Antibodies binding to human CCR8

Abstract

The present disclosure provides isolated antibodies that specifically bind to C-C motif chemokine receptor 8 (CCR8) expressed on the surface of a cell and exhibit a variety of functional characteristics, including the characteristics required in diagnosing antibodies. These properties include binding with high affinity and specificity to cells expressing CCR8, such as tumor infiltrating, activated CD4 + FOXP3 high Treg; and binding a human CCR8 (hCAR8) epitope other than the hCAR8 N-terminal domain to which the majority of the therapeutic anti-CCR8 antibodies bind.

Description

[0001] Throughout this application, various publications are cited in parentheses with the author's name and date, or patent number or patent publication number. Full citations for these publications can be found at the end of the specification, before the claims. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the prior art known to those skilled in the art before the invention date described and claimed herein. However, these disclosures are incorporated into this application only if there is no conflict between the information incorporated by reference and the information provided by the explicit disclosure in this application. It is noteworthy that the citation of references herein should not be construed as admitting that such references are prior art of the present invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 365,255, filed on May 24, 2022, the contents of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing submitted electronically through the Patent Center in XML format that complies with ST.26, and is incorporated herein by reference in its entirety. The ST.26 copy was created on May 18, 2023, named 20230518_SEQL_13429WOPCT.xml, and is 48,452 bytes in size. Field of the Invention

[0006] The present invention generally relates to monoclonal antibodies (mAbs) that specifically bind to human C-C motif chemokine receptor 8 (hCCR8), and related methods using such antibodies (Abs), including detecting and quantifying expression of hCCR8 on the surface of cells, evaluating engagement of the receptor by therapeutic anti-CCR8 mAbs, and measuring therapeutic anti-CCR8 mAb-mediated depletion of regulatory T cells (Tregs) expressing CCR8. The mAbs of the present invention (Abs of the present invention), disclosed for the first time herein, bind to an epitope that is different from an epitope located in the N-terminal region of CCR8 that has been shown to bind to certain therapeutic anti-CCR8 mAbs; therefore, binding of any Ab of the present invention to CCR8 is not affected by the presence of an Ab that binds to an N-terminal epitope. Background Art

[0007] Considerable success has been achieved in treating various solid tumors and hematological malignancies with immunotherapies that stimulate the activity of cytotoxic T cells by blocking immune checkpoint molecules such as PD-1, PD-L1, CTLA-4, or LAG-3, which are known to inhibit host antitumor immunity in the tumor microenvironment (Pardoll, 2012; Lesokhin et al., 2015; Baumeister et al., 2016; Pianko et al., 2017). However, among cancers suitable for checkpoint inhibitor treatment, typically less than about 15% of patients benefit from the treatment over the long term (Haslam and Prasad, 2019), and checkpoint inhibitors have been shown to be less effective in certain cancers, including breast and prostate cancer. Therefore, there is an urgent need for biomarkers that can be reliably used to predict whether a particular cancer or patient is suitable for treatment with a particular immunotherapy and to monitor the course of treatment at the molecular level.

[0008] The persistence of immunosuppressive mechanisms, especially those mediated by regulatory T cells (Tregs), may contribute to resistance to checkpoint inhibitor therapy in some cancers or in some patients (Fares et al., 2019; Han et al., 2019). Therefore, reducing the activity or number of tumor-infiltrating Tregs is considered an attractive approach to reverse immunosuppression and enhance anti-tumor immunity (Finotello and Trajanoski, 2017; Han et al., 2019). It has recently been demonstrated that CCR8 expression is selectively upregulated in tumor-resident Tregs in a variety of cancers (De Simone et al., 2016; Plitas et al., 2016), making CCR8 an attractive target for achieving depletion of tumor-resident Tregs in order to enhance anti-tumor immunity.

[0009] PCT Publication No. WO 2021 / 194942 discloses several human or humanized mAbs that specifically bind to hCCR8 expressed on the cell surface with high affinity and mediate CCR8 inhibition when administered to mice as monotherapy or in combination with checkpoint blockade. + Depletion of tumor-infiltrating Tregs and potent inhibition of tumor growth in multiple mouse tumor models. One of these mAbs, A419, is currently in a phase 1 / 2 clinical trial (NCT04895709; https: / / clinicaltrials.gov / ct2 / show / NCT04895709) and has been shown to bind to an epitope in the N-terminal region of hCCR8.

[0010] As disclosed herein, most mAbs generated against hCCR8 immunogens expressed on the cell surface bind to the N-terminal epitope. Recent publications describing the generation of therapeutic anti-CCR8 antibodies also describe antibodies that bind to epitopes in the N-terminal domain, such as PCT Publication Nos. WO 2020 / 138489, WO 2021 / 142002, WO 2021 / 152186, WO 2021 / 163064, WO 2021 / 194942, WO 2021 / 260209, and WO 2022 / 136649. 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. The mAbs of the present invention, referred to herein as "non-competitive" mAbs due to their inability to compete with therapeutic mAb4A19 for binding to hCCR8, can be used in many medical applications, including detecting, measuring the expression of hCCR8 on the surface of cells and measuring its receptor occupancy (RO), and detecting the depletion of cells expressing CCR8, even in the presence of therapeutic anti-hCCR8 Abs that bind to the N-terminal epitope. These properties of the mAbs disclosed herein make them useful for several purposes, including measuring the depletion of Tregs expressing CCR8 mediated by anti-CCR8 therapeutic Abs, as well as various diagnostic and biomarker applications. Summary of the invention

[0011] The present disclosure provides isolated Abs, preferably mAbs, of the present invention that specifically bind to CCR8 expressed on the surface of cells, such as human CCR8 (hCCR8), and exhibit various functional properties, including those desirable in diagnostic Abs that can be used to measure CCR8 expression in patients treated with therapeutic anti-CCR8 Abs. These properties include binding with high affinity and specificity to cells expressing CCR8, such as tumor-infiltrating, activated CD4 + FOXP3 高 Tregs, and bind an hCCR8 epitope that is distinct from the epitope in the N-terminal domain of hCCR8 bound by therapeutic anti-CCR8 Abs such as mAb4A19.

[0012] Specifically, the present disclosure provides mAbs or antigen-binding portions thereof that specifically bind to hCCR8 expressed on the surface of a cell, wherein the mAbs or antigen-binding portions thereof bind to an epitope other than an epitope in the N-terminal domain of hCCR8. MAb4A19 (WO 2021 / 194942) has been shown to bind to an N-terminal epitope comprising at least one amino acid, and in a preferred embodiment, comprising a sequence V 12 T 13 D14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 In certain embodiments, the N-terminal epitope to which the anti-hCCR8 mAbs of the present disclosure do not bind comprises 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 (SEQ ID NO: 73), for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 amino acids in a peptide having the sequence shown in SEQ ID NO: 73. In certain preferred embodiments, the N-terminal epitope to which the anti-hCCR8 mAbs of the present disclosure do not bind comprises a peptide having the sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 In other preferred embodiments, the N-terminal epitope to which the anti-hCCR8 mAbs of the present disclosure do not bind comprises 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 In other preferred embodiments, the N-terminal epitope to which the anti-hCCR8 mAbs of the present disclosure do not bind is composed of a peptide having sequence V 12 T 13 D 14 Y15Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73).

[0013] In certain preferred embodiments, the binding of an Ab of the invention, or an antigen binding portion thereof, to hCCR8 is not affected by the presence of an Ab that binds to an N-terminal epitope, e.g., in the presence of an Ab having 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) comprises at least one amino acid epitope, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 amino acids. In certain embodiments, amino acid Y in the N-terminal epitope 15 or Y 17 , preferably Y 15 and Y 17 Both are sulfated. In other embodiments, the Ab that binds to the N-terminal epitope is an Ab comprising the 6 CDRs (SEQ ID NOs: 53-58), heavy and / or light chain variable regions (SEQ ID NOs: 9 and / or 16), or heavy and / or light chains (SEQ ID NOs: 65 and / or 72) of mAb 4A19 described in WO 2021 / 194942. In other embodiments, the Ab that binds to the N-terminal epitope is an mAb 4A19 comprising the heavy and / or light chains (SEQ ID NOs: 65 and / or 72) of mAb 4A19.

[0014] 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, that comprises the six CDRs, heavy and / or light chain variable regions, or heavy and / or light chains of a mAb designated herein as 25T40, 21C17, 28P3, 22B13, 33H18, or 23A14.

[0015] The present invention also provides a labeled Ab or antigen binding thereof, which comprises the mAb of the present invention and a detectable label. In various embodiments, the detectable label is a fluorophore, a chromophore, an enzyme, a radioisotope, a micropolymer or a metal.

[0016] The present disclosure also provides a method for generating a first Ab (e.g., an Ab of the present invention) that does not bind to or does not cross-compete with a second Ab (e.g., anti-hCCR8 clone L263G8 commercialized by BioLegend, mAb433H sold by BD Biosciences, or any of mAbs 4A19, 18Y12, 8D55, 10R3, 14S15, and 14S15h described in WO 2021 / 194942) for binding to a defined epitope on an antigen (e.g., the N-terminal epitope of hCCR8). The method includes immunizing a vertebrate with an immunogen that comprises a cell line or a component of the cell line that expresses an antigen and also expresses a second Ab or an antigen-binding portion thereof that specifically binds to an epitope, wherein the binding of the second Ab or its antigen-binding portion to the epitope will shield the epitope from attack by the vertebrate immune system, reduce the generation of Abs that bind to the epitope, thereby resulting in the generation of a first Ab that does not bind to or does not cross-compete with the second Ab for binding to the epitope.

[0017] The present disclosure also provides a method for measuring the exhaustion of the number of Tregs in a subject, comprising: (a) determining a baseline percentage of T cells expressing Tregs as CCR8 in a first test tissue of a subject or taken from a subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering treatment to the subject; and (c) determining the percentage of T cells expressing Tregs as CCR8 in a second test tissue of a subject or taken from a subject during or after treatment; wherein a decrease in the percentage of T cells expressing Tregs as CCR8 in the second test tissue indicates that the number of Tregs in the test tissue has been exhausted. In certain preferred embodiments, the treatment administered to the subject is a treatment of cancer. In other preferred embodiments, the treatment of cancer comprises administering a Treg-depleting therapeutic anti-CCR8 Ab or an antigen-binding portion thereof to the subject, as a monotherapy or in combination with another anti-cancer therapy.

[0018] The present disclosure also provides a method for predicting the effectiveness of a therapeutic Treg-depleting anti-CCR8 Ab or an antigen-binding portion thereof in treating cancer in a subject, the method comprising: (a) determining the percentage of T cells that are CCR8-expressing Tregs in a test tissue of the subject 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 predetermined threshold; and (c) predicting the effectiveness of the therapeutic anti-CCR8 Ab, wherein the percentage of T cells that are CCR8-expressing Tregs exceeds the threshold, indicating that the therapeutic Ab or its antigen-binding portion will effectively treat the subject, and further wherein the percentage of T cells that are CCR8-expressing Tregs is less than the threshold, indicating that the therapeutic Ab or its antigen-binding portion will not be able to effectively treat the subject.

[0019] The present invention also relates to a method for treating cancer in a patient. Therefore, the present disclosure provides a method for treating cancer in a subject, the method comprising: (a) selecting a subject, the subject is a suitable candidate for immunotherapy with a Treg-depleting therapeutic anti-CCR8 Ab or its antigen-binding portion, the selection comprising: (i) determining the percentage of T cells that are CCR8-expressing Tregs in a test tissue of the subject 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 predetermined threshold; and (iii) based on an assessment that the percentage of T cells that are CCR8-expressing Tregs in cells of the test tissue exceeds a predetermined threshold, selecting the subject as a suitable candidate for immunotherapy with the therapeutic anti-CCR8 Ab or its antigen-binding portion; and (b) administering a composition comprising a therapeutically effective amount of a therapeutic anti-CCR8 Ab or its antigen-binding portion to the selected subject.

[0020] The present disclosure also provides a method for treating cancer in a subject, the method comprising: (a) selecting a subject who is not a suitable candidate for immunotherapy with a Treg-depleting therapeutic anti-CCR8 Ab or an antigen-binding portion thereof, the selection comprising: (i) determining the percentage of T cells that are CCR8-expressing Tregs in a test tissue of the subject 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 predetermined threshold; and (iii) selecting a subject who is not suitable for immunotherapy with the 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 cells of the test tissue is less than the predetermined threshold; and (b) administering a standard-of-care therapeutic other than the therapeutic anti-CCR8 Ab or an antigen-binding portion thereof to the selected subject.

[0021] The present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a Treg-depleting therapeutic anti-CCR8 Ab, or an antigen-binding portion thereof, wherein the subject is selected based on the percentage of T cells that are CCR8-expressing Tregs in cells of a test tissue of the subject or taken from the subject being determined to exceed a predetermined threshold level, wherein the test tissue comprises tumor cells and tumor-infiltrating Tregs.

[0022] The present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject a standard-of-care treatment in addition to a Treg-depleting therapeutic anti-CCR8 Ab, or an antigen-binding portion thereof, wherein the subject is selected based on the percentage of T cells that are CCR8-expressing Tregs in cells of a test tissue of the subject or taken from the subject being determined to be less than a predetermined threshold level, wherein the test tissue contains tumor cells and tumor-infiltrating Tregs.

[0023] In certain embodiments of any of these methods for treating cancer, the Treg-depleting therapeutic anti-CCR8 Ab or its antigen-binding portion comprises 6 CDRs, heavy and / or light chain variable regions, or heavy and / or light chains of mAb 4A19 described in WO 2021 / 194942, which specifically binds to CCR8 expressed on the cell surface with high affinity and mediates the depletion of cells, or its antigen-binding portion. In other embodiments, the Treg-depleting therapeutic anti-CCR8 Ab or its antigen-binding portion is a mAb named 4A19, 18Y12, 10R3, 8D55, 14S15 or 14S15h, or its antigen-binding portion. These Abs disclosed in WO 2021 / 194942 bind to the N-terminal epitope of hCCR8.

[0024] In other embodiments, the method of treatment also includes administering to the subject a therapeutically effective amount of an additional therapy for treating cancer. This additional anticancer therapy can be a small molecule agent, a polypeptide, an antibody, an immunomodulator, chemotherapy, targeted therapy, radiotherapy, surgery or any combination thereof. In certain embodiments, immunotherapy includes an agent that reduces the suppression of the immune system or increases the stimulation of the immune system. In certain preferred embodiments, the immunomodulator that reduces immune system suppression is an immune checkpoint inhibitor. In other preferred embodiments, the immunomodulator that reduces immune system suppression is an immune checkpoint inhibitor, such as an antagonist of PD-1, PD-L1, CTLA-4, LAG-3, TIGIT and / or TIM-3. In certain embodiments, chemotherapy comprises an alkylating agent such as dacarbazine, ifosfamide, cyclophosphamide or the platinum chemotherapeutic agents cisplatin, bendamustine, carboplatin and oxaliplatin; a mitotic inhibitor such as the vinca alkaloids vinblastine and vincristine, or the taxanes docetaxel, paclitaxel and cabazitaxel; a topoisomerase inhibitor such as etoposide or irinotecan; an antimetabolite such as 5-fluorouracil, azacytidine or gemcitabine; or an antitumor antibiotic such as bleomycin, mitomycin C or the anthracyclines daunorubicin, doxorubicin and mitoxantrone. In certain preferred embodiments, the additional anticancer therapy is an anti-PD-1 antibody or docetaxel.

[0025] The present disclosure also provides a variety of kits for performing the methods described herein, including kits for the following methods: measuring receptor (e.g., CCR8) occupancy of Treg-depleting therapeutic Abs (e.g., anti-CCR8, Treg-depleting therapeutic Abs); measuring the depletion of Treg numbers in a subject; predicting the effectiveness of therapeutic anti-CCR8 Abs; selecting a subject with cancer as a suitable candidate for immunotherapy with therapeutic anti-CCR8 Abs; and treating cancer in a subject. For example, the present disclosure provides a kit for measuring the depletion of Treg numbers in a subject, the kit comprising: (a) mAb or an antigen-binding portion thereof that specifically binds to hCCR8 expressed on the surface of a cell, wherein the mAb or its antigen-binding portion binds to an epitope other than an epitope in the N-terminal domain of hCCR8; and (b) instructions for using mAb or a portion thereof in any of the methods for measuring Treg depletion disclosed herein.

[0026] As another example, the present disclosure provides a kit for 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, wherein the mAb or its antigen binding portion binds to an epitope other than an epitope in the N-terminal domain of hCCR8; (b) a Treg-depleting therapeutic anti-CCR8 Ab or its antigen binding portion; and (c) instructions for using the mAb or portion thereof and the therapeutic anti-CCR8 Ab or its antigen binding portion in any of the methods for treating cancer in a subject disclosed herein.

[0027] Other features and advantages of the present invention will be apparent from the following detailed description and examples, but these detailed description and examples should not be considered as limiting. The contents of all references cited in this application, including scientific articles, GenBank entries, patents and patent applications are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1A-1C : Flow cytometry graphs showing the comparison of the two tested mAbs with those obtained with unlabeled mAb 4A19 ( Figure 1A )、21C17( Figure 1B ) or isotype control ( Figure 1C ) pre-treated gastric tumor Treg compartment, where the two tested mAbs were BV421-labeled 21C17 on the x-axis and phycoerythrin (PE)-labeled clone L263G8 (BioLegend) on the y-axis. Binding to 4A19 pre-treated Tregs was observed with the diagnostic BV421-labeled 21C17 mAb ( Figure 1A, quadrant 3), while the commercial PE-labeled L263G8 mAb did not bind to 4A19-pretreated Tregs ( Figure 1A ). Binding to 21C17 pre-treated Tregs was observed using PE-L263G8 Ab ( Figure 1B , Q1), while BV421-21C17 did not bind to 21C17 pre-treated Tregs ( Figure 1B ), indicating self-blocking of 21C17. Both labeled 4A19 and 21C17 mAbs bound to the same Tregs pretreated with isotype control Ab ( Figure 1C , Q2). These results indicate that mAb 21C17 is able to bind to CCR8-expressing tumor Tregs pretreated with 4A19 mAb.

[0029] Figures 2A and 2B Specific binding of six mAbs of the invention (25T40, 21C17, 28P3, 22B13, 33H18, and 23A14) to hCCR8-overexpressing Raji cells ( FIG. 2A ) and parental Raji cells not expressing CCR8 ( FIG. 2B ) was shown, as assessed by FACS using fluorescently labeled secondary antibodies. Four of the six mAbs had an EC value below 1 nM. 50 Binding to cells expressing hCCR8 (Fig. 2A; see Table 1), whereas no nonspecific binding was observed on parental Raji cells (Fig. 2B).

[0030] Figures 3A-3C show the evaluation of competition between Abs of the present invention and Abs that bind to N-terminal epitopes for binding to hCCR8. A: Mouse IgG2a variant of mAb 4A19 (4A19-mG2a) binds to activated human Tregs at a saturation concentration of 200 nM, followed by the addition of 23A14-hG1 mAbs at concentrations ranging from 200 nM to 0.0034 nM. Analysis of bound 4A19-mG2a and 23A14-hG1 (Figure 3A) measured by FACS using fluorescently labeled secondary antibodies showed that at saturation concentrations, both 4A19-mG2a and 23A14-hG1 were able to simultaneously bind to the same high percentage of CCR8 + B, C: Activated human Tregs were incubated with mAb 4A19 at titration concentrations from 200 nM to 0.0034 nM, and then with 100 nM 21C17-mG2a (Figure 3B) or 22B13-mG2a (Figure 3B). Figure 3C FACS competition assay analysis using fluorescently labeled secondary antibodies showed that 4A19 was able to simultaneously bind to the same high percentage of CCR8 when paired with 21C17-mG2a or 22B13-mG2a at saturating concentrations. + Treg binding.

[0031] Figures 4A and 4B The specific binding of mAb 21C17-mG2a directly conjugated with BV421 fluorophore to Raji cells (Fig. 4A) overexpressing hCCR8 and parental Raji cells (Fig. 4B) not expressing CCR8 is shown, as evaluated by FACS. Raji parental cells and Raji cells overexpressing CCR8 are incubated with titrated unlabeled 21C17-mG2a and 21C17-mG2a or keyhole limpet hemocyanin (KLH)-mg2a control Abs. Phycoerythrin (PE)-labeled anti-mouse IgG secondary antibodies are used to detect the binding of unlabeled 21C17-mG2a mAb to cell surface hCCR8. Relative cell binding is measured as the geometric mean fluorescence intensity (GMFI) of the total cells positive for the secondary antibodies conjugated with fluorescence and the fluorescence from the Ab directly conjugated. Both labeled and unlabeled parental 21C17-mG2a antibodies specifically bound to cell surface CCR8 on the Raji overexpressing cell line with similar efficiency ( FIG. 4A ), but did not bind to the Raji parental cell line ( FIG. 4B ).

[0032] Figure 5 Representative graphs showing percent CCR8 receptor occupancy (%RO) response curves are shown, where CCR8 %RO is plotted against mAb 4A19 concentration in healthy donor blood (n=3). The %RO curves for each donor were plotted using both direct and indirect RO assay formats. Response curves from blood assays of donors ##1199, 0612, and 398 are shown.

[0033] Figure 6 The anti-hCCR8 mAb, 4A19, is shown to deplete Tregs in dissociated human tumors. After treatment of dissociated tumor tissue with mAb4A19 or anti-KLH isotype Ab control for 48 hours, cells were stained with a viability stain to identify live cells and then stained with non-competing anti-CCR8 mAb 21C17, as well as conjugated CD4, FoxP3, and CD25 Abs to identify CCR8 + Tregs. MAb 4A19 depleted Tregs, but the KLH isotype control did not deplete Tregs, even at the highest dose tested. DETAILED DESCRIPTION

[0034] The present invention relates to mAbs that do not specifically bind to the N-terminal epitope of hCCR8, and the use of such mAbs. That is, the present invention relates to mAbs that specifically bind to hCCR8 epitopes, which are different from the epitopes in the N-terminal domains that Treg depletion therapeutic Abs (such as mAb 4A19) bind to, and methods for detecting and / or measuring the expression level of hCCR8 on cell surfaces using such mAbs, for example. MAbs that bind to the epitope of hCCR8 outside the N-terminal domain do not compete with MAbs that bind to the N-terminal epitope, and therefore, the binding of the former MAb to CCR8 is not affected by the presence of Abs that bind to the N-terminal domain. Therefore, even in the presence of therapeutic mAbs that bind to the N-terminal epitope, mAbs of the present invention can also be used to detect and / or measure the expression level of hCCR8 on cell surfaces. This feature is very useful in the use of mAbs of the present invention for diagnosis or biomarker applications.

[0035] the term

[0036] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms has the meaning described below. Additional definitions are listed throughout the application.

[0037] "Administering" and its various parts of speech (administering, administering, administration) refer to the physical introduction of a composition containing an agent, such as a therapeutic agent or a diagnostic agent, into a subject using any of the various methods and delivery systems known to those skilled in the art. The preferred route of administration for treating or diagnosing Abs, such as anti-CCR8 Abs, is intravenous (IV) administration. Other routes of administration include subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the Ab of the invention can be administered by a non-parenteral route, such as a topical, epidermal or mucosal administration route, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. Administration can also be performed, for example, once, multiple times and / or over one or more extended time periods.

[0038] "Antibody" (Ab) shall include, but is not limited to, a glycoprotein immunoglobulin (Ig) or an antigen-binding portion thereof that specifically binds to an antigen and comprises at least two heavy (h) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as V H ) and heavy chain constant region. The heavy chain constant region of IgG Ab contains three constant domains C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (abbreviated herein as V L ) and the light chain constant region. The light chain constant region of IgG Ab contains a constant domain C L . V H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. Various methods have been used to describe the CDR domains in Ab, including Kabat, Chothia, AbM, contact, and IMGT definitions. The constant region of Ab can mediate the binding of Ig to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0039] As used herein, according to conventional usage, an Ab described as comprising a heavy chain and / or a light chain refers to an Ab comprising "at least one" of the heavy chain and / or the light chain, thus encompassing an Ab with two or more heavy chains and / or light chains. Specifically, the Ab described in this manner will encompass a conventional Ab with two substantially identical heavy chains and two substantially identical light chains. If the Ab chains differ due to post-translational modifications (including, for example, C-terminal cleavage of lysine residues and optional glycosylation patterns), they may be substantially identical but not identical.

[0040] Ig can be derived from any commonly 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, including but not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the Ab category or subclass (e.g., IgM, IgG1 or IgG4) encoded by the heavy chain constant region gene. The term "antibody" includes, for example, naturally occurring and non-naturally occurring Ab, monoclonal and polyclonal Ab, chimeric and humanized Ab, human or non-human Ab, fully synthetic Ab and single-chain Ab. Non-human Ab can be partially or fully humanized by recombinant methods to reduce its immunogenicity in humans. If not explicitly stated, and unless otherwise indicated in the context, the term "antibody" also includes the antigen-binding fragment or antigen-binding portion of any of the above-mentioned Ig, and includes monovalent and divalent fragments or portions, and single-chain Ab.

[0041] "Isolated" Ab refers to an Ab that is substantially free of other Abs with different antigenic 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 have cross-reactivity with other antigens (e.g., CCR8 polypeptides from different species such as mice and cynomolgus monkeys). In addition, in some cases, an isolated Ab may also refer to an Ab that is purified to be substantially free of other cellular materials and / or chemicals. By comparison, an "isolated" nucleic acid refers to a nucleic acid composition of a substance that is significantly different from nucleic acids present in nature, i.e., has unique chemical properties, properties, and uses. For example, unlike natural DNA, isolated DNA is an independent part of natural DNA, rather than a component of a larger structural complex (chromosome) found in nature. In addition, unlike natural DNA, isolated DNA can be used as a PCR primer or hybridization probe for measuring gene expression and detecting biomarker genes or mutations to diagnose diseases or predict the efficacy of therapeutic agents, etc. Additionally, in certain cases, an isolated nucleic acid can refer to a nucleic acid that has been purified to be substantially free of other cellular components or other contaminants (eg, other cellular nucleic acids or proteins) using standard techniques well known in the art.

[0042] The term "monoclonal" Ab (mAb) refers to a non-naturally occurring preparation of Ab molecules of single molecular composition, i.e., Ab molecules whose primary sequences are substantially identical and which exhibit a single binding specificity and affinity for a particular epitope. mAbs are examples of isolated Abs. mAbs can be produced by hybridomas, recombination, transgenics, or other techniques known to those skilled in the art.

[0043] A "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.

[0044] "Human" mAb (HuMAb) refers to mAb with variable regions, wherein the framework region and CDR region are all from human germline immunoglobulin sequences. In addition, if Ab comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human Ab 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 mutations in vitro or by somatic mutations in vivo). However, the term "human" Ab as used herein is not intended to include Abs in which CDR sequences derived from another mammalian species (e.g., mouse) germline have been transplanted to human framework sequences. The terms "human" Ab and "full human" Ab are used synonymously.

[0045] A "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 corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an Ab, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions are not changed. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not eliminate the ability of the Ab to bind a specific antigen. A "humanized" Ab retains antigenic specificity similar to that of the original Ab.

[0046] "Anti-antigen" Ab refers to an Ab that specifically binds to an antigen. For example, an anti-CCR8 Ab is an Ab that specifically binds to CCR8.

[0047] An "antigen binding portion" or "antigen binding fragment" of an Ab refers to one or more fragments of an Ab, such as a mAb, that retain the ability to specifically bind to an antigen bound by the intact Ab. An anti-CCR8 antigen binding portion or fragment that mediates depletion of cells expressing CCR8 by, for example, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) necessarily comprises a portion of the Fc region of the Ab, which is required to mediate these effector functions through its interaction with Fc receptors on immune cells or with C1q.

[0048] "Antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to an in vitro or in vivo cell-mediated cytotoxic activity in which nonspecific effector cells (e.g., natural killer (NK) cells, macrophages, neutrophils, and eosinophils) expressing Fc receptors (FcRs) on the effector cell surface recognize the Fc region of an Ab bound to a surface antigen on a target cell and actively lyse the target cell. In principle, any effector cell with an activating FcR can be triggered to mediate ADCC.

[0049] Antibody-dependent cell-mediated phagocytosis ("ADCP") is an immunological mechanism of cell elimination by which phagocytic immune cells (e.g., monocytes, macrophages, and neutrophils) expressing Fc receptors (FcRs) on their cell surfaces recognize the Fc region of Abs bound to surface antigens of target cells to induce phagocytosis, leading to internalization and degradation of the target cells through phagosomal acidification.

[0050] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors, which can invade neighboring tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0051] "CC motif chemokine receptor 8" ("CCR8"; also known in the art 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 primarily expressed in tumors by FOXP3 hi The term "CCR8" as used herein includes human CCR8 (hCCR8), variants, isoforms, species homologs of hCCR8, such as mouse CCR8 (mCCR8), and analogs that share at least one common epitope with hCCR8. The complete amino acid sequences of hCCR8 and mCCR8 can be found in Found under accession numbers AAI07160.1 and NP_031746.1.

[0052] "Cell surface receptor" refers to a molecule or complex of molecules expressed on the surface of a cell that is capable of receiving a signal and transmitting the signal across the cytoplasmic membrane.

[0053] Complement dependent cytotoxicity ("CDC") is an immune response in which target cells are lysed by activation of the complement cascade and recruitment to the target cell surface. It is an effector function of IgG (mainly IgG1 and IgG3) and IgM Abs. Binding of C1q to these Abs (when bound to surface antigens on target cells) triggers activation of the classical complement pathway, leading to the formation of the membrane attack complex (MAC) and target cell lysis.

[0054] "Immune response" refers to a biological response in vertebrates to foreign agents that protects the organism from these agents and the diseases caused by them. The immune response is mediated by the action of one or more 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 Ab, cytokines, and complement) produced by any of these cells or the liver, resulting in selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues in the vertebrate body.

[0055] The term "immunotherapy" refers to treating a disease in a subject, or treating a subject at risk of contracting a disease or suffering a recurrence of a disease, by methods that include inducing, enhancing, suppressing or otherwise modifying an immune response. "Cancer immunotherapy" refers to the use of immunotherapy to treat or prevent cancer, typically by inducing or enhancing an immune response, such as by blocking an immunosuppressive pathway or mechanism in a subject.

[0056] The term "positron emission tomography" or "PET" refers to a non-invasive imaging technique that uses radioactive materials to visualize molecular targets and measure metabolic processes in a subject. The technique detects gamma-ray pairs emitted indirectly at biologically active molecules by positron-emitting radionuclides (tracers) introduced into the body and produces a three-dimensional image of the location of the tracer in the body. Exemplary applications of PET imaging tools in drug development include direct visualization of targets and changes in target numbers, monitoring drug uptake and metabolism in different tissues to predict toxicity or patient-to-patient variability, quantifying diseased tissue, assessing tumor metastasis, and monitoring drug efficacy or resistance over time.

[0057] "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, and rodents such as mice, rats, and guinea pigs. In preferred embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0058] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug or agent that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, prevention or reduction of impairment or disability due to disease affliction, or otherwise ameliorates disease symptoms in a subject.

[0059] "Treatment" or "therapy" of a subject refers to any type of intervention or method performed on a subject, including the administration of an active agent to the subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions or biochemical markers associated with a disease.

[0060] As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any stated or listed components.

[0061] The term "about" when applied to a numerical value refers to a value that is reasonably close to the value and within an acceptable error range determined by one skilled in the art, which may depend in part on how the value is measured or determined, such as on the limits of the measurement system. For example, "about" may refer to a range of plus or minus 50%, preferably a range of plus or minus 25%, or more preferably a range of plus or minus 10% of the reference value. According to the practice in the art, these ranges generally fall within an acceptable error range for the particular value.

[0062] The term "substantially the same" or "essentially the same" refers to a sufficiently high degree of similarity between two or more values, substances, compositions of matter, or features that a person skilled in the art would consider the differences between these values, substances, compositions of matter, or features to be minimal or non-existent biologically and / or statistically significant in the context of the properties measured. The differences between the measured values ​​may be, for example, less than about 50%, preferably less than about 25%, and more preferably less than about 10%.

[0063] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range, and fractions thereof (e.g., tenths and hundredths of integers) where appropriate.

[0064] Various aspects of the invention are described in more detail in the following subsections.

[0065] Targeting CCR8 due to its specific expression on tumor-infiltrating Tregs

[0066] It has been shown that CCR8 expression is selectively upregulated in tumor-resident Tregs in a variety of cancers (De Simone et al., 2016; Plitas et al., 2016) and is expressed in the most active and repressed FOXP3 Tregs associated with poor survival. hi CCR8 is expressed in tumor Treg subsets (Plitas et al., 2016; Wang et al., 2019; WO 2021 / 194942). The gene-gene correlation of human CCR8 and FOXP3 in the Cancer Genome Atlas Project (TCGA) further showed that in most cancer types, CCR8 expression has the highest correlation with FOXP3 (the main transcriptional regulator of Tregs), with CCR8 being expressed in tumor FOXP3. hi CCR8 is expressed on Tregs but rarely observed on Tregs and Teffs in peripheral blood (WO 2021 / 194942). CCR8 is also selectively expressed on: FOXP3 in hepatocellular carcinoma tumor samples hi Lymphocytes, but not in patient tumors, FOXP3 mid and FOXP3 neg CD8 and CD4 are expressed in effector T cells; a high proportion is found in tumor-resident Tregs, but tumor-infiltrating CD4 + T cells and CD8 + The proportion in T cells is much lower; and it is expressed in a small proportion of peripheral Tregs but in a large proportion of tumor-infiltrating Tregs (WO2021 / 194942).

[0067] These patterns of CCR8 expression make CCR8 a highly desirable target for the use of anti-CCR8 Abs to mediate depletion of these highly immunosuppressive Tregs through ADCC and ADCP, and because CCR8 is rarely expressed on Tregs and Teffs in peripheral blood or other tissues, targeting Tregs has minimal risk of toxicity. WO 2021 / 194942 describes the generation and characterization of a variety of anti-hCCR8 mAbs that exhibit desirable properties in therapeutic Abs for the treatment of cancer, including highly efficient mediating depletion of tumor-associated Tregs expressing CCR8. These Abs bind to epitopes in the extracellular N-terminal domain of hCCR8, and therefore require Abs that bind to epitopes outside the N-terminal domain. Such Abs that do not affect or are not affected by the binding of therapeutic Abs to the N-terminal domain of CCR8 can be used for diagnostic applications, including measuring or monitoring CCR8 expression on Tregs and the number of CCR8-expressing Tregs, even in the presence of therapeutic Abs that bind to the N-terminus of CCR8.

[0068] Generation of anti-hCCR8 MAbs that do not bind to the N-terminal domain

[0069] By using the immunogen immunization mouse containing the plasma membrane material of the cell that is derived from hCCR8 overexpression to generate mAb.Because it has been found that most anti-hCCR8 Abs bind one or more epitopes in the N-terminal domain of hCCR8, a strategy for preferentially generating Abs that bind epitopes different from these N-terminal epitopes has been developed.This strategy (see Example 1) involves using the proteoliposomes derived from the cells that overexpress hCCR8 as an immunogen, and the cells also express the anti-hCCR8 Ab clone L263G8 (Biolegend) that binds the N-terminal epitope.It is expected that the Ab of this expression will bind the N-terminal epitope of CCR8.Therefore, when using the proteoliposomes prepared by cells as an immunogen, the N-terminal epitope will be shielded by the Ab that is bound, so that it is protected from the attack of the mouse immune system, and the Ab generated will preferentially target the epitope other than the N-terminal epitope.

[0070] This strategy has been successfully demonstrated to generate mouse Abs that bind to epitopes on hCCR8 other than the N-terminal epitope. Specifically, as described in Example 1, this method was used to generate Abs that bind to epitopes other than the N-terminal domain of hCCR8 by immunizing mice with an immunogen comprising a proteoliposome material derived from cells overexpressing chimeric hCCR8 / hCCR5 proteins and an anti-hCCR8 Ab (clone L263G8) that binds to an epitope in the N-terminal domain of hCCR8. However, this method is broadly applicable to the generation of Abs that do not bind to a predetermined epitope of an antigen.

[0071] Thus, the present disclosure describes a method for generating a first Ab that does not bind or does not cross-compete with a second Ab for binding to a determined epitope on an antigen, the method comprising immunizing a vertebrate with an immunogen comprising a cell line or a component of the cell line that expresses the antigen and also expresses a second Ab or an antigen-binding portion thereof, wherein the second Ab specifically binds to the epitope, wherein the binding of the second Ab or the antigen-binding portion thereof to the epitope will shield the epitope from attack by the vertebrate immune system, reduce the generation of Abs that bind to the epitope, thereby preferentially resulting in the generation of a first Ab that does not bind or does not cross-compete with the second Ab for binding to the epitope.

[0072] In certain embodiments, the vertebrate is a mouse, as demonstrated in Example 1; or another mammal, such as a rat, hamster, rabbit, dog, goat, sheep or horse; or a bird, such as a chicken. In certain embodiments, the antigen is a CCR8 receptor, such as a human, cynomolgus monkey, mouse or rat CCR8 receptor. In a preferred embodiment, the antigen is an hCCR8 receptor. In certain embodiments of the method for generating an Ab for hCCR8, the epitope is an epitope in the N-terminal domain of the hCCR8 receptor. In other embodiments, the second Ab or its antigen-binding portion is a mAb named clone L263G8 (BioLegend); mAb named 433H (BD Biosciences) or mAb4A19, 18Y12, 10R3, 8D55, 14S15 or 15S15h as described in WO 2021 / 194942. In certain other embodiments, the immunogen is a detergent-stabilized proteoliposome component of a cell line.

[0073] MAbs of the invention that do not bind to the N-terminal epitope of hCCR8

[0074] Using a variety of immunization activities to generate anti-hCCR8 mAbs, including the above-mentioned method of shielding the N-terminal epitope from the mouse immune system, mouse Abs that bind to epitopes other than the N-terminal epitope on hCCR8 were generated. The immunogen was a detergent-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).

[0075] Hybridoma supernatants were screened by flow cytometry against at least two cell lines, one overexpressing hCCR8 and the other corresponding control cell line that did not overexpress CCR8, to identify mAbs that specifically bind to hCCR8 (see Example 2). To characterize the epitope bound by the hCCR8-specific Abs, hybridoma culture supernatants were screened by ELISA to measure binding to a BSA-conjugated peptide corresponding to the N-terminus of CCR8 (SEQ ID NO: 74). Several mAbs were identified by flow cytometry that specifically bind to hCCR8, but did not bind to the BSA-conjugated CCR8 N-terminal peptide by ELISA, indicating that the epitope that binds to hCCR8 is different from the N-terminal epitope (Example 2).

[0076] Thus, the present disclosure describes an isolated Ab, preferably a mAb or an antigen binding portion thereof, which specifically binds to hCCR8 expressed on the surface of a cell, wherein the Ab or its antigen binding portion binds to an epitope that is not located in the N-terminal domain of hCCR8, i.e., an epitope different from the epitope in the N-terminal domain of hCCR8 bound by the anti-CCR8 mAb disclosed in WO 2021 / 194942. The amino acid sequence of hCCR8 is shown in SEQ ID NO: 1. In certain embodiments, the N-terminal epitope comprises a peptide having the sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 (SEQ ID NO: 2). In certain other embodiments, the N-terminal epitope comprises 2, 3, 4, 5, 6 or all 7 amino acids within a peptide having a sequence of SEQ ID NO: 2. In certain preferred embodiments, the N-terminal epitope comprises all 7 amino acids within a peptide having a sequence of SEQ ID NO: 2. In certain other embodiments, the N-terminal epitope comprises a peptide having a sequence of 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). In certain other embodiments, the N-terminal epitope comprises 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 comprises all 11 amino acids within the peptide having SEQ ID NO: 73. In other preferred embodiments, amino acid Y 15 and / or Y 17 It is sulfated.

[0077] Abs binding to the N-terminal epitope do not interfere with the binding of mAbs of the invention binding to non-N-terminal epitopes of hCCR8

[0078] To determine whether binding of mAb 4A19 (Ab that binds to an N-terminal domain epitope of hCCR8 (see WO 2021 / 194942)) blocks binding of mAb 21C17 of the present invention to CCR8, tissue from dissociated gastric tumors was pre-incubated with unlabeled mAb 4A19 or unlabeled mAb 21C17 and then stained with immune marker Abs for CD3, CD8, CD4, FOXP3 and two anti-hCCR8 Abs (clone L263G8 (BioLegend) or mAb 21C17). Flow cytometric analysis (Example 3) showed that binding of mAb 4A19 to CCR8 blocked subsequent binding of L263G8, but did not block binding of mAb 21C17 to CCR8 on Tregs. In contrast, binding of 21C17 to CCR8 blocks subsequent binding of 21C17 itself, but does not block binding of L263G8 to Tregs. These results suggest that mAb 21C17 binds to an epitope on CCR8 that is distinct from the N-terminal epitope bound by mAb 4A19 and that binding of 4A19 does not interfere with 21C17 binding to Tregs expressing CCR8.

[0079] Therefore, the present disclosure provides an isolated Ab of the present invention, preferably a mAb or an antigen binding portion thereof, whose binding to hCCR8 is not affected by the presence of an Ab that binds to the N-terminal epitope of hCCR8. In certain embodiments, the mAbs of the present invention disclosed herein do not compete with any of the multiple mAbs mAb 4A19, 18Y12, 10R3, 8D55, 14S15 and 15S15h described in WO 2021 / 194942 for binding to hCCR8. In certain other embodiments, the binding of the mAbs of the present invention to hCCR8 is not affected by the presence of an Ab that binds to an epitope comprising an amino acid sequence V 12 T 13 D 14Y15Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 amino acids within the peptide domain of hCCR8 of (SEQ ID NO:73). In certain preferred embodiments, the binding of mAb 21C17 to hCCR8 is not affected by the presence of an Ab that binds to an epitope comprising 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 (SEQ ID NO: 73) or consist of all 11 amino acids within the peptide domain of hCCR8. In other preferred embodiments, the binding of mAb21C17 to hCCR8 is not affected by the presence of mAb4A19 (WO 2021 / 194942) that binds to hCCR8.

[0080] In certain aspects of the invention, the Ab that binds the N-terminal epitope comprises:

[0081] (a) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 53; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 54; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 55; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 56; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 57; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 58;

[0082] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 9; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 16; or

[0083] (c) a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:65; and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:72.

[0084] In certain embodiments, the Ab that does not interfere with the binding of the Ab of the present invention and binds to the N-terminal epitope is mAb 4A19, 18Y12, 10R3, 8D55, 14S15 or 14S15h (see WO 2021 / 194942) or L263G8 (BioLegend). Preferably, the Ab that does not interfere with the binding of the Ab of the present invention and binds to the N-terminal epitope is mAb 4A19.

[0085] MAbs that bind with high affinity to non-N-terminal epitopes on hCCR8

[0086] Certain anti-CCR8 mAbs of the present invention specifically bind to hCCR8 with high affinity. The binding affinity of the Ab to a target such as hCCR8 can be determined by measuring the dissociation constant (K) for binding to a cell line expressing CCR8. D ) or half maximal effective concentration (EC 50 ) is used to determine. The term "K D ” refers to the dissociation constant of a specific Ab-antigen interaction, which is determined by k off With k on The ratio (i.e., k off / k on ) and expressed as a molar concentration (e.g., nM). EC 50 Also expressed as molar concentration (eg, nM), it is the concentration of Ab that achieves half-maximal binding.

[0087] The binding specificity of the Abs of the invention to CCR8 was measured by fluorescence activated cell sorting (FACS) (see Example 4). The six mAbs tested showed binding to Raji cells expressing hCCR8, but not to parental Raji cells that do not express CCR8 (Example 4). FACS analysis identified four mAbs that bound to Raji-hCCR8 cells, of which EC 50 Less than 1 nM (see Table 1).

[0088] Thus, in certain embodiments, the mAbs or antigen binding portions thereof of the invention specifically bind to Raji cells expressing hCCR8, wherein EC 50 for:

[0089] (a) about 50 nM or less;

[0090] (b) from about 3 nM or less to about 0.5 nM or less;

[0091] (c) about 0.5 nM or less;

[0092] (d) about 0.1 nM or less;

[0093] (e) about 0.01 nM or less;

[0094] (f) about 0.005 nM or less;

[0095] (g) about 0.1 nM;

[0096] (h) about 0.005 nM to about 50 nM;

[0097] (i) about 0.02 nM to about 3 nM; or

[0098] (j) from about 0.08 nM to about 2 nM.

[0099] In certain preferred embodiments, EC 50 Measured by the binding assay described in Example 4.

[0100] When the binding affinity of an Ab to an antigen is expressed as a K of a specific value "or lower", D or EC 50 When used to express, it does not mean that K D or EC 50 There is no lower limit to the value, nor does it mean that the value is infinitely low. In fact, the K of an Ab that binds with very high affinity is D or EC 50 The value is not less than the picomolar range, i.e., about 0.001 nM. Therefore, it should be understood by those skilled in the art that EC 50 For example, about 0.5 nM or less refers to the EC of Ab binding to antigen. 50 is about 0.5 nM, or EC 50 Less than about 0.5 nM but not less than about 0.001 nM.

[0101] In certain other embodiments, the mAb of the invention, or antigen binding portion thereof, binds to hCCR8 polypeptide expressed on the surface of cells in formalin-fixed, paraffin-embedded (FFPE) tissue samples.

[0102] Competitive binding between mAbs that do not bind to the N-terminus of CCR8 and mAbs that bind to the N-terminus

[0103] Competition for binding to CCR8 on the surface of activated Tregs was determined by FACS between an anti-CCR8 mAb (4A19-mIgG2a) that binds to the N-terminus of CCR8 and mAbs that do not bind to the N-terminus (23A14-hIgG1, 21C17-mIgG2a, and 22B13-mIgG2a) (see Example 4). Binding of mAb 4A19-mIgG2a to CCR8 did not interfere with subsequent binding of any of 23A14-hIgG1, 21C17-mIgG2a, or 22B13-mIgG2a, and both types of mAbs tested at saturating amounts were able to detect CCR8. + Equal populations of Tregs.

[0104] Thus, the mAbs of the invention bind to one or more epitopes of hCCR8 that are different from the epitopes in the N-terminal domain of hCCR8 bound by the therapeutic anti-CCR8 mAbs described in WO 2021 / 194942 (e.g., mAb 4A19), and the presence of the Ab that binds to the N-terminal epitope does not interfere with the binding of the Ab to another epitope. mAbs of the invention that bind to hCCR8 epitopes outside the N-terminal region include 25T40, 21C17, 28P3, 22B13, 33H18, and 23A14.

[0105] Anti-CCR8 mAb that cross-competes with the reference Ab for binding to CCR8

[0106] Also within the scope of the disclosed invention is an isolated Ab, preferably a mAb or an antigen binding portion thereof, which specifically binds to hCCR8 expressed on the surface of a cell and cross-competes with a reference Ab or a reference antigen binding portion thereof for binding to hCCR8. The ability of a pair of Abs to "cross-compete" for binding to an antigen (e.g., CCR8) indicates that the first Ab binds to substantially the same epitope region of the antigen as the second Ab and sterically hinders the binding of the second Ab to the specific epitope region, and conversely, the second Ab binds to substantially the same epitope region of the antigen as the first Ab and sterically hinders the binding of the first Ab to the epitope region. Therefore, the ability of a test Ab to competitively inhibit, for example, the binding of mAb 25T40 or 21C17 to hCCR8 indicates that the test Ab binds to substantially the same epitope region of hCCR8 as mAb25T40 or 21C17.

[0107] If the first Ab reduces the binding of the second Ab to the antigen by at least about 40%, then the first Ab is considered to bind to "substantially the same epitope region" as the second Ab. 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 Ab and the second Ab can be reversed, i.e., the "second" Ab can be first bound to the surface and the "first" Ab is contacted with the surface in the presence of the "second" Ab. If a reduction in competitiveness for binding to the antigen is observed regardless of the order in which the antibodies are added to the immobilized antigen, then the Abs are considered to "cross-compete".

[0108] "Epitope region" refers to the spatial region around the epitope and the epitope. It is worth noting that the Abs that bind to the substantially identical "epitope region" of the antigen may not necessarily bind to the same epitope (although the Abs that bind to the same epitope can be identified by first screening the Abs that bind to the same epitope region, and then using techniques well known in the art to carry out epitope mapping (epitopemapping) on ​​these Abs, the techniques include array-based oligopeptide scanning, fixed-point scanning mutagenesis mapping (e.g., alanine scanning epitope mapping), high-throughput shotgun mutagenesis epitope mapping (high-throughput shotgun mutagenesis epitopemapping), hydrogen-deuterium exchange (HDX) and X-ray crystallography). For example, two cross-competing mAbs that bind to the substantially identical epitope region of the antigen can bind to adjacent or overlapping but not identical epitopes, and spatially hinder the binding of each other to their cognate epitopes (cognate epitope). Alternatively, the binding of mAb to an epitope can induce conformational changes in the antigen, which reduces the binding of another mAb to different epitopes in the substantially identical epitope region. However, cross-competitive Abs are generally expected to have very similar functional properties due to their 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 properties are expected to be. For example, if two cross-competitive Abs each inhibit the binding of the other to the epitope by at least about 80%, more if they each inhibit the binding of the other to the epitope by at least about 90%, and even more if they each inhibit the binding of the other to the epitope by about 100%, then they are expected to have substantially the same functional properties. If the binding of the two cross-competitive Abs to the epitope is determined by K D or EC 50 If cross-competing Abs are measured to exhibit similar binding affinity to the epitope, then the functional similarity is expected to be even closer.

[0109] Cross-competing anti-antigen Abs can be identified based on their performance in standard antigen binding assays including The ability of a test Ab to compete with mAb 21C17 for hCCR8 binding can be easily identified by the ability to detectably compete with recombinant antigen molecules or antigen molecules expressed on the cell surface in a quantitative assay (e.g., ELISA assay, or flow cytometry). For example, a simple competition assay to identify whether a test Ab competes with mAb 21C17 for binding to hCCR8 may include: (1) measuring the binding of 21C17 applied at saturating concentrations to mAb immobilized with hCCR8; The binding of 21C17 to hCCCR8-coated cells pre-bound with the test Ab is measured by: (1) measuring the binding of 21C17 to a cell-free hCCCR8-coated ... Chip (or other suitable medium). Compare the binding of 21C17 to the surface coated with hCCR8-1 in the presence and absence of the test Ab. The significant (e.g., greater than about 40%) reduction in 21C17 binding in the presence of the test Ab indicates that the two Abs recognize substantially the same epitope region, so that they compete for binding to the hCCR8 target. The percentage of the first Ab binding to the antigen 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. In order to determine whether the Abs cross-compete, the competitive binding assay is repeated, except that the binding of the test Ab to the hCCR8-coated chip is measured in the presence of pre-bound mAb 21C17.

[0110] Any anti-CCR8 antibody disclosed herein that binds to an epitope of hCCR8 outside the N-terminal domain can be used as a reference Ab in a cross-competition assay. Thus, for example, certain aspects of the present disclosure relate to an isolated Ab, preferably a mAb or an antigen-binding portion thereof, that cross-competes with a reference Ab for binding to hCCR8, wherein the reference Ab comprises:

[0111] (a)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 3; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 10;

[0112] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 4; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 11;

[0113] (c)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 5; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 12;

[0114] (d)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 6; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 13;

[0115] (e)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 7; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 14; or

[0116] (f)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 8; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO:15.

[0117] Anti-CCR8 mAb that binds to the same non-N-terminal binding CCR8 epitope as the reference Ab

[0118] Certain other aspects of the invention relate to an isolated Ab, preferably a mAb, or an antigen binding portion thereof, that binds to the same epitope as a reference Ab, wherein the reference Ab comprises:

[0119] (a)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 3; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 10;

[0120] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 4; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 11;

[0121] (c)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 5; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 12;

[0122] (d)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 6; and V L, which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 13;

[0123] (e)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 7; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 14; or

[0124] (f)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 8; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO:15.

[0125] Structurally defined mAbs of the invention

[0126] Certain other aspects of the invention relate to an isolated Ab, preferably a mAb, or an antigen binding portion thereof, that specifically binds hCCR8 expressed on the surface of a cell and comprises the CDR1, CDR2, and CDR3 domains of:

[0127] (a)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 3; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 10;

[0128] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 4; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 11;

[0129] (c)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 5; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 12;

[0130] (d)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 6; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 13;

[0131] (e)V H, which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 7; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 14; or

[0132] (f)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 8; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO:15.

[0133] Different methods have been developed to describe CDR domains within Abs. The method of Kabat and colleagues (Wu and Kabat, 1970; Kabat et al., 1983) was based on the assumption that the CDRs comprise the most variable positions in the Ab and could therefore be identified by aligning the rather limited number of Ab sequences available at the time. Based on this alignment, Kabat et al. introduced a numbering scheme for residues in the hypervariable regions and determined which positions marked the start and end of each CDR (http: / / bioinf.org.uk / abs / simkab.html).

[0134] In addition to the widely used Kabat definition, other definitions, including 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 MGT (Lefranc et al., 2003; http: / / www.imgt.org) definitions, have been used in an attempt to address the deficiencies of the Kabat definition. The Kabat definition remains the most commonly used method for predicting CDR domains, although it was developed when no structural information about Abs was available.

[0135] In the absence of explicit instructions, unless the context indicates otherwise, the CDRs disclosed herein have been identified using the Kabat definition. The amino acid sequences of the six CDR domains defined using the Kabat method, as well as the V domains of mAb 25T40, 21C17, 28P3, 22B13, 33H18, and 23A14 are shown in Table 3. H 、V L , amino acid sequences of the heavy and light chains.

[0136] In certain other embodiments, an Ab, preferably a mAb, or antigen binding portion thereof, of the invention comprises the following CDR domains as defined by the Kabat method:

[0137] (a) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 17; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 18; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 19; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 20; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 21; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 22;

[0138] (b) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 23; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 24; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 25; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 26; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 27; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 28;

[0139] (c) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 29; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 30; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 31; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 32; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 33; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 34;

[0140] (d) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 35; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 36; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 37; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 38; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 39; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 40;

[0141] (e) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:41; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:42; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:43; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:44; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:45; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:46; or

[0142] (f) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:47; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:48; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:49; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:50; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:51; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:22.

[0143] In other embodiments, the Ab, preferably a mAb, or an antigen binding portion thereof, comprises:

[0144] (a)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 3; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 10;

[0145] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 4; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 11;

[0146] (c)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 5; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 12;

[0147] (d)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 6; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 13;

[0148] (e)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 7; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 14; or

[0149] (f)V H, which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 8; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO:15.

[0150] Contains V H and V L The anti-CCR8 Ab of V H and V L The region has an amino acid sequence that is highly similar or homologous to the amino acid sequence of any of the above-mentioned anti-CCR8 Abs and retains the functional properties of these Abs. For example, suitable Abs include those comprising V H and / or V L mAb in each V H and / or V L The region comprises consecutively linked amino acids, each of which has a sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID No. 4 and / or 11. In other embodiments, for example, V H and / or V L The amino acid sequence exhibits at least about 85%, at least about 90%, at least about 95% or at least about 99% identity to the sequence shown in 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 shared by the sequences relative to the length of the compared sequences (i.e., % identity = number of identical positions / total number of compared positions × 100), taking into account the number of any gaps and the length of each such gap, introduced to maximize the degree of sequence identity between the two sequences. The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using a mathematical algorithm well known to those of ordinary skill in the art.

[0151] In the case of comprising V sequences that exhibit high sequence identity (e.g., at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the Ab structurally defined herein), H and / or V L In certain embodiments of an Ab (preferably a mAb) having an amino acid sequence, compared to the CDR sequence of a defined antibody, in V H and V L There are no more than 3 amino acid modifications in each of the CDR domains. In certain preferred embodiments, in V H and V L In each of the CDR domains there are no more than 2 amino acid modifications. In certain more preferred embodiments, in V H and V LIn each of the CDR domains there is no more than one amino acid modification. In certain even more preferred embodiments, in V H and V L There are no amino acid modifications in each of the CDR domains.

[0152] In the preferred embodiment of the Ab that comprises one or more amino acid modifications in the CDR, these modifications are "conservative" amino acid modifications. As used herein, "conservative" amino acid modifications refer to amino acid modifications that do not significantly affect or change the binding properties of the Ab containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Conservative amino acid substitutions are substitutions in which the amino acid residues are substituted by amino acid residues with similar side chains. The amino acid residue family with similar side chains has been defined in the art, including 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in conservative amino acid substitutions, one or more amino acid residues within the CDR region of an Ab are substituted with another amino acid residue from the same side chain family, and the altered Ab is tested using assays well known in the art to confirm whether the Ab function, such as binding specificity and affinity, is substantially the same as that of the unmodified Ab.

[0153] In certain other embodiments, the Ab, preferably a mAb, or an antigen binding portion thereof, comprises:

[0154] (a) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 59; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 66;

[0155] (b) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 60; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 67;

[0156] (c) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 61; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 68;

[0157] (d) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 62; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 69;

[0158] (e) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 63; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 70; or

[0159] (f) a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:64; and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:71.

[0160] In certain preferred embodiments, the mAb, or antigen binding portion thereof, is an Ab designated herein as 25T40, 21C17, 28P3, 22B13, 33H18, or 23A14, or an antigen binding portion thereof.

[0161] In certain embodiments, the isolated anti-CCR8 Ab of the present invention, preferably mAb or its antigen binding portion is a human Ab or a fragment thereof. In other embodiments, it is a humanized Ab or a fragment thereof. In other embodiments, it is a chimeric Ab or a fragment thereof. In other embodiments, the isolated anti-CCR8 Ab or its antigen binding portion is a mouse Ab or a fragment thereof. For use in human subjects, Ab is preferably a chimeric Ab, or more preferably a humanized or human Ab. Such chimeric mAb, humanized mAb, human mAb or mouse mAb can be prepared and isolated by methods known in the art.

[0162] In certain other embodiments, the Ab, preferably a mAb, or an antigen binding portion thereof, comprises a heavy chain constant region of a human IgG1, IgG2, IgG3, or IgG4 isotype. In other embodiments, the Ab, or an antigen binding portion thereof, comprises a heavy chain constant region of a human IgG2 or IgG4 isotype.

[0163] MAbs labeled with detectable tags

[0164] MAbs can be labeled with various substances or tags, including small molecules, enzymes, radioisotopes, and fluorescent dyes, to aid 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 (ELISA) and immunohistochemistry (IHC). These markers, as well as fluorescent markers, are used in Western blotting, while fluorescent markers are used in flow cytometry and immunofluorescence (IF) staining. Radiolabeled mAbs are used in positron emission tomography (PET) (Aluicio-Sarduy et al., 2018), and rare earth metal isotopes are used in mass cytometry.

[0165] The present invention provides a labeled Ab, preferably mAb or its antigen binding, which comprises any of the above Ab of the present invention or its antigen binding portion and 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 other embodiments, the fluorophore is BRILLIANT VIOLET TM Dyes (e.g. BV-421), AmCyan dyes, ALEXA dye, dye, In some embodiments, the enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase or β-galactosidase. In other embodiments, the chromophore is porphyrin, pyropheophorbide-α, benzoporphyrin monoacid ring A (BPDMA) or chlorin e6.

[0166] Radiolabeling mAbs with positron emitters for PET imaging (immunoPET) can provide valuable information about the in vivo biodistribution of these molecules and their related therapeutics (Aluicio-Sarduy et al., 2018). For each PET application, the selection of the optimal radioisotope is critical. The half-life of the radionuclide must first be matched to the pharmacokinetic profile of the mAb in vivo. This ensures that the time course of the radioactivity matches that of the mAb. Typically, accumulation of mAbs in tumors tends to peak several days after injection due to their extended half-life in the bloodstream, which necessitates the use of isotopes with long half-lives (e.g., 89 Zr, 64 Cu and86 Y), rather than more traditional options (e.g., 11 C. 18 F or 68 Ga). In cases where conventional isotopes are unsuitable for a desired application, other radionuclides that offer more suitable chemical or decay properties have been 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). 89 Zr is used more extensively in clinical trials than any other PET-radiometal. Its 78-hour half-life matches the typical pharmacokinetic time scale of mAbs, making it 89 Zr is very suitable for centralized production and domestic and international transportation. 64 Cu represents another convenient alternative for Ab and protein labeling because it has an intermediate half-life of 12.7 h and negligible contaminating gamma-ray emission. 86 Yttrium is another isotope with good properties for use in immunoPET, but it is a significant gamma-ray emitter, limiting the dose that can be injected into a subject.

[0167] In certain preferred embodiments of labeling mAbs for PET, the radioisotope is 89 Zr, 64 Cu or 86 In certain other embodiments, the radioisotope is 11 C. 18 F. 68 Ga, 52 Mn, 55 Co. 152 Tb, 90 Nb, 66 Ga, 72 As, 69 Ge or I 125 .

[0168] Mass cytometry, also known as time-of-flight cytometry (CyTOF), is a new generation of flow cytometry platforms that utilizes elemental mass spectrometry to detect rare metal isotopes conjugated to Abs that bind to target antigens on single cells either intracellularly or extracellularly (Spitzer and Nolan, 2016). In this technique, cells are stained with Abs conjugated to metal isotope reporters with different masses. The fixed, stained cells are then separated into single-cell droplets, atomized, and analyzed by mass spectrometry. The number of cell parameters that can be monitored simultaneously by conventional fluorescence flow cytometry experiments is essentially limited by the overlap of fluorophore emission spectra, but mass cytometry accurately distinguishes metal isotopes of different atomic masses without channel overlap. This allows for the simultaneous quantification of more than 40 protein parameters in each single cell, far exceeding the approximately 20 cell features that can be analyzed simultaneously by fluorescence flow cytometry. Compared to flow cytometry, mass spectrometry provides similar quantification of cell lineages along with markers of cell differentiation, function, activation, and exhaustion for fresh and cryopreservable PBMCs or tumor tissues (Gadalla et al., 2019). However, the throughput (~1,000 cells / s) and sensitivity of mass cytometry are still approximately 10-fold lower than conventional FACS, and cells analyzed by mass cytometry cannot be recovered for downstream analysis because the cells completely "evaporate" during the assay (Spitzer and Nolan, 2016).

[0169] In certain embodiments of Ab labeled with a metal isotope, the metal is yttrium (Y), indium (In), lanthanide (Ln, from La to Lu, excluding Pm), iodine (i), cadmium (Cd), tellurium (Te), silver (Ag), palladium (Pd), rhodium (Rh), iridium (Ir), platinum (Pt), ruthenium (Ru), osmium (Os) or bismuth (Bi).

[0170] Anti-CCR8 immunoconjugate

[0171] On the other hand, the present invention relates to any one of the isolated anti-hCAR8 Ab of the present invention disclosed herein or an antigen binding portion thereof, which is connected to a cytolytic agent such as a cytotoxin, a radioisotope or a photosensitizer (PS). Such conjugates are referred to herein as "immunoconjugates". Cytotoxins can be conjugated to the Ab of the present invention using linker technology available in the art. Methods for preparing radioimmunoconjugates have also been established in the art.

[0172] Photodynamic therapy (PDT) is a non-invasive treatment that involves accumulation of PS in solid tumors, followed by local delivery of light of the correct wavelength to activate the PS, which in the presence of oxygen leads to the in situ generation of reactive oxygen species (ROS), which cause damage to cellular components and ultimately lead to necrosis or apoptosis. PDT is a promising tool in oncology, but is often limited by side effects caused by insufficient targeting of the photosensitizer. This problem can often be circumvented by conjugation of PS to tumor-specific mAbs. The use of antigen-binding Ab fragments (e.g., Fab or scFv fragments) can be advantageous because, while maintaining the same binding specificity, they penetrate tumor masses more effectively due to their smaller size and are more effectively cleared from the circulation due to the lack of an Fc domain.

[0173] Porphyrins are ubiquitous in the fields of photodynamic therapy and photodiagnosis and are one of the most prominent classes of photosensitizers in these fields of biomedical science (Sandland and Boyle, 2019). In certain embodiments of the photosensitizer-Ab conjugate, the photosensitizer is a tetrapyrrolic macrocycle. In other embodiments, the tetrapyrrolic macrocycle is a porphyrin, a chlorin, a bacteriochlorin, or a phthalocyanine.

[0174] Anti-CCR8 chimeric antigen receptor (CAR) and T cell receptor (TCR)

[0175] On the other hand, the present invention relates to a chimeric antigen receptor (CAR), which comprises any one of the isolated anti-hCCR8 antibodies of the present invention disclosed herein or an antigen binding portion thereof, which specifically binds to an hCCR8 epitope outside the N-terminal domain of hCCR8. In certain embodiments, CAR also comprises a transmembrane domain. In certain other embodiments, CAR also comprises an intracellular signaling domain. In other embodiments, CAR also comprises a hinge region and / or a spacer region.

[0176] 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 hCCR8 epitope 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.

[0177] Bispecific molecules

[0178] In another aspect, the invention relates to a bispecific molecule comprising any of the anti-hCCR8 mAbs of the invention disclosed herein, or an antigen binding portion thereof, linked to a binding domain having a different binding specificity than the anti-hCCR8 mAb or antigen binding portion thereof. The binding domain can be a functional molecule, e.g., another Ab, an antigen binding portion of an Ab, or a ligand of a receptor, such that the resulting bispecific molecule binds to at least two different binding sites or target molecules.

[0179] Pharmaceutical composition

[0180] Ab disclosed herein and used for any diagnosis, patient selection and treatment methods described can constitute a composition, such as a pharmaceutical composition comprising any Ab of the present invention and a pharmaceutically acceptable carrier. The present disclosure also provides a composition, such as a pharmaceutical composition, which includes any disclosed immunoconjugate, bispecific molecule, CAR and TCR, and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents that are physiologically compatible. Preferably, the carrier of the composition containing Ab is suitable for intravenous (IV), intramuscular, subcutaneous (SC), parenteral, spinal or epidermal administration (e.g., by injection or infusion). The pharmaceutical composition may include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers and / or adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants.

[0181] The choice of SC injection is based on Halozyme Therapeutics' Drug delivery technology involving co-formulation of Ab with recombinant human hyaluronidase (rHuPH20) eliminates traditional limitations on the volume of biologics and drugs that can be delivered subcutaneously due to the extracellular matrix (U.S. Pat. No. 7,767,429). Two Abs for combination therapy can be co-formulated into a single composition for SC administration.

[0182] Nucleic acids encoding anti-hCCR8 MAbs and use thereof for expressing Abs

[0183] Another aspect of the invention relates to a nucleic acid encoding any isolated anti-hCCR8 antibody of the invention (nucleic acid of the invention). The present disclosure provides an isolated nucleic acid encoding any anti-CCR8 mAb of the invention described herein or an antigen binding portion thereof, any bispecific Ab disclosed herein, a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0184] "Isolated" nucleic acid refers primarily to a nucleic acid composition of a substance that is significantly different from nucleic acids found in nature (i.e., has unique chemical properties, properties, and uses). For example, unlike natural DNA, isolated DNA is an independent part of natural DNA, rather than a component of a larger structural complex (chromosome) found in nature. In addition, unlike natural DNA, isolated DNA can be used as a PCR primer or hybridization probe to measure gene expression and detect biomarker genes or mutations to diagnose diseases or predict the efficacy of therapeutic agents, etc. In a narrower sense, when the context indicates, the term "isolated" can also be used to describe nucleic acids that are different from nucleic acids found in nature, which means that they are purified using standard techniques well known in the art to be substantially free of other cellular components or other contaminants, such as other cellular nucleic acids or proteins.

[0185] The nucleic acids of the present invention can be obtained using standard molecular biology techniques. For Abs expressed by hybridomas (e.g., hybridomas prepared from wild-type or transgenic mice carrying human Ig genes as described in Example 1), cDNAs encoding the light and heavy chains or variable regions of the Abs prepared by the hybridomas can be obtained by standard PCR amplification techniques. Once the V H and V L The DNA fragments of the fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region DNA into full-length Ab chain genes, Fab fragment genes or scFv genes. For the Ab obtained by an Ig gene library (e.g., using phage display technology), the nucleic acid encoding the Ab can be recovered from the library.

[0186] The nucleic acid of the present invention can be, for example, RNA or DNA, such as cDNA or genomic DNA. In a preferred embodiment, the nucleic acid is cDNA.

[0187] The present disclosure also provides an expression vector comprising an isolated nucleic acid encoding an anti-CCR8 mAb disclosed herein or its antigen binding portion thereof, any bispecific Ab disclosed herein, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). The present disclosure also provides a host cell comprising the expression vector or any CAR or TCR disclosed herein. Eukaryotic cells, most preferably mammalian host cells, are preferably used as host cells for expressing Ab, because such eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete correctly folded immunoactive Abs than prokaryotic cells. Preferred mammalian host cells for expressing the recombinant Ab 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 other embodiments, the host cell is a T cell or a NK cell.

[0188] The 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 its antigen binding portion, a bispecific Ab, a CAR or a TCR in a host cell and isolating the mAb or its antigen binding portion, a bispecific Ab, a CAR or a TCR from the host cell. The host cell can be used in vitro or in vivo. The DNA encoding the heavy and light chains of the Ab can be inserted into different expression vectors, or more typically, both are inserted into the same vector. The V of the Ab H and V L The segments can be used to generate full-length Abs of any isotype by inserting DNA encoding these variable regions into expression vectors that already encode the heavy and light chain constant regions of the desired isotype, such that V H C in the segment and vector H segments are effectively connected, and V κ C in the segment and vector L Anti-CCR8 antibodies suitable for the disclosed treatment methods

[0189] Disclosed herein are therapeutic methods comprising screening or diagnostic steps using the anti-CCR8 Ab of the present invention. Anti-CCR8 Abs suitable for use in these methods are isolated Abs, preferably mAbs or antigen binding portions thereof, which specifically bind to epitopes located outside the N-terminal domain of hCCR8 expressed on the cell surface. Such Abs exhibit one or more properties important for the screening or diagnostic applications described herein. Specifically, the isolated Abs or antigen binding portions thereof:

[0190] (a) binding to an epitope outside the N-terminal domain of hCCR8, optionally binding to a region comprising a region having sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 The epitope of the peptide of (SEQ ID NO:2) is different from the epitope;

[0191] (b) binding to an epitope outside the N-terminal domain of hCCR8, optionally binding to a region comprising a region having sequence V 12 T 13 D14Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (SEQ ID NO: 73); or

[0192] (c) binding to an epitope outside the N-terminal domain of hCCR8, optionally binding to a peptide having sequence V 12 T 13 D 14 Y15Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 The epitope composed of the peptide of (SEQ ID NO: 73) is different from the epitope.

[0193] In further embodiments, the isolated Ab or antigen binding portion thereof preferably exhibits at least one, at least two, or at least three of the following properties:

[0194] (d) Specific binding to hCCR8 expressed on the cell surface, EC 50 is about 3 nM or lower, such as EC 50 From about 0.01 to about 3 nM; preferably EC 50 is about 0.1 nM or lower, such as EC 50 From about 0.001 to about 0.1 nM;

[0195] (e) binding to hCCR8 polypeptide expressed on the cell surface in formalin-fixed, paraffin-embedded (FFPE) tissue samples;

[0196] (f) its binding to hCCR8 is not affected by the presence of an Ab or antigen-binding portion thereof that binds to the N-terminal epitope (e.g., mAb4A19 (WO 2021 / 194942)); and

[0197] (g) Its binding to hCCR8 does not affect the binding of Ab or its antigen-binding portion (e.g., mAb 4A19 (WO 2021 / 194942)) to the N-terminal epitope.

[0198] In a preferred embodiment, the isolated Ab or antigen-binding portion thereof exhibits at least 6 of the aforementioned properties (a) to (g), for example, exhibits properties ((a) to (d), (f), and (g).

[0199] Diagnostic, theranostic, and other methods using anti-CCR8 MAbs

[0200] PCT Publication No. WO 2021 / 194942 describes humanized and human mAbs that specifically bind to hCCR8 expressed on the surface of tumor-infiltrating Tregs expressing CCR8 with high affinity, and mediate the depletion of these Tregs through mechanisms including ADCC. The application also describes a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an anti-CCR8 mAb that results in the depletion of immunosuppressive tumor-infiltrating Tregs, thereby enhancing the subject's immune response to effectively treat cancer. At least one therapeutic Treg-depleting mAb, mAb 4A19, binds to an epitope in the N-terminal domain of hCCR8, including residues 12-22 of hCCR8 (SEQ ID NO: 1) and including sulfated tyrosine-15 and tyrosine-17 residues.

[0201] The mAbs of the invention specifically bind to hCCR8, but bind to a different epitope outside the N-terminal domain, and therefore different from the N-terminal epitope bound by the therapeutic anti-CCR8 mAbs disclosed in WO 2021 / 194942. These mAbs of the invention do not interfere with the binding of therapeutic Abs that bind to the N-terminal domain when binding to hCCR8; similarly, the binding of therapeutic N-terminally bound mAbs does not interfere with the binding of mAbs of the invention. Therefore, the mAbs of the invention can be used in a variety of different methods, which can even be performed in the presence of bound therapeutic mAbs, including methods for measuring CCR8 in the blood or tumor environment of a subject. + The frequency of cells such as Treg; measuring CCR8 in the subject's blood or tumor environment + Depletion of the number of cells such as Tregs; measuring receptor occupancy (RO), i.e., the proportion of hCCR8 receptors bound by an anti-CCR8 therapeutic antibody, in a subject treated with a therapeutic antibody; predicting the effectiveness of a therapeutic anti-CCR8 Ab in treating cancer in a subject; selecting a subject with cancer as a suitable candidate for immunotherapy with a therapeutic Treg-depleting anti-CCR8 Ab; and a method of treatment comprising determining the expression level of CCR8 in a test tissue of or taken from a subject prior to administration of a therapeutic Treg-depleting anti-CCR8 mAb.

[0202] Example 6 describes a method for detecting and quantifying CCR8 expression on peripheral Tregs. To detect and quantify CCR8 in tumor tissues, single staining immunohistochemistry (IHC) was used (see Example 7).

[0203] As described in WO 2021 / 194942, CCR8 + Cells are primarily Tregs. Advantageously, CCR8+ Anti-CCR8 mAbs of cell frequency do not compete with therapeutic anti-CCR8 mAbs for binding to CCR8. In certain embodiments, therapeutic anti-CCR8 mAbs bind to an epitope in the N-terminal domain of hCCR8. In other embodiments, therapeutic anti-CCR8 mAbs are one of mAbs 4A19, 18Y12, 8D55, 10R3, 14S15, and 14S15h described in WO 2021 / 194942. In certain preferred embodiments, therapeutic anti-CCR8 mAbs are mAb4A19, which comprises 6 CDRs (SEQ ID NO.53-58 herein), heavy and / or light chain variable regions (SEQ ID NO:9 and / or 16 herein) and / or heavy and / or light chains (SEQ ID NO:65 and / or 72 herein) of mAb 4A19.

[0204] In certain embodiments of these methods including the use of therapeutic anti-CCR8 mAbs that bind to an epitope in the N-terminal domain of hCCR8, a non-competitive mAb is an Ab that binds to an epitope outside the N-terminal domain and does not compete with a mAb that binds to an epitope in the N-terminal domain for binding to hCCR8. Examples include the Abs of the invention disclosed herein, i.e., one of the mAbs designated herein as 25T40, 21C17, 28P3, 22B13, 33H18, or 23A14, or an antigen binding portion thereof. In certain preferred embodiments, the non-competitive mAb is mAb 21C17 or an antigen binding portion thereof, comprising the 6 CDRs (SEQ ID NO. 23-28) of mAb 21C17, or heavy and / or light chain variable regions (SEQ ID NO: 4 and / or 11), or heavy and / or light chains (SEQ ID NO: 60 and / or 67).

[0205] In certain other embodiments, the therapeutic anti-CCR8 mAb binds to an epitope outside the N-terminal domain of hCCR8. For example, in other embodiments, the therapeutic anti-CCR8 mAb is mAb VHH-01 disclosed in WO 2022 / 003156, or mAb VHH-65, VHH-74, VHH-62, and VHH-56 disclosed in WO 2022 / 136647.

[0206] In certain embodiments of the method comprising the use of a therapeutic anti-CCR8 mAb that binds to an epitope outside the N-terminal domain of hCCR8, a non-competing mAb is an Ab that binds to an epitope in the N-terminal domain and does not compete with a mAb that binds to an epitope outside the N-terminal domain for binding to hCCR8. Examples of such Abs that bind to an epitope in the N-terminal domain include clone L263G8 (Biolegend) and mAb 433H disclosed in WO 2007 / 044756 (and sold by BD Biosciences, https: / / www.bdbiosciences.com / content / bdb / paths / generate-tds-document.us.566899.pdf).

[0207] 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 previously exposed to various concentrations of the therapeutic mAb; (b) incubating aliquots of free (i.e., blood samples from indirect / total test tubes), bound (i.e., blood samples from direct test tubes), or fluorescence minus one [FMO] (i.e., a control tube of blood samples stained with all Abs except anti-Id Ab for direct RO format) with a buffer that does not contain the therapeutic mAb; (c) staining the samples with a core Ab panel to identify basal T cell markers, T cell differentiation markers, and Treg markers; (d) adding to the core mixture panel (i) for direct / total RO assays, an anti-idiotypic Ab to detect bound CCR8 receptors, or (ii) for indirect / total assays, two additional Abs, comprising an allophycocyanin (APC)-conjugated anti-hCCR8 Ab that competes with the therapeutic Ab to detect free CCR8 receptors; and an anti-hCCR8 Ab that does not compete with the therapeutic Ab. Ab to detect total CCR8 receptors; (e) lyse red blood cells (to clean the sample while leaving target cells intact) and analyze the cleaned samples by flow cytometry; and (f) determine the %RO for each concentration of therapeutic Ab using the following formula:

[0208] %RO=100×[1-((free amount after administration / free amount before administration) / (total amount after administration / total amount before administration))].

[0209] In certain embodiments of the method, therapeutic Ab specifically binds to an epitope in the N-terminal domain of hCCR8. In certain other embodiments, therapeutic Ab is one of Abs that specifically bind to an epitope in the N-terminal domain of hCCR8 described in WO 2021 / 194942. In other embodiments, therapeutic Ab comprises 6 CDRs (SEQ ID NO.53-58 herein) or heavy and / or light chain variable regions (SEQ ID NO herein: 9 and / or 16) of mAb 4A19 described in WO 2021 / 194942. In certain preferred embodiments, therapeutic Ab is mAb 4A19 comprising a heavy chain and / or light chain (SEQ ID NO: 65 and / or 72) of mAb 4A19.

[0210] In certain embodiments, when a therapeutic Ab is administered to a subject, such as during a clinical trial or during treatment with an anti-CCR8 mAb, exposure of a whole blood sample to various doses of the therapeutic mAb occurs. In other embodiments, whole blood samples are exposed to different concentrations of the therapeutic Ab in vitro to simulate different drug doses in patients.

[0211] In certain embodiments, the basic T cell markers for the core marker panel include anti-human CD3, anti-human CD4 and / or anti-human CD8 Abs conjugated to UV-excitable fluorescent dyes. In certain other embodiments, T cell differentiation markers include fluorescently labeled anti-human CD45RA Abs. In certain other embodiments, Treg markers include fluorescently labeled anti-human CD25, anti-human CD127 and / or anti-human CCR4 Abs. In other embodiments, the competitive anti-hCCR8 Ab added to the group for indirect / total determination is a fluorescently labeled mAb 433H (BD Biosciences) for detecting free CCR8 receptors, and the non-competitive Ab for detecting total CCR8 receptors is a fluorescently labeled mAb 21C17.

[0212] As described in Example 6, CCR8 engagement measured in direct and indirect assay formats using the RO assay showed that hCCR8 targets expressed on the cell surface were specifically engaged by the therapeutic mAb 4A19. This engagement of the receptor with the therapeutic Ab is consistent with the potential mechanism of action expected for anti-CCR8 immunotherapy, which requires the anti-CCR8 Ab to bind to CCR8 on Tregs and mediate the depletion of these immunosuppressive Tregs through processes including ADCC, ADCP and / or CDC (see Example 8). The RO assay is also quantitative and further demonstrates that CCR8 +The %RO on Tregs correlates with increasing doses of therapeutic Ab. These %RO data can guide the selection of appropriate dosing regimens, i.e., dosing amount and frequency, to optimize efficacy and minimize potential safety issues by avoiding doses that exceed those required for effective receptor occupancy.

[0213] 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 receiving anti-CCR8 mAb treatment, the method comprising: (a) determining a baseline level of the frequency of CCR8-expressing cells in a first sample of whole blood or PBMC from the subject; (b) administering treatment with an anti-CCR8 mAb to the subject; and (c) determining the frequency of CCR8-expressing cells in a second sample of whole blood or PBMC taken from the subject after administration of the anti-CCR8 mAb; wherein a decrease in the frequency of CCR8-expressing cells in the second sample indicates that the number of CCR8-expressing cells in the blood has been depleted. The method allows monitoring the depletion of CCR8 in the peripheral blood of a subject treated with an anti-CCR8 mAb. + Depletion of Tregs. Since anti-CCR8 Ab immunotherapy is premised on the depletion of immunosuppressive Tregs expressing CCR8 by Ab, measuring Treg depletion mediated by administration of anti-CCR8 therapeutic Abs may allow for the evaluation of the efficacy of treatment with anti-CCR8 therapeutic Abs even before clinical signs of efficacy are apparent. + Tregs make up a very small percentage of PBMCs, approximately 0.5-2%, so the availability of anti-CCR8 Abs that do not compete with therapeutic anti-CCR8 Abs for binding to CCR8 (e.g., anti-CCR8 mAbs of the present invention that do not compete with therapeutic Abs for binding to the N-terminal domain of hCCR8) is essential for detecting and quantifying these low levels of CCR8. + The cells are very important because they do not compete for binding to the N-terminal epitope to which the therapeutic mAb already binds.

[0214] The present disclosure also provides a method for measuring the number of tumor-infiltrating CCR8-expressing Tregs depleted in a subject receiving anti-CCR8 mAb treatment, the method comprising: (a) determining a baseline level of the frequency of CCR8-expressing Tregs in a first sample of a test tissue in or from a subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering an anti-CCR8 mAb treatment to the subject; and (c) determining the frequency of CCR8-expressing Tregs in a second sample of a test tissue in or from a subject during or after treatment; wherein a decrease in the frequency of CCR8-expressing Tregs in the second sample of the test tissue indicates that the number of Tregs in the test tissue has been depleted. The method provides a means of monitoring the depletion of tumor-infiltrating Tregs in a subject's tumor environment. Since the intended mechanism of action of anti-CCR8 immunotherapy is that effective anti-CCR8 Abs lead to the depletion of immunosuppressive Tregs expressing CCR8, measuring Treg depletion in the tumor environment mediated by administration of anti-CCR8 therapeutic Abs allows for the assessment of the possible effectiveness of treatment with anti-CCR8 therapeutic Abs even before clinical signs of efficacy are apparent.

[0215] In certain embodiments of the method, depletion of the number of tumor-infiltrating Tregs in the test tissue indicates an enhancement of the immune response in the subject, and / or the efficacy of treating a disease that can be treated by an enhanced immune response. In certain embodiments, the treatment administered to the subject is a treatment of cancer. In other embodiments, the treatment of cancer comprises administering a therapeutic anti-CCR8 Ab or an antigen-binding portion thereof.

[0216] In certain other embodiments, the anti-CCR8 antibody therapy administered to the subject is a treatment of an infectious disease.

[0217] CCR8 is expressed on 60-80% of all skin T cells, not just on Tregs. Therefore, the method of the present invention can also be used to measure the depletion of the number of skin T cells in a subject receiving treatment, the method comprising: (a) determining a baseline level of the frequency of skin T cells expressing CCR8 in a first sample of a test tissue of the subject or taken from the subject, the test tissue comprising skin T cells; (b) administering treatment with an anti-CCR8 mAb to the subject; and (c) determining the frequency of skin T cells expressing CCR8 in a second sample of a test tissue of the subject or taken from the subject during or after treatment; wherein a decrease in the frequency of skin T cells expressing CCR8 in the second test tissue indicates that the number of skin T cells in the test tissue has been depleted.

[0218] Since Treg is immunosuppressive, it is expected that subjects expressing high levels of Treg (i.e., T cells expressing CCR8 at high levels) in the tumor environment will benefit most from treatment with Treg-depleting Abs, and will therefore be suitable candidates for immunotherapy with therapeutic Treg-depleting Abs. Therefore, the present disclosure also provides a method for predicting the effectiveness of therapeutic Treg-depleting Abs or their antigen-binding portions in treating cancer in a subject, the method comprising: (a) determining the frequency of CCR8-expressing Tregs in a test tissue of a subject or taken from a subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) comparing the frequency of CCR8-expressing Tregs with a predetermined threshold; and (c) predicting the effectiveness of therapeutic Treg-depleting Abs or their antigen-binding portions, wherein the frequency of CCR8-expressing Tregs is higher than the threshold indicating that the therapeutic Ab or its antigen-binding portion will effectively treat the subject, and wherein the frequency of CCR8-expressing Tregs is lower than the threshold indicating that the therapeutic Ab or its antigen-binding portion will not be able to effectively treat 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 other embodiments, the Treg-depleting Ab is an anti-CCR4 or anti-CD25 Ab.

[0219] The present disclosure also provides a method for predicting the effectiveness of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof in treating a subject's cancer, the method comprising: (a) determining the frequency of CCR8-expressing Tregs in a test tissue obtained from the subject or from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (b) administering a therapeutic Treg-depleting Ab or an antigen-binding portion thereof to the subject; (c) determining whether the frequency of CCR8-expressing Tregs decreases after administering the therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (d) if the frequency of CCR8-expressing Tregs decreases by more than a predetermined threshold, predicting that the therapeutic Treg-depleting Ab or an antigen-binding portion thereof will effectively treat the subject's cancer, or (e) if the frequency of CCR8-expressing Tregs decreases by less than a predetermined threshold, predicting that the therapeutic Treg-depleting Ab or an antigen-binding portion thereof will not effectively treat the subject's cancer. 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 other embodiments, the Treg-depleting Ab is an anti-CCR4 or anti-CD25 Ab.

[0220] The present disclosure also provides a method for selecting a subject with cancer as a suitable candidate for cancer immunotherapy with therapeutic Treg depletion Ab or its antigen binding portion, the method comprising: (a) determining the frequency of CCR8 expression Treg in a test tissue of the subject or taken from the subject, the test tissue comprising tumor cells and tumor infiltrating Treg; (b) comparing the frequency of CCR8 expression Treg with a predetermined threshold; and (c) based on the assessment that the frequency of CCR8 expression Treg in the test tissue exceeds the predetermined threshold, selecting the subject as a suitable candidate for immunotherapy with therapeutic Treg depletion Ab or its antigen binding portion. In certain preferred embodiments of the method, Treg depletion Ab is anti-CCR8 Ab. In certain other embodiments, Treg depletion Ab is anti-CTLA-4Ab. In other embodiments, Treg depletion Ab is anti-CCR4 or anti-CD25 Ab.

[0221] The present disclosure also provides a method for selecting a subject with cancer as a suitable candidate for cancer immunotherapy with therapeutic Treg depletion Ab or its antigen binding portion, the method comprising: (a) determining the frequency of CCR8 expressing Treg in a test tissue taken from a subject or from a subject, the test tissue comprising tumor cells and tumor infiltrating Treg; (b) administering a therapeutic Treg depletion Ab or its antigen binding portion to the subject; (c) determining whether the frequency of CCR8 expressing Treg is reduced after administering the Ab or its antigen binding portion of therapeutic depletion Treg; and (d) based on the assessment that the frequency of CCR8 expressing Treg is reduced by more than a predetermined threshold, selecting the subject as a suitable candidate for immunotherapy with therapeutic Treg depletion Ab or its antigen binding portion. In certain preferred embodiments of the method, Treg depletion Ab is anti-CCR8 Ab. In certain other embodiments, Treg depletion Ab is anti-CTLA-4 Ab. In other embodiments, Treg depletion Ab is anti-CCR4 or anti-CD25 Ab.

[0222] The present disclosure provides a method for treating cancer in a subject, the method comprising: (a) selecting a subject who is a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, the selection comprising: (i) determining the frequency of CCR8-expressing Tregs in a test tissue of the subject or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) comparing the frequency of CCR8-expressing Tregs with a predetermined threshold; and (iii) based on an assessment that the frequency of CCR8-expressing Tregs in the test tissue exceeds a predetermined threshold, selecting the subject as a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (b) administering a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof to the selected subject. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab.

[0223] The present disclosure also provides a method for treating cancer in a subject, the method comprising: (a) selecting a subject who is a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, the selection comprising: (i) determining the frequency of CCR8-expressing Tregs in a test tissue of the subject or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) administering a therapeutic Treg-depleting Ab or an antigen-binding portion thereof to the subject; (iii) determining whether the frequency of CCR8-expressing Tregs is reduced after administering the therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (iv) selecting the subject as a suitable candidate for immunotherapy with the therapeutic agent based on an assessment that the reduction in the frequency of CCR8-expressing Tregs exceeds a predetermined threshold; and (b) administering a composition comprising a therapeutically effective amount of a therapeutic Treg-depleting Ab or an antigen-binding portion thereof to the selected subject. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab.

[0224] The present disclosure also provides a method for treating cancer in a subject, the method comprising (a) selecting a subject who is not a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, the selection comprising: (i) determining the frequency of CCR8-expressing Tregs in the subject or in a test tissue from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) comparing the frequency of CCR8-expressing Tregs with a predetermined threshold; and (iii) selecting a subject who is not suitable for immunotherapy with the therapeutic Treg-depleting Ab or an antigen-binding portion thereof based on an assessment that the frequency of CCR8-expressing Tregs in cells of the test tissue is lower than the predetermined threshold; and (b) administering standard treatment other than the therapeutic anti-CCR8 Ab or an antigen-binding portion thereof to the selected subject.

[0225] The present disclosure also provides a method for treating cancer in a subject, the method comprising (a) selecting a subject who is not a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting Ab or an antigen-binding portion thereof, the selection comprising: (i) determining the frequency of CCR8-expressing Tregs in a test tissue of the subject or taken from the subject, the test tissue comprising tumor cells and tumor-infiltrating Tregs; (ii) administering the therapeutic Treg-depleting Ab or an antigen-binding portion thereof to the subject; (iii) determining whether the frequency of CCR8-expressing Tregs decreases after administering the therapeutic Treg-depleting Ab or an antigen-binding portion thereof; and (iv) selecting a subject who is not suitable for immunotherapy with the therapeutic Treg-depleting Ab or an antigen-binding portion thereof based on an assessment that the frequency of CCR8-expressing Tregs decreases below a predetermined threshold; and (b) administering standard treatment other than a therapeutic anti-CCR8 Ab or an antigen-binding portion thereof to the selected subject. In certain preferred embodiments of the method, the Treg-depleting Ab is an anti-CCR8 Ab.

[0226] The present disclosure also provides a method of treating cancer in a subject, the method 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 the frequency of CCR8-expressing Tregs in cells in the subject or in a test tissue from the subject being determined to exceed a predetermined threshold level, wherein the test tissue comprises tumor cells and tumor-infiltrating Tregs.

[0227] The present disclosure also provides a method of treating cancer in a subject, the method 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 the therapeutic Treg-depleting Ab, or the antigen-binding portion thereof, causing a decrease in the frequency of CCR8-expressing Tregs exceeding a predetermined threshold.

[0228] The present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject a standard of care in addition to a therapeutic Treg-depleting Ab, or an antigen-binding portion thereof, wherein the subject is selected based on the frequency of CCR8-expressing Tregs in the subject or in a test tissue from the subject being determined to be below a predetermined threshold level, wherein the test tissue contains tumor cells and tumor-infiltrating Tregs.

[0229] The present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject a standard of care in addition to a therapeutic Treg-depleting Ab, or an antigen-binding portion thereof, wherein the subject is selected based on the therapeutic Treg-depleting Ab, or an antigen-binding portion thereof, causing a decrease in the frequency of CCR8-expressing Tregs below a predetermined threshold level.

[0230] 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 other preferred embodiments, the Treg depleting Ab or its antigen binding portion is a mAb comprising 6 CDRs, heavy and light chain variable regions, or heavy and light chains of the mAb named 4A19. In certain other embodiments, the Treg depleting Ab or its antigen binding portion is 4A19, 18Y12, 8D55, 10R3, 14S15 or 14S15h or its antigen binding portion. In certain preferred embodiments, the Treg depleting Ab is mAb 4A19. In certain other embodiments, the Treg depleting Ab is an anti-CTLA-4 Ab. In other embodiments, the Treg depleting Ab is an anti-CCR4 or anti-CD25 Ab.

[0231] In some cases, such as when the availability of tissue samples from tumor biopsies is limited, or when it is necessary to predict or monitor the efficacy before clinical indications are shown in tumors, the use of blood or PBMC samples rather than tumor samples to measure the frequency of cells expressing CCR8 and the depletion of CCR8 expressing cells induced by administering Treg depleting treatment can be faster, more efficient and / or more practical. As disclosed herein, the non-competitive anti-CCR8 mAbs of the present invention enable quantification of CCR8 expressing cells, and the depletion of cells expressing CCR8 in whole blood or PBMCs, although the frequency of such cells in peripheral blood is very low. Therefore, in any of the above methods for predicting the effectiveness of therapeutic Treg depleting Abs in treating cancer in patients, selecting cancer patients as suitable candidates for cancer immunotherapy with therapeutic Treg depleting Abs, and / or treating such patients with therapeutic Treg depleting Abs, the step of determining the frequency of CCR8 expressing Tregs, and / or determining whether the frequency of CCR8 expressing Tregs is reduced after administering therapeutic Treg depleting Abs is performed using blood or PBMC samples of or taken from the subject.

[0232] In certain embodiments, Treg depletion Ab is administered to a subject in combination with a therapeutically effective amount of an additional therapeutic agent for treating cancer. In certain embodiments, the additional anticancer agent is a small molecule, a polypeptide, an antibody, an immunomodulator, a chemotherapeutic agent, a targeted therapeutic agent, or any combination thereof. In certain embodiments, immunotherapy comprises an agent that reduces the suppression of the immune system or increases the stimulation of the immune system. In certain preferred embodiments, the additional therapeutic agent is a compound that reduces the suppression of the immune system or increases the stimulation of the immune system. This additional therapeutic agent can be, for example, a small molecule compound, a macrocyclic peptide, a fusion protein, or an Ab, such as a mAb. In certain embodiments, the additional therapeutic agent that reduces suppression of the immune system is an antagonist (e.g., an antagonist mAb) that specifically binds to an immunosuppressive receptor, such as programmed death receptor-1 (PD-1), programmed 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 cell 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 inhibitor of T cell activation (VISTA), proto-oncogene tyrosine protein kinase MER (MerTK), natural killer cell receptor 2B4 (CD244), or CD160.

[0233] In certain preferred embodiments, the additional therapeutic agent is an antagonist Ab or an antigen binding portion thereof that specifically binds to PD-1. In other embodiments, the Ab that specifically binds to PD-1 is selected from nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tisellizumab, toripalimab, dostarlimab, retifanlimab, zimberelimab, pimivalimab, serplullimab, vopratelimab and acrixolimab. In certain preferred embodiments, Ab is selected from nivolumab and pembrolizumab.

[0234] In other preferred embodiments, the additional therapeutic agent is an antagonist that specifically binds to PD-L1, such as Ab or its antigen binding portion. In other embodiments, the agent that specifically binds to PD-L1 is selected from atezolizumab, durvalumab, avelumab, envafolimab, 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.

[0235] In other preferred embodiments, the additional therapeutic agent is an antagonist Ab or antigen binding portion thereof that specifically binds to CTLA-4. In other embodiments, the Ab that specifically binds to CTLA-4 is ipilimumab or tremelimumab. In certain preferred embodiments, the Ab is ipilimumab.

[0236] In other preferred embodiments, the additional therapeutic agent is an antagonist that specifically binds to LAG-3. In other embodiments, the agent that specifically binds to LAG-3 is selected from the Ab relatlimab, favezelimab, tiragolumab, fianlimab or tebotelimab, or the soluble protein eftilagimod alpha. In certain preferred embodiments, the Ab is relatlimab.

[0237] In certain embodiments, the additional therapeutic agent that increases immune system stimulation is an agonist, such as an agonist mAb that specifically binds to an immunostimulatory receptor, such as inducible T-cell co-stimulator (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-induced TNFR-related protein (GITR), or herpes virus entry mediator (HVEM).

[0238] In certain embodiments of the above method, determining the expression level of CCR8 in the test tissue includes evaluating the expression level of CCR8 on the surface of Treg in the test tissue. In certain other embodiments, determining the expression level of CCR8 in the test tissue includes evaluating the proportion of Treg expressing CCR8 on the surface of Treg cells in the test tissue.

[0239] In other embodiments, the expression level of CCR8 in a test tissue of a subject is determined by an in vivo method. In other embodiments, the in vivo method comprises PET tracing, for example, using an anti-CCR8 Ab of the invention that binds to an epitope of CCR8 outside the N-terminal domain.

[0240] In other embodiments, the expression level of CCR8 is determined in vitro in a test tissue sample obtained from a subject. For example, in certain embodiments of an ex vivo method, the anti-CCR8 Ab or its antigen binding portion of the label of the present invention is combined with the CCR8 expressed on the cell surface in the tissue, and the expression level of CCR8 is determined by immunohistochemistry (IHC), flow cytometry or mass spectrometry coupled flow cytometry. In certain embodiments, IHC is performed on fresh frozen tissue or formalin-fixed paraffin embedded (FFPE) tissue. In certain preferred embodiments, the expression level of CCR8 is determined using the anti-CCR8 Ab of the present invention without being affected by the combination of therapeutic Ab or its antigen binding portion with the N-terminal domain of CCR8.

[0241] In certain embodiments of any of the methods described above, the therapeutic anti-CCR8 Ab, or antigen binding portion thereof, comprises:

[0242] (a) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 53; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 54; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 55; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 56; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 57; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 58;

[0243] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 9; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 16; and / or

[0244] (c) a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:65; and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:72.

[0245] In certain other embodiments, the therapeutic anti-CCR8 Ab, or an antigen binding portion thereof, is an Ab designated 4A19, 18Y12, 8D55, 10R3, 14S15, or 14S15h in WO 2021 / 194942, or an antigen binding portion thereof.

[0246] In certain embodiments of the methods described herein requiring a therapeutic anti-CCR8 Ab or an antigen binding portion thereof, the therapeutic Ab or an antigen binding portion thereof is a mAb comprising 6 CDRs, heavy and light chain variable regions and / or heavy and light chains of the mAb named 4A19, 18Y12, 8D55, 10R3, 14S15 or 14S15h in WO 2021 / 194942. In other embodiments, the therapeutic Ab or an 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.

[0247] In certain embodiments of such methods where an anti-CCR8 Ab that does not compete with a therapeutic Ab for binding to CCR8 is desired, the non-competitive Ab is a mAb comprising the 6 CDRs, heavy and light chain variable regions and / or heavy and light chains of the mAbs designated herein as 23A14, 21C17, 22B13, 25T40, 28P3, and 33H18. In certain preferred embodiments, the non-competitive Ab is 21C17.

[0248] In certain preferred embodiments of any of these methods, the subject is a human.

[0249] Cancers that can be treated by depletion of Tregs

[0250] Immuno-oncology relies on using the almost unlimited flexibility of the immune system to attack and destroy cancer cells, which is applicable to the treatment of a variety 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, the anti-PD-1 Ab nivolumab 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; WO 2013 / 173223; Drugs.com-Opdivo Approval History), and is currently in clinical trials in a variety of solid cancers and hematological cancers. Similarly, anti-PD-L1 drugs such as atezolizumab Durvalumab and avelumab It has been approved in a variety of indications. Therefore, immunotherapeutic methods that use depletion of Tregs to treat cancer, such as the CCR8-mediated depletion of tumor-infiltrating Tregs disclosed herein, can be used to treat a variety of solid tumors and liquid tumors.

[0251] For example, in certain embodiments of a Treg depletion therapy method for treating cancer, wherein an Ab of the invention is used to monitor changes in Treg numbers, the cancer is a solid tumor.

[0252] Based on the demonstration of effective treatment of different cancers with anti-CCR8 in mouse models (see, WO 2021 / 194942), it is expected that certain tumor types are particularly suitable for treatment with anti-CCR8 Ab. Therefore, in certain embodiments of the methods disclosed herein, the solid tumor is a cancer selected from the following: colon adenocarcinoma, bladder cancer, breast cancer and fibrosarcoma.

[0253] In certain embodiments, based on CCR8 identified by single cell RNA-seq analysis + Relatively high expression of CCR8 and CD8A in Tregs and a high ratio of CCR8 / CD8A, the solid tumor is a cancer selected from the group consisting of 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 endocervical adenocarcinoma (CESC).

[0254] High levels of CCR8 expression in certain tumors can be identified by IHC in FFPE tissue samples, and tumors expressing high levels of CCR8 are more likely to respond to treatment with anti-CCR8 Abs. Therefore, in certain embodiments, the solid tumor is a cancer selected from the following based on the level of CCR8 expression: 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 cancer, gastric cancer, bladder cancer, and breast cancer.

[0255] For a Phase 1 / 2 clinical trial of 4A19 anti-CCR8 mAb as monotherapy and in combination with the anti-PD-1 mAb, nivolumab, certain solid tumors were selected based on the demonstrated efficacy of anti-CCR8 in mouse tumor models, RNA expression of CCR8 and CD8A in tumor types represented in The Cancer Genome Atlas (National Cancer Institute, 2021), tumor types with relatively high CCR8 expression and enrichment of CD8A expression, and CCR8 expression levels in different tumor types or subtypes measured by IHC. Based on the tumor type selected for the clinical trial, in certain embodiments of the disclosed methods, the solid tumor is a cancer selected from the following: non-small cell lung cancer (NSCLC), SCCHN, microsatellite stable colorectal cancer (MSS-CRC), gastric / gastroesophageal (GE) junction adenocarcinoma, 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).

[0256] In other embodiments, the solid tumor is a cancer selected from the group consisting of squamous cell carcinoma, small cell lung cancer (SCLC), NSCLC, squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, esophageal cancer, stomach cancer, gastrointestinal cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), kidney cancer, RCC, bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, colon carcinoma, anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinonasal natural killer cell, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium, fallopian tube cancer, ovarian cancer, carcinoma of the cervix (carcinoma of the endometrium), cervix), vaginal cancer, vulvar cancer, testicular cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, penile cancer, carcinoma of the renal pelvis, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, solid tumors of children, environmentally induced cancers, virus-related cancers, and cancers of viral origin. In certain embodiments, the cancer is advanced, unresectable, metastatic, refractory cancer, or recurrent cancer, or any combination thereof.

[0257] In certain embodiments of the disclosed methods, the cancer is a hematological malignancy. Hematological malignancies include liquid tumors derived from either of the two major blood cell lineages: the myeloid lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid lineage (which produces B cells, T cells, NK cells, and plasma cells), including all types of leukemias, lymphomas, and myelomas.

[0258] TARGET (Therapeutically Applicable Research to Generate Effective Treatments, https: / / ocg.cancer.gov / programs / target) analysis showed that among the blood malignancies examined, follicular lymphoma (FL) and acute lymphocytic leukemia and lymphoma were found to have the highest relative expression of CCR8, and anti-CCR8 mAbs should be used for treatment first (see WO 2021 / 194942). Therefore, in certain embodiments of the treatment methods of the present invention, the blood malignancy is FL or acute lymphocytic leukemia and lymphoma.

[0259] In certain other embodiments, the hematological malignancy is a cancer selected from the group consisting of acute, chronic lymphocytic (lymphoblastic) and / or myeloid leukemias, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML); lymphomas, such as Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), 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, and / or myeloid leukemia; The main types of NHL include lymphomas (mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, and splenic marginal zone B-cell lymphoma), Burkitt's lymphoma, lymphoplasmacytoid lymphoma (LPL; also known as Waldenstrom's macroglobulinemia (WM)), hairy cell lymphoma, and primary central nervous system (CNS) lymphomas; NHLs that are T-cell lymphomas, including precursor T-lymphoblastic lymphoma / leukemia and T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL); peripheral T-cell lymphomas, such as cutaneous T-cell lymphoma (CTLC, also known as mycosis fungoides, Sezary syndrome, and syndrome), adult T-cell lymphoma / leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer cell / T-cell lymphoma, nasal type, enteropathy-associated intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), and nonspecific peripheral T-cell lymphoma, acute myeloid lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphomas, post-transplant lymphoproliferative disorders, true histiocytic lymphomas, primary effusion lymphomas, diffuse histiocytic lymphomas (DHL), immunoblastic large cell lymphomas, and precursor B-lymphoblastic lymphomas; myelomas, such as multiple myeloma, smoldering myeloma (also known as indolent myeloma), monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma, IgG myeloma, light chain myeloma, non-secretory myeloma, and amyloidosis; and any combination of the foregoing hematologic malignancies.

[0260] The method is also applicable to the treatment of advanced, metastatic, refractory and / or relapsed hematological malignancies, as well as any combination of said hematological malignancies.

[0261] Reagent test kit

[0262] The scope of the present invention also includes a kit comprising an anti-CCR8 Ab that specifically binds to an epitope outside the N-terminal domain of hCCR8 and other components required for performing any method disclosed herein. The kit generally includes a label and instructions for use indicating the intended use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit or otherwise accompanying the kit. Therefore, the present disclosure provides a kit for monitoring and / or quantifying the number of Tregs in a subject, wherein the kit is used to measure the exhaustion of the number of Tregs in a subject, predict the effectiveness of therapeutic Treg-depleting Abs in treating cancer in a subject, select a subject with cancer as a suitable candidate for immunotherapy with therapeutic Treg-depleting Abs, or treat cancer in a subject with therapeutic Treg-depleting Abs, or treat cancer in a subject with a standard-of-care therapeutic other than therapeutic Treg-depleting Abs, as described in the methods disclosed herein. In certain embodiments, the kit comprises (a) a 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, a therapeutic Treg-depleting Ab, or an antigen binding portion thereof, that binds to the N-terminal domain of hCCR8 for use in a treatment method, or alternatively a standard therapeutic agent other than the 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 Treg numbers in a subject, predicting the effectiveness of a therapeutic Treg-depleting Ab, or an antigen binding portion thereof, in treating cancer in a subject, selecting a subject having cancer as a suitable candidate for immunotherapy with 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 therapy other than the therapeutic Treg-depleting Ab, or an antigen binding portion thereof.

[0263] In certain preferred embodiments of the above-mentioned kit, the therapeutic Treg depletion Ab is an anti-CCR8 Ab. In other preferred embodiments, the Treg depletion Ab is an anti-CCR8 Ab that binds to an epitope in the N-terminal domain of hCCR8. For this Treg depletion Ab, the kit comprises a mAb of the present invention or an antigen binding portion thereof, which specifically binds to an epitope outside the N-terminal domain of hCCR8. In certain variants, the kit comprises a Treg depletion 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 a mAb that specifically binds to an epitope within the N-terminal domain of hCCR8.

[0264] In certain other embodiments, the Treg depletion Ab is an anti-CTLA-4 Ab. In other embodiments, the Treg depletion Ab is an anti-CCR4 or anti-CD25 Ab. In certain other embodiments, the kit comprises an additional anti-cancer therapeutic agent or therapy, which can be, for example, a small molecule agent, a polypeptide, an antibody, an immunomodulator, a chemotherapeutic agent, a targeted therapeutic agent, or a combination thereof. In certain preferred embodiments, other anti-cancer therapeutic agents are checkpoint inhibitors, such as anti-PD-1, anti-PD-L1, anti-CTLA-4 or anti-LAG-3 Ab, and / or chemotherapeutic agents such as docetaxel.

[0265] In certain embodiments, the kit comprises (a) one or more doses ranging from about 0.1 to about 20 mg / kg body weight of a therapeutic mAb, or an antigen-binding portion thereof, that specifically binds to CCR8; and optionally (b) one or more doses of a checkpoint inhibitor, e.g., about 3 mg / kg body weight or 200 to about 1600 mg of an anti-PD-1 / anti-PD-L1 mAb, or an antigen-binding portion thereof.

[0266] The present invention is further illustrated by the following examples, which should not be construed as limiting in any way.The contents of all references cited in this application are expressly incorporated herein by reference.

[0267] Example 1

[0268] Generation of anti-CCR8 mAb

[0269] Mouse or human anti-hCCR8 mAbs were generated by immunizing C57BL / 6 mice or transgenic mouse strains expressing the human Ig repertoire, respectively, with the hCCR8 immunogen.

[0270] Immunization with CCR8 antigen

[0271] Cohorts of 2-5 C57BL / 6 mice or human Ig transgenic mice were immunized every 5-7 days by footpad or tail base injection for 8-12 immunizations over 7-10 weeks with material derived from cells overexpressing hCCR8 to generate anti-hCCR8 Abs. In some cases, 2.5 × 10 6The plasma membrane fraction was isolated from BA / F3 cells overexpressing hCCR8 to enrich the hCCR8 protein in the plasma membrane and reduce the administration of non-CCR8 cell antigens. In other cases, detergent-stabilized proteoliposome materials were derived from HEK293 cells, which overexpressed chimeric hCCR8 / hCCR5 proteins and also overexpressed anti-hCCR8 Ab clone L263G8 (BioLegend). Proteoliposomes were delivered in a 5 μg dose in a phosphate buffered saline (PBS) solution of dodecyl-BD-maltoside (DDM) detergent. Water-in-oil RIBI adjuvant (Sigma-Aldrich, St. Louis, MO) was injected at sites adjacent to antigen injection to enhance immune response without destabilizing the proteoliposomes or denaturing the extracellular conformation of CCR8.

[0272] To monitor immune responses, titrated sera from retro-orbital or caudal bleeds were screened, typically 4-6 weeks after immunization, by flow cytometry and ELISA as described in Example 2. Sera were screened for Ab binding to multiple CCR8 overexpressing cell lines, corresponding negative control cell lines that did not overexpress CCR8, and sulfated N-terminal peptide of CCR8 conjugated to bovine serum albumin (BSA). CCR8-specific and CCR8-nonspecific Ab responses were measured in each animal, and animals with adequate anti-CCR8 Ig titers were selected for final immunization 6 and 3 days before sacrifice and tissue harvest to generate hybridoma fusions.

[0273] Generation of hybridomas producing monoclonal antibodies against CCR8

[0274] Collect the lymphoid organs targeted by the immunization strategy. Most typically, popliteal, inguinal, and iliac lymph nodes from mice immunized with the CCR8 immunogen via the footpad and tail base are used. The tissue is homogenized into a cell suspension and mixed with an equal amount of immortalized mouse myeloma cells derived from the P3X63AgU.1 cell line (ATCC CRL-1597). Cells are cultured at 1 × 10 7 The combined cells were prepared at a density of 10 cells / ml. The B cells secreting Ab were fused with immortalized myeloma cells using an electrofusion unit (BTX) to generate hybridomas. The resulting cells were cultured in flat-bottomed 96-well cell culture plates and hybridomas were selected using medium E (StemCell Technologies, Cambridge, MA) containing hypoxanthine and thymidine supplemented with aminopterin (Sigma-Aldrich).

[0275] Example 2

[0276] Screening and selection of anti-human CCR8 mAbs

[0277] Screening for mAbs that selectively bind to human CCR8

[0278] To generate mAbs that bind to hCCR8, conventional mice or human Ig transgenic mice were immunized with antigens derived from hCCR8 overexpressing cells, and hybridomas were generated as described in Example 1. After 10-13 days of culture and replacement of the culture medium, hybridoma culture supernatants were collected from each well and screened to identify wells with secreted CCR8-specific Abs. All supernatants were initially screened for at least two cell lines, one overexpressing hCCR8, and the corresponding control cell line not overexpressing CCR8. Ab binding on cells was measured by image-based fluorescence microassay technology (FMAT) screening.

[0279] The hybridoma of the positive well is transferred to a 24-well plate with a new culture medium, grown for 2-3 days, and then screened by flow cytometry again to confirm the combination of Ab and CCR8. In brief, 75-100 μl hybridoma culture supernatant and CCR8 overexpressed cells (e.g., CCR8 overexpressed CHO or 293F cells) or control cells (e.g., GFP-CHO or parent 293F) are co-incubated for 30-60 minutes, washed, and incubated with anti-mouse IgG Fc or anti-human IgG Fc secondary antibodies (Jackson ImmunoResearch, West Grove, PA) conjugated to AF647, APC or PE. After incubation and washing, fluorescence is measured by flow cytometry.

[0280] Hybridomas secreting anti-CCR8 Ab were subcloned once or twice to ensure monoclonality. Briefly, approximately 700 live hybridoma cells were inoculated in 5 ml semisolid methylcellulose medium (StemCell Technologies) and IgG secreted by hybridomas was detected with AF488-conjugated anti-human or anti-mouse IgG Ab (Jackson ImmunoResearch). After 7 days, hybridoma colonies from single cells were imaged using the ClonePix2 system (Molecular Devices, San Jose, CA). Individual colonies with desired properties (including distance to other colonies, IgG secretion levels, colony size, and colony circularity) were picked into wells of 96-well plates and cultured for 2-4 days. Culture supernatants were screened by flow cytometry to confirm CCR8 Ab binding as described previously. Stable hybridoma subclones were cultured in vitro to generate Abs for affinity purification and further characterization. DNA sequences encoding the Ab heavy and light chains were obtained by standard sequencing techniques (Sanger sequencing and next-generation sequencing). The predicted mass of the Ab amino acid sequence was compared with the known mass of purified Ab obtained by mass spectrometry to ensure the accuracy of the sequencing.

[0281] Characterization of the epitope of anti-hCCR8 Ab

[0282] In order to extensively characterize the epitopes of the confirmed CCR8-specific Abs, hybridoma culture supernatants were screened by ELISA to measure binding to the N-terminal peptide of hCCR8. Briefly, a BSA-conjugated peptide (SEQ ID NO:74; 2 μg / ml) corresponding to the N-terminal 35 amino acid residues of hCCR8 representing the N-terminus of appropriately sulfated hCCR8 was coated overnight at 4°C on a high-binding 96-well plate (Corning, Corning, NY). The plate was blocked with BSA and washed, and then 100 μl of culture supernatant was added on a plate shaker for 30-60 minutes. After washing, a suitable anti-Fc secondary antibody conjugated to horseradish peroxidase (HRP) was added and placed for 30-60 minutes. The plate was developed with ABTS or HRP substrate (SurModics, Eden Prairie, MN) and on a Sunrise microplate reader (TECAN; The absorbance was measured at 405 or 650 nm on a 400 nm Spectrum Scanner (Switzerland).

[0283] ELISA showed that the generated Abs against hCCR8 mainly, but not exclusively, bind to BSA-conjugated N-terminal peptide sequences with two sulfated tyrosine residues. Several Ab competition experiments based on flow cytometry showed that this group of major hCCR8 N-terminal binding Abs always blocked each other from binding to CCR8 on cells at the same time. However, 6 mAbs (23A14, 21C17, 22B13, 25T40, 28P3 and 33H18) were identified by flow cytometry, which specifically bind to hCCR8, but were found not to bind to BSA-conjugated CCR8 N-terminal peptides by ELISA, indicating that they bind to epitopes on hCCR8 that are not located in the N-terminal domain. These 6 Abs were further characterized by binding competition experiments based on flow cytometry.

[0284] Example 3

[0285] CD4 T cells in tumors pretreated with anti-CCR8 MAb 21C17 (but not commercial clone L263G8) and MAb 4A19

[0286] Treg binding

[0287] PCT Publication No. WO 2021 / 194942 describes humanized mAbs and human mAbs that bind to hCCR8 expressed on the cell surface with high affinity and mediate the depletion of tumor-infiltrating Tregs expressing CCR8 through mechanisms including ADCC. One of these mAbs (designated 4A19) was demonstrated to bind to an epitope in the N-terminal domain of CCR8, including residues 12-22 and including sulfated tyrosine-15 and tyrosine-17 residues.

[0288] A study was conducted to determine whether binding of mAb 4A19 to hCCR8 blocks binding of 21C17. Tissue from gastric tumors was dissociated using a tumor dissociation kit (Miltenyi Biotec, Sunnyvale, CA) in conjunction with a gentleMACS dissociator (Miltenyi Biotec) and stored in liquid nitrogen. For this study, thawed cells were incubated on ice for 15 minutes with unlabeled mAb4A19, unlabeled mAb 21C17, or unlabeled isotype control (each at a concentration of 10 μg / ml). After incubation, samples were stained with an immune profiling panel that included Abs against CD3, CD8, CD4, FOXP3, and two Abs against CCR8: commercially available anti-CCR8 Ab L263G8 (BioLegend) conjugated to phycoerythrin (PE), and BRILLIANT VIOLET TM After staining the cells, they were processed on a flow cytometer and analyzed using FLOWJO TM Data were analyzed and plotted using BD Biosciences software (BD Biosciences, San Jose, CA).

[0289] The three flow cytometry graphs in Figure 1 show the binding of the gastric tumor Treg compartment to mAb 21C17 conjugated to BV421 (x-axis) and to clone L263G8 conjugated to PE (y-axis). Figure 1A Tumor Tregs pre-treated with 4A19 are shown. The results suggest that PE-L263G8 was blocked from binding to 4A19 pre-treated Tregs, as no CCR8 was observed in the Q1 or Q2 quadrants. + cells, indicating that 4A19 binding to CCR8 blocks subsequent binding of L263G8. However, in contrast, BV421-21C17 binds to these 4A19 pre-treated cells, as CCR8 is observed in the Q3 quadrant. + group( Figure 1A ). Figure 1BFigure 2 represents an unlabeled 21C17 treated sample showing the opposite situation, where BV421-21C17 Ab is blocked from binding to 21C17 pre-treated Tregs (quadrants Q2 and Q3), whereas PE-L263G8 binds to 21C17 pre-treated Tregs because most CCR8 + The population is located in the Q1 quadrant. Thus, 21C17 does not block the binding of L263G8, but blocks its own binding to CCR8 as expected. Figure 1C The effect of pretreatment with isotype control is shown, indicating that both 21C17 and PE-L263G8 are able to bind to the same cells, as most CCR8 + The cell is located in the Q2 quadrant. In summary, the results indicate that the 21C17 mAb binds to a different epitope on CCR8 than 4A19, as it can bind to tumor Tregs expressing CCR8 that have previously bound the 4A19 mAb. The lack of cross-competition between mAbs 4A9 and 21C17 for binding to hCCR8 suggests that they do not bind to substantially the same epitope region of hCCR8, i.e., their epitopes are not adjacent to or overlapping each other, such that binding of one Ab to its epitope sterically hinders binding of the other Ab to its epitope.

[0290] Example 4

[0291] Antibodies that bind with high affinity to epitopes outside the N-terminus of CCR8

[0292] EC for mAb binding to epitopes other than the N-terminus of hCCR8 50 value

[0293] The binding specificity of Ab binding to CCR8 was assessed using FACS. Transfected Raji cells overexpressing hCCR8 and parental Raji cells not expressing CCR8 were incubated with the active dye on ice for 30 min. Unbound dye was washed away and cells were plated at 5 × 10 5 The cells were plated in 96-well round-bottom plates at a concentration of 10 cells per well. Serial dilutions of mAbs that bind to epitopes other than the N-terminal epitope of CCR8 were added at a starting concentration of 20 nM and incubated on ice for 30 minutes. Unbound antibody was washed away, and a secondary detection antibody was added and incubated on ice for 30 minutes. Additional washing was performed and Ab binding affinity was determined by measuring the fluorescence intensity of the secondary antibody on an LSRFortessa X-20 cell analyzer (BD Biosciences).

[0294] Figure 2A shows the binding curves of different concentrations of mAb binding to Raji cells expressing hCCR8, while Figure 2B shows the lack of binding to parental Raji cells. This binding analysis identified four mAbs that bound to Raji-hCCR8 cells, EC 50 The concentration of 5-mercaptoethanol was about 0.1 nM or lower (see Table 1), while no nonspecific binding was observed on parental Raji cells.

[0295] Table 1. CCR8 Abs bound to Raji-hCCR8

[0296]

[0297] Competitive binding between N-terminally and non-N-terminally binding MAbs

[0298] Competition for hCCR8 binding between an anti-CCR8 mAb that does not bind to the N-terminus of CCR8 (23A14-hIgG1) and a mAb that binds to the N-terminus (4A19-mIgG2a) was determined by FACS. To isolate human Tregs (hTeg), PBMCs were separated from leukocytes (AllCells) using Ficoll density gradients (GE Healthcare). CD25 was 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 低 CD25 高 CD45RA + Treg.

[0299] In the presence of 100 U / ml rhIL-2, DYNABEADS TM Isolated naive Tregs were activated in vitro with human T-activator CD3 / CD28 microbeads at a 1:3 cell to microbead ratio to stimulate CCR8 expression. Activated hTREGs were incubated with the viability dye on ice for 30 min. Unbound dye was washed away and cells were plated at 5 × 10 5The cells were plated in 96-well round-bottom plates at a concentration of 10 cells per well. The anti-CCR8 mAb 4A19 (4A19-mIgG2a), a mouse IgG2a native form described in WO 2021 / 194942, was added to the cells at a saturating concentration of 200 nM and incubated on ice for 30 minutes. Unbound antibody was washed away and the cells were incubated with mAb 23A14-hIgG1 at concentrations continuously titrated from 200 nM to 0.0034 nM. Additional washes were performed, and fluorophore-conjugated secondary antibodies specific for human and mouse Ig were added and incubated for 30 minutes. A final wash was performed and the samples were analyzed on an LSRFortessa X-20 cell analyzer.

[0300] As shown in Figure 3A, in the presence of saturating amounts of bound 4A19-mIgG2a, the binding of mAb 23A14-hIgG1 increased, indicating that the binding of mAb 4A19-mIgG2a did not hinder the binding of mAb 23A14-hIgG1. This competition assay analysis showed that the mAbs 4A19-mIgG2a and 23A14-hIgG1 tested at saturating concentrations were able to detect CCR8 + Equal populations of Tregs.

[0301] Two other anti-CCR8 mAbs (21C17-mIgG2a and 22B13-mIgG2a) that do not bind to the N-terminal epitope were tested by incubating activated hTregs stained with a vital dye and continuously titrating mAb 4A19 (WO 2021 / 194942) at concentrations from 200nM to 0.0034nM. After incubation on ice for 30 minutes and washing, a fixed concentration (100nM) of 21C17-mIgG2a or 22B13-mIgG2a was added and incubated on ice for 30 minutes. The cells were washed, detection antibodies specific for human and mouse Ig were added, and incubated on ice for 30 minutes. A final wash step was performed, and the samples were analyzed on an LSRFortessa X-20 cell analyzer.

[0302] The results obtained with 21C17-mIgG2a and 22B13-mIgG2a are shown in Figure 3B and C, respectively. Competition assay analysis showed that mAb 4A19 could detect CCR8 at saturating concentrations when paired with 21C17-mIgG2a or 22B13-mIgG2a. + Similar populations of human Tregs.

[0303] Example 5

[0304] Fluorophore-labeled Ab binding to CCR8 expressing cells

[0305] Flow cytometry is used to assess the combination of anti-hCCR8 Ab on the cell line overexpressing hCCR8. In this assay, the Raji cells overexpressing hCCR8 are used to determine the combination of mAb 21C17-mIgG2a directly conjugated with BV-421 by BioLegend. Unlabeled 21C17-mIgG2a is used as a positive control. The combination with the parent Raji cells not expressing CCR8 is also tested to assess the specificity of mAb. Parent Raji cells and the Raji cells overexpressing hCCR8 are mixed with unlabeled 21C17-mIgG2a, 21C17-mIgG2a or KLH-mIgG2a control mAb of serial dilution (20 to 0.0034nM) respectively, BV-421-labeled. Binding of unlabeled 21C17-mIgG2a to cell surface hCCR8 was detected using PE-labeled anti-mouse IgG (Fab')2 fragments (Jackson ImmunoResearch).Relative cell binding was measured as the geometric mean fluorescence intensity (GMFI) of total cells positive for PE-conjugated anti-mouse IgG Ab or directly BV-421-labeled 21C17-mIgG2a Ab.

[0306] As shown in Figure 4A, both unlabeled and directly BV-421 labeled 21C17-mIgG2a antibodies bind to cell surface CCR8 on hCCR8 overexpressing Raji cell lines. These Abs were also confirmed to specifically bind to hCCR8, as indicated by the absence of binding to parental Raji cells that do not express hCCR8 (Figure 4B).

[0307] Example 6

[0308] Receptor occupancy assay for evaluating target engagement of therapeutic anti-CCR8 antibodies

[0309] Receptor occupancy (RO) assays are used in clinical trials to assess target engagement and determine dose selection, providing valuable insights into the pharmacodynamics and safety assessment of biotherapeutics. A flow cytometric assay using peripheral blood was developed and validated to measure CCR8 RO in subjects following administration of an anti-CCR8 therapeutic Ab.

[0310] Two separate RO assay formats were developed: (1) direct / total format (measuring bound and total receptors), and (2) indirect / total format (measuring free and total receptors). The latter represents a combination of the indirect RO and total RO assay formats; the indirect RO format uses competitive anti-CCR8 Ab to detect free CCR8 receptors that are not occupied by anti-CCR8 therapeutic Ab after treatment to indirectly obtain RO, while the total RO assay format uses non-competitive anti-CCR8 Ab to measure the total CCR8 receptors available on Tregs for binding therapeutic anti-CCR8 Ab after treatment. Both free and total CCR8 receptor information are used to obtain %RO in the indirect / total assay format using the following formula.

[0311] Because the total number of CCR8 receptors on Tregs may fluctuate throughout the course of treatment with a therapeutic anti-CCR8 Ab, for example in clinical studies, the use of a non-competitive anti-CCR8 Ab is advantageous because it enables continuous monitoring of total CCR8 receptor levels and allows for any changes in total receptor levels in response to anti-CCR8 Ab treatment to be taken into account when deriving the %RO during treatment. Therefore, the use of a non-competitive anti-CCR8 Ab, i.e., an Ab that does not compete with the therapeutic Ab for binding to CCR8, is important to determine modulation of total CCR8 expression levels and aid in accurate %RO calculations.

[0312] The measurement of CCR8 RO is particularly challenging due to the limited assay range caused by low CCR8 expression on peripheral Tregs. However, by utilizing a non-competitive anti-CCR8 Ab, specific CCR8 + Treg subsets rather than total Tregs were used as the target cell population for the assay. This is an effective strategy that increases the dynamic range of the assay by 5-fold, making the indirect / total RO assay more robust and reliable.

[0313] A head-to-head comparison of two different RO assay formats was performed. Whole blood samples (180 μl) were pretreated with different concentrations of the therapeutic mAb, mAb 4A19 (WO 2021 / 194942), simulating different 4A19 dose treatment levels of subjects in the dose escalation group of a Phase I clinical study (NCT04895709), and pre-incubated at 37°C for 1 hour. Aliquots of the treated samples were transferred to 4°C for 24, 48, and 72 hours for sample stability experiments.

[0314] After pre-incubation, the tube containing the background sample was treated with a saturating concentration (10 μg / ml) of mAb 4A19, while the other tubes (containing free or bound or FMO [fluorescence minus one] samples) were treated with PBS (equal volume with mAb4A19) for 30 minutes at room temperature (RT). The samples were centrifuged at 500 x g for 5 minutes at 4°C and the pellet was precipitated with GIBCO TM The cells were washed three times with Dulbecco's phosphate buffered saline (w / oCa / Mg) (DPBS; Thermo Fisher Scientific, Waltham, MA) and transferred to a new tube, then stained with a mixture of seven markers in the following core panel: basal T cell markers: anti-human CD3 AF488 (BioLegend), anti-human CD4 BV510 (BioLegend), and anti-human CD8 BV605 (BD Biosciences); T cell differentiation markers: anti-human CD45RA APC-CY7 (BioLegend); Treg markers: anti-human CD25 PE (BioLegend), anti-human CD127 APC-R700 (BD Biosciences), and anti-human CCR4 PE-Cy7 (BioLegend). One additional Ab was added to the core mixture set for direct / total RO assay (custom-conjugated anti-idiotypic Ab anti-Id AF647, Bristol Myers Squibb) for detection of bound CCR8 receptors, while two additional Abs were added to the mixture set for indirect / total assay, competitive anti-hCCCR8 Ab APC, clone 433H (BD Biosciences) for detection of free CCR8 receptors, non-competitive anti-hCCCR8 mAb, 21C17, for detection of total CCR8 receptors, on ice for 30 minutes. Once staining was complete, the samples were washed twice with MACS buffer (MiltenyiBiotec) and then lysed with FACS lysing solution (dedicated buffer solution containing <10% formaldehyde and <50% diethylene glycol; BD Biosciences) at room temperature in the dark for 15 minutes. After lysis, the samples were washed and then stored at 4°C before being collected on the flow cytometer on the same day. The %RO for each 4A19 concentration point was obtained using the following formula:

[0315] %RO=100×[1-((free amount after administration / free amount before administration) / (total amount after administration / total amount before administration))].

[0316] Among them, "free amount after administration" is the MFI of the competing CCR8 Ab at each therapeutic mAb concentration (after exposure to therapeutic mAb) determined by flow cytometry; "free amount before administration" is the MFI of the competing CCR8 Ab when not exposed to therapeutic mAb (before exposure to therapeutic mAb); "total amount after administration" is the MFI of the non-competitive anti-CCR8 mAb at each concentration of therapeutic mAb (after exposure to therapeutic mAb); and "total amount before administration" is the MFI of the non-competitive anti-CCR8 mAb when not exposed to therapeutic mAb (before exposure to therapeutic mAb).

[0317] Response curves, ie, %RO versus drug (eg, mAb 4A19) concentration, were plotted, for example Figure 5 Both methods produced similar %RO drug dose-response curves and EC50 (eg, 0.022 nM vs. 0.023 nM for a particular donor, n=3), indicating the reliability of the developed assay. Figure 5 is a representative graph showing the change in CCR8%RO with increasing concentrations of mAb 4A19 in healthy donor blood (n=3) collected in sodium heparin (NaHep) and stored at 4°C for 24 hours after processing. The %RO curve for each donor was plotted using direct and indirect RO assay formats. Both formats were independently validated to ensure sample stability for up to 72 hours after collection with acceptable intra-assay precision and inter-assay precision (coefficient of variation [CV] ≤ 30%). In addition, consistent RO data were observed over the intra-subject longitudinal drug dose range (CV ≤ 25%). The consistency of these data suggests that these CCR8 RO assays are robust enough to be used in clinical trials of therapeutic anti-CCR8 Abs to evaluate and quantify the engagement of therapeutic Abs with the CCR8 target.

[0318] Example 7

[0319] Detection of CCR8 by immunohistochemistry (IHC)

[0320] 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 automatic stainer (Leica Biosystems, Buffalo Grove, IL). The samples were sliced ​​at 4 μm thickness, fixed on positively charged slides, dried with a fan for at least 1 hour, baked in an oven at 60 ° C for 30 minutes, dewaxed, and rehydrated offline. The tissue was then placed on an automatic stainer and pretreated with ER2 (Leica, Buffalo Grove, IL) at 100 ° C for 30 minutes, followed by rinsing and incubation in Bond Wash Buffer (Leica) for 3 minutes. The tissue was incubated with peroxide blocking solution (Bond Polymer Refine Detection Kit, Leica), rinsed in Bond Wash Buffer (Leica), incubated with human-human protein blocking solution (Sigma; St. Louis, MO), and then incubated with primary Ab (mouse IgG2a, κ clone 433H, BD Biosciences) or mouse IgG1 (κ negative control antibody) for 30 minutes. Normal thymus tissue containing CCR8 positive and negative cell characteristics was used as positive and negative controls.

[0321] After incubation with Ab, tissues were processed using the Bond Polymer Refine Detection Kit according to the manufacturer's instructions and incubated with 3,3′-diaminobenzidine (DAB) for 10 minutes, followed by rinsing in distilled water. Tissues were incubated with hematoxylin for 5 minutes, followed by rinsing in distilled water, rinsing in Bond Wash Buffer, and finally rinsing in distilled water.

[0322] Coverslipping was performed with an automated glass coverslipper, and slides were scanned with an Aperio AT Turbo system (Aperio, Vista, CA) to generate whole slide micrographs. Images were analyzed by pathologist tumor cell scoring, by using the CytoNuclear algorithm from Indica (Corrales, NM), and / or by pathologist visual immunoscores.

[0323] Example 8

[0324] Depletion of Tregs by MAb 4A19 in dissociated human tumors

[0325] The ability of the therapeutic anti-CCR8 mAb 4A19 to induce depletion of CCR8+ Tregs was evaluated ex vivo in dissociated human tumors.

[0326] Human tumor dissociation

[0327] Excised human solid tumors were shipped overnight at 4°C in HypoThermosol biopreservation medium (Charles River Laboratories, Wilmington, MA). Whole tumor samples were minced with laboratory scissors before dissociation. TM Tumors were mechanically and enzymatically dissociated in Roswell Park Memorial Institute (RPMI)-1640 medium at 37°C for 30 minutes using C tubes, dissociators, and tumor dissociation kits. The resulting single-cell suspension was filtered through a 70 μm cell strainer and then processed for flow cytometry.

[0328] Processing cells for flow cytometry

[0329] After generating single cell suspension from tumor tissue, cells were washed with PBS and then stained with amine reactive active dye on ice for 20 minutes. Cells were washed and blocked on ice for 20 minutes in 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 blocking solution (BioLegend), which was diluted in FACS staining buffer (PBS w / o Ca / Mg, 2mM EDTA, 0.2% BSA). After blocking, cells were stained with UV-excitable dye-conjugated anti-human CD3, CD4, and CD8 mAbs (BD Biosciences) to identify T cell subsets, while anti-human CD14 and CD56 mAbs (BD Biosciences) were used to identify macrophages, monocytes, and NK cells. In addition to lineage markers, CCR8, CCR4, and CD16 Abs were used to identify CCR8. + Tumor Tregs. The cells were then fixed and permeabilized with FOXP3 transcription factor staining buffer for 30 min at 4°C, followed by incubation with anti-FOXP3 Ab for 30 min at 4°C for intracellular staining. Finally, the stained cells were washed and filtered using a 40-μm cell strainer.

[0330] Using BD LSRFortessa TM Flow cytometric analysis was performed using an X-20 flow cytometer and FLOWJO TMThe software analyzed and calculated the population percentage and mean fluorescence intensity values.

[0331] Depletion of Tregs by MAb 4A19 in dissociated human tumors from cancer patients

[0332] Single cells were collected by filtration from dissociated tumor tissue and washed with complete RPMI-1640 medium. Cells were resuspended in complete RPMI-1640 medium and cultured with serial dilutions of mAb 4A19 or anti-KLH isotype control mAb for 48 hours. Cells were seeded at a concentration of 500,000 cells / well in flat-bottom plates. After 48 hours of incubation, cells were processed for flow cytometry analysis as described above. + FoxP3 + 、CD25 高 and CD45RA - Gating within the cell to determine depletion of CCR8 + The percentage of Treg. Using FLOWJO TM Software analysis and calculation CCR8 + The frequency of cells was determined and depletion graphs were generated using PRISM software (GraphPad Software, San Diego, CA).

[0333] Treatment with mAb 4A19 resulted in dissociation of CCR8 in tumor tissues isolated from cancer patient donors (n=5). + Table 2 summarizes the CCR8 expression in these tumor tissues at the concentrations (1 μg / ml or 0.1 μg / ml) at which maximal depletion was observed. + The percentage of Treg depletion was calculated using the following formula: CCR8 compared with KLH isotype control + Percentage of Treg depletion:

[0334] (1-(CCR8 of parental phylum in A419-treated samples + Treg) / (CCR8 of the parental gate in KLH isotype treated samples + Treg)×100%.

[0335] like Figure 6 As shown, mAb 4A19 induced CCR8 + Dose-dependent depletion of tumor Tregs, whereas anti-KLH isotype Ab did not deplete Tregs even at the highest dose tested. This depletion plot is from a single patient and is representative of similar depletion assays from 5 patient donors. Parental gate represents CD3 + CD4 + FoxP3 + CD45RA population.

[0336] Table 2. MAb 4A19 depletes Tregs in dissociated human tumors

[0337]

[0338]

[0339] Exemplary embodiments

[0340] The disclosed invention includes the following non-exhaustive list of embodiments. The list should not be interpreted as any limitation, and those skilled in the art will appreciate that various modifications can be made to these embodiments without changing the nature and scope of the invention disclosed herein.

[0341] 1. A monoclonal antibody or an antigen-binding portion thereof that specifically binds to human C-C motif chemokine receptor 8 (hCCR8) expressed on the surface of a cell, wherein the antibody or the antigen-binding portion thereof binds to an epitope that is not in the N-terminal domain of hCCR8 and does not cross-compete with an antibody that binds to the N-terminal epitope for binding to hCCR8.

[0342] 2. The monoclonal antibody or antigen-binding portion thereof according to embodiment 1, wherein the sequence of hCCR8 is as shown in SEQ ID NO:1.

[0343] 3. The monoclonal antibody or antigen binding portion thereof of embodiment 1 or 2, wherein the N-terminal epitope comprises a 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 At least one amino acid within the peptide of (SEQ ID NO: 73).

[0344] 4. The monoclonal antibody or antigen binding portion thereof according to any one of the preceding embodiments, wherein the N-terminal epitope comprises a 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 At least 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 amino acids within the peptide of (SEQ ID NO: 73).

[0345] 5. The monoclonal antibody or antigen binding portion thereof according to any one of the preceding embodiments, wherein the N-terminal epitope comprises a 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).

[0346] 6. The monoclonal antibody or antigen binding portion thereof according to any one of the preceding embodiments, wherein the N-terminal epitope is composed of 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 The amino acid composition of (SEQ ID NO:73).

[0347] 7. The monoclonal antibody or antigen binding portion thereof according to any one of embodiments 3 to 6, wherein amino acid Y 15 and / or Y 17 It is sulfated.

[0348] 8. The monoclonal antibody or antigen binding portion thereof of any one of the preceding embodiments, wherein binding to hCCR8 is not affected by the presence of an antibody that binds to an N-terminal epitope comprising a region having sequence V 12 T 13 D 14 Y 15 Y 16 Y17P 18 D 19 I 20 F 21 S 22 At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 amino acids within the peptide of (SEQ ID NO: 73).

[0349] 9. The monoclonal antibody, or antigen binding portion thereof, of any one of the preceding embodiments, wherein the antibody that binds to the N-terminal epitope is an antibody comprising:

[0350] (a) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 53; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 54; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 55; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 56; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 57; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 58;

[0351] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 9; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 16; and / or

[0352] (c) a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:65; and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:72.

[0353] 10. The monoclonal antibody, or antigen binding portion thereof, of any preceding embodiment, which binds hCCR8 polypeptide expressed on the cell surface in a formalin-fixed paraffin-embedded (FFPE) tissue sample.

[0354] 11. The monoclonal antibody or antigen binding portion thereof of any one of the preceding embodiments, which specifically binds to Raji cells expressing human CCR8, having the following EC values ​​as measured by the binding assay described in Example 4: 50 :

[0355] (a) about 50 nM or less;

[0356] (b) about 3 nM or less;

[0357] (c) about 0.5 nM or less;

[0358] (d) about 0.1 nM or less;

[0359] (e) about 0.01 nM or less;

[0360] (f) about 0.005 nM or less;

[0361] (g) about 0.1 nM;

[0362] (h) about 0.005 nM to about 50 nM;

[0363] (i) about 0.02 nM to about 3 nM; or

[0364] (j) from about 0.08 nM to about 2 nM.

[0365] 12. The monoclonal antibody, or antigen binding portion thereof, of any one of the preceding embodiments, which cross-competes with a reference antibody for binding to hCCR8, wherein the reference antibody comprises:

[0366] (a)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 4; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 11;

[0367] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 6; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 13; or

[0368] (c)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 8; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO:15.

[0369] 13. The monoclonal antibody, or antigen binding portion thereof, of any one of the preceding embodiments, which binds to the same epitope as a reference antibody, wherein the reference antibody comprises:

[0370] (a)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 4; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 11;

[0371] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 6; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 13; or

[0372] (c)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 8; and V L, which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO:15.

[0373] 14. The monoclonal antibody, or antigen binding portion thereof, of any one of the preceding embodiments, comprising the following CDR domains as defined by the Kabat method:

[0374] (a) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 23; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 24; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 25; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 26; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 27; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 28;

[0375] (b) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 35; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 36; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 37; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 38; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 39; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 40; or

[0376] (c) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:47; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:48; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:49; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:50; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:51; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO:22.

[0377] 15. The monoclonal antibody or antigen binding portion thereof of embodiment 14, comprising:

[0378] (a)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 4; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 11;

[0379] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 6; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 13; or

[0380] (c)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 8; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO:15.

[0381] 16. The monoclonal antibody or antigen binding portion thereof of embodiment 14 or 15, comprising:

[0382] (a) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 60; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 67;

[0383] (b) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 62; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 69; or

[0384] (c) a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:64; and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:71.

[0385] 17. The monoclonal antibody or antigen binding portion thereof of any one of embodiments 14-16, which is a monoclonal antibody or antigen binding portion thereof designated herein as 21C17, 22B13 or 23A14.

[0386] 18. The monoclonal antibody or antigen binding portion thereof according to any one of the preceding embodiments, which is a chimeric antibody, a humanized antibody, a human antibody or a fragment thereof.

[0387] 19. The monoclonal antibody, or antigen binding portion thereof, of any one of embodiments 1-15, which comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotype.

[0388] 20. The monoclonal antibody, or antigen binding portion thereof, of any one of embodiments 1-15, which comprises a heavy chain constant region of the human IgG2 or IgG4 isotype.

[0389] 21. A labeled antibody or antigen conjugate thereof, comprising the monoclonal antibody or antigen conjugate thereof according to any one of the preceding embodiments and a detectable label.

[0390] 22. The labeled antibody or antigen binding agent of embodiment 21, wherein the detectable label is a fluorophore, a chromophore, an enzyme, a radioisotope, a micropolymer or a metal.

[0391] 23. The labeled antibody of embodiment 21, wherein the detectable label is biotin.

[0392] 24. The labeled antibody of embodiment 22, wherein the fluorophore is Brilliant Violet TM Dyes (e.g. BV-421), AmCyan dyes, Alexa dye, dye, dyes, fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), phycoerythrin (PE), allophycocyanin (APC), or peridinin-chlorophyll protein (PerCP).

[0393] 25. The labeled antibody of embodiment 22, wherein the chromophore is porphyrin, pyropheophorbide-α, benzoporphyrin monoacid ring A (BPDMA) or chlorin e6.

[0394] 26. The labeled antibody of embodiment 22, wherein the enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase or β-galactosidase.

[0395] 27. The labeled antibody of embodiment 22, wherein 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.

[0396] 28. The labeled antibody of embodiment 22, wherein the metal label is yttrium (Y), indium (In), lanthanide (Ln, from La to Lu, excluding Pm), iodine (i), cadmium (Cd), tellurium (Te), silver (Ag), palladium (Pd), rhodium (Rh), iridium (Ir), platinum (Pt), ruthenium (Ru), osmium (Os) or bismuth (Bi).

[0397] 29. An immunoconjugate comprising the monoclonal antibody, or antigen binding portion thereof, of any one of embodiments 1-20 linked to a lytic agent.

[0398] 30. The immunoconjugate of embodiment 28, wherein the cytolytic agent is a cytotoxin, a radioisotope or a photosensitizer.

[0399] 31. A chimeric antigen receptor (CAR) comprising the monoclonal antibody or antigen binding portion thereof of any one of embodiments 1-20.

[0400] 32. A T cell receptor (TCR) comprising the monoclonal antibody, or antigen binding portion thereof, of any one of embodiments 1-20.

[0401] 33. A bispecific molecule comprising the monoclonal antibody, or antigen binding portion thereof, of any one of embodiments 1-20 linked to a binding domain having a different binding specificity than the monoclonal antibody, or antigen binding portion thereof.

[0402] 34. A composition comprising:

[0403] (a) the monoclonal antibody or antigen-binding portion thereof according to any one of embodiments 1-20;

[0404] (b) the labeled antibody or antigen-binding portion thereof according to any one of embodiments 21 to 28;

[0405] (c) the immunoconjugate of embodiment 29 or 30;

[0406] (d) the CAR according to embodiment 31,

[0407] (e) the TCR of embodiment 32, or

[0408] (f) the bispecific molecule of embodiment 33,

[0409] and a pharmaceutically acceptable carrier.

[0410] 35. An isolated nucleic acid encoding the monoclonal antibody, or antigen binding portion thereof, of any one of embodiments 1-20.

[0411] 36. An expression vector comprising the isolated nucleic acid of embodiment 35.

[0412] 37. A host cell comprising the expression vector of embodiment 36.

[0413] 38. A method for preparing an anti-CCR8 antibody or an antigen-binding portion thereof, comprising expressing the antibody or the antigen-binding portion thereof in the host cell of embodiment 37, and isolating the antibody or the antigen-binding portion thereof from the host cell.

[0414] 39. A method for generating a first antibody that does not bind or does not cross-compete with a second antibody for binding to a determined epitope on an antigen, comprising immunizing a vertebrate with an immunogen, wherein the immunogen comprises a cell line or a component of the cell line that expresses the antigen and also expresses a second antibody or its antigen-binding portion that specifically binds to the epitope, wherein the binding of the second antibody or its antigen-binding portion to the epitope will shield the epitope from attack by the vertebrate immune system, reduce the generation of antibodies that bind to the epitope, and thereby preferentially result in the generation of a first antibody that does not bind or does not cross-compete with the second antibody for binding to the epitope.

[0415] 40. The method of embodiment 39, wherein the antigen comprises a CCR8 receptor.

[0416] 41. The method of embodiment 40, wherein the CCR8 receptor is hCCR8 receptor.

[0417] 42. The method of any one of embodiments 39-41, wherein the vertebrate is a mammal or a bird.

[0418] 43. The method of embodiment 42, wherein the mammal is a mouse, rat, hamster, rabbit, dog, goat, sheep or horse.

[0419] 44. The method of any one of embodiments 39-43, wherein the immunogen is a detergent-stabilized proteoliposome component of the cell line.

[0420] 45. The method of embodiment 41, wherein the epitope is an epitope in the N-terminal domain of the hCCR8 receptor.

[0421] 46. ​​The method of embodiment 44, wherein the second antibody, or antigen-binding portion thereof, is a monoclonal antibody designated L263G8 (BioLegend), a monoclonal antibody designated 433H (BD Biosciences), 4A19, 18Y12, 10R3, 8D55, 14S15, or 14S15h.

[0422] 47. A method for measuring receptor occupancy (RO) of a cell membrane-bound CCR8 receptor to which a therapeutic Treg-depleting anti-CCR8 antibody binds, the method comprising:

[0423] (a) adding a saturating concentration of a therapeutic antibody to a background whole blood sample previously exposed to various concentrations of the therapeutic antibody;

[0424] (b) incubating aliquots of free, bound or fluorescence minus one (FMO) samples with buffer without therapeutic antibody;

[0425] (c) staining samples with a core panel of antibodies to identify basal T cell markers, T cell differentiation markers, and Treg markers;

[0426] (d) Join the core group

[0427] (i) For direct / total RO assays, anti-idiotypic antibodies to detect bound CCR8 receptors, or

[0428] (ii) for an indirect / total assay, two additional antibodies comprising an allophycocyanin (APC)-conjugated anti-hCCR8 antibody that competes with the therapeutic antibody to detect free CCR8 receptors and an anti-hCCR8 antibody that does not compete with the therapeutic antibody to detect total CCR8 receptors;

[0429] (e) lysing red blood cells to clear the sample and analyzing the cleared sample by flow cytometry; and

[0430] (f) Determine the %RO for each concentration of the therapeutic antibody using the following formula:

[0431] %RO=100×[1-((free amount after administration / free amount before administration) / (total amount after administration / total amount before administration))].

[0432] 48. A method for measuring the depletion of the frequency of cells expressing CCR8 in the peripheral blood of a subject receiving anti-CCR8 mAb treatment, the method comprising:

[0433] (a) determining a baseline level of CCR8 expression and / or frequency of CCR8 expressing cells in a first sample of whole blood or PBMCs from a subject;

[0434] (b) administering treatment with an anti-CCR8 mAb to the subject; and

[0435] (c) determining the level of CCR8 expression and / or the frequency of CCR8 expressing cells in a second sample of whole blood or PBMC taken from the subject after administration of the anti-CCR8 mAb;

[0436] Wherein a decrease in the level of CCR8 expression and / or a decrease in the frequency of CCR8 expressing cells in the second sample indicates that the number of CCR8 expressing cells in the blood has been depleted.

[0437] 49. A method for measuring the depletion of the number of tumor-infiltrating CCR8-expressing Tregs in a subject receiving treatment, the method comprising:

[0438] (a) determining a baseline level of CCR8 expression and / or frequency of CCR8 expressing Tregs in the subject or in a first sample of a test tissue from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0439] (b) administering treatment with an anti-CCR8 antibody to the subject; and

[0440] (c) determining the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs in the subject or in a second sample of a test tissue from the subject during or after treatment;

[0441] Wherein a decrease in the level of CCR8 expression and / or a 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.

[0442] 50. The method of embodiment 49, wherein depletion of the number of Tregs in the test tissue indicates an enhancement of the immune response in the subject.

[0443] 51. The method of embodiment 49 or 50, wherein the treatment administered to the subject is a treatment of cancer.

[0444] 52. The method of embodiment 51, wherein the treatment of cancer comprises administering a therapeutic Treg-depleting antibody or antigen-binding portion thereof.

[0445] 53. The method of embodiment 52, wherein the therapeutic Treg-depleting antibody or antigen-binding portion thereof is an anti-CCR8 antibody or antigen-binding portion thereof.

[0446] 54. A method for measuring depletion of skin T cell populations in a subject receiving treatment, the method comprising:

[0447] (a) determining a baseline level of CCR8 expression in a first sample of a test tissue of a subject or taken from the subject, the test tissue comprising skin T cells;

[0448] (b) administering treatment with an anti-CCR8 antibody to the subject; and

[0449] (c) determining the expression level of CCR8 in the subject or in a second test tissue from the subject during or after treatment;

[0450] A decrease in the level of CCR8 expression in the second test tissue indicates that the number of skin T cells in the test tissue has been depleted.

[0451] 55. A method for predicting the effectiveness of a therapeutic Treg-depleting antibody, or antigen-binding portion thereof, in treating cancer in a subject, the method comprising:

[0452] (a) determining the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0453] (b) comparing the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs with a predetermined threshold; and

[0454] (c) predicting the effectiveness of a therapeutic Treg-depleting antibody or antigen-binding portion thereof,

[0455] wherein the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs exceeds a threshold value indicates that the therapeutic antibody, or antigen-binding portion thereof, will be effective in treating the subject, and

[0456] Wherein the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs is below a threshold value, indicating that the therapeutic antibody, or antigen-binding portion thereof, will not be able to effectively treat the subject.

[0457] 56. A method for predicting the effectiveness of a therapeutic Treg-depleting antibody, or antigen-binding portion thereof, in treating cancer in a subject, the method comprising:

[0458] (a) determining the level of CCR8 surface expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue taken from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0459] (b) administering to the subject a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof;

[0460] (c) determining whether the frequency of CCR8 expressing Tregs is reduced following administration of a therapeutic Treg depleting antibody or antigen binding portion thereof; and

[0461] (d) predicting that the therapeutic Treg-depleting antibody, or antigen-binding portion thereof, will be effective in treating cancer in the subject if the frequency of CCR8-expressing Tregs is reduced by more than a predetermined threshold, or

[0462] (e) if the frequency of CCR8-expressing Tregs decreases below a predetermined threshold, then predicting that the therapeutic Treg-depleting antibody, or antigen-binding portion thereof, will not be effective in treating cancer in the subject.

[0463] 57. A method of selecting a subject having cancer as a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody, or antigen-binding portion thereof, the method comprising:

[0464] (a) determining the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0465] (b) comparing the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs with a predetermined threshold; and

[0466] (c) selecting the subject as a suitable candidate for immunotherapy with the therapeutic Treg-depleting antibody, or antigen-binding portion thereof, based on an assessment that the level of CCR8 surface expression and / or the frequency of CCR8-expressing Tregs in cells of the test tissue exceeds a predetermined threshold.

[0467] 58. A method of selecting a subject having cancer as a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody or antigen-binding portion thereof, the method comprising:

[0468] (a) determining the level of CCR8 surface expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue taken from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0469] (b) administering to the subject a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof;

[0470] (c) determining whether the frequency of CCR8 expressing Tregs is reduced following administration of a therapeutic Treg depleting antibody or antigen binding portion thereof; and

[0471] (d) selecting the subject as a suitable candidate for immunotherapy with a therapeutic Treg-depleting antibody, or antigen-binding portion thereof, based on the assessment that the frequency of expressing CCR8 is reduced by more than a predetermined threshold.

[0472] 59. A method for treating cancer in a subject, the method comprising:

[0473] (a) selecting a subject who is a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody or antigen-binding portion thereof, the selecting comprising:

[0474] (i) determining the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0475] (ii) comparing the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs with a predetermined threshold; and

[0476] (iii) selecting the subject as a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody, or antigen-binding portion thereof, based on an assessment that the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs in cells of the test tissue exceeds a predetermined threshold; and

[0477] (b) administering to the selected subject a composition comprising a therapeutically effective amount of the therapeutic Treg-depleting antibody, or antigen-binding portion thereof.

[0478] 60. A method for treating a subject having cancer, the method comprising:

[0479] (a) selecting a subject who is a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody or antigen-binding portion thereof, the selecting comprising:

[0480] (i) determining the level of surface expression of CCR8 and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue taken from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0481] (ii) administering to the subject a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof;

[0482] (iii) determining whether the frequency of CCR8 expressing Tregs is reduced following administration of a therapeutic Treg depleting antibody or antigen binding portion thereof; and

[0483] (iv) selecting the subject as a suitable candidate for immunotherapy with a therapeutic agent based on the assessment that the frequency of CCR8-expressing Tregs is reduced by more than a predetermined threshold; and

[0484] (b) administering to the selected subject a composition comprising a therapeutically effective amount of the therapeutic Treg-depleting antibody, or antigen-binding portion thereof.

[0485] 61. A method for treating cancer in a subject, the method comprising:

[0486] (a) selecting a subject who is not a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody or antigen-binding portion thereof, the selecting comprising:

[0487] (i) determining the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0488] (ii) comparing the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs with a predetermined threshold; and

[0489] (iii) selecting a subject who is not suitable for immunotherapy with the therapeutic Treg-depleting antibody or antigen-binding portion thereof based on an assessment 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; and

[0490] (b) administering to the selected subject a standard of care in addition to the therapeutic Treg-depleting antibody, or antigen-binding portion thereof.

[0491] 62. A method for treating cancer in a subject, the method comprising:

[0492] (a) selecting a subject who is not a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody or antigen-binding portion thereof, the selecting comprising:

[0493] (i) determining the level of surface expression of CCR8 and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue taken from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs;

[0494] (ii) administering to the subject a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof;

[0495] (iii) determining whether the frequency of CCR8 expressing Tregs is reduced following administration of a therapeutic Treg depleting antibody or antigen binding portion thereof; and

[0496] (iv) selecting a subject who is not suitable for immunotherapy with the therapeutic Treg-depleting antibody, or antigen-binding portion thereof, based on an assessment that the frequency of CCR8-expressing Tregs is reduced below a predetermined threshold; and

[0497] (b) administering to the selected subject a standard of care treatment in addition to the therapeutic anti-CCR8 antibody, or antigen-binding portion thereof.

[0498] 63. A method for treating cancer in a subject, the method 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 level of expression and / or frequency of CCR8-expressing Tregs in the subject or in a test tissue from the subject being determined to exceed a predetermined threshold level, the test tissue comprising tumor cells and tumor-infiltrating Tregs.

[0499] 64. A method of treating cancer in a patient, the method comprising administering to the patient 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 therapeutic Treg-depleting antibody, or the antigen-binding portion thereof, causing a decrease in the frequency of CCR8-expressing Tregs exceeding a predetermined threshold.

[0500] 65. A method of treating cancer in a subject, the method comprising administering to the subject a standard of care in addition to a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof, wherein the subject is selected based on the level of CCR8 and / or CCR8-expressing Treg frequency in cells in the subject or from a test tissue of the subject being determined to be below a predetermined threshold level, the test tissue comprising tumor cells and tumor-infiltrating Tregs.

[0501] 66. A method of treating cancer in a subject, the method comprising administering to the subject a standard of care in addition to a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof, wherein the subject is selected based on the therapeutic Treg-depleting Ab, or an antigen-binding portion thereof, causing a reduction in the frequency of CCR8-expressing Tregs below a predetermined threshold level.

[0502] 67. The method of any one of embodiments 55-66, wherein the Treg-depleting antibody is an anti-CCR8, anti-CTLA-4, anti-CCR4 or anti-CD25 antibody.

[0503] 68. The method of any one of embodiments 48-67, wherein determining the expression level of CCR8 in the test tissue comprises assessing the expression level of CCR8 on the surface of Tregs in the test tissue.

[0504] 69. The method of any one of embodiments 48-67, wherein determining the expression level of CCR8 in the test tissue comprises assessing the proportion of Tregs expressing CCR8 on the surface of Treg cells in the test tissue.

[0505] 70. The method according to any one of embodiments 48 to 69, wherein the expression level of CCR8 in a test tissue of the subject is determined by an in vivo method.

[0506] 71. The method of embodiment 70, wherein the in vivo method comprises PET tracking using the anti-CCR8 antibody of embodiment 1.

[0507] 72. The method of any one of embodiments 48-70, wherein the expression level of CCR8 is determined ex vivo in a test tissue sample obtained from the subject.

[0508] 73. The method of embodiment 72, wherein a labeled anti-CCR8 antibody or an antigen-binding portion thereof according to any one of embodiments 21-28 is used to bind to CCR8 expressed on the surface of cells in a tissue, and the expression level of CCR8 is determined by immunohistochemistry (IHC), flow cytometry, or mass spectrometry-coupled flow cytometry.

[0509] 74. The method of embodiment 73, wherein IHC is performed on fresh frozen tissue or formalin-fixed paraffin-embedded (FFPE) tissue.

[0510] 75. The method of embodiment 70 or 73, wherein determining the level of CCR8 expression using the anti-CCR8 antibody of embodiment 1 is not affected by binding of the therapeutic antibody or antigen binding portion thereof to the N-terminal domain of CCR8.

[0511] 76. The method of any one of embodiments 52-66 and 75, wherein the therapeutic anti-CCR8 antibody, or antigen-binding portion thereof, comprises:

[0512] (a) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 53; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 54; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 55; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 56; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 57; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 58;

[0513] (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 9; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 16; or

[0514] (c) a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:65; and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:72.

[0515] 77. The method of any one of embodiments 52-66 and 75, wherein the therapeutic anti-CCR8 antibody, or antigen binding portion thereof, is an antibody, or antigen binding portion thereof, designated 4A19, 18Y12, 8D55, 10R3, 14S15, or 14S15h.

[0516] 78. The method of any one of embodiments 49-77, wherein the subject is a human.

[0517] 79. The method of any one of embodiments 51-78, wherein the cancer is a solid tumor.

[0518] 80. The method of embodiment 79, wherein the solid tumor is a cancer selected from the group consisting of squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, esophageal cancer, stomach cancer, gastrointestinal cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, colon carcinoma, anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinonasal natural killer cell, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium. endometrium), fallopian tube cancer, ovarian cancer, carcinoma of the cervix, vaginal cancer, vulvar cancer, testicular cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, penile cancer, carcinoma of the renal pelvis, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, solid tumors of childhood, environmentally induced cancers, virus-related cancers, cancers of viral origin, advanced cancers, unresectable cancers, metastatic cancers, refractory cancers, recurrent cancers, and any combination thereof.

[0519] 81. The method of embodiment 79, wherein the solid tumor is a cancer selected from the group consisting of head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), stomach 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 endocervical adenocarcinoma (CESC).

[0520] 82. The method of embodiment 79, wherein the solid tumor is a cancer selected from the group consisting of colon adenocarcinoma, bladder cancer, breast cancer, and fibrosarcoma.

[0521] 83. The method of embodiment 79, wherein the solid tumor is a cancer selected from the group consisting of NSCLC, SCCHN, microsatellite stable colorectal cancer (MSS-CRC), gastric / gastroesophageal (GE) junction adenocarcinoma, and cervical cancer.

[0522] 84. The method of any one of embodiments 51-78, wherein the cancer is a hematological malignancy.

[0523] 85. The method of embodiment 84, wherein the hematological malignancy is selected from follicular lymphoma (FL) and acute lymphoblastic leukemia and lymphoma.

[0524] 86. The method of embodiment 84, wherein the hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), T-cell lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), and any combination of said hematological malignancies.

[0525] 87. The method of embodiment 84, wherein the hematological malignancy is selected from 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's 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 cell / T-cell lymphoma, nasal type, enteropathy-associated intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), nonspecific peripheral T-cell lymphoma, 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 above hematological malignancies.

[0526] 88. The method of embodiment 84, wherein the hematological malignancy is an advanced, metastatic, refractory and / or relapsed hematological malignancy, and any combination of said hematological malignancies.

[0527] 89. The method of any one of embodiments 59-88, further comprising administering to the subject a therapeutically effective amount of an additional therapeutic agent for treating cancer.

[0528] 90. The method of embodiment 89, wherein the additional therapeutic agent is a compound that reduces suppression of the immune system or increases stimulation of the immune system.

[0529] 91. The method of embodiment 90, wherein the additional therapeutic agent is:

[0530] (a) an antagonist that specifically binds to programmed death receptor-1 (PD-1), programmed 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 cell 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 inhibitor of T cell activation (VISTA), proto-oncogene tyrosine protein kinase MER (MerTK), natural killer cell receptor 2B4 (CD244) or CD160; or

[0531] (b) Agonists that specifically bind to: inducible T-cell co-stimulator (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-induced TNFR-related protein (GITR), or herpes virus entry mediator (HVEM).

[0532] 92. The method of embodiment 91, wherein the antagonist that specifically binds to PD-1 is selected from the group consisting of: nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tisellizumab, toripalimab, dostarlimab, retifanlimab, zimberelimab, pimivalimab, serplullimab, vopratelimab and acrixolimab.

[0533] 93. The method of embodiment 91, wherein the antagonist that specifically binds to PD-L1 is selected from: atezolizumab, durvalumab, avelumab, envafolimab, cosibelimab (CK-301), BMS-936559, BMS-986189, CS-1001, SHR-1316, CBT-502, BGB-A333, KN035, AUNP12 and CA-170.

[0534] 94. The method of embodiment 91, wherein the antagonist that specifically binds to CTLA-4 is ipilimumab or tremelimumab.

[0535] 95. The method of embodiment 91, wherein the antagonist that specifically binds to LAG3 is relatlimab, favezelimab, tiragolumab, fianlimab, tebotelimab, or eftilagimod alpha

[0536] 96. A kit for measuring depletion of Treg population in a subject, the kit comprising:

[0537] (a) a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to hCCR8 expressed on the surface of a cell, wherein the antibody, or the antigen-binding portion thereof, binds to an epitope that is different from an epitope in the N-terminal domain of hCCR8; and

[0538] (b) instructions for using the monoclonal antibody, or portion thereof, in the method of any one of embodiments 49-53.

[0539] 97. A kit for predicting the effectiveness of a therapeutic anti-CCR8 antibody that binds to the N-terminal domain of hCCR8 in treating cancer in a subject, the kit comprising:

[0540] (a) a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to hCCR8 expressed on the surface of a cell, wherein the antibody, or the antigen-binding portion thereof, binds to an epitope that is different from an epitope in the N-terminal domain of hCCR8; and

[0541] (b) instructions for using the monoclonal antibody or portion thereof in the method of embodiment 55.

[0542] 98. A kit for selecting a subject having cancer as a suitable candidate for immunotherapy with a therapeutic anti-CCR8 antibody that binds to the N-terminal domain of hCCR8, the kit comprising:

[0543] (a) a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to hCCR8 expressed on the surface of a cell, wherein the antibody, or the antigen-binding portion thereof, binds to an epitope that is different from an epitope in the N-terminal domain of hCCR8; and

[0544] (b) instructions for using the monoclonal antibody or portion thereof in the method of embodiment 56.

[0545] 99. A kit for treating cancer in a subject, the kit comprising:

[0546] (a) a monoclonal antibody, or an antigen-binding portion thereof, which specifically binds to hCCR8 expressed on the surface of a cell, wherein the antibody, or the antigen-binding portion thereof, binds to an epitope that is different from an epitope in the N-terminal domain of hCCR8;

[0547] (b) a therapeutic anti-CCR8 antibody, or an antigen-binding portion thereof, which binds to the N-terminal domain of hCCR8; and

[0548] (c) instructions for using the monoclonal antibody or portion thereof and the therapeutic anti-CCR8 antibody or antigen-binding portion thereof in the method of embodiment 57 or 63.

[0549] 100. A kit for treating cancer in a subject, the kit comprising:

[0550] (a) a monoclonal antibody, or an antigen-binding portion thereof, which specifically binds to hCCR8 expressed on the surface of a cell, wherein the antibody, or the antigen-binding portion thereof, binds to an epitope that is different from an epitope in the N-terminal domain of hCCR8;

[0551] (b) a therapeutic anti-CCR8 antibody, or an antigen-binding portion thereof, which binds to the N-terminal domain of hCCR8; and

[0552] (c) instructions for using the monoclonal antibody or portion thereof and the therapeutic anti-CCR8 antibody or antigen-binding portion thereof in the method of embodiment 61 or 65.

[0553] The disclosed amino acid sequence

[0554] Table 3 summarizes the SEQ ID NOs and the corresponding amino acid sequences mentioned in this application.

[0555] Table 3. Summary of SEQ ID NOs and amino acid sequences

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

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Claims

1. A monoclonal antibody or an antigen-binding portion thereof that specifically binds to human CC motif chemokine receptor 8 (hCCR8) expressed on the surface of a cell, wherein the sequence of hCCR8 is as shown in SEQ ID NO: 1, and wherein the antibody or the antigen-binding portion thereof binds to an epitope that is not in the N-terminal domain of hCCR8 and does not cross-compete with an antibody that binds to the N-terminal epitope for binding to hCCR8.

2. The monoclonal antibody or antigen binding portion thereof according to claim 1, wherein the N-terminal epitope comprises at least 15 Y 16 Y 17 P 18 D 19 I 20 F 21 (SEQ ID NO: 2), or comprising an amino acid having sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D19I 20 F 21 S 22 (SEQ ID NO: 73), wherein amino acid Y 15 and / or Y 17 It is sulfated. Optionally, wherein the antibody that binds to the N-terminal epitope is an antibody comprising: (a) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 53; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 54; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 55; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 56; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 57; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as set forth in SEQ ID NO: 58; (b)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO:9; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 16; and / or (c) a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:65; and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:

72.

3. The monoclonal antibody or antigen-binding portion thereof according to claim 1 or 2, which: Contains the following CDR domains as defined by the Kabat method, (a) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 23; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 24; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 25; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 26; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 27; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 28; (b) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 35; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 36; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 37; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 38; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 39; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 40; or (c) a heavy chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 47; a heavy chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 48; a heavy chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 49; a light chain variable region CDR1 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 50; a light chain variable region CDR2 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 51; and a light chain variable region CDR3 comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 22; comprising the following heavy chain variable region and light chain variable region, (d)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 4; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 11; (e)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 6; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 13; or (f)V H , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 8; and V L , which comprises consecutively linked amino acids having a sequence as shown in SEQ ID NO: 15; and / or It contains the following heavy and light chains, (g) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 60; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 67; (h) a heavy chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 62; and a light chain comprising consecutively linked amino acids having a sequence as shown in SEQ ID NO: 69; or (i) a heavy chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:64; and a light chain comprising consecutively linked amino acids having the sequence shown in SEQ ID NO:

71.

4. A labeled antibody or antigen combination thereof, comprising the monoclonal antibody or antigen combination thereof according to any one of the preceding claims and a detectable label, Optionally, wherein the detectable label is a fluorophore, a chromophore, an enzyme, a radioisotope, a micropolymer or a metal, Optionally, wherein the detectable label is biotin, Optionally, wherein the fluorophore is Brilliant Violet TM Dyes (e.g. BV-421), AmCyan dyes, Alexa dye, dye, dyes, fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), phycoerythrin (PE), allophycocyanin (APC), or peridinin-chlorophyll protein (PerCP), Optionally, wherein the chromophore is porphyrin, pyropheophorbide-α, benzoporphyrin monoacid ring A (BPDMA) or chlorin e6, Optionally, wherein the enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase or β-galactosidase, Optionally, wherein 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 Optionally, the metal label is yttrium (Y), indium (In), lanthanide (Ln, from La to Lu, excluding Pm), iodine (i), cadmium (Cd), tellurium (Te), silver (Ag), palladium (Pd), rhodium (Rh), iridium (Ir), platinum (Pt), ruthenium (Ru), osmium (Os) or bismuth (Bi).

5. An immunoconjugate comprising a monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3 linked to a cytolytic agent, Optionally, wherein the lytic agent is a cytotoxin, a radioisotope or a photosensitizer; or A chimeric antigen receptor (CAR) comprising the monoclonal antibody, or antigen binding portion thereof, of any one of claims 1 to 3; or A T cell receptor (TCR) comprising the monoclonal antibody, or antigen binding portion thereof, of any one of claims 1 to 3; or A bispecific molecule comprising the monoclonal antibody, or antigen binding portion thereof, of any one of claims 1 to 20 linked to a binding domain having a different binding specificity than the monoclonal antibody, or antigen binding portion thereof.

6. A composition comprising: (a) the monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 3; (b) the labeled antibody or antigen-binding portion thereof of claim 4; (c) the immunoconjugate of claim 5; (d) the CAR of claim 5, (e) the TCR of claim 5, or (f) the bispecific molecule of claim 5, and a pharmaceutically acceptable carrier.

7. An isolated nucleic acid encoding the monoclonal antibody or antigen binding portion thereof of any one of claims 1 to 3; or An expression vector comprising the nucleic acid; or A host cell comprising the expression vector; or A method for preparing an anti-CCR8 antibody or an antigen-binding portion thereof, comprising expressing the antibody or the antigen-binding portion thereof in a host cell and isolating the antibody or the antigen-binding portion thereof from the host cell.

8. A method for generating a first antibody that does not bind or does not cross-compete with a second antibody for binding to a defined epitope on an antigen, comprising immunizing a vertebrate with an immunogen, wherein the immunogen comprises a cell line or a component of the 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 the binding of the second antibody or its antigen-binding portion to the epitope will shield the epitope from attack by the vertebrate immune system, reduce the generation of antibodies that bind to the epitope, and thereby preferentially result in the generation of a first antibody that does not bind or does not cross-compete with the second antibody for binding to the epitope. Optionally, wherein the antigen comprises a CCR8 receptor, Optionally, wherein the CCR8 receptor is a human CCR8 receptor, Optionally, wherein the vertebrate is a mammal or a bird, Optionally, wherein the mammal is a mouse, rat, hamster, rabbit, dog, goat, sheep or horse, Optionally, wherein the immunogen is a detergent-stabilized proteoliposome component of a cell line, Optionally, wherein the epitope is an epitope in the N-terminal domain of the hCCR8 receptor, Optionally, the second antibody or antigen binding portion thereof is the monoclonal antibody designated L263G8 (BioLegend), the monoclonal antibody designated 433H (BD Biosciences), 4A19, 18Y12, 10R3, 8D55, 14S15 or 14S15h.

9. A method for measuring receptor occupancy (RO) of a cell membrane-bound CCR8 receptor to which a therapeutic Treg-depleting anti-CCR8 antibody binds, the method comprising: (a) adding a saturating concentration of a therapeutic antibody to a background whole blood sample previously exposed to various concentrations of the therapeutic antibody; (b) incubating aliquots of free, bound or fluorescence minus one (FMO) samples with buffer without therapeutic antibody; (c) staining samples with a core panel of antibodies to identify basal T cell markers, T cell differentiation markers, and Treg markers; (d) Join the core group (i) For direct / total RO assays, anti-idiotypic antibodies to detect bound CCR8 receptors, or (ii) for an indirect / total assay, two additional antibodies comprising an allophycocyanin (APC)-conjugated anti-hCCR8 antibody that competes with the therapeutic antibody to detect free CCR8 receptors and an anti-hCCR8 antibody that does not compete with the therapeutic antibody to detect total CCR8 receptors; (e) lysing red blood cells to clean the sample and analyzing the cleaned sample by flow cytometry; as well as (f) Determine the %RO for each concentration of the therapeutic antibody using the following formula: %RO=100×[1-((free amount after administration / free amount before administration) / (total amount after administration / total amount before administration))].

10. A method for measuring the depletion of the frequency of cells expressing CCR8 in the peripheral blood of a subject receiving anti-CCR8 mAb treatment, the method comprising: (a) determining a baseline level of CCR8 expression and / or frequency of CCR8 expressing cells in a first sample of whole blood or PBMCs from a subject; (b) administering treatment with an anti-CCR8 mAb to the subject; and (c) determining the level of CCR8 expression and / or the frequency of CCR8 expressing cells in a second sample of whole blood or PBMC taken from the subject after administration of the anti-CCR8 mAb; Wherein a decrease in the level of CCR8 expression and / or a decrease in the frequency of CCR8 expressing cells in the second sample indicates that the number of CCR8 expressing cells in the blood has been depleted.

11. A method for measuring the depletion of the number of tumor-infiltrating CCR8-expressing Tregs in a subject receiving treatment, the method comprising: (a) determining a baseline level of CCR8 expression and / or frequency of CCR8 expressing Tregs in the subject or in a first sample of a test tissue from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs; (b) administering treatment with an anti-CCR8 antibody to the subject; as well as (c) determining the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs in the subject or in a second sample of a test tissue from the subject during or after treatment; wherein a decrease in the level of CCR8 expression and / or a 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, Optionally, wherein depletion of the number of Tregs in the test tissue indicates an enhancement of an immune response in the subject, Optionally, wherein the treatment administered to the subject is treatment of cancer, Optionally, wherein the treatment of cancer comprises administration of a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof, and optionally, wherein the therapeutic Treg-depleting antibody, or an antigen-binding portion thereof, is an anti-CCR8 antibody, or an antigen-binding portion thereof.

12. A method for predicting the effectiveness of a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof, in treating cancer in a subject, the method comprising: (a) determining the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue 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; and (c) predicting the effectiveness of a therapeutic Treg-depleting antibody or antigen-binding portion thereof, wherein the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs exceeds a threshold value indicates that the therapeutic antibody, or antigen-binding portion thereof, will be effective in treating the subject, and Wherein the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs is below a threshold value, indicating that the therapeutic antibody, or antigen-binding portion thereof, will not be able to effectively treat the subject.

13. A method for predicting the effectiveness of a therapeutic Treg-depleting antibody, or an antigen-binding portion thereof, in treating cancer in a subject, the method comprising: (a) determining the level of CCR8 surface expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue 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 whether the frequency of CCR8 expressing Tregs is reduced following administration of a therapeutic Treg depleting antibody or antigen binding portion thereof; and (d) predicting that the therapeutic Treg-depleting antibody, or antigen-binding portion thereof, will be effective in treating cancer in the subject if the frequency of CCR8-expressing Tregs is reduced by more than a predetermined threshold, or (e) if the frequency of CCR8-expressing Tregs decreases below a predetermined threshold, predicting that the therapeutic Treg-depleting antibody, or antigen-binding portion thereof, will not be effective in treating cancer in the subject.

14. A method for treating cancer in a subject, the method comprising: (a) selecting a subject who is a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody or antigen-binding portion thereof, the selecting comprising: (i) determining the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue from the subject, the test tissue comprising tumor cells and tumor infiltrating Tregs; (ii) comparing the level of CCR8 expression and / or the frequency of CCR8 expressing Tregs with a predetermined threshold; and (iii) selecting the subject as a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody, or antigen-binding portion thereof, based on an assessment that the level of CCR8 expression and / or the frequency of CCR8-expressing Tregs in cells of the test tissue exceeds a predetermined threshold; and (b) administering to the selected subject a composition comprising a therapeutically effective amount of the therapeutic Treg-depleting antibody, or antigen-binding portion thereof.

15. A method for treating a subject suffering from cancer, the method comprising: (a) selecting a subject who is a suitable candidate for cancer immunotherapy with a therapeutic Treg-depleting antibody or antigen-binding portion thereof, the selecting comprising: (i) determining the level of surface expression of CCR8 and / or the frequency of CCR8 expressing Tregs in the subject or in a test tissue taken from the subject, the test tissue 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 the frequency of CCR8 expressing Tregs is reduced following administration of a therapeutic Treg depleting antibody or antigen binding portion thereof; and (iv) selecting the subject as a suitable candidate for immunotherapy with a therapeutic agent based on the assessment that the frequency of CCR8-expressing Tregs is reduced by more than a predetermined threshold; and (b) administering to the selected subject a composition comprising a therapeutically effective amount of the therapeutic Treg-depleting antibody, or an antigen-binding portion thereof, Optionally, the Treg-depleting antibody is an anti-CCR8, anti-CTLA-4, anti-CCR4 or anti-CD25 antibody.

16. The method according to claim 14 or 15, wherein: (a) the cancer is a solid tumor, Optionally, the solid tumor is a cancer selected from the group consisting of squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, esophageal cancer, stomach cancer, gastrointestinal cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, colon carcinoma, anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinonasal natural killer cell, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, carcinoma of the endometrium, fallopian tube cancer, ovarian cancer, carcinoma of the cervix (carcinoma of the endometrium), cervix), vaginal cancer, vulvar cancer, testicular cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, penile cancer, carcinoma of the renal pelvis, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, solid tumors of childhood, environmentally induced cancers, virus-related cancers, cancers of viral origin, advanced cancers, unresectable cancers, metastatic cancers, refractory cancers, recurrent cancers, and any combination thereof; or (b) the cancer is a hematological malignancy, Optionally, the hematological malignancy is selected from 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's lymphoma, lymphoplasmacytoid lymphoma (LPL), hairy cell lymphoma, primary central nervous system (CNS) lymphoma, precursor T-lymphoblastic lymphoma / leukemia, T-lymphoblastic lymphoma / leukemia, T-cell lymphoma / leukemia (T-Lbly / T-ALL), cutaneous T-cell lymphoma, adult T-cell lymphoma / leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer cell / T-cell lymphoma, nasal type, enteropathy-associated intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), nonspecific peripheral T-cell lymphoma, 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 above hematological malignancies.

17. The method of any one of claims 14-16, further comprising administering to the subject a therapeutically effective amount of an additional therapeutic agent for treating cancer, Optionally, wherein the additional therapeutic agent is a compound that reduces suppression of the immune system or increases stimulation of the immune system.

18. A kit for treating cancer in a subject, the kit comprising: (a) a monoclonal antibody, or an antigen-binding portion thereof, which specifically binds to hCCR8 expressed on the surface of a cell, wherein the antibody, or the antigen-binding portion thereof, binds to an epitope that is different from an epitope in the N-terminal domain of hCCR8; (b) a therapeutic anti-CCR8 antibody, or an antigen-binding portion thereof, which binds to the N-terminal domain of hCCR8; and (c) instructions for using the monoclonal antibody or portion thereof and the therapeutic anti-CCR8 antibody or antigen-binding portion thereof in the method of any one of claims 14 to 17.

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