Accordingly, the present disclosure describes a method for generating a first Ab that does not bind to a defined epitope on an antigen or does not cross-compete with a second Ab for binding to that epitope, the method comprising immunizing a vertebrate with an immunogen comprising a cell line that expresses the antigen and also expresses the second Ab or an antigen-binding portion thereof, or a component of said cell line, wherein the second Ab specifically binds to the epitope and binding of the second Ab or an antigen-binding portion thereof to the epitope shields the epitope from the vertebrate immune system, reducing the generation of Abs that bind to the epitope, thereby preferentially causing the generation of a first Ab that does not bind to the epitope or does not cross-compete with the second Ab for binding.

[0062] In certain embodiments, the vertebrate is a mouse as shown in Example 1, or another mammal such as a rat, hamster, rabbit, dog, goat, sheep, horse, etc., or a bird such as a chicken, etc. In certain embodiments, the antigen is a CCR8 receptor such as a human, cynomolgus monkey, mouse, rat CCR8 receptor, etc. In a preferred embodiment, the antigen is the hCCR8 receptor. In certain embodiments of a method for generating an Ab targeting hCCR8, the epitope is an epitope within the N-terminal domain of the hCCR8 receptor. In a further embodiment, the second Ab or an antigen-binding portion thereof is a mAb named Clone L263G8 (BioLegend), a mAb named 433H (BD Biosciences), or 4A19, 18Y12, 10R3, 8D55, 14S15, 15S15h of the mAbs described in WO2021 / 194942. In certain other embodiments, the immunogen is a surfactant-stabilized proteoliposome component of the cell line.

[0063] The mAb of the present invention that does not bind to the N-terminal epitope of hCCR8 Using multiple immunization campaigns to generate anti-hCCR8 mAbs, including the above method for shielding N-terminal epitope(s) from the mouse immune system, generated mouse Abs that bind to epitopes on hCCR8 other than the N-terminal epitope(s). The immunogen was a surfactant-stabilized proteoliposome material derived from HEK293 cells engineered to overexpress hCCR8 and an anti-CCR8 mAb named Clone L263G8 (BioLegend) that binds to the N-terminal epitope of hCCR8. Ab-secreting B cells from immunized mice were fused with immortalized myeloma cells to generate hybridomas that produce mAbs (see Example 1).

[0064] For at least two cell lines, a cell line overexpressing hCCR8 and a corresponding control cell line not overexpressing CCR8, the hybridoma supernatants were screened by flow cytometry to identify mAbs that specifically bind to hCCR8 (see Example 2). To characterize the epitope to which the hCCR8-specific Ab binds, the hybridoma culture supernatants were screened by ELISA to measure binding to a BSA-conjugated peptide (SEQ ID NO: 74) corresponding to the N-terminus of CCR8. Several mAbs were identified that specifically bind to hCCR8 by flow cytometry but do not bind to the BSA-conjugated CCR8 N-terminal peptide by ELISA, suggesting binding to an epitope of hCCR8 different from the N-terminal epitope (Example 2).

[0065] Accordingly, the present disclosure describes an isolated Ab, preferably an mAb or an antigen-binding site thereof, that specifically binds to hCCR8 expressed on the surface of a cell, wherein the Ab or its antigen-binding site binds to an epitope that is not located within the N-terminal domain of hCCR8, i.e., an epitope different from the epitope within the N-terminal domain of hCCR8 to which the anti-CCR8 mAb disclosed in WO2021 / 194942 binds. The amino acid sequence of hCCR8 is represented as SEQ ID NO: 1. In certain embodiments, the N-terminal epitope is the sequence Y 15 Y16 Y 17 P 18 D 19 I 20 F 21 (SEQ ID NO: 2) contains at least one amino acid within the peptide. In certain embodiments, the N-terminal epitope contains 2, 3, 4, 5, 6, or all 7 amino acids within the peptide having the sequence of SEQ ID NO: 2. In certain preferred embodiments, the N-terminal epitope contains all 7 amino acids within the peptide having the sequence of SEQ ID NO: 2. In certain other embodiments, the N-terminal epitope is the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73) contains at least one amino acid within the peptide. In certain other embodiments, the N-terminal epitope contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 amino acids within the peptide having the sequence of SEQ ID NO: 73. In certain preferred embodiments, the N-terminal epitope contains all 11 amino acids within the peptide having the sequence of SEQ ID NO: 73. In a more preferred embodiment, the amino acid Y 15 and / or Y 17 is sulfated.

[0066] The Ab that binds to the N-terminal epitope does not interfere with the binding of the mAb of the present invention that binds to the non-N-terminal epitope of hCCR8 To determine whether mAb4A19 (see WO2021 / 194942), an Ab that binds to the N-terminal domain epitope of hCCR8, blocks the binding of the mAb21C17 of the present invention to CCR8, tissue derived from fragmented gastric tumors was pre-incubated with unlabeled mAb4A19 or unlabeled mAb21C17, and then stained with CD3, CD8, CD4, FOXP3, and two anti-hCCR8 Abs, Clone L263G8 (BioLegend) or an immune marker Ab against mAb21C17. Analysis by flow cytometry (Example 3) revealed that when mAb4A19 binds to CCR8, subsequent binding of L263G8 is blocked, but binding of mAb21C17 to CCR8 on Tregs is not blocked. Conversely, when 21C17 binds to CCR8, subsequent binding of 21C17 itself is blocked, but binding of L263G8 to Tregs is not blocked. These results indicate that mAb21C17 binds to an epitope on CCR8 distinct from the N-terminal epitope to which mAb4A19 binds, and that binding of mAb4A19 does not interfere with binding of 21C17 to CCR8-expressing Tregs.

[0067] Accordingly, the present disclosure provides an isolated Ab according to the present invention, preferably an mAb or an antigen-binding site thereof, whose binding to hCCR8 is not affected in the presence of an Ab that binds to the N-terminal epitope of hCCR8. In certain embodiments, the mAbs according to the present invention disclosed herein do not compete with any of the mAbs 4A19, 18Y12, 10R3, 8D55, 14S15, and 15S15h, which are the mAbs described in WO2021 / 194942, for binding to hCCR8. In certain embodiments, the binding of the mAb according to the present invention to hCCR8 is amino acid sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22It is not affected even in the presence of an Ab that binds to an epitope containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of the amino acids within the peptide domain of hCCR8 having (SEQ ID NO: 73). In certain preferred embodiments, the binding of mAb21C17 to hCCR8 is the amino acid sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It is not affected even in the presence of an Ab that binds to an epitope consisting of or containing all 11 of the amino acids within the peptide domain of hCCR8 having (SEQ ID NO: 73). In other preferred embodiments, the binding of mAb21C17 to hCCR8 is not affected even in the presence of mAb4A19 (WO2021 / 194942) bound to hCCR8.

[0068] In certain aspects of the invention, an Ab that binds to an N-terminal epitope is (a) Heavy chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 53; heavy chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 54; heavy chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 55; light chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 56; light chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 57; and light chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 58; (b) V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 9 H and V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 16 L ; or (c) A heavy chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 65 and a light chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 72 comprising.

[0069] In certain embodiments, Abs that do not interfere with the binding of the Ab of the present invention even when binding to the N-terminal epitope are each mAb 4A19, 18Y12, 10R3, 8D55, 14S15, or 14S15h (see WO2021 / 194942), or L263G8 (BioLegend). Preferably, the Ab that does not interfere with the binding of the Ab of the present invention even when binding to the N-terminal epitope is mAb 4A19.

[0070] mAbs that bind with high affinity to non-N-terminal epitopes on hCCR8 Certain anti-CCR8 mAbs of the present invention specifically bind to hCCR8 with high affinity. The binding affinity for Abs that bind to targets such as hCCR8 is the dissociation constant (K D ), or the 50% effective concentration (EC 50 ). The term "K D " as used herein is intended to refer to the dissociation constant for a particular Ab-antigen interaction obtained from the ratio of k on to k off (i.e., k off / k on ) and expressed as molar concentration (e.g., nM). Also expressed as molar concentration (e.g., nM), EC 50 is the concentration of Ab that achieves half of the maximum binding.

[0071] The binding specificity of the Ab of the present invention when binding to CCR8 was measured by fluorescence-activated cell sorting (FACS) (see Example 4). It was revealed that the six mAbs tested bind to hCCR8-expressing Raji cells but not to parental Raji cells that do not express CCR8 (Example 4). From FACS analysis, an EC 50Four types of mAbs that bind to Raji-hCCR8 cells were identified (see Table 1).

[0072] Thus, in certain embodiments, the mAb of the present invention or its antigen-binding portion is: (a) about 50 nM or less, (b) about 3 nM or less to about 0.5 nM or less, (c) about 0.5 nM or less, (d) about 0.1 nM or less, (e) about 0.01 nM or less, (f) about 0.005 nM or less, (g) about 0.1 nM, (h) about 0.005 nM to about 50 nM, (i) about 0.02 nM to about 3 nM, or (j) about 0.08 nM to about 2 nM of EC 50 and specifically binds to Raji cells expressing hCCR8.

[0073] In certain preferred embodiments, EC 50 is measured by the binding assay described in Example 4.

[0074] When the binding affinity of the Ab for the antigen has a specific value "or less" represented by K D or EC 50 , it does not mean that there is no lower limit to the K D or EC 50 value, or that this value is infinitely low. In fact, even when the Ab binds with very high affinity, its K D or EC 50 value will not be below the picomolar range, i.e., about 0.001 nM. Thus, one of ordinary skill in the art will understand that, for example, an EC 50 of about 0.5 nM or less means that the Ab binds to the antigen with an EC 50 of about 0.5 nM or lower, but still not lower than about 0.001 nM, i.e., an EC 50 in the range of about 0.001 nM to about 0.5 nM.

[0075] In certain other embodiments, the mAb or antigen-binding portion thereof of the invention binds to cell surface-expressed hCCR8 polypeptide in formalin-fixed paraffin-embedded (FFPE) tissue samples.

[0076] Competitive binding between an mAb that does not bind to the N-terminus of CCR8 and an N-terminus-binding mAb Competition regarding binding to CCR8 that occurs between an anti-CCR8 mAb (4A19-mIgG2a) that binds to the N-terminus of CCR8 and mAbs (23A14-hIgG1, 21C17-mIgG2a, and 22B13-mIgG2a) that do not bind to the N-terminus was assayed by FACS on the surface of activated Tregs (see Example 4). The binding of mAb 4A19-mIgG2a to CCR8 did not interfere with the subsequent binding of any of 23A14-hIgG1, 21C17-mIgG2a, or 22B13-mIgG2a, but both types of mAbs tested, at saturating amounts, detected an equal population of + Tregs.

[0077] Thus, the mAb of the invention binds to one or more epitopes of hCCR8 distinct from the epitope(s) located in the N-terminal domain of hCCR8 to which therapeutic anti-CCR8 mAbs described in WO2021 / 194942, such as mAb 4A19, etc., bind, and does not interfere with the binding of the Ab to the other epitope even if an Ab that binds to the N-terminal epitope is present. Examples of the mAb of the invention that binds to an hCCR8 epitope outside the N-terminal region include 25T40, 21C17, 28P3, 22B13, 33H18, and 23A14.

[0078] An anti-CCR8 mAb that cross-competes with a reference Ab for binding to CCR8 Also included within the scope of the disclosed invention are isolated Abs, preferably mAbs, or antigen-binding portions thereof, that specifically bind to hCCR8 expressed on the surface of cells and cross-compete with the reference Ab or its reference antigen-binding portion for binding to hCCR8. The ability of a set of Abs to "cross-compete" for binding to an antigen, such as CCR8, means that the first Ab binds to a region that is substantially identical to the epitope region of the antigen of the second Ab and sterically hinders the second Ab from binding to that specific epitope region, and vice versa, the second Ab binds to a region that is substantially identical to the epitope region of the antigen of the first Ab and sterically hinders the first Ab from binding to that epitope region. Thus, if a test Ab has the ability to competitively inhibit the binding of, for example, mAb25T40 or 21C17 to hCCR8, the test Ab demonstrates binding to a region that is substantially identical to the epitope region of hCCR8 to which mAb25T40 or 21C17 binds.

[0079] If the first Ab reduces the binding of the second Ab to the antigen by at least about 40%, the first Ab is considered to bind to an "epitope region that is substantially identical" to the epitope region to which the second Ab binds. Preferably, the first Ab reduces the binding of the second Ab to the antigen by more than about 50% (e.g., at least about 60% or at least about 70%). In a more preferred embodiment, the first Ab reduces the binding of the second Ab to the antigen by more than about 70% (e.g., at least about 80%, at least about 90%, or about 100%). The order of the first and second Abs can be reversed, i.e., the "second" Ab can bind to the surface first and the "first" Ab can then contact the surface in the presence of the "second" Ab. Regardless of the order in which the Abs are added to the immobilized antigen, if a competitive reduction in binding to the antigen is observed, both Abs are considered to "cross-compete".

[0080] The "epitope region" refers to the epitope and the spatial region around the epitope. In particular, Abs that bind to substantially the same "epitope region" of an antigen may not necessarily bind to the same epitope [however, Abs that bind to the same epitope can be initially screened for some Abs that bind to the same epitope region, and subsequently, for those Abs, techniques well-known in the art including array-based oligo-peptide scanning, site-directed scanning mutagenesis mapping (e.g., alanine scanning epitope mapping), high-throughput shotgun mutagenesis epitope mapping, hydrogen-deuterium exchange method (HDX), and X-ray crystallography can be used to identify them by epitope mapping]. For example, two cross-competitive mAbs that bind to substantially the same epitope region of an antigen may bind to adjacent or overlapping but non-identical epitopes and may sterically hinder each other's binding to their cognate epitopes. Alternatively, the binding of one mAb to an epitope may induce a conformational change in the antigen and reduce the binding of another mAb to a different epitope within the substantially same epitope region. Nevertheless, cross-competitive Abs are generally expected to have very similar functional characteristics due to their property of binding to substantially the same epitope region of an antigen, such as the CCR8 receptor. The higher the degree of cross-competition, the more similar the functional characteristics are expected to be. For example, two cross-competitive Abs are expected to have essentially the same functional characteristics if they inhibit each other's binding to the epitope by at least about 80% each, even more so if they inhibit each other's binding to the epitope by at least about 90% each, and even more so if they inhibit each other's binding to the epitope by about 100% each. K D or EC 50 When measured by, if cross-competitive Abs show similar affinities for binding to the epitope, the similarity in this function is expected to be closer.

[0081] Cross-competitive anti-antigen Abs can be readily identified based on their ability to competitively detectably in standard antigen-binding assays including BIACORE® analysis, ELISA assays, or flow cytometry using either recombinant antigen molecules or cell surface-expressed antigen molecules. As an example, a simple competition assay to determine whether a test Ab competes with mAb21C17 for binding to hCCR8 can involve (1) measuring the binding of 21C17 applied at a saturating concentration to a BIACORE® chip (or other suitable medium for SPR analysis) to which hCCR8 is immobilized, and (2) measuring the binding of 21C17 to an hCCR8-coated BIACORE® chip (or other suitable medium) pre-bound with the test Ab. The binding of 21C17 to the hCCR8-1-coated surface in the presence / absence of the test Ab is compared. If the binding of 21C17 is significantly (e.g., by more than about 40%) reduced in the presence of the test Ab, it indicates that both Abs recognize substantially the same epitope region, e.g., they compete for binding to the hCCR8 target, etc. The percentage by which the binding of the first Ab to the antigen is inhibited by the second Ab can be calculated as [1 - (binding of the first Ab detected in the presence of the second Ab) / (binding of the first Ab detected in the absence of the second Ab)] × 100. The competition binding assay is repeated to determine whether the Abs cross-compete, except when the binding of the test Ab to the hCCR8-coated chip is measured in the presence of pre-bound mAb21C17.

[0082] Any of the anti-CCR8 Abs disclosed herein that bind to an epitope of hCCR8 outside the N-terminal domain can serve as a reference Ab in a cross-competition assay. Thus, for example, certain embodiments of the invention disclosed herein relate to an isolated Ab, preferably an mAb, or an antigen-binding portion thereof that cross-competes with a reference Ab for binding to hCCR8, where the reference Ab is (a) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 3 Hand V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 10 L , (b) V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 4 H and V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 11 L , (c) V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 5 H and V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 12 L , (d) V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 6 H and V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 13 L , (e) V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 7 H and V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 14 L , or (f) V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 8 H and V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 15 L comprises.

[0083] An anti-CCR8 mAb that binds to the same non-N-terminal binding CCR8 epitope to which reference Ab binds Certain other aspects of the invention relate to an isolated Ab, preferably an mAb, or an antigen-binding portion thereof, that binds to the same epitope to which reference Ab binds, where the reference Ab is (a) V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 3 H and V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 10 L , (b) V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 4H and a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 11 L , (c) a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 5 H and a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 12 L , (d) a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 6 H and a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 13 L , (e) a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 7 H and a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 14 L , or (f) a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 8 H and a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 15 L comprising

[0084] The structurally defined mAb of the present invention Certain other aspects of the present invention relate to an isolated Ab, preferably an mAb, or an antigen-binding portion thereof, that specifically binds to hCCR8 expressed on the surface of a cell, and (a) a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 3 H and a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 10 L , (b) a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 4 H and a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 11 L , (c) a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 5 Hand V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 12 L , (d) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 6 H and V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 13 L , (e) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 7 H and V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 14 L , or (f) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 8 H and V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 15 L each containing CDR1, CDR2, and CDR3 domains present therein.

[0085] Several different methods have been developed to represent CDR domains within Abs. The approach of Kabat and co-workers (Wu and Kabat, 1970; Kabat et al., 1983) was based on the hypothesis that CDRs occupy the most variable positions within Abs and are thus identified by aligning a much more limited number of Ab sequences than the available Ab sequences. Based on this alignment, Kabat et al. introduced a numbering scheme for residues within the hypervariable regions and determined which positions corresponded to the start and end points of each CDR (http: / / bioinf.org.uk / abs / simkab.html).

[0086] In addition to the widely used Kabat definition, other definitions have also been adopted, including those of Chothia (Chothia et al., 1987; 1989; Al-Lazikani et al., 1997; http: / / bioinf.org.uk / abs / chothia.html), AbNum (Abhinandan and Martin, 2008; see AbNum; available at http: / / www.bioinf.org.uk / abs / abnum / ), AbM (http: / / www.bioinf.org.uk / abs; Martin et al., 1989), contact (http: / / bioinf.org.uk / abs / ; MacCallum et al., 1996), and IMGT (Lefranc et al., 2003; http: / / www.imgt.org), which were considered to address the deficiencies of the Kabat definition. Despite being developed when structural information about Abs was not available, the Kabat definition remains the most commonly used method for predicting CDR domains.

[0087] Unless explicitly stated otherwise and unless the context indicates otherwise, the CDRs disclosed herein are identified using the Kabat definition. The amino acid sequences corresponding to the six CDR domains defined using the Kabat method, as well as the V H V L amino acid sequences corresponding to the heavy and light chains are shown in Table 3.

[0088] In certain other embodiments, the Ab of the invention, preferably an mAb, or an antigen-binding portion thereof, comprises the following CDR domains defined by the Kabat method: (a) The heavy-chain variable-region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 17; the heavy-chain variable-region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 18; the heavy-chain variable-region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 19; the light-chain variable-region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 20; the light-chain variable-region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 21; and the light-chain variable-region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 22. (b) The heavy-chain variable-region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 23; the heavy-chain variable-region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 24; the heavy-chain variable-region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 25; the light-chain variable-region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 26; the light-chain variable-region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 27; and the light-chain variable-region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 28. (c) The heavy-chain variable-region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 29; the heavy-chain variable-region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 30; the heavy-chain variable-region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 31; the light-chain variable-region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 32; the light-chain variable-region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 33; and the light-chain variable-region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 34. (d) The heavy-chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 35; the heavy-chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 36; the heavy-chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 37; the light-chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 38; the light-chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 39; and the light-chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 40, (e) The heavy-chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 41; the heavy-chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 42; the heavy-chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 43; the light-chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 44; the light-chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 45; and the light-chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 46, or (f) The heavy-chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 47; the heavy-chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 48; the heavy-chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 49; the light-chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 50; the light-chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 51; and the light-chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 22.

[0089] In a further embodiment, the Ab, preferably the mAb, or an antigen-binding portion thereof, (a) V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 3 Hand V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 10 L , (b) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 4 H and V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 11 L , (c) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 5 H and V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 12 L , (d) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 6 H and V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 13 L , (e) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 7 H and V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 14 L , or (f) V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 8 H and V comprising contiguous amino acids having the sequence represented as SEQ ID NO: 15 L is included.

[0090] V having an amino acid sequence that is very similar to or identical to any of the amino acid sequences of the anti-CCR8 Ab and that retains the functional properties of such Ab H and V L regions are also suitable for use in this method. For example, suitable Abs include mAbs each comprising V H and / or V L regions and having contiguous amino acids each having a sequence that is at least about 80% identical to the amino acid sequence represented by SEQ ID NO: 4 and / or 11. In a further embodiment, for example, V H and / or V LThe amino acid sequence shows at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with the sequence represented by SEQ ID NO: 4 and / or 11, respectively. As used herein, the percentage of sequence identity between two amino acid sequences is a function of the number of identical positions common to both sequences relative to the length of the sequences being compared, taking into account the number of any gaps introduced to maximize the degree of sequence identity between the two sequences and the length of each such gap (i.e., % identity = number of identical positions / total number of positions compared × 100). Comparison of sequences and determination of the percentage of sequence identity between two sequences can be accomplished using mathematical algorithms well known to those of skill in the art.

[0091] For an Ab structurally defined herein, a V that exhibits a high sequence identity, e.g., at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity H and / or V L amino acid sequence-containing Ab, preferably in certain embodiments of an mAb, compared to the CDR sequences of the defined Ab, V H and V L each of the CDR domains has no more than 3 amino acid modifications. In certain preferred embodiments, V H and V L each of the CDR domains has no more than 2 amino acid modifications. In certain more preferred embodiments, V H and V L each of the CDR domains has no more than 1 amino acid modification. In certain even more preferred embodiments, V H and V L each of the CDR domains has no amino acid modifications.

[0092] In a preferred embodiment of an Ab that contains one or more amino acid modifications in the CDR, such modifications are "conservative" amino acid modifications. As used herein, "conservative" amino acid modifications refer to amino acid modifications that do not significantly affect or result in a change in the binding properties of the Ab containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in a conservative amino acid substitution, one or more amino acid residues within the CDR region of the Ab are replaced with another amino acid residue derived from the same side chain family, and the modified Ab is also tested using assays well known in the art to verify that the Ab function, such as binding specificity and affinity, is substantially the same as that of the unmodified Ab.

[0093] In certain other embodiments, the Ab, preferably an mAb, or an antigen-binding portion thereof (a) a heavy chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 59 and a light chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 66, (b) a heavy chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 60 and a light chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 67, (c) A heavy chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 61 and a light chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 68, (d) A heavy chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 62 and a light chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 69, (e) A heavy chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 63 and a light chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 70, or (f) A heavy chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 64 and a light chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 71 is included.

[0094] In certain preferred embodiments, the mAb or its antigen-binding portion is an Ab or its antigen-binding portion designated herein as 25T40, 21C17, 28P3, 22B13, 33H18, or 23A14.

[0095] In certain embodiments, the isolated anti-CCR8 Ab of the invention, preferably an mAb, or its antigen-binding portion is a human Ab or its fragment. In other embodiments, it is a humanized Ab or its fragment. In further embodiments, it is a chimeric Ab or its fragment. In other embodiments, the isolated anti-CCR8 Ab or its antigen-binding portion is a mouse Ab or its fragment. When used in human subjects, the Ab is preferably a chimeric Ab, or more preferably a humanized or human Ab. Such chimeric, humanized, human, or mouse mAbs can be prepared and isolated by methods well known in the art.

[0096] In certain other embodiments, the Ab, preferably an mAb, or an antigen-binding portion thereof, comprises a heavy chain constant region that is a heavy chain constant region of a human IgG1, IgG2, IgG3, or IgG4 isotype. In further embodiments, the Ab or an antigen-binding portion thereof comprises a heavy chain constant region that is a heavy chain constant region of a human IgG2 or IgG4 isotype.

[0097] mAb labeled with a detectable tag The mAb can be labeled with various substances or tags, including small molecules, enzymes, radioisotopes, and fluorescent dyes, to aid in detection. The type of label used depends on the downstream application. For example, reporter enzymes or biotin are commonly used in enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry (IHC). These labels, as well as fluorescent labels, are used in Western blots, while fluorescent labels are used in flow cytometry and immunofluorescence (IF) staining. Radio-labeled mAbs are used in positron emission tomography (PET) (Aluicio-Sarduy et al., 2018), and rare earth metal isotopes are used in mass cytometry.

[0098] The present disclosure provides a labeled Ab, preferably an mAb, or an antigen-binding portion thereof, comprising either the above Ab of the present invention, or an antigen-binding portion thereof, and any one of a detectable label. In certain embodiments, the detectable label is a fluorophore, an enzyme, a micropolymer, a radioisotope, or a metal. In certain other embodiments, the detectable label is biotin. In further embodiments, the fluorophore is a BRILLIANT VIOLET™ dye (e.g., BV-421), an AmCyan dye, an ALEXA FLUOR® dye, a CY® dye, a CF® dye, fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC) phycoerythrin (PE), allophycocyanin (APC), or peridinin-chlorophyll protein (PerCP). In certain other embodiments, the enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase, or β-galactosidase. In further embodiments, the chromophore is porphyrin, pyropheophorbide-α, benzoporphyrin monoacid ring A (BPDMA), or chlorin e6.

[0099] Radioactive labeling of mAbs with positron emitters for positron emission tomography (immunoPET) can provide useful information about the in vivo biodistribution of such molecules and their associated therapeutic agents (Aluicio-Sarduy et al., 2018). It is important to select the optimal radioisotope for each PET application. Start by matching the half-life of the radionuclide to the pharmacokinetic profile of the mAb in vivo. This ensures that the time course of the radioactivity matches that of the mAb. Generally, due to the prolonged half-life in the bloodstream, the intratumoral accumulation of mAbs tends to peak several days after injection, compared to more conventional options such as 11 C, 18 F, or 68 Ga etc., instead of long half-life isotopes (e.g., 89 Zr, 64 Cu, and 86The use of (Y) is required. In cases where conventional isotopes are not suitable for the desired application, other radionuclides that provide more suitable chemical or attenuation properties were investigated. Notable examples of such alternative radionuclides include 52 Mn, 55 Co, 152 Tb, 90 Nb, 66 Ga, 72 As, and 69 Ge (Aluicio-Sarduy et al., 2018). Currently, 89 Zr is being used much more extensively in clinical trials than any other PET-radiometal. Its 78-hour half-life is consistent with the typical pharmacokinetic timescale of mAbs and is well-suited for 89 Zr in single-site production and domestic and international transportation. With an intermediate half-life of 12.7 hours and negligible contaminating gamma-ray emission, 64 Cu represents another convenient alternative for Ab and protein labeling. 86 Y, with a half-life of 14.7 hours, is another isotope with promising characteristics for immunoPET but is a significant gamma-ray emitter that limits the amount that can be injected into a subject.

[0100] In certain preferred embodiments of mAbs labeled for PET, the radionuclide is 89 Zr, 64 Cu, or 86 Y. In certain other embodiments, the radionuclide is 11 C, 18 F, 68 Ga, 52 Mn, 55 Co, 152 Tb, 90 Nb, 66 Ga, 72 As, 69 Ge, or I 125 .

[0101] Mass cytometry is a next-generation flow cytometry platform, also known as cytometry by time-of-flight (CyTOF), which utilizes elemental analysis mass spectrometry to detect rare metal isotopes conjugated to Abs that are bound intracellularly or extracellularly to the target antigen on a single cell (Spitzer and Nolan, 2016). In this technique, cells are stained using Abs conjugated to metal isotope reporters with different masses. Next, the fixed and stained cells are split into single-cell droplets, atomized, and analyzed by mass spectrometry. The number of cellular parameters that can be simultaneously monitored by a conventional fluorescence flow cytometry assay is essentially limited by the overlap of fluorophore emission spectra. However, mass cytometry can accurately distinguish metal isotopes of different atomic masses without overlap between channels. This enables simultaneous quantification within each single cell for over 40 protein parameters, far exceeding the approximately 20 cellular characteristics that can be simultaneously analyzed by fluorescence flow cytometry. Compared to flow cytometry, mass spectrometry achieves similar quantification for cell lineages while associating with markers of cell differentiation, function, activation, and exhaustion when used with fresh and cryopreserved PBMCs or tumor tissue (Gadalla et al., 2019). However, the throughput (about 1,000 cells / second) and sensitivity of mass cytometry are still about 1 / 10 lower than those of conventional FACS, and the cells are completely "evaporated" during the assay, so cells analyzed by mass cytometry cannot be recovered for downstream analysis (Spitzer and Nolan, 2016).

[0102] In certain embodiments of the Ab labeled with metal isotopes, the metal is yttrium (Y), indium (In), a series of lanthanide elements (Ln, excluding La-Lu, Pm), iodine (I), cadmium (Cd), tellurium (Te), silver (Ag), palladium (Pd), rhodium (Rh), iridium (Ir), platinum (Pt), ruthenium (Ru), osmium (Os), or bismuth (Bi).

[0103] Anti-CCR8 immunoconjugate In another aspect, the present invention relates to any one of the isolated anti-hCCR8 Ab or antigen-binding portion thereof disclosed herein conjugated to a cytolytic agent, such as a cytotoxin, a radioisotope, or a photosensitizer (PS). Such conjugates are referred to herein as "immunoconjugates". The cytotoxin can be conjugated to the Ab of the present invention using linker technologies available in the art. Methods for preparing radioimmunoconjugates are also established in the art.

[0104] Photodynamic therapy (PDT) is a non-invasive treatment involving the accumulation of PS within solid tumors and the subsequent activation of PS induced by the local delivery of light of an exact wavelength. The PS induces, in the presence of oxygen, the in situ generation of reactive oxygen species (ROS) that cause damage to cellular components and ultimately necrosis or apoptosis. PDT is a promising tool in oncology but is frequently limited by side effects caused by inappropriate targeting of the photosensitizer. This problem can often be avoided by conjugating the PS to a tumor-specific mAb. The use of antigen-binding Ab fragments, such as Fab or scFv fragments, can be advantageous as they retain the same binding specificity, are more efficient in tumor penetration due to their smaller size, and are more efficiently cleared from the circulation due to the absence of the Fc domain.

[0105] Porphyrins are widely used in the fields of photodynamic therapy and photodiagnosis and belong to one of the most prominent classes of photosensitizers in such areas of biomedical science (Sandland and Boyle, 2019). In certain embodiments of the photosensitizer-Ab conjugate, the photosensitizer is a tetrapyrrole macrocyclic compound. In further embodiments, the tetrapyrrole macrocyclic compound is porphyrin, chlorin, bacteriochlorin, or phthalocyanine.

[0106] Anti-CCR8 chimeric antigen receptor (CAR) and T cell receptor (TCR) In another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising any one of the isolated anti-hCCR8 Abs or antigen-binding portions thereof disclosed herein that specifically bind to an epitope of hCCR8 outside the N-terminal domain of hCCR8. In certain embodiments, the CAR further comprises a transmembrane domain. In certain other embodiments, the CAR further comprises an intracellular signaling domain. In further embodiments, the CAR further comprises a hinge region and / or a spacer region.

[0107] In another aspect, the present invention relates to a T cell receptor (TCR) comprising an antigen-binding region disclosed herein that specifically binds to an epitope of hCCR8 outside the N-terminal domain of hCCR8. In certain embodiments, the TCR further comprises a transmembrane domain. In certain other embodiments, the TCR further comprises an intracellular signaling domain.

[0108] Bispecific molecule In another aspect, the invention relates to a bispecific molecule comprising any one of the anti-hCCR8 mAbs or antigen-binding portions thereof disclosed herein linked to a binding domain having a binding specificity different from that of the anti-hCCR8 mAb or antigen-binding portion thereof. The binding domain can be a functional molecule, such as another Ab, an antigen-binding portion of an Ab, or a ligand for a receptor, and the bispecific molecule produced thereby will bind to at least two different binding sites or target molecules.

[0109] Pharmaceutical composition The Abs used in any of the diagnostic, patient selection, and treatment methods disclosed and described herein can be formulated as a pharmaceutical composition comprising a composition, such as any of the Abs of the invention, and a pharmaceutically acceptable carrier. The present disclosure also provides a composition comprising any of the disclosed immunoconjugates, bispecific molecules, CARs, and TCRs, and a pharmaceutically acceptable carrier, such as a pharmaceutical composition. As used herein, "pharmaceutically acceptable carrier" includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., having physiological compatibility. Preferably, the carrier for the composition containing the Ab is suitable for intravenous (IV), intramuscular, subcutaneous (SC), parenteral, spinal, or epidermal administration (e.g., by injection or infusion). The pharmaceutical composition can include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents, etc.

[0110] An option for SC injection relates to the co-formulation of an Ab with recombinant human hyaluronidase enzyme (rHuPH20) based on Halozyme Therapeutics' ENHANZE® drug delivery technology, which enzyme removes the conventional limitations due to the extracellular matrix on the volume of biological agents and drugs deliverable subcutaneously (U.S. Patent No. 7,767,429). It is also possible to co-formulate two Abs used in combination therapy into a single composition for SC administration.

[0111] Nucleic acid encoding anti-hCCR8 mAb and use for expressing the Ab Another aspect of the disclosed invention relates to a nucleic acid encoding any of the isolated anti-hCCR8 Abs of the invention (the nucleic acids of the invention). This disclosure provides isolated nucleic acids encoding any of the anti-CCR8 mAbs of the invention described herein or antigen-binding portions thereof, any bispecific Ab, chimeric antigen receptor (CAR), or T cell receptor (TCR) disclosed herein.

[0112] An "isolated" nucleic acid refers primarily to a nucleic acid composition that is distinct from nucleic acids as they exist in nature, i.e., having a unique chemical identity, properties, and use. For example, an isolated DNA is different from natural DNA in that it is an autonomous part of natural DNA and is not an essential part of a larger structural complex such as a chromosome found in nature. Further, an isolated DNA is different from natural DNA in that it can be used as a PCR primer or hybridization probe, among other things, for measuring gene expression and detecting biomarker genes or mutations for the purpose of diagnosing diseases or predicting the efficacy of therapeutic agents. Narrowly, when the context indicates, the term "isolated" can also be used to describe a nucleic acid that is different from nucleic acids as they exist in nature in that the nucleic acid is purified using standard techniques well known in the art so as to be substantially free of other cellular components or other contaminants, such as other cellular nucleic acids or proteins.

[0113] The nucleic acids of the invention can be obtained using standard molecular biology techniques. In the case of Abs expressed by hybridomas (e.g., hybridomas prepared from wild-type or transgenic mice carrying human Ig genes as described in Example 1), the cDNAs encoding the light and heavy chains or variable regions of the Abs produced by the hybridomas can be obtained by standard PCR amplification techniques. V H and V LOnce a DNA fragment encoding a segment is obtained, this DNA fragment can be further manipulated using standard recombinant DNA techniques, for example, to convert the variable region DNA into a full-length Ab heavy chain gene, a Fab fragment gene, or a scFv gene. In the case of Abs obtained from an Ig gene library (e.g., using phage display technology), the nucleic acid encoding the Ab can be recovered from the library.

[0114] The nucleic acids of the present invention can be, for example, RNA, or DNA, such as cDNA or genomic DNA, etc. In a preferred embodiment, the nucleic acid is cDNA.

[0115] The present disclosure also provides an expression vector comprising an isolated nucleic acid encoding an anti-CCR8 mAb or an antigen-binding portion thereof, any bispecific Ab, chimeric antigen receptor (CAR), or T cell receptor (TCR) disclosed herein. The present disclosure further provides a host cell comprising the expression vector or any CAR or TCR disclosed herein. Eukaryotic cells, particularly mammalian cells, are more likely to fold properly and assemble and secrete immunologically active Abs than prokaryotic cells, and thus such eukaryotic cells, most preferably mammalian host cells, are preferred as host cells for expressing Abs. Preferred mammalian host cells for expressing the recombinant Abs of the present invention include Chinese hamster ovary (CHO) cells (Kaufman and Sharp, 1982), NSO myeloma cells, COS cells, and SP2 cells. In certain embodiments, the host cell is an immune cell. In further embodiments, the host cell is a T cell or an NK cell.

[0116] A host cell can be used in a method for preparing an anti-CCR8 mAb or an antigen-binding portion thereof, a bispecific Ab, a CAR, or a TCR, the method comprising expressing the mAb or an antigen-binding portion thereof, a bispecific Ab, a CAR, or a TCR in the host cell, and isolating the mAb or an antigen-binding portion thereof, a bispecific Ab, a CAR, or a TCR from the host cell. The host cell can be used ex vivo or in vivo. The DNAs encoding the heavy and light chains of the Ab may be inserted into separate expression vectors, but more commonly both are inserted into the same vector. The V H and V L segments are such that the V H segment is operably linked to the C H segment(s) in the vector and the V K segment is operably linked to the C L segment in the vector, and the DNAs encoding these variable regions are inserted into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype, so as to create a full-length Ab of any isotype.

[0117] An anti-CCR8 Ab suitable for use in the therapies of the present disclosure Therapies are disclosed herein that include screening or diagnostic steps involving the use of the anti-CCR8 Ab of the invention. Anti-CCR8 Abs suitable for use in these methods are isolated Abs, preferably mAbs, or antigen-binding portions thereof, that specifically bind to an epitope located outside the N-terminal domain of hCCR8 expressed on the surface of cells. Such Abs exhibit one or more properties that are important for the screening or diagnostic uses described herein. In particular, the isolated Ab or antigen-binding portion thereof (a) an epitope outside the N-terminal domain of hCCR8, optionally the sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21Binds to an epitope different from the epitope containing the peptide having [SEQ ID NO: 2], (b) An epitope outside the N-terminal domain of hCCR8, optionally sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 Binds to an epitope different from the epitope containing the peptide having [SEQ ID NO: 73], or (c) An epitope outside the N-terminal domain of hCCR8, optionally sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 Binds to an epitope different from the epitope composed of the peptide having [SEQ ID NO: 73].

[0118] In a further embodiment, the isolated Ab or its antigen-binding portion preferably has the following characteristics: (d) An EC of about 3 nM or less, 50 e.g., an EC of about 0.01 to about 3 nM, 50 preferably an EC of about 0.1 nM or less, 50 e.g., an EC of about 0.001 to about 0.1 nM, 50 and specifically binds to hCCR8 expressed on the surface of cells, (e) Binds to cell surface-expressed hCCR8 polypeptide in a formalin-fixed paraffin-embedded (FFPE) tissue sample, (f) Its binding to hCCR8 is not affected by the presence of an Ab or its antigen-binding portion that binds to the N-terminal epitope, e.g., mAb4A19 (WO2021 / 194942), and (g) Its binding to hCCR8 does not affect the binding of an Ab or an antigen-binding portion thereof that binds to the N-terminal epitope, such as the binding of mAb4A19 (WO2021 / 194942). Exhibit at least one, at least two, or at least three of such properties.

[0119] In a preferred embodiment, the isolated Ab or an antigen-binding portion thereof exhibits at least six of the above properties (a)-(g), for example, exhibits properties (a)-(d), (f), and (g).

[0120] Diagnostic methods, seranostic methods, and other methods employing anti-CCR8 mAbs PCT Publication No. WO2021 / 194942 describes humanized and human mAbs that specifically bind with high affinity to hCCR8 expressed on the surface of CCR8-expressing tumor-infiltrating Tregs and mediate the depletion of these Tregs by mechanisms including ADCC. The application also describes a method of treating cancer in a subject, including administering to the subject a therapeutically effective amount of an anti-CCR8 mAb, which causes depletion of immunosuppressive tumor-infiltrating Tregs upon administration, thereby enhancing the immune response in the subject to effectively treat cancer. At least one of the therapeutic Treg-depleting mAbs, mAb4A19, binds to an epitope within the N-terminal domain of hCCR8 that includes residues 12-22 (SEQ ID NO: 1) of hCCR8 and includes sulfated tyrosine 15 and tyrosine 17 residues.

[0121] The mAb of the present invention specifically binds to an epitope on hCCR8 that is distinct from the epitope to which the therapeutic anti-CCR8 mAb disclosed in WO2021 / 194942 binds, namely the epitope outside the N-terminal domain. This mAb of the present invention, when bound to hCCR8, does not interfere with the binding of a therapeutic Ab that binds to the N-terminal domain; similarly, it does not interfere with the binding of the mAb of the present invention even when a therapeutic N-terminal-binding mAb is bound. Thus, this mAb can be used in a variety of different ways that are practicable even when present in a state where a therapeutic mAb is bound, such ways including measuring the frequency of CCR8 + cells, such as Tregs, etc., in the blood or tumor environment of a subject; measuring the depletion of the number of CCR8 + cells, such as Tregs, etc., in the blood or tumor environment of a subject; measuring the receptor occupancy (RO), i.e., the proportion of receptors of hCCR8 bound by an anti-CCR8 therapeutic antibody, in a subject being treated with a therapeutic antibody; predicting the effectiveness of a therapeutic anti-CCR8 Ab in treating cancer in a subject; a method for selecting a subject suffering from cancer as a suitable candidate for immunotherapy using a therapeutic Treg-depleting anti-CCR8 Ab; and a treatment method including determining the level of expression of CCR8 in a test tissue within or taken from a subject before administering a therapeutic Treg-depleting anti-CCR8 mAb.

[0122] A method for detecting and quantifying CCR8 expression on peripheral Tregs is described in Example 6. When detecting and quantifying CCR8 in tumor tissue, single-staining immunohistochemistry (IHC) was used (see Example 7).

[0123] As described in WO2021 / 194942, CCR8 + cells in the tumor environment or peripheral blood are mainly Tregs. Advantageously, CCR8 +The anti-CCR8 mAb used to measure the frequency of cells can be mentioned as not competing with the therapeutic anti-CCR8 mAb for binding to CCR8. In certain embodiments, the therapeutic anti-CCR8 mAb binds to an epitope within the N-terminal domain of hCCR8. In further embodiments, the therapeutic anti-CCR8 mAb is one of mAb4A19, 18Y12, 8D55, 10R3, 14S15, and 14S15h described in WO2021 / 194942. In certain preferred embodiments, the therapeutic anti-CCR8 mAb is mAb4A19 comprising six CDRs of mAb4A19 (SEQ ID NOs: 53-58 herein), heavy and / or light chain variable regions (SEQ ID NOs: 9 and / or 16 herein), and / or heavy and / or light chains (SEQ ID NOs: 65 and / or 72 herein).

[0124] In certain embodiments of such methods that include the use of a therapeutic anti-CCR8 mAb that binds to an epitope within the N-terminal domain of hCCR8, the non-competing mAb is an Ab that binds to an epitope outside the N-terminal domain and does not compete with the mAb that binds to an epitope within the N-terminal domain for binding to hCCR8. By way of example, one of the Abs of the invention disclosed herein, i.e., the mAb or antigen-binding portion thereof named herein as 25T40, 21C17, 28P3, 22B13, 33H18, or 23A14 is mentioned. In certain preferred embodiments, the non-competing mAb is mAb21C17, or an antigen-binding portion thereof comprising six CDRs of mAb21C17 (SEQ ID NOs: 23-28), or heavy and / or light chain variable regions (SEQ ID NOs: 4 and / or 11), or heavy and / or light chains (SEQ ID NOs: 60 and / or 67).

[0125] In certain other embodiments, the therapeutic anti-CCR8 mAb binds to an epitope outside the N-terminal domain of hCCR8. For example, in a further embodiment, the therapeutic anti-CCR8 mAb is mAbVHH-01 disclosed in WO2022 / 003156, or each of the mAbs VHH-65, VHH-74, VHH-62, and VHH-56 disclosed in WO2022 / 136647.

[0126] In certain embodiments of a method comprising the use of a therapeutic anti-CCR8 mAb that binds to an epitope outside the N-terminal domain of hCCR8, the non-competing mAb is an Ab that binds to an epitope within the N-terminal domain and does not compete with the mAb that binds to an epitope outside the N-terminal domain for binding to hCCR8. Examples of such Abs that bind to an epitope within the N-terminal domain include Clone L263G8 (BioLegend) and mAb433H disclosed in WO2007 / 044756 (and available from BD Bioscience, https: / / www.bdbiosciences.com / content / bdb / paths / generate-tds-document.us.566899.pdf).

[0127] The present disclosure also provides a method for measuring the RO of a cell membrane-bound CCR8 receptor to which a therapeutic Treg-depleting anti-CCR8 Ab binds, as exemplified in Example 6. The method comprises: (a) adding a saturating concentration of a therapeutic mAb to a background whole blood sample that has been previously exposed to various concentrations of the therapeutic mAb; (b) incubating a fixed volume of a free-type (i.e., blood sample from an indirect / whole experimental tube), a bound-type (i.e., blood sample from a direct experimental tube), or a fluorescence minus one [FMO] (i.e., blood sample from a control tube stained with all Abs except the anti-Id Ab for the direct RO format) sample with a buffer containing no therapeutic mAb; (c) staining the sample with a core panel of Abs to identify basic T cell markers, T cell differentiation markers, and Treg markers; (d) adding to the core cocktail panel two additional Abs, namely, (i) in a direct / whole RO assay, an anti-idiotype Ab for detecting the bound CCR8 receptor, or (ii) in an indirect / whole assay, an allophycocyanin (APC)-conjugated anti-hCCR8 Ab that competes with the therapeutic Ab for detecting the free CCR8 receptor and an anti-hCCR8 Ab that does not compete with the therapeutic Ab for detecting the total CCR8 receptor; (e) lysing the red blood cells (while keeping the target cells intact and clarifying the sample), and analyzing the clarified sample by flow cytometry; (f) using the formula: %RO = 100 × {1 - [(free type after administration / free type before administration) / (total amount after administration / total amount before administration)]} to determine the %RO for each concentration of the therapeutic Ab.

[0128] In certain embodiments of this method, the therapeutic Ab specifically binds to an epitope within the N-terminal domain of hCCR8. In certain other embodiments, the therapeutic Ab is one of the Abs described in WO2021 / 194942 that specifically binds to an epitope within the N-terminal domain of hCCR8. In further embodiments, the therapeutic Ab comprises the six CDRs of mAb4A19 described in WO2021 / 194942 (SEQ ID NOs: 53-58 herein), or the heavy and / or light chain variable regions (SEQ ID NOs: 9 and / or 16 herein). In certain preferred embodiments, the therapeutic Ab is mAb4A19 comprising the heavy and / or light chains of mAb4A19 (SEQ ID NOs: 65 and / or 72).

[0129] In certain embodiments, when the therapeutic Ab is administered to a subject, for example, during a clinical trial or in the course of treatment with an anti-CCR8 mAb, whole blood samples are exposed to various doses of the therapeutic mAb. In other embodiments, whole blood samples are exposed in vitro to different concentrations of the therapeutic Ab to mimic different drug doses in a patient.

[0130] In certain embodiments, basic T cell markers used in the core panel of markers include UV-excitable, fluorescent dye-conjugated anti-human CD3, anti-human CD4, and / or anti-human CD8 Abs. In certain other embodiments, the T cell differentiation marker comprises a fluorescently labeled anti-human CD45RA Ab. In certain other embodiments, Treg markers include fluorescently labeled anti-human CD25, anti-human CD127, and / or anti-human CCR4 Abs. In still further embodiments, the competitive anti-hCCR8 Ab added to the panel for indirect / whole assays is the fluorescently labeled mAb433H (BD Biosciences) for detecting the free CCR8 receptor, and the non-competitive Ab for detecting the total CCR8 receptor is the fluorescently labeled mAb21C17.

[0131] As described in Example 6, from the association of CCR8 measured using the RO assay in both direct and indirect assay formats, it was revealed that the hCCR8 target expressed on the cell surface was specifically associated by the therapeutic mAb4A19. This association of the receptor by the therapeutic Ab is consistent with the expected potential mechanism of action for anti-CCR8 immunotherapy, which requires the anti-CCR8 Ab to bind to CCR8 on Tregs and mediate the depletion of such immunosuppressive Tregs (see Example 8) by a process involving ADCC, ADCP, and / or CDC. Also, the RO assay is quantitative, and CCR8 +

[0132] The present disclosure also provides a method for measuring the depletion of the frequency of CCR8-expressing cells in the peripheral blood of a subject undergoing treatment with an anti-CCR8 mAb, comprising: (a) determining the baseline level of the frequency of CCR8-expressing cells in a first sample of whole blood or PBMCs from the subject; (b) administering treatment with the anti-CCR8 mAb to the subject; and (c) determining the frequency of CCR8-expressing cells in a second sample of whole blood or PBMCs from the subject taken after administration of the anti-CCR8 mAb, wherein a decrease in the frequency of CCR8-expressing cells in the second sample indicates depletion of the number of CCR8-expressing cells in the blood. By this method, the peripheral blood CCR8 of a subject being treated with an anti-CCR8 mAb +It becomes possible to monitor the depletion of Tregs. Since anti-CCR8 Ab immunotherapy is based on an antibody that causes depletion of CCR8-expressing immunosuppressive Tregs, measuring the depletion of Tregs mediated by the administration of anti-CCR8 therapeutic Ab can enable evaluation of the effectiveness of treatment with anti-CCR8 therapeutic Ab even before clinical signs of effectiveness appear. CCR8 + Since Tregs account for a very small proportion of only about 0.5 - 2% of PBMCs, anti-CCR8 mAbs of the present invention that do not compete with therapeutic anti-CCR8 Ab for binding to CCR8, for example, do not compete with therapeutic Abs that bind to the N-terminal domain of hCCR8, do not compete for binding to the N-terminal epitope to which the therapeutic mAb is already bound, and thus such low levels of CCR8 + are important for the detection and quantification of cells.

[0133] The present disclosure also provides a method for measuring the depletion of the number of tumor-infiltrating CCR8-expressing Tregs in a subject undergoing treatment with anti-CCR8 mAb, comprising: (a) determining a baseline level of the frequency of CCR8-expressing Tregs in a first sample of a test tissue within or derived from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering a treatment of anti-CCR8 mAb to the subject; and (c) determining the frequency of CCR8-expressing Tregs in a second sample of a test tissue within or derived from the subject, taken during or after the treatment, wherein a decrease in the frequency of CCR8-expressing Tregs in the second sample of the test tissue indicates depletion of the number of Tregs in the test tissue. This method provides a means for monitoring the depletion of tumor-infiltrating Tregs in the tumor environment of a subject. Since the expected mechanism of action of anti-CCR8 immunotherapy is that an effective anti-CCR8 Ab causes depletion of CCR8-expressing immunosuppressive Tregs, measuring the depletion of Tregs mediated by the administration of anti-CCR8 therapeutic Ab in the tumor environment can enable evaluation of whether treatment with anti-CCR8 therapeutic Ab is effective even before clinical signs of effectiveness appear.

[0134] In certain embodiments of the method, depletion of the number of tumor-infiltrating Tregs in a test tissue indicates enhancement of the immune response in a subject and / or efficacy in treating a disease treatable by enhancement of the immune response. In certain embodiments, the treatment administered to the subject is a treatment for cancer. In further embodiments, the treatment for cancer comprises administration of a therapeutic anti-CCR8 Ab or an antigen-binding portion thereof.

[0135] In certain other embodiments, the anti-CCR8 antibody treatment administered to the subject is a treatment for an infectious disease.

[0136] CCR8 is expressed in 60-80% of all skin T cells as well as Tregs. Thus, the method may also be adapted to measure depletion of skin T cell numbers in a subject being treated, the method comprising (a) determining a baseline level of the frequency of CCR8-expressing skin T cells in a first sample of test tissue within or taken from the subject, the test tissue comprising skin T cells, (b) administering a treatment of anti-CCR8 mAb to the subject, and (c) determining the frequency of CCR8-expressing skin T cells in a second sample of test tissue within or taken from the subject during or after the treatment, wherein a decrease in the frequency of CCR8-expressing skin T cells in the second test tissue indicates depletion of the number of skin T cells in the test tissue.

[0137] Since Tregs are immunosuppressive, subjects expressing high levels of Tregs in the tumor microenvironment, i.e., high levels of CCR8-expressing T cells, are expected to benefit most from treatment with Treg-depleting Abs and are thus suitable candidates for immunotherapy with therapeutic Treg-depleting Abs. Accordingly, the present disclosure provides a method for predicting the efficacy of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof in treating cancer in a subject, the method comprising: (a) determining the frequency of CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) comparing the frequency of CCR8-expressing Tregs to a pre-determined threshold; and (c) predicting the efficacy of the therapeutic Treg-depleting Ab or an antigen-binding portion thereof, wherein a frequency of CCR8-expressing Tregs higher than the threshold indicates that the therapeutic Ab or an antigen-binding portion thereof is effective in treating the subject, and a frequency of CCR8-expressing Tregs lower than the threshold indicates that the therapeutic Ab or an antigen-binding portion thereof is not effective in treating the subject. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab. In certain other embodiments, the Treg-depleting Ab is an anti-CTLA-4 Ab. In further embodiments, the Treg-depleting Ab is an anti-CCR4 or anti-CD25 Ab.

[0138] The present disclosure provides a method for predicting the efficacy of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof in treating cancer in a subject, comprising: (a) determining the frequency of CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering to the subject a therapeutic Treg-depleting Ab or an antigen-binding portion thereof; (c) determining whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of the therapeutic Treg-depleting Ab or an antigen-binding portion thereof; (d) predicting that the therapeutic Treg-depleting Ab or an antigen-binding portion thereof is effective in treating cancer in the subject if the decrease in the frequency of CCR8-expressing Tregs exceeds a pre-determined threshold; or (e) predicting that the therapeutic Treg-depleting Ab or an antigen-binding portion thereof is not effective in treating cancer in the subject if the decrease in the frequency of CCR8-expressing Tregs is less than a pre-determined threshold. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab. In certain other embodiments, the Treg-depleting Ab is an anti-CTLA-4 Ab. In further embodiments, the Treg-depleting Ab is an anti-CCR4 or anti-CD25 Ab.

[0139] The present disclosure also provides a method for selecting a subject suffering from cancer as a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, comprising: (a) determining the frequency of CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) comparing the frequency of CCR8-expressing Tregs with a pre-determined threshold; and (c) selecting the subject as a suitable candidate for immunotherapy using a therapeutic Treg-depleting Ab or an antigen-binding portion thereof based on an assessment that the frequency of CCR8-expressing Tregs in the test tissue exceeds the pre-determined threshold. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab. In certain other embodiments, the Treg-depleting Ab is an anti-CTLA-4 Ab. In further embodiments, the Treg-depleting Ab is an anti-CCR4 or anti-CD25 Ab.

[0140] The present disclosure provides a method for selecting a subject suffering from cancer as a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, the method comprising: (a) determining the frequency of CCR8-expressing Tregs in a test tissue within or taken from a subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering to the subject a therapeutic Treg-depleting Ab or an antigen-binding portion thereof; (c) determining whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of the therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (d) selecting the subject as a suitable candidate for immunotherapy using the therapeutic Treg-depleting Ab or an antigen-binding portion thereof based on an assessment that the decrease in the frequency of CCR8-expressing Tregs exceeds a pre-determined threshold. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab. In certain other embodiments thereof, the Treg-depleting Ab is an anti-CTLA-4 Ab. In further embodiments, the Treg-depleting Ab is an anti-CCR4 or anti-CD25 Ab.

[0141] The present disclosure provides a method for treating cancer in a subject, the method comprising: (a) (i) determining the frequency of CCR8-expressing Tregs in a test tissue within or taken from a subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) comparing the frequency of CCR8-expressing Tregs with a pre-determined threshold; and (iii) selecting the subject as a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting Ab or an antigen-binding portion thereof based on an assessment that the frequency of CCR8-expressing Tregs in the test tissue exceeds a pre-determined threshold, and (b) administering to the selected subject a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab.

[0142] The present disclosure relates to a method for treating cancer in a subject, comprising: (a) (i) determining the frequency of CCR8-expressing Tregs in a test tissue within or derived from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) administering to the subject a therapeutic Treg-depleting Ab or an antigen-binding portion thereof; (iii) determining whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of the therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (iv) selecting the subject as a suitable candidate for immunotherapy using a therapeutic agent based on an evaluation that the decrease in the frequency of CCR8-expressing Tregs exceeds a pre-determined threshold, thereby selecting a subject who is a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (b) administering to the selected subject a composition comprising a therapeutically effective amount of the therapeutic Treg-depleting Ab or an antigen-binding portion thereof. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab.

[0143] The present disclosure also relates to a method for treating cancer in a subject, comprising: (a) (i) determining the frequency of CCR8-expressing Tregs in a test tissue within or derived from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) comparing the frequency of CCR8-expressing Tregs with a pre-determined threshold; and (iii) selecting the subject as not suitable for immunotherapy using a therapeutic Treg-depleting Ab or an antigen-binding portion thereof based on an evaluation that the frequency of CCR8-expressing Tregs in the cells of the test tissue is less than the pre-determined threshold, thereby selecting a subject who is not a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (b) administering to the selected subject a standard therapeutic agent other than the therapeutic anti-CCR8 Ab or an antigen-binding portion thereof.

[0144] The present disclosure is a method for treating cancer in a subject, comprising: (a) (i) determining the frequency of CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) administering to the subject a therapeutic Treg-depleting Ab or an antigen-binding portion thereof; (iii) determining whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of the therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (iv) selecting the subject as not suitable for immunotherapy using the therapeutic Treg-depleting Ab or an antigen-binding portion thereof based on an assessment that the decrease in the frequency of CCR8-expressing Tregs is less than a pre-determined threshold, thereby selecting a subject who is not a suitable candidate for cancer immunotherapy using the therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (b) administering to the selected subject a standard therapeutic agent other than the therapeutic anti-CCR8 Ab or an antigen-binding portion thereof. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab.

[0145] The present disclosure also provides a method for treating cancer in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, wherein the subject is selected based on a determination that the frequency of CCR8-expressing Tregs in cells of a test tissue within or derived from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs, exceeds a pre-determined threshold level.

[0146] The present disclosure also provides a method for treating cancer in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, wherein the subject is selected based on a determination that the therapeutic Treg-depleting Ab or an antigen-binding portion thereof causes a decrease in the frequency of CCR8-expressing Tregs that exceeds a pre-determined threshold.

[0147] The present disclosure further provides a method for treating cancer in a subject, comprising administering to the subject a therapeutic Treg-depleting Ab or a standard therapeutic treatment other than its antigen-binding portion, wherein the subject is selected based on the determination that the frequency of CCR8-expressing Tregs in a test tissue within or derived from the subject, which comprises tumor cells and tumor-infiltrating Tregs, is less than a predetermined threshold level.

[0148] The present disclosure further provides a method for treating cancer in a subject, comprising administering to the subject a therapeutic Treg-depleting Ab or a standard therapeutic treatment other than its antigen-binding portion, wherein the subject is selected based on the ability of the therapeutic Treg-depleting Ab or its antigen-binding portion to cause a decrease in the frequency of CCR8-expressing Tregs that is less than a predetermined threshold level.

[0149] In certain preferred embodiments of any of the above methods for treating cancer in a subject, the therapeutic Treg-depleting Ab is an anti-CCR8 Ab. In a more preferred embodiment, the Treg-depleting Ab or its antigen-binding portion is an mAb comprising the six CDRs, the heavy and light chain variable regions, of the mAb named 4A19, or the heavy and light chains. In certain other embodiments, the Treg-depleting Ab or its antigen-binding portion is 4A19, 18Y12, 8D55, 10R3, 14S15, or 14S15h, or an antigen-binding portion thereof. In certain preferred embodiments, the Treg-depleting Ab is mAb4A19. In certain other embodiments, the Treg-depleting Ab is an anti-CTLA-4 Ab. In a further embodiment, the Treg-depleting Ab is an anti-CCR4 or anti-CD25 Ab.

[0150] In certain situations, for example, when there are constraints on the availability of tissue samples obtained from tumor biopsies, or when it is desirable to predict or monitor efficacy before clinical signs appear within the tumor, it may be quicker, more efficient, and / or more practical to measure the frequency of CCR8-expressing cells, and the depletion of CCR8-expressing cells induced by administration of a Treg depletion treatment, using a blood or PBMC sample rather than a tumor sample. As disclosed herein, the non-competing anti-CCR8 mAbs of the invention enable quantification of CCR8-expressing cells in whole blood or PBMCs, and depletion of CCR8-expressing cells, despite the very low frequency of such cells in peripheral blood. Thus, in any of the above methods for predicting the efficacy of a therapeutic Treg depletion Ab when treating cancer in a patient, selecting a cancer patient as a suitable candidate for cancer immunotherapy using a therapeutic Treg depletion Ab and / or treatment of such a patient with a therapeutic Treg depletion Ab, determining the frequency of CCR8-expressing Tregs, and / or determining whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of a therapeutic Treg depletion Ab is carried out using a blood or PBMC sample taken from or within the subject.

[0151] In certain embodiments, the Treg-depleting Ab is administered to a subject in a therapeutically effective amount in combination with an additional therapeutic agent for treating cancer. In certain embodiments, the additional anti-cancer agent is a small molecule, polypeptide, antibody, immunomodulatory agent, chemotherapeutic agent, agent for targeted therapy, or any combination thereof. In certain embodiments, the immunotherapy comprises an agent that reduces the inhibition or increases the stimulation of the immune system. In certain preferred embodiments, the additional therapeutic agent is a compound that reduces the inhibition or increases the stimulation of the immune system. Such additional therapeutic agents can be, for example, small molecule compounds, macrocyclic peptides, fusion proteins, or Abs, such as mAbs. In certain embodiments, the additional therapeutic agent that reduces the inhibition of the immune system is an antagonist agent that specifically binds to an immune inhibitory receptor, such as programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), B and T lymphocyte attenuator (BTLA), T cell immunoglobulin and mucin domain 3 (TIM-3), killer immunoglobulin-like receptor (KIR), killer cell lectin-like receptor G1 (KLRG-1), adenosine A2a receptor (A2aR), T cell immune receptor with Ig and ITIM domains (TIGIT), V domain Ig suppressor of T cell activation (VISTA), proto-oncogene tyrosine protein kinase MER (MerTK), natural killer cell receptor 2B4 (CD244), or CD160, such as an antagonistic mAb, etc.

[0152] In certain preferred embodiments, the additional therapeutic agent is an antagonistic Ab or an antigen-binding portion thereof that specifically binds to PD-1. In further embodiments, the Ab that specifically binds to PD-1 is selected from nivolumab, pembrolizumab, semipramab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, zimberelimab, pimavlimab, serplulimab, boptrelizumab, and acrixolimab. In certain preferred embodiments, the Ab is selected from nivolumab and pembrolizumab.

[0153] In other preferred embodiments, the additional therapeutic agent is an antagonistic agent, such as an Ab that specifically binds to PD-L1 or an antigen-binding portion thereof. In further embodiments, the agent that specifically binds to PD-L1 is selected from atezolizumab, durvalumab, avelumab, enoblituzumab, cosibelimab (CK-301), BMS-936559, BMS-986189, CS-1001, SHR-1316, CBT-502, BGB-A333, KN035, AUNP12, and CA-170. In certain preferred embodiments, the agent is an Ab selected from atezolizumab, durvalumab, and avelumab.

[0154] In other preferred embodiments, the additional therapeutic agent is an antagonistic Ab or an antigen-binding portion thereof that specifically binds to CTLA-4. In further embodiments, the Ab that specifically binds to CTLA-4 is ipilimumab or tremelimumab. In certain preferred embodiments, the Ab is ipilimumab.

[0155] In other preferred embodiments, the additional therapeutic agent is an antagonistic agent that specifically binds to LAG-3. In further embodiments, the agent that specifically binds to LAG-3 is selected from each Ab of relatlimab, fabzolimab, tiragolumab, fianlimab, or tebotelimab, or the soluble protein eftilagimod alpha. In certain preferred embodiments, the Ab is relatlimab.

[0156] In certain embodiments, the additional therapeutic agent that increases stimulation of the immune system is an activating agent, such as an activating mAb, that is an activating agent that specifically binds to an immune-stimulatory receptor, such as inducible T cell co-stimulator molecule (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-induced TNFR-related protein (GITR), or herpes virus entry mediator (HVEM).

[0157] In certain embodiments of the above method, determining the level of CCR8 expression in a test tissue comprises assessing the level of CCR8 expression on the surface of Tregs in the test tissue. In certain other embodiments, determining the level of CCR8 expression in a test tissue comprises assessing the proportion of Tregs in the test tissue that express CCR8 on the surface of the Treg cells.

[0158] In further embodiments, the level of CCR8 expression in a subject's test tissue is determined by an in vivo method. In still further embodiments, the in vivo method comprises, for example, a PET tracking method using the anti-CCR8 Ab of the present invention that binds to an epitope of CCR8 located outside the N-terminal domain.

[0159] In other embodiments, the level of CCR8 expression is determined ex vivo in a test tissue sample obtained from a subject. For example, in certain embodiments of the ex vivo method, the level of CCR8 expression is determined by immunohistochemistry (IHC), flow cytometry, or mass spectrometry-coupled flow cytometry using the labeled anti-CCR8 Ab of the present invention or an antigen-binding portion thereof that binds to CCR8 expressed on the surface of cells in the tissue. In certain embodiments, the IHC is performed on fresh frozen tissue or formalin-fixed paraffin-embedded (FFPE) tissue. In certain preferred embodiments, determining the level of CCR8 expression using the anti-CCR8 Ab of the present invention is not affected by the binding of a therapeutic Ab or an antigen-binding portion thereof to the N-terminal domain of CCR8.

[0160] In certain embodiments of any of the above methods, the therapeutic anti-CCR8 Ab or an antigen-binding portion thereof is (a) Heavy chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 53; heavy chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 54; heavy chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 55; light chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 56; light chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 57; and light chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 58 (b) V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 9 H and V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 16 L and / or (c) A heavy chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 65 and a light chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 72 comprising.

[0161] In certain other embodiments, the therapeutic anti-CCR8 Ab or antigen-binding portion thereof is an Ab or antigen-binding portion thereof designated as 4A19, 18Y12, 8D55, 10R3, 14S15, or 14S15h in WO2021 / 194942.

[0162] In certain embodiments of the methods described herein that require a therapeutic anti-CCR8 Ab or antigen-binding portion thereof, the therapeutic Ab or antigen-binding portion thereof is the six CDRs, heavy and light chain variable regions, and / or heavy and light chains of an mAb designated as 4A19, 18Y12, 8D55, 10R3, 14S15, or 14S15h in WO2021 / 194942. In further embodiments, the therapeutic Ab or antigen-binding portion thereof is 4A19, 18Y12, 8D55, 10R3, 14S15, or 14S15h, or an antigen-binding portion thereof. In certain preferred embodiments, the therapeutic Ab is 4A19.

[0163] In certain embodiments of such methods that require an anti-CCR8 Ab that does not compete with the therapeutic Ab for binding to CCR8, the non-competing Ab is an mAb comprising the six CDRs, heavy and light chain variable regions, and / or heavy and light chains of the mAbs named herein as 23A14, 21C17, 22B13, 25T40, 28P3, and 33H18. In certain preferred embodiments, the non-competing Ab is 21C17.

[0164] In certain preferred embodiments of any of such methods, the subject is human.

[0165] Cancer treatable by depletion of Tregs Immuno - oncology, which is based on using the virtually infinite flexibility of the immune system to attack and destroy cancer cells, is applied to the treatment of a very wide range of cancers (see, e.g., Yao et al., 2013; Callahan et al., 2016; Pianko et al., 2017; Farkona et al., 2016; Kamta et al., 2017; Drugs.com - Opdivo Approval History: https: / / www.drugs.com / history / opdivo.html). For example, nivolumab, an anti - PD - 1 Ab, has been shown to be effective in treating many different types of cancers (see, e.g., Brahmer et al., 2015; Guo et al., 2017; Pianko et al., 2017; WO2013 / 173223; Drugs.com - Opdivo Approval History), and its clinical trials are currently ongoing in multiple solid and hematological cancers. Similarly, anti - PD - L1 drugs, such as atezolizumab [TECENTRIQ (registered trademark)], durvalumab [IMFINZI (registered trademark)], and avelumab [BAVENCIO (registered trademark)] etc., are seeking approval in various indications. Thus, an immunotherapeutic method for treating cancer that utilizes depletion of Tregs, such as CCR8 - mediated depletion of tumor - infiltrating Tregs as disclosed herein, is applicable to treating both solid tumors and liquid tumors with their diverse types of tumors.

[0166] For example, in certain embodiments of a Treg - depletion therapy for treating cancer in which the Abs of the invention are used to monitor changes in the number of Tregs, the cancer is a solid tumor.

[0167] Based on the demonstration of the efficacy of treating different cancers with anti-CCR8 in a mouse model (see WO2021 / 194942), a particular tumor type is expected to be particularly effective against treatment with anti-CCR8 Ab. Thus, in certain embodiments of the methods disclosed herein, the solid tumor is a cancer selected from colon adenocarcinoma, bladder cancer, breast cancer, and fibrosarcoma.

[0168] In certain embodiments, CCR8 identified by RNA sequence analysis of a single cell + Based on the relatively high expression of CCR8 and CD8A in Tregs, and the high ratio of CCR8 / CD8A, the solid tumor is a cancer selected from head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), gastric adenocarcinoma (STAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), rectal adenocarcinoma (READ), esophageal cancer (ESCA), invasive breast cancer (BRCA), colon adenocarcinoma (COAD), and cervical squamous cell carcinoma and cervical adenocarcinoma (CESC).

[0169] High levels of CCR8 expression may be identified by IHC in FFPE tissue samples within a particular tumor, and tumors expressing high levels of CCR8 are more likely to respond to treatment with anti-CCR8 Ab. Thus, in certain embodiments, the solid tumor is a cancer selected based on CCR8 expression level from head and neck squamous cell carcinoma (HNSCC) [also referred to herein as squamous cell carcinoma of the head and neck (SCCHN)], cervical cancer, colorectal cancer (CRC), non-small cell lung cancer - squamous cell carcinoma (NSCLC-SCC), NSCLC - adenocarcinoma (NSCLC-ADC), pancreatic, gastric, bladder cancer, and breast cancer.

[0170] In the case of the Phase I / II clinical trial of anti-CCR8 mAb 4A19, administered as a single agent and in combination with nivolumab, an anti-PD-1 mAb, certain solid tumors were selected based on the demonstrated efficacy of anti-CCR8 in a mouse tumor model, the RNA expression of CCR8 and CD8A within tumor types presented in The Cancer Genome Atlas (National Cancer Institute, 2021), tumor types with relatively high expression of CCR8 and enriched CD8A expression, and the level of CCR8 expression within different tumor types or subtypes as measured by IHC. Based on the tumor types selected for the clinical trial, in certain embodiments of the disclosed methods, the solid tumors are cancers selected from non-small cell lung cancer (NSCLC), SCCHN, microsatellite stable colorectal cancer (MSS-CRC), adenocarcinoma of the stomach / gastroesophageal (GE) junction, cervical cancer [squamous cell carcinoma (SCC) or adenocarcinoma], renal cell carcinoma (RCC), urothelial carcinoma (UC), pancreatic ductal adenocarcinoma (PDAC), melanoma, ovarian cancer (OC), and triple negative breast cancer (TNBC).

[0171] In further embodiments, the solid tumor is a cancer selected from squamous cell carcinoma, small cell lung cancer (SCLC), NSCLC, squamous cell NSCLC, non-squamous cell 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, RCC, bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, colon carcinoma, anal area cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, nasal natural killer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, endometrial carcinoma, fallopian tube cancer, ovarian cancer, cervical cancer, vaginal cancer, vulvar cancer, testicular cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, penile cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid cancer, squamous cell cancer, pediatric solid tumor, environmentally induced cancer, virus-related cancer, and virus-originated cancer. In certain embodiments, the cancer is an advanced unresectable metastatic refractory cancer, or a recurrent cancer, or any combination thereof.

[0172] In certain embodiments of the disclosed methods, the cancer is a hematological malignancy. Hematological malignancies include liquid tumors derived from either of two major blood cell lineages, namely the myeloid cell lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells), or the lymphoid cell lineage (which produces B, T, NK, and plasma cells), including all types of leukemia, lymphoma, and myeloma.

[0173] In the TARGET (Therapeutically Applicable Research to Generate Effective Treatments, https: / / ocg.cancer.gov / programs / target) analysis, among the hematological malignancies tested, follicular lymphoma (FL), as well as acute lymphoblastic leukemia and lymphoma, have been found to have the highest relative expression levels of CCR8 and have been shown to be the top priorities for treatment with anti-CCR8 mAb (see WO2021 / 194942). Thus, in certain embodiments of this treatment method, the hematological malignancies are FL or acute lymphoblastic leukemia and lymphoma.

[0174] In certain other embodiments, the hematological malignancy is acute, chronic, lymphocytic (lymphoblastic), and / or myelogenous leukemia, such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), etc.; lymphoma, such as Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), etc., about 85% of which are B-cell lymphomas including diffuse large B-cell lymphoma (DLBCL), FL, CLL / small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphoma [lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, and splenic marginal zone B-cell lymphoma], Burkitt lymphoma, lymphoplasmacytic lymphoma [also known as Waldenström macroglobulinemia (WM)], hairy cell lymphoma, and primary central nervous system (CNS) lymphoma, precursor T-lymphoblastic lymphoma / leukemia, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, such as cutaneous T-cell lymphoma, etc. (CTLC, i.e., mycosis fungoides, Sézary syndrome, etc.), adult T-cell lymphoma / leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma nasal type, enteropathy-associated intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), and peripheral T-cell lymphoma of unspecified type; NHL including acute myeloid lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, and precursor B-lymphoblastic lymphoma; myeloma, such as multiple myeloma, smoldering myeloma (also called indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma, IgG myeloma, light chain myeloma, non-secretory myeloma, and amyloidosis, etc.; and cancers selected from any combination of the foregoing hematological malignancies.

[0175] This method is also applicable to the treatment of advanced, metastatic, refractory, and / or relapsed hematological malignancies, and any combination of said hematological malignancies.

[0176] Kit Also included within the scope of the present invention are kits comprising an anti-CCR8 Ab that specifically binds to an epitope located outside the N-terminal domain of hCCR8, and other components necessary for carrying out any of the methods disclosed herein. Kits generally include a label and instructions indicating the intended use of the kit contents. The term "label" includes any written or recorded material supplied on or with the kit, or otherwise attached thereto. Accordingly, the present disclosure provides a kit for monitoring and / or quantifying the number of Tregs in a subject, measuring depletion of the number of Tregs in a subject, predicting the efficacy of a therapeutic Treg-depleting Ab in treating cancer in a subject, selecting a subject afflicted with cancer as a suitable candidate for immunotherapy using a therapeutic Treg-depleting Ab, or treating cancer in a subject with a therapeutic Treg-depleting Ab or treating cancer in a subject with a standard therapeutic agent other than a therapeutic Treg-depleting Ab, as described in the methods disclosed herein. In certain embodiments, the kit comprises (a) an mAb of the invention or an antigen-binding portion thereof that specifically binds to an epitope located outside the N-terminal domain of hCCR8, (b) optionally, as a treatment method, a therapeutic Treg-depleting Ab or an antigen-binding portion thereof that binds to the N-terminal domain of hCCR8, or alternatively a standard therapeutic agent other than a therapeutic Treg-depleting Ab, and (c) instructions for using the mAb of the invention or a portion thereof in any of the methods disclosed herein for measuring depletion of the number of Tregs in a subject, predicting the efficacy of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof in treating cancer in a subject, selecting a subject afflicted with cancer as a suitable candidate for immunotherapy using a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, treating cancer in a subject with a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, or treating cancer in a subject with a standard therapeutic agent other than a therapeutic Treg-depleting Ab or an antigen-binding portion thereof.

[0177] In a certain preferred embodiment of the above kit, the therapeutic Treg-depleting Ab is an anti-CCR8 Ab. In a more preferred embodiment, the Treg-depleting Ab is an anti-CCR8 Ab that binds to an epitope within the N-terminal domain of hCCR8. In the case of such a Treg-depleting Ab, the kit comprises the mAb of the present invention or an antigen-binding portion thereof that specifically binds to an epitope located outside the N-terminal domain of hCCR8. In a certain variant form, the kit comprises a Treg-depleting Ab that is an anti-CCR8 Ab that binds to an epitope outside the N-terminal domain of hCCR8, in which case the kit comprises an mAb that specifically binds to an epitope within the N-terminal domain of hCCR8.

[0178] In certain other embodiments, the Treg-depleting Ab is an anti-CTLA-4 Ab. In further embodiments, the Treg-depleting Ab is an anti-CCR4 or anti-CD25 Ab. In certain other embodiments, the kit comprises additional anti-cancer therapeutic agents or therapies that can be, for example, small molecule agents, polypeptides, antibodies, immunomodulators, chemotherapeutic agents, agents for targeted therapy, or combinations thereof. In certain preferred embodiments, the additional anti-cancer therapeutic agents are checkpoint inhibitors, such as anti-PD-1, anti-PD-L1, anti-CTLA-4, or anti-LAG-3 Abs, etc., and / or chemotherapeutic agents, such as docetaxel, etc.

[0179] In certain embodiments, the kit comprises (a) one or more dosages in the range of about 0.1 to about 20 mg per kg of body weight for a therapeutic mAb or an antigen-binding portion thereof that specifically binds to CCR8; and optionally (b) one or more dosages for a checkpoint inhibitor, for example, a dosage such as about 3 mg per kg of body weight, or 200 to about 1600 mg, etc., for an anti-PD-1 / anti-PD-L1 mAb or an antigen-binding portion thereof.

[0180] The present invention is further illustrated by the following examples, which should not be construed as further limitations. The contents of all references cited throughout this application are hereby expressly incorporated by reference herein.

Examples

[0181] [Example 1]

[0182] Generation of mAbs targeting CCR8 Mouse or human anti-hCCR8 mAbs were generated by immunizing C57BL / 6 mice or a strain of transgenic mice expressing the human Ig repertoire with the hCCR8 immunogen, respectively.

[0183] Immunization with CCR8 antigen To generate Abs targeting hCCR8, a cohort of 2 - 5 C57BL / 6 mice or human Ig transgenic mice was immunized 8 - 12 times over 7 - 10 weeks with materials derived from hCCR8 overexpressing cells, immunizing every 5 - 7 days via injection into the footpad or the base of the tail while performing 8 - 12 immunization procedures. In some cases, to enrich the hCCR8 protein in the plasma membrane and reduce the number of non-CCR8 cell antigen administrations, the plasma membrane fraction was isolated from 2.5×10 6 individual hCCR8 overexpressing BA / F3 cells using differential centrifugation. In other cases, the surfactant-stabilized proteoliposome material was derived from HEK293 cells that overexpressed the chimeric hCCR8 / hCCR5 protein and also overexpressed the anti-hCCR8 Ab Clone L263G8 (BioLegend). The 5 μg dose of proteoliposome was delivered with the n-dodecyl-β-D-maltoside (DDM) surfactant dissolved in phosphate buffered saline (PBS). Another injection of water-in-oil RIBI adjuvant (Sigma-Aldrich, St. Louis, MO) was administered at a site adjacent to the antigen injection to enhance the immune response without destabilizing the proteoliposome or denaturing the extracellular conformation of CCR8.

[0184] To monitor the immune response, as described in Example 2, generally 4 to 6 weeks after the immunization, titrated sera from retro-orbital or tail bleeds were screened by flow cytometry and ELISA. The sera were screened for binding of Abs to a plurality of CCR8 overexpressing cell lines, corresponding negative control cell lines that do not overexpress CCR8, and sulfated N-terminal peptides of CCR8 conjugated to bovine serum albumin (BSA). CCR8-specific and CCR8-nonspecific Ab responses were measured in each animal, and animals having a sufficient titer for anti-CCR8 Ig were selected for the final immunization, sacrificed 6 and 3 days later, and tissues were harvested to create hybridoma fusions.

[0185] Generation of hybridomas producing mAbs against CCR8 Lymphoid organs targeted by the immunization strategy were collected. Most commonly, popliteal, inguinal, and iliac lymph nodes from mice immunized with CCR8 immunogen via the plantar and caudal bases were used. Tissues were homogenized into cell suspensions and integrated with an equal number of immortalized mouse myeloma cells derived from the P3X63AgU.1 cell line (ATCC CRL-1597). The integrated cells were prepared at a density of 1×10 cells / ml in low conductivity Cytofusion Medium C (BTX, Holliston, MA). Hybridomas were generated using an electrofusion unit (BTX) to fuse Ab-secreting B cells with immortalized myeloma cells. The resulting cells were grown in flat-bottom 96-well cell culture plates using hypoxanthine and thymidine-containing Medium E (StemCell Technologies, Cambridge, MA) supplemented with aminopterin (Sigma-Aldrich) for hybridoma selection. 7 [Example 2]

[0186] Screening and selection of anti-human CCR8 mAbs Screening of mAbs that selectively bind to human CCR8 To generate mAbs that bind to hCCR8, normal mice or human Ig transgenic mice were immunized with an antigen derived from hCCR8 overexpressing cells, and hybridomas were generated as described in Example 1. 10 - 13 days after culture and growth medium replacement, hybridoma culture supernatants were collected from individual wells and screened to identify wells containing secreted CCR8 - specific Abs. All supernatants were first screened against at least two cell lines, a line overexpressing hCCR8 and the corresponding control cell line that did not overexpress CCR8. Ab binding to the cells was measured through an image - based fluorescence quantitative micro - volume assay technology (FMAT) screening.

[0187] Hybridomas from positive wells were transferred to a 24 - well plate containing fresh culture medium and grown for 2 - 3 days, and then rescreened by flow cytometry to confirm Ab binding to CCR8. Briefly, 75 - 100 μl of hybridoma culture supernatant and CCR8 overexpressing cells (e.g., CCR8 overexpressing CHO or 293F cells, etc.) or control cells (e.g., GFP - CHO or parental 293F, etc.) were co - incubated for 30 - 60 minutes, washed, and then incubated with an anti - mouse IgG Fc or anti - human IgG Fc secondary Ab conjugated to AF647, APC, or PE (Jackson ImmunoResearch, West Grove, PA). After incubation and washing, fluorescence was measured by flow cytometry.

[0188] To ensure monoclonality, anti-CCR8 Ab-secreting hybridomas were subcloned one or two times. Briefly, approximately 700 viable hybridoma cells were seeded into 5 ml of semi-solid methylcellulose medium (StemCell Technologies) containing AF488-conjugated anti-human or anti-mouse IgG Ab (Jackson ImmunoResearch), which is used to detect hybridoma-secreted IgG. After 7 days, hybridoma colonies arising from single cells were imaged using the ClonePix2 system (Molecular Devices, San Jose, CA). A single colony with desirable characteristics (including distance from other colonies, IgG secretion level, colony size, and colony circularity) was picked into a well of a 96-well plate and cultured for 2 - 4 days. To confirm CCR8 Ab binding, culture supernatants were screened by flow cytometry as previously described. Stable hybridoma subclones were cultured in vitro to generate Ab for affinity purification and further characterization. DNA sequences encoding the heavy and light chains of the Ab were obtained by standard sequencing techniques (Sanger sequencing and next-generation sequencing). To ensure sequencing accuracy, the mass predicted from the Ab amino acid sequence was compared with the known mass of the purified Ab obtained by mass spectrometry.

[0189] Characterization of the anti-hCCR8Ab Epitope To roughly characterize the epitopes of the confirmed CCR8-specific Abs, hybridoma culture supernatants were screened by ELISA to measure binding to the hCCR8 N-terminal peptide. Briefly, a BSA-conjugated peptide corresponding to the N-terminal 35 amino acid residues of hCCR8 (SEQ ID NO: 74; 2 μg / ml) that presents the appropriately sulfated N-terminus of hCCR8 was coated overnight at 4° C. onto high-binding 96-well plates (Corning, Corning, NY). The plates were blocked with BSA, washed, and then 100 μl of the culture supernatant was added on a plate shaker for 30-60 minutes. After washing, the appropriate anti-Fc secondary Ab conjugated to horseradish peroxidase (HRP) was added and left for 30-60 minutes. The plates were developed using ABTS or an HRP substrate (SurModics, Eden Prairie, MN), and absorbance was measured at 405 or 650 nm on a Sunrise microplate reader (TECAN; Mannedorf, Switzerland).

[0190] Abs generated against hCCR8 were shown by ELISA to bind predominantly, but not exclusively, to the BSA-conjugated N-terminal peptide sequence having two sulfated tyrosine residues. From several flow cytometry-based Ab competition experiments, this major group of hCCR8 N-terminal-binding Abs was shown to consistently block each other's simultaneous binding to CCR8 on cells. However, six mAbs (23A14, 21C17, 22B13, 25T40, 28P3, and 33H18) were found to specifically bind to hCCR8 by flow cytometry but not to the N-terminal peptide of BSA-conjugated CCR8 by ELISA, suggesting that they bind to epitopes on hCCR8 that are not located within the N-terminal domain. These six Abs were further characterized by flow cytometry-based binding competition experiments. [Example 3]

[0191] The anti-CCR8 mAb 21C17 binds to tumor CD4 Tregs pretreated with the mAb 4A19, while the commercial Clone L263G8 does not bind. PCT Publication No. WO2021 / 194942 describes humanized and human mAbs that bind with high affinity to hCCR8 expressed on the cell surface and mediate depletion of CCR8-expressing tumor-infiltrating Tregs by mechanisms including ADCC. One of those mAbs (designated 4A19) was shown to bind to an epitope within the N-terminal domain of CCR8 that includes residues 12 - 22 and contains the sulfated tyrosine 15 and leucine 17 residues.

[0192] To determine whether the binding of mAb4A19 to hCCR8 blocks the binding of 21C17, a test was conducted. By using the Tumor Dissociation Kit (Miltenyi Biotec, Sunnyvale, CA) in conjunction with the gentleMACS Dissociator (Miltenyi Biotec), tissue derived from gastric tumors was fragmented and stored in liquid nitrogen. In this test, thawed cells were incubated on ice for 15 minutes with unlabeled mAb4A19, unlabeled mAb21C17, or unlabeled isotype control (10 μg / ml each). After incubation, the samples were stained with an immunoprofiling panel containing Abs targeting CD3, CD8, CD4, FOXP3, and two Abs targeting CCR8, the commercially available anti-CCR8 Ab L263G8 (BioLegend) conjugated to phycoerythrin (PE), and mAb21C17 conjugated to BRILLIANT VIOLET™ 421. After the cells were stained, they were processed on a flow cytometer, the data were analyzed, and plotted using FLOWJO™ software (BD Biosciences, San Jose, CA).

[0193] The three flow cytometry plots in Figure 1 show the gastric tumor Treg compartment that binds to the BV421 conjugate mAb21C17 on the x-axis and the PE conjugate Clone L263G8 on the y-axis. Tumor Tregs pretreated with 4A19 are shown in Figure 1A. The results show that no CCR8 + cells are observed in the Q1 or Q2 quadrants, indicating that PE-L263G8 is blocked from binding to Tregs pretreated with 4A19, and that binding of subsequent L263G8 is blocked by the binding of 4A19 to CCR8. In contrast, however, since the CCR8 + population is observed in the Q3 quadrant, BV421-21C17 binds to cells pretreated with such 4A19 (Figure 1A). Figure 1B represents a sample treated with unlabeled 21C17. Conversely, BV421-21C17Ab has its binding blocked (quadrants Q2 and Q3), while most of the CCR8 + population is in the Q1 quadrant, indicating that PE-L263G8 binds to Tregs pretreated with 21C17. Thus, 21C17 does not block the binding of L263G8, but, as expected, blocks its own binding to CCR8. Figure 1C represents the effect of pretreatment with an isotype control. Since most of the CCR8 + cells are in the Q2 quadrant, it shows that both 21C17 and PE-L263G8 can bind to the same cells. Overall, the results show that the 21C17 mAb can bind to tumor CCR8-expressing Tregs to which the 4A19 mAb has previously bound, indicating that it binds to an epitope different from that of 4A19 on CCR8. Regarding binding to hCCR8, no cross-competition is observed between mAb4A9 and 21C17, suggesting that they do not bind to substantially the same epitope region of hCCR8, i.e., their epitopes are not adjacent or overlapping with each other, and binding of one Ab to its epitope does not sterically hinder the binding of the other Ab to its epitope. [Example 4]

[0194] An antibody that binds with high affinity to an epitope located outside the N-terminus of CCR8 EC regarding the binding of mAb to an epitope located outside the N-terminus of hCCR8 50 value FACS was used to evaluate the binding specificity of the Ab that binds to CCR8. Transfected Raji cells overexpressing hCCR8 and parental Raji cells not expressing CCR8 were incubated on ice for 30 minutes with a cell viability dye. Unbound dye was washed away, and the cells were seeded in 96-well round-bottom plates at a concentration of 5×10 5 cells per well. mAbs that bind to epitopes other than the N-terminal epitope of CCR8 were serially diluted, starting at a concentration of 20 nM, and the dilutions were added and incubated on ice for 30 minutes. Unbound Ab was washed away, a secondary Ab for detection was added, and the mixture was incubated on ice for 30 minutes. Additional washes were performed, and Ab binding affinity was determined by measuring the fluorescence intensity of the secondary Ab on an LSR Fortessa X-20 cytometer (BD Biosciences).

[0195] A binding curve showing the range when different concentrations of mAb bind to Raji cells expressing hCCR8 is shown in Figure 2A, while Figure 2B shows that no binding to parental Raji cells was observed. By this binding analysis, four mAbs that bind to Raji-hCCR8 cells with an EC 50 accompanied by approximately 0.1 nM or less (see Table 1) and show no non-specific binding on parental Raji cells were identified.

[0196]

Table 1

[0197] Competitive binding between N-terminal binding mAb and non-N-terminal binding mAb For binding to hCCR8, competition between an anti-CCR8 mAb (23A14-hIgG1) that does not bind to the N-terminus of CCR8 and an mAb (4A19-mIgG2a) that binds to the N-terminus was assayed by FACS. To isolate human Tregs (hTregs), PBMCs were isolated from leukopak (AllCells) using the Ficoll density gradient method (GE Healthcare). CD25 + cells were magnetically enriched using anti-CD25 MicroBeads II (Miltenyi Biotec). The enriched cells were stained for CD25 (4E3, Miltenyi Biotec), CD127 (A019D5, BioLegend), CD45RA (HI100, BioLegend), and CD4 (SK3, BD Biosciences). CD4 + CD127 low CD25 high CD45RA + Tregs were sorted on a BD FACSAria II.

[0198] To stimulate CCR8 expression, naive Tregs after isolation were activated in vitro at a cell:bead ratio of 1:3 in the presence of 100 U / ml of rhIL-2 using DYNABEADS™ Human T-Activator CD3 / CD28 beads. The activated hTregs were incubated on ice for 30 minutes with a cell viability dye. Unbound dye was washed away, and the cells were resuspended at 5×10 cells per well in FACS staining buffer 5Cells were seeded in 96-well round-bottom plates at various concentrations. The mouse IgG2a original version of anti-CCR8 mAb4A19 (4A19-mIgG2a) described in WO2021 / 194942 was added to the cells at a saturating concentration of 200 nM and incubated on ice for 30 minutes. Unbound Ab was washed away, and the cells were incubated with mAb23A14-hIgG1 whose concentration was continuously decreased from 200 nM to 0.0034 nM. Additional washes were performed, a fluorophore-conjugated secondary Ab specific for human and mouse Ig was added, and the mixture was incubated for 30 minutes. Final washes were performed, and the samples were analyzed on an LSR Fortessa X-20 cytometer.

[0199] As shown in Figure 3A, the binding of mAb23A14-hIgG1 increased even in the presence of a saturating amount of bound 4A19-mIgG2a, indicating that the binding of mAb23A14-hIgG1 was not inhibited even when mAb4A19-mIgG2a was bound. From this competitive assay analysis, although mAb4A19-mIG2a and 23A14-hIgG1 were tested at saturating concentrations, it was still found that CCR8 + It was revealed that it was possible to equally detect the population of Tregs.

[0200] Two additional anti-CCR8 mAbs (21C17-mIgG2a and 22B13-mIgG2a) that do not bind to the N-terminal epitope were tested by incubating them with activated hTregs stained with a cell viability determination dye and mAb4A19 (WO2021 / 194942) at concentrations continuously decreased in the range of 200 nM to 0.0034 nM. After incubation and washing on ice for 30 minutes, a fixed concentration (100 nM) of 21C17-mIgG2a or 22B13-mIgG2a was added and incubated on ice for 30 minutes. The cells were washed, a detection Ab specific for human and mouse Ig was added, and the mixture was incubated on ice for 30 minutes. A final washing step was performed, and the samples were analyzed on an LSR Fortessa X-20 cytometer.

[0201] The results obtained for 21C17-mIgG2a and 22B13-mIgG2a are shown in Figures 3B and 3C, respectively. From the competitive assay analysis, when mAb4A19 pairs with either 21C17-mIgG2a or 22B13-mIgG2a, CCR8 at saturation concentration + It is clear that a similar population of human Tregs can be detected. [Example 5]

[0202] Fluorescent dye-labeled Ab that binds to hCCR8-expressing cells Flow cytometry assays were used to evaluate the binding of anti-hCCR8 Ab on cell lines overexpressing hCCR8. In this assay, Raji cells overexpressing hCCR8 were used to determine the binding of mAb21C17-mIgG2a directly conjugated with BV-421 from BioLegend. Unlabeled 21C17-mIgG2a was used as a positive control. To evaluate the specificity of the mAb, binding to parental Raji cells that do not express CCR8 was also tested. Parental Raji cells and Raji cells overexpressing hCCR8 were individually mixed with serial dilutions (20 - 0.0034 nM) of unlabeled 21C17-mIgG2a, BV-421-labeled 21C17-mIgG2a, or KLH-mIgG2a control mAb. Binding of unlabeled 21C17-mIgG2a to cell surface hCCR8 was detected using a PE-labeled anti-mouse IgG (Fab’)2 fragment (Jackson ImmunoResearch). Relative cell binding was measured as the geometric mean fluorescence intensity (GMFI) of all cells positive for the PE-conjugated anti-mouse IgG Ab or the directly BV-421-labeled 21C17-mIgG2a Ab.

[0203] As shown in Figure 4A, both unlabeled and directly BV-421-labeled 21C17-mIgG2a Ab bind to cell surface CCR8 on the hCCR8-overexpressing Raji cell line. It was confirmed that these Abs also specifically bind to hCCR8, as evidenced by the lack of binding to parental Raji cells that do not express hCCR8 (Figure 4B). [Example 6]

[0204] Receptor occupancy assay for evaluating target engagement by a therapeutic anti-CCR8 antibody Receptor occupancy (RO) assays are utilized in clinical trials to evaluate target engagement, define dose selection, and thereby provide useful insights into the pharmacodynamics and safety assessment of biotherapeutics. A flow cytometry assay using peripheral blood was developed and validated for measuring CCR8 RO in subjects after administration of anti-CCR8 therapeutic Ab.

[0205] Two independent RO assay formats were developed: (1) a direct / gross format (measuring bound and total receptors), and (2) an indirect / gross format (measuring free and total receptors). The latter represents a combination of an indirect RO and a gross RO assay format; the indirect RO format employs a competitive anti-CCR8 Ab to detect free CCR8 receptors not occupied by the anti-CCR8 therapeutic Ab after treatment and indirectly derive RO, while the gross RO assay format utilizes a non-competitive anti-CCR8 Ab for binding of the therapeutic anti-CCR8 Ab after treatment to measure total CCR8 receptors available on Tregs. Information on both free CCR8 receptors and total CCR8 receptors is used in calculating %RO in the indirect / gross assay format using the formula described below.

[0206] The total number of CCR8 receptors on Tregs can vary throughout the course of treatment with therapeutic anti-CCR8 Ab, for example, in clinical trials. Thus, continuous monitoring of overall CCR8 receptor levels is enabled, and it becomes possible to account for any changes in overall receptor levels that occur in response to anti-CCR8 Ab therapy when calculating %RO throughout the course of treatment. Therefore, it is advantageous to use non-competitive anti-CCR8 Ab, i.e., an Ab that does not compete with the therapeutic Ab for binding to CCR8. Thus, using a non-competitive anti-CCR8 Ab, i.e., an Ab that does not compete with the therapeutic Ab for binding to CCR8, is important for determining the regulation of overall CCR8 expression levels and for assisting in accurate %RO calculations.

[0207] Measurement of CCR8 RO is particularly challenging due to the limited assay range resulting from low CCR8 expression levels on peripheral Tregs. However, by leveraging non-competitive anti-CCR8 Ab, a specific CR8 + Treg subset was selected as the target cell population for the assay. This served as an effective strategy to improve the assay dynamic range 5-fold, thereby enhancing the robustness and reliability of the indirect / overall RO assay.

[0208] A direct comparison of two separate RO assay formats was performed. Whole blood samples (180 μl) were pre-treated with various concentrations of therapeutic mAb, i.e., mAb4A19 (WO2021 / 194942), mimicking different 4A19 dosing treatment levels administered to subjects within the dose escalation cohort of a Phase I clinical trial (NCT04895709), and pre-incubated at 37 °C for 1 hour. Aliquots of the treated samples were transferred to 4 °C for sample stability experiments and held for 24 hours, 48 hours, and 72 hours.

[0209] After pre-incubation, tubes containing background samples were treated with mAb4A19 at a saturation concentration (10 μg / ml), while the other tubes [containing free, bound, or FMO (fluorescence minus one sample)] were treated with PBS (same volume as mAb4A19) for 30 minutes at room temperature (RT). Samples were centrifuged at 4°C, 500 × g for 5 minutes, the pellet was washed 3 times with GIBCO (trademark) Ca / Mg-free Dulbecco's phosphate-buffered saline (DPBS, Thermo Fisher Scientific, Waltham, MA), transferred to a new tube, and then stained with a cocktail consisting of 7 markers within the following core panel: anti-human CD3 AF488 (BioLegend) as a basic T cell marker, anti-human CD4 BV510 (BioLegend), and anti-human CD8 BV605 (BD Biosciences); anti-human CD45RA APC-CY7 (BioLegend) as a T cell differentiation marker; and anti-human CD25 PE (BioLegend), anti-human CD127 APC-R700 (BD Biosciences), and anti-human CCR4 PE-Cy7 (BioLegend) as Treg markers. For the direct / whole RO assay, to detect the bound CCR8 receptor, one additional Ab (anti-Id AF647, a custom-conjugated anti-idiotype Ab, Bristol Myers Squibb) was added to the core cocktail panel, while for the indirect / whole assay, two additional Abs, namely clone 433H (BD Biosciences), a competitive anti-hCCR8 Ab APC for detecting the free CCR8 receptor, and 21C17, a non-competitive anti-hCCR8 mAb for detecting the total CCR8 receptor, were added on ice for 30 minutes. Once staining was complete, samples were washed twice with MACS buffer (Miltenyi Biotec), followed by erythrocyte lysis with FACs lysis solution (a special buffer containing less than 10% formaldehyde and less than 50% diethylene glycol, BD Biosciences) for 15 minutes in the dark at RT.After dissolution, the samples were washed and then stored at 4 °C before being acquired on the flow cytometer on the same day. For each 4A19 concentration point, %RO was calculated using the following formula:. %RO = 100×{1 - [(free form after administration / free form before administration) / (total amount after administration / total amount before administration)]}, wherein, "free form after administration" is the MFI determined by flow cytometry of the competitive CCR8Ab at each concentration of the therapeutic mAb (after exposure to the therapeutic mAb); "free form before administration" is the MFI of the competitive CCR8Ab when not exposed to the therapeutic mAb (before exposure to the therapeutic mAb); "total amount after administration" is the MFI of the non-competitive anti-CCR8 mAb at each concentration of the therapeutic mAb (after exposure to the therapeutic mAb); and "total amount before administration" is the MFI of the non-competitive anti-CCR8 mAb when not exposed to the therapeutic mAb (before exposure to the therapeutic mAb).

[0210] For example, as shown in Figure 5, the response curve, i.e., %RO vs. drug (e.g., mAb4A19) concentration, was plotted. From either approach, similar %RO drug dose response curves and EC 50 (e.g., for a particular donor, 0.022 nM and 0.023 nM, n = 3) were obtained, demonstrating the reliability of the developed assay. Figure 5 is a representative diagram showing the changes in CCR8 %RO in healthy donor blood collected in sodium heparin (NaHep) and stored at 4 °C for 24 hours after treatment when the concentration of mAb4A19 was increased (n = 3). For each donor, the %RO curve was plotted using the direct vs. indirect RO assay format. Both formats were independently verified for sample stability up to 72 hours after collection, as well as acceptable within-assay and between-assay precision (coefficient of variation [CV] ≤ 30%). Furthermore, consistent RO data were observed over the entire drug dose range from within the same subject over time (CV ≤ 25%). Given the consistency of these data, these CCR8 RO assays demonstrate sufficient robustness even when used in clinical trials of therapeutic anti-CCR8 Abs to evaluate and quantify the engagement of the CCR8 target by the therapeutic Ab. [Example 7]

[0211] Detection of CCR8 by immunohistochemistry (IHC) Single-staining IHC on formalin-fixed paraffin-embedded (FFPE) samples was used to detect and monitor CCR8 expression in tumor samples. IHC analysis was performed at room temperature using a Leica Bond Rx automated stainer (Leica Biosystems, Buffalo Grove, IL). Samples were sectioned at a thickness of 4 μm, mounted on positively charged glass slides, dried using a fan for at least 1 hour, baked in an oven at 60 °C for 30 minutes, deparaffinized, and rehydrated offline. The tissue was then placed in the automated stainer, pretreated at 100 °C for 30 minutes using ER2 (Leica, Buffalo Grove, IL), then rinsed and incubated in Bond Wash Buffer (Leica) for 3 minutes. The tissue was incubated with Peroxide Block (Bond Polymer Refine Detection Kit, Leica), rinsed in Bond Wash Buffer (Leica), incubated with Human-to-Human protein block (Sigma; St. Louis, MO), and then incubated with the primary Ab (mouse IgG2a, kappa clone 433H, BD Biosciences) or mouse IgG1, kappa isotype control antibody for 30 minutes. Normal thymus tissue with cell characteristics positive and negative for CCR8 was used as positive and negative controls.

[0212] After incubation with the Ab, the tissue was processed using the Bond Polymer Refine Detection Kit according to the manufacturer's instructions, incubated with 3,3'-diaminobenzidine (DAB) for 10 minutes, and then rinsed in distilled water. The tissue was incubated with hematoxylin for 5 minutes, followed by rinsing in distilled water, rinsing in Bond Wash Buffer, and a final rinse in distilled water.

[0213] The cover slip was mounted using an automatic glass cover slipper, and slides were scanned using the Aperio AT Turbo system (Aperio, Vista, CA) to generate microscopic photographs of the entire slide. The images were analyzed by a pathologist's tumor cell score, by analysis using the CytoNuclear algorithm of Indica (Corrales, NM), and / or by a pathologist's visual immune score. [Example 8]

[0214] Depletion of Tregs by mAb4A19 in fragmented human tumors The ability of the therapeutic anti-CCR8 mAb 4A19 to + induce Treg depletion was evaluated ex vivo in fragmented human tumors.

[0215] Fragmentation of human tumors Human solid tumors after resection were transported overnight at 4 °C in HypoThermosol biopreservation medium (Charles River Laboratories, Wilmington, MA). After the whole tumor sample was dissected, it was fragmented with laboratory scissors. Next, the tumor was mechanically and enzymatically fragmented in Roswell Park Memorial Institute (RPMI)-1640 medium at 37 °C for 30 minutes using a Miltenyi BioTec gentleMACS (trademark) C tube, a fragmentation device, and a tumor fragmentation kit. The resulting single cell suspension was filtered through a 70 μm cell strainer and then processed for flow cytometry.

[0216] Cell processing for flow cytometry After generating a single-cell suspension from tumor tissue, the cells were washed with PBS and stained on ice for 20 minutes using an amine-reactive cell viability dye. The cells were washed and blocked on ice for 20 minutes in a blocking solution containing 2% rat serum (MilliporeSigma, St. Louis, MO), 2% mouse serum (SouthernBiotech, Birmingham, AL), 10% human A / B serum (Gemini Bio, West Sacramento, CA), 1:250 dilution of human FcX (BioLegend), and 1:100 dilution of monocyte block (BioLegend), and diluted in FACS staining buffer (PBS without Ca / Mg, 2 mM EDTA, 0.2% BSA). After blocking, each mAb of anti-human CD3, CD4, and CD8 of UV-excitable dye-conjugated type (BD Biosciences) was used for staining to define T cell subsets, while each mAb of anti-human CD14 and CD56 (BD Biosciences) was used to define macrophages, monocytes, and NK cells. In addition to lineage markers, CCR8 + To identify tumor Tregs, each Ab of anti-human CCR8, CCR4, and CD16 was also used. Next, the cells were fixed and permeabilized at 4°C for 30 minutes using a FOXP3 transcription factor staining buffer set, followed by incubation with an anti-FOXP3 Ab for intracellular staining at 4°C for an additional 30 minutes. Finally, the stained cells were washed and filtered using a 40-μm cell strainer.

[0217] Flow cytometry analysis was performed using a BD LSRFortessa™ X-20 flow cytometer, and the values of the population percentages and mean fluorescence intensities were calculated using FLOWJO™ software analysis.

[0218] Depletion of Tregs by mAb4A19 in fragmented human tumors from cancer patients Filtered single cells derived from fragmented tumor tissue were collected and washed with complete RPMI-1640 medium. The cells were resuspended in complete RPMI-1640 medium and cultured for 48 hours with serial dilutions of mAb4A19 or anti-KLH isotype control mAb. The cells were seeded in flat-bottom plates at a concentration of 500,000 cells per well. After incubating for 48 hours, the cells were processed for flow cytometry analysis as described above. Depleted CCR8 + The percentage of Tregs was determined by gating on CCR8 + cells within the parental gate FoxP3 high , CD25 - and CD45RA + . The frequency of CCR8 + cells was calculated using FLOWJO™ software, and depletion graphs were generated using PRISM software (GraphPad Software, San Diego, CA).

[0219] As a result of treatment with mAb4A19, CCR8 + Tregs were depleted in fragmented tumor tissue from cancer patient donors (n = 5). The percentage of CCR8 + Treg depletion in these tumor tissues at the concentration with the greatest depletion (1 μg / ml or 0.1 μg / ml) is summarized in Table 2. The percentage of depletion of CCR8 + Tregs compared to the KLH isotype control was calculated using the following formula: [1 - (percentage of CCR8 + Tregs in the parental gate in the A419-treated sample) / (percentage of CCR8 + Tregs in the parental gate in the KLH isotype-treated sample)] × 100%.

[0220] As shown in Figure 6, mAb4A19 +While inducing dose-dependent depletion of tumor Tregs, anti-KLH isotype Ab does not deplete Tregs even at the highest dose tested. This depletion plot is from a single patient but is representative of similar depletion assays from 5 patient donors. The parent gate is CD3 + CD4 + FoxP3 + represents the CD45RA population.

[0221]

Table 2

[0222] Exemplary embodiments The disclosed invention includes the following non-exhaustive list of embodiments. This list should not be construed as limiting in any way, and those skilled in the art will understand that various modifications can be made to these embodiments without departing from the essence and scope of the invention disclosed herein. 1. A monoclonal antibody or antigen-binding portion thereof that specifically binds to human C-C motif chemokine receptor 8 (hCCR8) expressed on the surface of a cell, binds to an epitope that does not exist within the N-terminal domain of hCCR8, and does not cross-compete with an antibody that binds to an N-terminal epitope for binding to hCCR8. 2. The monoclonal antibody or antigen-binding portion thereof according to embodiment 1, wherein the sequence of hCCR8 is represented as SEQ ID NO: 1. 3. The N-terminal epitope is within the peptide having the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73) and includes at least one amino acid within the peptide, and the monoclonal antibody or antigen-binding portion thereof according to embodiment 1 or 2. 4. The N-terminal epitope is within the sequence V 12 T13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73) The monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 amino acids within the peptide having 5. The N-terminal epitope is the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73) The monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, comprising the amino acid having 6. The N-terminal epitope is the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73) The monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, consisting of the amino acid having 7. Amino acid Y 15 and / or Y 17 is sulfated, the monoclonal antibody or antigen-binding portion thereof according to any one of Embodiments 3 to 6. 8. The binding to hCCR8 is the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S22 A monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, which is not affected by the presence of an antibody that binds to an N-terminal epitope comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 amino acids within the peptide having [SEQ ID NO: 73]. 9. An antibody that binds to an N-terminal epitope is (a) a heavy chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 53]; a heavy chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 54]; a heavy chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 55]; a light chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 56]; a light chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 57]; and a light chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 58], (b) a V comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 9] H and a V comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 16] L and / or (c) a heavy chain comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 65] and a light chain comprising continuously linked amino acids having the sequence represented by [SEQ ID NO: 72] A monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, which is an antibody comprising the same. 10. A monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, which binds to a cell surface-expressed hCCR8 polypeptide in a formalin-fixed paraffin-embedded (FFPE) tissue sample. 11. When measured by the binding assay described in Example 4, (a) about 50 nM or less, (b) about 3 nM or less, (c) about 0.5 nM or less, (d) about 0.1 nM or less, (e) About 0.01 nM or less, (f) About 0.005 nM or less, (g) About 0.1 nM, (h) About 0.005 nM to about 50 nM, (i) About 0.02 nM to about 3 nM, or (j) About 0.08 nM to about 2 nM of the EC 50 and specifically binds to human CCR8-expressing Raji cells, the monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments. 12. Cross-competes with the reference antibody for binding to hCCR8, and the reference antibody (a) V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 4 H and V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 11 L , (b) V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 6 H and V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 13 L , or (c) V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 8 H and V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 15 L and the monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments. 13. Binds to the same epitope as the epitope to which the reference antibody binds, and the reference antibody (a) V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 4 H and V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 11 L , (b) V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 6 H and V containing continuously linked amino acids having the sequence represented by SEQ ID NO: 13 L , or (c) a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 8 H and a V comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 15 L The monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, comprising the same. 14. The following CDR domains defined by the Kabat method, (a) Heavy chain variable region CDR1 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 23; heavy chain variable region CDR2 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 24; heavy chain variable region CDR3 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 25; light chain variable region CDR1 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 26; light chain variable region CDR2 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 27; and light chain variable region CDR3 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 28 (b) Heavy chain variable region CDR1 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 35; heavy chain variable region CDR2 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 36; heavy chain variable region CDR3 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 37; light chain variable region CDR1 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 38; light chain variable region CDR2 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 39; and light chain variable region CDR3 comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 40, or (c) A heavy chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 47; a heavy chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 48; a heavy chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 49; a light chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 50; a light chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 51; and a light chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 22 The monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, comprising . 15. (a) A V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 4 H and a V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 11 L , (b) A V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 6 H and a V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 13 L , or (c) A V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 8 H and a V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 15 L The monoclonal antibody or antigen-binding portion thereof according to embodiment 14, comprising . 16. (a) A heavy chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 60 and a light chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 67, (b) A heavy chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 62 and a light chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 69, or (c) A heavy chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 64 and a light chain comprising continuously linked amino acids having the sequence represented as SEQ ID NO: 71 The monoclonal antibody or antigen-binding portion thereof according to embodiment 14 or 15, comprising . The monoclonal antibody or antigen-binding portion thereof according to any one of embodiments 14 to 16, which is the monoclonal antibody or antigen-binding portion thereof named herein as 17.21C17, 22B13, or 23A14. 18. The monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments, which is a chimeric antibody, humanized antibody, human antibody, or fragment thereof. 19. The monoclonal antibody or antigen-binding portion thereof according to any one of embodiments 1 to 15, comprising a heavy chain constant region that is a human IgG1, IgG2, IgG3, or IgG4 isotype. 20. The monoclonal antibody or antigen-binding portion thereof according to any one of embodiments 1 to 15, comprising a heavy chain constant region that is a heavy chain constant region of a human IgG2 or IgG4 isotype. 21. A labeled antibody or antigen-binding portion thereof, comprising the monoclonal antibody or antigen-binding portion thereof according to any one of the preceding embodiments and a detectable label. 22. The labeled antibody or antigen-binding thereof according to embodiment 21, wherein the detectable label is a fluorophore, chromophore, enzyme, radioisotope, micropolymer, or metal. 23. The labeled antibody according to embodiment 21, wherein the detectable label is biotin. 24. The labeled antibody according to embodiment 22, wherein the fluorophore is a Brilliant Violet™ dye (e.g., BV-421), AmCyan dye, Alexa Fluor® dye, Cy® dye, CF® dye, fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC) phycoerythrin (PE), allophycocyanin (APC), or peridinin-chlorophyll protein (PerCP). 25. The labeled antibody according to embodiment 22, wherein the chromophore is porphyrin, pyropheophorbide-α, benzoporphyrin monoacid ring A (BPDMA), or chlorin e6. 26. The labeled antibody according to embodiment 22, wherein the enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase, or β-galactosidase. 27. The radioisotope is 89 Zr, 64 Cu, 86 Y, 11 C, 18 F, 68 Ga, 52 Mn, 55 Co, 152 Tb, 90 Nb, 66 Ga, 72 As, I 125 , or 69 Ge, and is the labeled antibody according to embodiment 22. 28. The labeled antibody according to embodiment 22, wherein the metal label is yttrium (Y), indium (In), a series of lanthanide elements (excluding Ln, La-Lu, Pm), iodine (I), cadmium (Cd), tellurium (Te), silver (Ag), palladium (Pd), rhodium (Rh), iridium (Ir), platinum (Pt), ruthenium (Ru), osmium (Os), or bismuth (Bi). 29. An immunoconjugate comprising the monoclonal antibody according to any one of embodiments 1 to 20 or an antigen-binding portion thereof linked to a cytolytic agent. 30. The immunoconjugate according to embodiment 28, wherein the cytolytic agent is a cytotoxin, a radioisotope, or a photosensitizer. 31. A chimeric antigen receptor (CAR) comprising the monoclonal antibody according to any one of embodiments 1 to 20 or an antigen-binding portion thereof. 32. A T cell receptor (TCR) comprising the monoclonal antibody according to any one of embodiments 1 to 20 or an antigen-binding portion thereof. A bispecific molecule comprising the monoclonal antibody or an antigen-binding portion thereof according to any one of Embodiments 1 to 20, linked to a binding domain having a binding specificity different from that of the monoclonal antibody or the antigen-binding portion thereof. 34. (a) The monoclonal antibody or an antigen-binding portion thereof according to any one of Embodiments 1 to 20, (b) The labeled antibody or an antigen-binding portion thereof according to any one of Embodiments 21 to 28, (c) The immunoconjugate according to Embodiment 29 or 30, (d) The CAR according to Embodiment 31, (e) The TCR according to Embodiment 32, or (f) The bispecific molecule according to Embodiment 33, and a pharmaceutically acceptable carrier A composition comprising the same. 35. An isolated nucleic acid encoding the monoclonal antibody or an antigen-binding portion thereof according to any one of Embodiments 1 to 20. 36. An expression vector comprising the nucleic acid according to Embodiment 35. 37. A host cell comprising the expression vector according to Embodiment 36. 38. A method for preparing an anti-CCR8 antibody or an antigen-binding portion thereof, comprising expressing an antibody or an antigen-binding portion thereof in the host cell according to Embodiment 37, and isolating the antibody or an antigen-binding portion thereof from the host cell. 39. A method for generating a first antibody that does not bind to a defined epitope on an antigen or does not cross-compete with a second antibody for binding, the method comprising immunizing a vertebrate with an immunogen comprising a cell line that expresses the antigen and also expresses a second antibody or an antigen-binding portion thereof that specifically binds to the epitope, wherein binding of the second antibody or an antigen-binding portion thereof to the epitope shields the epitope from the vertebrate immune system and reduces the production of antibodies that bind to the epitope, thereby preferentially causing the production of a first antibody that does not bind to the epitope or does not cross-compete with the second antibody for binding. 40. The method according to embodiment 39, wherein the antigen comprises the CCR8 receptor. 41. The method according to embodiment 40, wherein the CCR8 receptor is the hCCR8 receptor. 42. The method according to any one of embodiments 39 to 41, wherein the vertebrate is a mammal or a bird. 43. The method according to embodiment 42, wherein the mammal is a mouse, a rat, a hamster, a rabbit, a dog, a goat, a sheep, or a horse. 44. The method according to any one of embodiments 39 to 43, wherein the immunogen is a surfactant-stabilized proteoliposome component of a cell line. 45. The method according to embodiment 41, wherein the epitope is an epitope within the N-terminal domain of the hCCR8 receptor. 46. The method according to embodiment 44, wherein the second antibody or antigen-binding portion thereof is a monoclonal antibody named L263G8 (BioLegend), a monoclonal antibody named 433H (BD Biosciences), 4A19, 18Y12, 10R3, 8D55, 14S15, or 14S15h. 47. A method for measuring the receptor occupancy (RO) of a cell membrane-bound CCR8 receptor to which a therapeutic Treg-depleting anti-CCR8 antibody binds, comprising: (a) adding a saturating concentration of a therapeutic antibody to background whole blood samples that have been previously exposed to various concentrations of the therapeutic antibody; and (b) incubating aliquots of free, bound, or fluorescence minus one (FMO) samples with a buffer that does not contain the therapeutic antibody; and (c) staining the samples using a core panel of antibodies to identify basic T cell markers, T cell differentiation markers, and Treg markers; and (d) in the core panel, (i) in a direct / global RO assay, an anti-idiotype antibody for detecting the bound CCR8 receptor, or (ii) In an indirect / global assay, add two additional antibodies including an allophycocyanin (APC)-conjugated anti-hCCR8 antibody that competes with the therapeutic antibody for detecting the free CCR8 receptor and an anti-hCCR8 antibody that does not compete with the therapeutic antibody for detecting the total CCR8 receptor, (e) Lyse red blood cells to clarify the sample and analyze the clarified sample by flow cytometry, (f) The formula: %RO = 100 × {1 - [(free form after administration / free form before administration) / (total amount after administration / total amount before administration)]} is used to determine %RO for each concentration of the therapeutic antibody and a method comprising the same. 48. A method for measuring the depletion of the frequency of CCR8-expressing cells in the peripheral blood of a subject being treated with an anti-CCR8 mAb, comprising: (a) determining the baseline level of the expression of CCR8 and / or the frequency of CCR8-expressing cells in a first sample of whole blood or PBMCs from the subject; (b) administering treatment with an anti-CCR8 mAb to the subject; (c) determining the level of the expression of CCR8 and / or the frequency of CCR8-expressing cells in a second sample of whole blood or PBMCs from the subject taken after administration of the anti-CCR8 mAb wherein a decrease in the level of the expression of CCR8 and / or the frequency of CCR8-expressing cells in the second sample indicates depletion of the number of CCR8-expressing cells in the blood. 49. A method for measuring the depletion of the number of tumor-infiltrating CCR8-expressing Tregs in a treated subject, comprising: (a) determining the baseline level of the expression of CCR8 and / or the frequency of CCR8-expressing Tregs in a first sample of test tissue from within or from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering treatment with an anti-CCR8 antibody to the subject; (c) determining the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs in a second sample of test tissue within or derived from the subject, taken during or after the treatment comprising a decrease in the level of CCR8 expression and / or a decrease in the frequency of CCR8-expressing Tregs in the second sample, indicating depletion of the number of Tregs in the test tissue. 50. The method according to embodiment 49, wherein depletion of the number of Tregs in the test tissue indicates enhancement of the immune response in the subject. 51. The method according to embodiment 49 or 50, wherein the treatment administered to the subject is a treatment for cancer. 52. The method according to embodiment 51, wherein the treatment for cancer comprises administration of a therapeutic Treg-depleting antibody or an antigen-binding portion thereof. 53. The method according to embodiment 52, wherein the therapeutic Treg-depleting antibody or an antigen-binding portion thereof is an anti-CCR8 antibody or an antigen-binding portion thereof. 54. A method for measuring depletion of skin T cells in a subject undergoing treatment, comprising: (a) determining a baseline level of CCR8 expression in a first sample of test tissue within or taken from the subject, comprising skin T cells; (b) administering a treatment of an anti-CCR8 antibody to the subject; (c) determining the level of CCR8 expression in a second test tissue within or derived from the subject, taken during or after the treatment. comprising a decrease in the level of CCR8 expression in the second test tissue indicating depletion of the number of skin T cells in the test tissue. 55. A method for predicting the efficacy of a therapeutic Treg-depleting antibody or an antigen-binding portion thereof in treating cancer in a subject, comprising: (a) determining the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs in a test tissue within or derived from the subject, comprising tumor cells and tumor-infiltrating Tregs; (b) comparing the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs with a pre-determined threshold. (c) predicting the effectiveness of a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, and comprising when the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs exceeds a threshold, indicating that the therapeutic antibody or an antigen-binding portion thereof is effective in treating a subject, when the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs is less than a threshold, indicating that the therapeutic antibody or an antigen-binding portion thereof is not effective in treating a subject, a method. 56. A method for predicting the effectiveness of a therapeutic Treg-depleting antibody or an antigen-binding portion thereof in treating cancer in a subject, (a) determining the level of surface expression of CCR8 and / or the frequency of CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering to the subject a therapeutic Treg-depleting antibody or an antigen-binding portion thereof; (c) determining, after administration of the therapeutic Treg-depleting antibody or an antigen-binding portion thereof, whether a decrease in the frequency of CCR8-expressing Tregs is observed; (d) predicting that the therapeutic Treg-depleting antibody or an antigen-binding portion thereof is effective in treating cancer in the subject if the decrease in the frequency of CCR8-expressing Tregs exceeds a pre-determined threshold, or (e) predicting that the therapeutic Treg-depleting antibody or an antigen-binding portion thereof is not effective in treating cancer in the subject if the decrease in the frequency of CCR8-expressing Tregs is less than a pre-determined threshold, and a method comprising. 57. A method for selecting a subject suffering from cancer as a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, (a) determining the level of expression of CCR8 and / or the frequency of CCR8-expressing Tregs in a test tissue within or derived from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) Comparing the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs with a predetermined threshold; (c) Based on the assessment that the level of surface expression of CCR8 and / or the frequency of CCR8-expressing Tregs in the cells of the test tissue exceeds a predetermined threshold, selecting the subject as a suitable candidate for immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof; A method comprising the steps of. 58. A method for selecting a subject suffering from cancer as a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, comprising: (a) Determining the level of surface expression of CCR8 and / or the frequency of CCR8-expressing Tregs in a test tissue within or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) Administering to the subject a therapeutic Treg-depleting antibody or an antigen-binding portion thereof; (c) After administration of the therapeutic Treg-depleting antibody or an antigen-binding portion thereof, determining whether a decrease in the frequency of CCR8-expressing Tregs is observed; (d) Based on the assessment that the decrease in the frequency of CCR8-expressing Tregs exceeds a predetermined threshold, selecting the subject as a suitable candidate for immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof; A method comprising the steps of. 59. A method for treating cancer in a subject, comprising: (a) (i) Determining the level of expression of CCR8 and / or the frequency of CCR8-expressing Tregs in a test tissue within or derived from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) Comparing the level of expression of CCR8 and / or the frequency of CCR8-expressing Tregs with a predetermined threshold; (iii) Based on the assessment that the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs in the cells of the test tissue exceeds a predetermined threshold, selecting the subject as a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof; Selecting a subject who is a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, which comprises b) Administering to the selected subject a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting antibody or an antigen-binding portion thereof A method comprising. 60. A method for treating a subject suffering from cancer, comprising (a) (i) Determining the level of surface expression of CCR8 and / or the frequency of CCR8-expressing Tregs in a test tissue within or taken from the subject, which comprises tumor cells and tumor-infiltrating Tregs (ii) Administering to the subject a therapeutic Treg-depleting antibody or an antigen-binding portion thereof (iii) Determining whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of the therapeutic Treg-depleting antibody or an antigen-binding portion thereof (iv) Selecting the subject as a suitable candidate for immunotherapy using a therapeutic agent based on the assessment that the decrease in the frequency of CCR8-expressing Tregs exceeds a pre-determined threshold Selecting a subject who is a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, which comprises (b) Administering to the selected subject a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting antibody or an antigen-binding portion thereof A method comprising. 61. A method for treating cancer in a subject, comprising (a) (i) Determining the level of expression of CCR8 and / or the frequency of CCR8-expressing Tregs in a test tissue within or derived from the subject, which comprises tumor cells and tumor-infiltrating Tregs (ii) Comparing the level of expression of CCR8 and / or the frequency of CCR8-expressing Tregs with a pre-determined threshold (iii) selecting a subject as not suitable for immunotherapy with a therapeutic Treg-depleting antibody or an antigen-binding portion thereof based on an evaluation that the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs in the cells of the test tissue is below a predetermined threshold selecting a subject who is not a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, comprising: (b) administering to the selected subject a standard therapeutic agent other than the therapeutic anti-CCR8 antibody or an antigen-binding portion thereof A method comprising: 62. A method for treating cancer in a subject, comprising: (a) (i) determining the level of surface expression of CCR8 and / or the frequency of CCR8-expressing Tregs in a test tissue within or taken from the subject, comprising tumor cells and tumor-infiltrating Tregs; (ii) administering to the subject a therapeutic Treg-depleting antibody or an antigen-binding portion thereof; (iii) determining whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of the therapeutic Treg-depleting antibody or an antigen-binding portion thereof; (iv) selecting a subject as not suitable for immunotherapy with a therapeutic Treg-depleting antibody or an antigen-binding portion thereof based on an evaluation that the decrease in the frequency of CCR8-expressing Tregs is below a predetermined threshold selecting a subject who is not a suitable candidate for cancer immunotherapy using a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, comprising: (b) administering to the selected subject a standard therapeutic agent other than the therapeutic anti-CCR8 antibody or an antigen-binding portion thereof A method comprising: 63. A method for treating cancer in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, wherein the subject is selected based on the determination that the level and / or frequency of CCR8-expressing Treg in a test tissue within or derived from the subject, which comprises tumor cells and tumor-infiltrating Treg, exceeds a pre-determined threshold level. 64. A method for treating cancer in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, wherein the subject is selected based on the determination that the therapeutic Treg-depleting antibody or an antigen-binding portion thereof causes a decrease in the frequency of CCR8-expressing Treg that exceeds a pre-determined threshold. 65. A method for treating cancer in a subject, comprising administering to the subject a standard therapeutic treatment other than a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, wherein the subject is selected based on the determination that the intracellular CCR8 level and / or frequency of CCR8-expressing Treg in a test tissue within or derived from the subject, which comprises tumor cells and tumor-infiltrating Treg, is less than a pre-determined threshold level. 66. A method for treating cancer in a subject, comprising administering to the subject a standard therapeutic treatment other than a therapeutic Treg-depleting antibody or an antigen-binding portion thereof, wherein the subject is selected based on the determination that the therapeutic Treg-depleting Ab or an antigen-binding portion thereof causes a decrease in the frequency of CCR8-expressing Treg that is less than a pre-determined threshold level. 67. The method according to any one of embodiments 55 to 66, wherein the Treg-depleting antibody is an anti-CCR8, anti-CTLA-4, anti-CCR4, or anti-CD25 antibody. 68. The method according to any one of embodiments 48 to 67, wherein determining the level of expression of CCR8 in a test tissue comprises assessing the level of expression of CCR8 on the surface of Treg in the test tissue. 69. The method according to any one of embodiments 48 to 67, comprising determining the level of CCR8 expression in a test tissue, which includes assessing the proportion of Tregs in a test tissue that expresses CCR8 on the surface of Treg cells. 70. The method according to any one of embodiments 48 to 69, wherein the level of CCR8 expression in a test tissue of a subject is determined by an in vivo method. 71. The method according to embodiment 70, wherein the in vivo method includes a PET tracking method using the anti-CCR8 antibody described in embodiment 1. 72. The method according to any one of embodiments 48 to 70, wherein the level of CCR8 expression is determined ex vivo in a test tissue sample obtained from a subject. 73. The method according to embodiment 72, wherein the level of CCR8 expression is determined by immunohistochemistry (IHC), flow cytometry, or mass spectrometry-linked flow cytometry using the labeled anti-CCR8 antibody or antigen-binding portion thereof described in any one of embodiments 21 to 28 that binds to CCR8 expressed on the surface of cells in the tissue. 74. The method according to embodiment 73, wherein the IHC is performed on fresh frozen tissue or formalin-fixed paraffin-embedded (FFPE) tissue. 75. The method according to embodiment 70 or 73, wherein determining the level of CCR8 expression using the anti-CCR8 antibody described in embodiment 1 is not affected by the binding of a therapeutic antibody or antigen-binding portion thereof to the N-terminal domain of CCR8. 76. The therapeutic anti-CCR8 antibody or antigen-binding portion thereof is (a) A heavy chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 53; a heavy chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 54; a heavy chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 55; a light chain variable region CDR1 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 56; a light chain variable region CDR2 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 57; and a light chain variable region CDR3 comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 58. (b) A V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 9 H and a V comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 16 L or (c) A method according to any one of embodiments 52 to 66 and 75, comprising a heavy chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 65 and a light chain comprising continuously linked amino acids having the sequence represented by SEQ ID NO: 72. 77. A method according to any one of embodiments 52 to 66 and 75, wherein the therapeutic anti-CCR8 antibody or antigen-binding portion thereof is an antibody or antigen-binding portion thereof named 4A19, 18Y12, 8D55, 10R3, 14S15, or 14S15h. 78. A method according to any one of embodiments 49 to 77, wherein the subject is human. 79. A method according to any one of embodiments 51 to 78, wherein the cancer is a solid tumor. 80. The method according to embodiment 79, wherein the solid tumor is selected from squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell NSCLC, non-squamous cell 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 region cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, natural killer of the paranasal sinuses, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, endometrial carcinoma, fallopian tube carcinoma, ovarian cancer, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, testicular cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, penile cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, solid tumor in childhood, environmentally induced cancer, virus-related cancer, virus-originated cancer, advanced cancer, inoperable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination thereof. 81. The method according to embodiment 79, wherein the solid tumor is selected from head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), gastric adenocarcinoma (STAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), rectal adenocarcinoma (READ), esophageal cancer (ESCA), invasive breast cancer (BRCA), colon adenocarcinoma (COAD), and cervical squamous cell carcinoma and cervical adenocarcinoma (CESC). 82. The method according to embodiment 79, wherein the solid tumor is selected from colon adenocarcinoma, bladder cancer, breast cancer, and fibrosarcoma. 83. The method according to embodiment 79, wherein the solid tumor is selected from NSCLC, SCCHN, microsatellite stable colorectal cancer (MSS-CRC), adenocarcinoma of the stomach / gastroesophageal (GE) junction, and cervical cancer. 84. The method according to any one of embodiments 51 to 78, wherein the cancer is a hematological malignancy. 85. The method according to embodiment 84, wherein the hematological malignancy is selected from follicular lymphoma (FL), and acute lymphoblastic leukemia and lymphoma. 86. The method according to embodiment 84, wherein the hematological malignancy is selected from 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 gammopathy of undetermined significance (MGUS), and any combination of the foregoing hematological malignancies. 87. The method according to embodiment 84, wherein the hematological malignancy is diffuse large B cell lymphoma (DLBCL), CLL / small lymphocytic lymphoma (SLL), mantle cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B cell lymphoma, splenic marginal zone B cell lymphoma, Burkitt lymphoma, lymphoplasmacytoid lymphoma (LPL), hairy cell lymphoma, primary central nervous system (CNS) lymphoma, precursor T lymphoblastic lymphoma / leukemia, T lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), cutaneous T cell lymphoma, adult T cell lymphoma / leukemia, angioimmunoblastic T cell lymphoma, extranodal natural killer / T cell lymphoma nasal type, enteropathy-associated intestinal T cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), peripheral T cell lymphoma, unspecified, lymphoplasmacytoid lymphoma, monocytoid B cell lymphoma, angiocentric lymphoma, intestinal T cell lymphoma, primary mediastinal B cell lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, solitary plasmacytoma, IgG myeloma, light chain myeloma, non-secretory myeloma, amyloidosis, and any combination of the foregoing hematological malignancies. 88. The method according to embodiment 84, wherein the hematological malignancy is a progressive, metastatic, refractory, and recurrent hematological malignancy, and any combination of the foregoing hematological malignancies. 89. The method according to any one of embodiments 59 to 88, further comprising administering to the subject an additional therapeutic agent in a therapeutically effective amount for treating cancer. 90. The method according to embodiment 89, wherein the additional therapeutic agent is a compound that reduces immune system inhibition or increases immune system stimulation. 91. The additional therapeutic agent is (a) an antagonistic agent that specifically binds to programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), B and T lymphocyte attenuator (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), killer immunoglobulin-like receptor (KIR), killer cell lectin-like receptor G1 (KLRG-1), adenosine A2a receptor (A2aR), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), proto-oncogene tyrosine-protein kinase MER (MerTK), natural killer cell receptor 2B4 (CD244), or CD160, or (b) an agonistic agent that specifically binds to inducible T cell co-stimulator molecule (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-induced TNFR-related protein (GITR), or herpesvirus entry mediator (HVEM). The method according to embodiment 90. 92. The method according to embodiment 91, wherein the antagonistic agent that specifically binds to PD-1 is selected from nivolumab, pembrolizumab, semiprimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retilimab, zimberelimab, pimavlimab, serplulimab, boptaterlimab, and acxolimab. 93. The method according to embodiment 91, wherein the antagonistic agent that specifically binds to PD-L1 is selected from atezolizumab, durvalumab, avelumab, enoblituzumab, cosibelimab (CK-301), BMS-936559, BMS-986189, CS-1001, SHR-1316, CBT-502, BGB-A333, KN035, AUNP12, and CA-170. 94. The method according to embodiment 91, wherein the antagonistic agent that specifically binds to CTLA-4 is ipilimumab or tremelimumab. 95. The method according to embodiment 91, wherein the antagonistic agent that specifically binds to LAG3 is selected from relatlimab, fabeselimab, tiragolumab, fianlimab, tebotelimab, or eftilagimod alpha. 96. A kit for use in measuring the depletion of the number of Tregs in a subject, (a) A monoclonal antibody or an antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of a cell and binds to an epitope different from the epitope within the N-terminal domain of hCCR8, (b) Instructions for using the monoclonal antibody or a portion thereof in the method according to any one of embodiments 49 to 53 and a kit comprising the same. 97. A kit for use in predicting the efficacy of a therapeutic anti-CCR8 antibody that binds to the N-terminal domain of hCCR8 in the treatment of cancer in a subject, (a) A monoclonal antibody or an antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of a cell and binds to an epitope different from the epitope within the N-terminal domain of hCCR8, (b) Instructions for using the monoclonal antibody or a portion thereof in the method according to embodiment 55 and a kit comprising the same. 98. A kit for use in selecting a subject suffering from cancer as a suitable candidate for immunotherapy using a therapeutic anti-CCR8 antibody that binds to the N-terminal domain of hCCR8, (a) A monoclonal antibody or antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of cells and binds to an epitope different from the epitope within the N-terminal domain of hCCR8, (b) Instructions for using the monoclonal antibody or portion thereof in the method of Embodiment 56 A kit comprising 99. A kit for use in treating cancer in a subject, (a) A monoclonal antibody or antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of cells and binds to an epitope different from the epitope within the N-terminal domain of hCCR8, (b) A therapeutic anti-CCR8 antibody or antigen-binding portion thereof that binds to the N-terminal domain of hCCR8, (c) Instructions for using the monoclonal antibody or portion thereof and the therapeutic anti-CCR8 antibody or antigen-binding portion thereof in the method according to Embodiment 57 or 63 A kit comprising 100. A kit for use in treating cancer in a subject, (a) A monoclonal antibody or antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of cells and binds to an epitope different from the epitope within the N-terminal domain of hCCR8, (b) A therapeutic anti-CCR8 antibody or antigen-binding portion thereof that binds to the N-terminal domain of hCCR8, (c) Instructions for using the monoclonal antibody or portion thereof and the therapeutic anti-CCR8 antibody or antigen-binding portion thereof in the method according to Embodiment 61 or 65 A kit comprising

[0223] The disclosed amino acid sequences The sequence numbers and corresponding amino acid sequences referred to in this application are summarized in Table 3.

[0224]

Table 3

[0225] References TIFF2025520063000009.tif235157 TIFF2025520063000010.tif250155 TIFF2025520063000011.tif184156

Claims

1. A monoclonal antibody or its antigen-binding moiety that specifically binds to human C-C motif chemokine receptor 8 (hCCR8) expressed on the surface of cells, wherein the sequence of hCCR8 is represented as Sequence ID No. 1, the antibody or its antigen-binding moiety binds to an epitope located outside the N-terminal domain of hCCR8, and does not cross-compete with a therapeutic Treg-depleted antibody that binds to the N-terminal epitope for binding to hCCR8; a non-competitive monoclonal antibody or its antigen-binding moiety.

2. The N-terminal epitope is (a) A peptide having sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (Sequence ID 73), comprising at least one amino acid in the peptide, (b) A peptide having the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (Sequence ID 73) containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 amino acids, (c) Contains an amino acid having the sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 (Sequence ID 2), wherein amino acid Y 15 or Y 17 is sulfated. (d) Contains an amino acid having the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (Sequence ID 73), wherein amino acids Y 15 and Y 17 are sulfated, or (e) Consists of amino acids having the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (Sequence ID 73), wherein amino acids Y 15 and Y 17 are sulfated. A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1.

3. A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, wherein the binding to hCCR8 is not affected by the presence of an antibody bound to an N-terminal epitope containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 amino acids in a peptide having the sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73).

4. Antibodies that bind to therapeutic N-terminal epitopes, (a) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 53; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 54; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 55; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 56; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 57; and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO:

58. (b) V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 9 Ｈ V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 16 Ｌ , and / or (c) A heavy chain containing continuously linked amino acids having the sequence represented by Sequence ID No. 65 and a light chain containing continuously linked amino acids having the sequence represented by Sequence ID No. 72 It is an antibody that contains A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2.

5. A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, which binds to a cell surface-expressed hCCR8 polypeptide in a formalin-fixed paraffin-embedded (FFPE) tissue sample.

6. When measured by a binding assay, (a) about 50 nM or less; (b) about 3 nM or less; (c) about 0.5 nM or less; (d) about 0.1 nM or less; (e) about 0.01 nM or less; (f) about 0.005 nM or less; (g) about 0.1 nM, (h) about 0.005 nM to about 50 nM, (i) Approximately 0.02 nM to approximately 3 nM, (j) about 0.08 nM to about 2 nM A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, which specifically binds to human CCR8-expressing Razi cells at EC 50.

7. The reference antibody cross-competes with the reference antibody for binding to hCCR8, or binds to the same epitope as the reference antibody, and the reference antibody: (a) V H containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 4, and V L containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO:

11. (b) V H containing a sequence of linked amino acids having the sequence represented as SEQ ID NO: 6 and V L containing a sequence of linked amino acids having the sequence represented as SEQ ID NO: 13, or (c) V H containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 8 and V L containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 15 A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, comprising:

8. A non-competitive monoclonal antibody or its antigen-binding portion according to claim 1 or 2, which is a chimeric antibody, a humanized antibody, a human antibody, or a fragment thereof.

9. A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, comprising a heavy chain constant region which is a human IgG1, IgG2, IgG3, or IgG4 isotype.

10. A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 9, comprising a heavy chain constant region which is a heavy chain constant region of a human IgG2 or IgG4 isotype.

11. The following CDR domains are defined by the Kabat method: (a) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 23; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 24; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 25; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 26; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 27; and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO:

28. (b) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 35; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 36; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 37; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 38; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 39; and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 40, or (c) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 47; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 48; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 49; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 50; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 51; and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 52 A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, comprising:

12. The following heavy chain and light chain variable regions, (a) V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 4 Ｈ V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 11 Ｌ , (b) V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 6 Ｈ V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 13 Ｌ ,or (c) V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 8 Ｈ V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 15 Ｌ A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 11, comprising:

13. The heavy and light chains listed below, (a) A heavy chain containing continuously linked amino acids having the sequence represented as SEQ ID NO: 60 and a light chain containing continuously linked amino acids having the sequence represented as SEQ ID NO: 67 (b) A heavy chain containing continuously linked amino acids having the sequence represented as SEQ ID NO: 62 and a light chain containing continuously linked amino acids having the sequence represented as SEQ ID NO: 69, or (c) A heavy chain containing continuously linked amino acids having the sequence represented as SEQ ID NO: 64 and a light chain containing continuously linked amino acids having the sequence represented as SEQ ID NO: 71 A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 12, comprising:

14. A non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, wherein the monoclonal antibody or its antigen-binding moiety is named herein as 21C17, 22B13, or 23A14.

15. A labeled non-competitive antibody or its antigen-binding moiety, comprising a non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2 and a detectable label.

16. The labeled non-competitive antibody or its antigen-binding moiety according to claim 15, wherein the detectable label is biotin, a fluorophore, a chromophore, an enzyme, a radioisotope, a micropolymer, or a metal.

17. A labeled non-competitive antibody or its antigen-binding moiety according to claim 16, The fluorophore is Brilliant Violet® dye (e.g., BV-421), AmCyan dye, Alexa Fluor® dye, Cy® dye, CF® dye, fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), phycoerythrin (PE), allophycocyanin (APC), or peridinin-chlorophyll protein (PerCP). The chromophore is porphyrin, pyropheophorbide-α, benzoporphyrin monoacid ring A (BPDMA), or chlorin e6. The enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase, or β-galactosidase. The radioisotope is ８９ Zr, ６４ Cu, ８６ Y, １１ C, １８ F, ６８ Ga, ５２ Mn, ５５ Co, １５２ Tb, ９０ Nb, ６６ Ga, ７２ As, I １２５ or ６９ Ge, The metallic labels are yttrium (Y), indium (In), a series of lanthanide elements (excluding Ln, La-Lu, and Pm), iodine (I), cadmium (Cd), tellurium (Te), silver (Ag), palladium (Pd), rhodium (Rh), iridium (Ir), platinum (Pt), ruthenium (Ru), osmium (Os), or bismuth (Bi). A labeled non-competitive antibody or its antigen-binding moiety.

18. An immunoconjugate comprising a non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, linked to a cell lysating agent.

19. The immunoconjugate according to claim 18, wherein the cell lysing agent is a cytotoxin, a radioisotope, or a photosensitizer.

20. A chimeric antigen receptor (CAR) comprising the non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2.

21. A T cell receptor (TCR) comprising the non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2.

22. A bispecific molecule comprising a non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, linked to a binding domain having different binding specificity from the non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2.

23. A composition comprising a non-competitive monoclonal antibody or its antigen-binding moiety and a pharmaceutically acceptable carrier as described in Claim 1.

24. A composition comprising a labeled non-competitive antibody or its antigen-binding moiety as described in Claim 15 and a pharmaceutically acceptable carrier.

25. A composition comprising the immunoconjugate described in claim 18 and a pharmaceutically acceptable carrier.

26. A composition comprising the CAR described in claim 20 and a pharmaceutically acceptable carrier.

27. ​​A composition comprising the TCR described in Claim 21 and a pharmaceutically acceptable carrier.

28. A composition comprising the bispecific molecule described in Claim 22 and a pharmaceutically acceptable carrier.

29. An isolated nucleic acid encoding a non-competitive monoclonal antibody or its antigen-binding moiety according to claim 1 or 2.

30. An expression vector comprising the nucleic acid described in Claim 29.

31. A host cell comprising the expression vector according to claim 30.

32. A method for preparing a non-competitive anti-CCR8 antibody or its antigen-binding moiety, comprising expressing the antibody or its antigen-binding moiety in a host cell as described in Claim 31, and isolating the antibody or its antigen-binding moiety from the host cell.

33. A method for measuring the receptor occupancy (RO) of a cell membrane-bound CCR8 receptor to which a Treg-depleted anti-CCR8 antibody that binds to a therapeutic N-terminal epitope binds, (a) Adding a saturated concentration of therapeutic antibody to background whole blood samples that have been previously exposed to various concentrations of therapeutic antibody, (b) Incubating a certain amount of free, conjugated, or fluorescence-minus-one (FMO) sample with a buffer that does not contain therapeutic antibodies, (c) Stain the sample using an antibody core panel to identify basic T cell markers, T cell differentiation markers, and Treg markers, (d) Core panel, (i) In direct / whole RO assays, an anti-idiotype antibody is used to detect the conjugated CCR8 receptor, or (ii) In an indirect / whole assay, two additional antibodies are added, comprising an allophycocyanin (APC) conjugate anti-hCCR8 antibody that competes with the therapeutic antibody for detecting free CCR8 receptors, and an anti-hCCR8 non-competitive antibody according to claim 1 or 2 that does not compete with the therapeutic antibody for detecting all CCR8 receptors. (e) Lysing red blood cells to clarify the sample, and analyzing the clarified sample by flow cytometry, (f) Formula: %RO = 100 × {1 - [(Free form after administration / Free form before administration) / (Total amount after administration / Total amount before administration)]} To determine the %RO for each concentration of the therapeutic antibody, A method that includes this.

34. A method for measuring the depletion of tumor-infiltrating CCR8-expressing Tregs in a subject treated with a Treg-depleting anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated moiety that binds to a therapeutic N-terminal epitope, wherein the method is (a) Determining the baseline level of CCR8 expression and / or the frequency of CCR8-expressing Tregs in a first sample of test tissue taken from or within a subject, including tumor cells and tumor-infiltrating Tregs, by measuring the binding of CCR8 to the non-competitive anti-CCR8 antibody according to claim 1 or 2, (b) administering a therapeutic anti-CCR8 antibody or a portion thereof to the subject, (c) Determining the level of CCR8 expression and / or the frequency of CCR8 expression in a second sample of test tissue taken from or within the subject during or after the procedure. Includes, Compared to the baseline level, the decrease in the level of CCR8 expression and / or the decrease in the frequency of CCR8-expressing Tregs in the second sample indicates that the number of Tregs in the test tissue has been depleted. method.

35. A method for predicting the effectiveness of a Treg-depleted anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated moiety that binds to a therapeutic N-terminal epitope when treating cancer in a subject, wherein the method is: (a) Determining the level of surface expression of CCR8 and / or the frequency of CCR8-expressing Tregs in or from a test tissue, including tumor cells and tumor-infiltrating Tregs, by measuring the binding of CCR8 to the non-competitive anti-CCR8 antibody according to claim 1 or 2, (b) Administering the subject a therapeutic Treg depletion anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated portion, (c) Determine whether a decrease in the frequency of CCR8 expression tregs is observed after administration of the therapeutic antibody or a portion thereof, (d) If the reduction in the frequency of CCR8-expressing Tregs exceeds a predetermined threshold, it is predicted that the therapeutic Treg-depleting antibody or a portion thereof will be effective in treating cancer in the target, or (e) If the reduction in the frequency of CCR8-expressing Tregs is below a predetermined threshold, it is predicted that the therapeutic Treg-depleting antibody or its portion will not be effective in treating cancer in the target. A method that includes this.

36. A method for selecting a cancer patient as a suitable candidate for cancer immunotherapy using a Treg-depleted anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated portion that binds to a therapeutic N-terminal epitope, wherein the method is: (a) Determining the level of surface expression of CCR8 and / or the frequency of CCR8-expressing Tregs in or from a test tissue, including tumor cells and tumor-infiltrating Tregs, by measuring the binding of CCR8 to the non-competitive anti-CCR8 antibody according to claim 1 or 2, (b) Administering the subject a therapeutic Treg depletion anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated portion, (c) Determine whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of a therapeutic Treg-depleting anti-CCR8 antibody or a portion thereof, (d) If the decrease in the frequency of CCR8-expressing Tregs exceeds a predetermined threshold, the subject shall be selected as a suitable candidate for immunotherapy using a therapeutic Treg-depleting anti-CCR8 antibody or a portion thereof. Methods that include...

37. A pharmaceutical composition comprising the composition according to claim 23 for use in a method for treating cancer in a subject, wherein the method is (a)(i) Determining the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs in or from test tissues of the subject, including tumor cells and tumor-infiltrating Tregs, by measuring the binding of CCR8 to a non-competitive anti-CCR8 antibody, (ii) Comparing the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs to a predetermined threshold, (iii) Based on the evaluation that the level of CCR8 expression in cells of the test tissue and / or the frequency of CCR8-expressing Tregs exceeds a predetermined threshold, subjects are selected as suitable candidates for cancer immunotherapy using a therapeutic Treg-depleting anti-CCR8 antibody or a portion thereof. Select subjects that are suitable candidates for cancer immunotherapy using therapeutic Treg depletion anti-CCR8 monoclonal antibodies or their antigen-binding ADCC-mediated moieties, (b) Administering a therapeutically effective amount of a composition containing a therapeutic Treg depletion antibody or a portion thereof to a selected subject. A pharmaceutical composition containing the above.

38. A pharmaceutical composition comprising the composition according to claim 23 for use in a method for treating cancer in a subject, wherein the method is (a)(i) Determining the level of surface expression of CCR8 and / or the frequency of CCR8-expressing Tregs in or from test tissues within the subject, including tumor cells and tumor-infiltrating Tregs, by measuring the binding of CCR8 to a non-competitive anti-CCR8 antibody, (ii) Administering a therapeutic Treg depletion antibody or a portion thereof to the subject, (iii) Determine whether a decrease in the frequency of CCR8-expressing Tregs is observed after administration of a therapeutic Treg-depleting antibody or a portion thereof, (iv) Based on the assessment that the reduction in the frequency of CCR8-expressing Tregs exceeds a predetermined threshold, subjects are selected as suitable candidates for immunotherapy using a therapeutic Treg-depleting anti-CCR8 antibody or a portion thereof. Select subjects that are suitable candidates for cancer immunotherapy using therapeutic Treg depletion anti-CCR8 monoclonal antibodies or their antigen-binding ADCC-mediated moieties, (b) Administering a therapeutically effective amount of a composition containing a therapeutic Treg depletion antibody or a portion thereof to a selected subject. A pharmaceutical composition containing the above.

39. A pharmaceutical composition comprising the composition according to claim 23 for use in a method for treating cancer in a subject, wherein the method comprises administering a therapeutic Treg-depleted anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated portion to a subject, the subject being selected on the criterion that the level and / or frequency of CCR8-expressing Treg expression in or from the subject, including tumor cells and tumor-infiltrating Treg, exceeds a predetermined threshold level when measured by the binding of CCR8 to a non-competitive anti-CCR8 antibody.

40. A pharmaceutical composition comprising the composition according to claim 23 for use in a method for treating cancer in a subject, wherein the method comprises administering a therapeutic Treg-depleting anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated moiety to a subject, the subject being selected on the criterion that the therapeutic Treg-depleting antibody or its antigen-binding ADCC-mediated moiety causes a decrease in the frequency of CCR8-expressing Tregs as measured by the binding of CCR8 to a non-competitive anti-CCR8 antibody.

41. The pharmaceutical composition according to any one of claims 37 to 40, wherein determining the level of CCR8 expression in a test tissue includes evaluating the level of CCR8 expression on the surface of Treg cells in the test tissue, or evaluating the proportion of Treg cells expressing CCR8 on the surface of Treg cells in the test tissue.

42. The pharmaceutical composition according to claim 41, wherein the level of CCR8 expression in the target test tissue is determined by an in vivo method.

43. The pharmaceutical composition according to claim 42, wherein the in vivo method includes a PET tracking method.

44. The pharmaceutical composition according to any one of claims 37 to 40, wherein the level of CCR8 expression is determined ex vivo in a test tissue sample obtained from a subject.

45. The pharmaceutical composition according to claim 44, wherein the level of CCR8 expression is determined by immunohistochemistry (IHC), flow cytometry, or mass spectrometry-coupled flow cytometry.

46. The pharmaceutical composition according to claim 45, wherein the IHC is performed on fresh frozen tissue or formalin-fixed paraffin-embedded (FFPE) tissue.

47. A therapeutic Treg-depleting anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated portion, (a) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 53; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 54; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 55; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 56; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO: 57; and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence represented as SEQ ID NO:

58. (b) V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 9 Ｈ V containing a sequence of continuously linked amino acids having the sequence represented as Sequence ID No. 16 Ｌ ,or (c) A heavy chain containing continuously linked amino acids having the sequence represented by Sequence ID No. 65 and a light chain containing continuously linked amino acids having the sequence represented by Sequence ID No. 72 A pharmaceutical composition according to any one of claims 37 to 40, comprising:

48. The pharmaceutical composition according to any one of claims 37 to 40, wherein the Treg-depleted anti-CCR8 monoclonal antibody or its antigen-binding ADCC-mediated portion is an antibody or its antigen-binding ADCC-mediated portion named 4A19, 18Y12, 8D55, 10R3, 14S15, or 14S15h.

49. The pharmaceutical composition according to any one of claims 37 to 40, wherein the target is a human.

50. The pharmaceutical composition according to any one of claims 37 to 40, wherein the cancer is a solid tumor or a hematological malignancy.

51. (a) Solid tumor is squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, lung adenocarcinoma (LUAD), head and neck cancer, breast cancer, invasive breast cancer (BRCA), breast cancer, esophageal cancer, esophageal cancer (ESCA), gastric cancer, adenocarcinoma of the gastric / gastroesophageal (GE) junction, gastric adenocarcinoma (STAD), gastrointestinal cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HC C) Pancreatic cancer (PAC), pancreatic adenocarcinoma (PAAD), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), microsatellite-stable colorectal cancer (MSS-CRC), colon cancer, colon adenocarcinoma (COAD), rectal adenocarcinoma (READ), anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, Glioblastoma, germ cell tumor, childhood sarcoma, natural killer sinus cancer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, endometrial cancer, fallopian tube cancer, ovarian cancer, cervical cancer, cervical squamous cell carcinoma, cervical adenocarcinoma (CESC), vaginal cancer, vulvar cancer, testicular cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, penile cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, childhood solid tumors, environmentally induced cancer, virus-associated cancer, viral-origin cancer, advanced cancer, unresectable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination thereof. (b) Hematological malignancies include diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma (FL), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), mantle cell lymphoma, and mucosa-associated lymphoid tissue (MAL). T) Lymphoma, Nodal marginal zone B-cell lymphoma, Splenic marginal zone B-cell lymphoma, Burkitt lymphoma, Lymphoplasmacytoid lymphoma (LPL), Pyloricell lymphoma, Primary central nervous system (CNS) lymphoma, Progenitor T-lymphoblastic lymphoma / leukemia, T-lymphoblastic lymphoma / leukemia (T-Lby / T-ALL), Cutaneous T-cell lymphoma, Adult T-cell lymphoma / leukemia, Angioimmunoblastic T-cell lymphoma, Extranodal Natural killer / T-cell lymphoma (T-cell lymphoma) of the nasal cavity type, enteric T-cell lymphoma (EATL) associated with intestinal disease, anaplastic large cell lymphoma (ALCL), unspecified peripheral T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, vascular central lymphoma, enteric T-cell lymphoma, primary mediastinal B-cell lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary exudative lymphoma, diffuse histiocytic lymphoma Hematological malignancies selected from DHL, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, solitary plasmacytoma, multiple myeloma, smoldering myeloma, monoclonal gammaglobulinemia of unknown significance (MGUS), IgG myeloma, light chain myeloma, nonsecretory myeloma, amyloidosis, progressive, metastatic, refractory, and / or recurrent hematological malignancies and any combination thereof. The pharmaceutical composition according to claim 50.

52. The pharmaceutical composition according to any one of claims 37 to 40, further comprising administering a therapeutically effective amount of an additional therapeutic agent to treat cancer.

53. The pharmaceutical composition according to claim 52, wherein the additional therapeutic agent is an antagonist that reduces the inhibition of the immune system or an agonist that increases its stimulation.

54. An additional therapeutic agent, (a) An antagonist that specifically binds to programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), B and T lymphocyte attenuators (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), killer immunoglobulin-like receptor (KIR), killer cell lectin-like receptor G1 (KLRG-1), adenosine A2a receptor (A2aR), T cell immune receptor having Ig and ITIM domains (TIGIT), T cell activation V-domain Ig suppressor (VISTA), proto-oncogene tyrosine protein kinase MER (MerTK), natural killer cell receptor 2B4 (CD244), or CD160, or (b) Agitated agents that specifically bind to inducible T cell costimulatory molecules (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-induced TNFR-related protein (GITR), or herpesvirus entry mediator (HVEM). The pharmaceutical composition according to claim 53.

55. The pharmaceutical composition according to claim 53, wherein the antagonist that specifically binds to PD-1 is selected from nivolumab, pembrolizumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, retifanlimab, zinbererimab, pimivalimab, serprulimab, voplaterimab, and acrixolimab.

56. The pharmaceutical composition according to claim 54, wherein the antagonist that specifically binds to PD-L1 is selected from atezolizumab, durvalumab, avelumab, emvafolimab, cosiberimab (CK-301), BMS-936559, BMS-986189, CS-1001, SHR-1316, CBT-502, BGB-A333, KN035, AUNP12, and CA-170.

57. The pharmaceutical composition according to claim 53, wherein the antagonist that specifically binds to CTLA-4 is ipilimumab or tremelimumab.

58. The pharmaceutical composition according to claim 53, wherein the antagonist that specifically binds to LAG-3 is selected from relatrimab, fabezerimab, tilagolumab, fianlimab, teboterimab, or eftiradimod alfa.

59. A kit for use in the treatment of cancer in a subject, (a) A non-competitive monoclonal anti-CCR8 antibody according to claim 1 or 2 or its antigen-binding moiety, (b) A therapeutic Treg-depleting anti-CCR8 antibody that binds to an epitope in the N-terminal domain of hCCR8, or its antigen-binding ADCC-mediated moiety, (c) Instructions for treating cancer in the subject and A kit that includes this.

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