Anti-CCR8 antibodies for treating cancer

Abstract

The present disclosure provides isolated antibodies, e.g., monoclonal antibodies, that specifically bind to CC motif chemokine receptor 8 (CCR8) expressed on the surface of cells and mediate depletion of CCR8-expressing cells by antibody-dependent cellular cytotoxicity (ADCC). The present disclosure provides methods for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an anti-CCR8 antibody, either as monotherapy or in combination with an anti-cancer agent, such as an immune checkpoint inhibitor, e.g., an anti-PD-1 or anti-PD-L1 antibody.

Description

[Technical Field]

[0001] Throughout this application, various publications are referenced in parentheses by author name and date, or by patent number or patent publication number. The complete sources of these publications will be found at the end of this specification immediately preceding the claims. Thus, the disclosures of these publications are incorporated into this application in their entirety by reference to more completely describe the latest art known to those skilled in the art as of the date of the invention described herein and claimed. However, these disclosures are incorporated into this application only to the extent that there is no conflict between the information incorporated by reference and the information provided by explicit disclosure in this application. Furthermore, references to references in this specification should not be construed as an admission that such references are prior art of the invention.

[0002] Cross-reference of related applications This application claims the benefits of U.S. Provisional Applications No. 63 / 157,618, filed March 5, 2021, No. 63 / 041,992, filed June 21, 2020, and No. 62 / 993,570, filed March 23, 2020, the entire contents of which are incorporated herein by reference.

[0003] Sequence List This application includes a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on 19 March 2021, named 20210319_SEQL_13358WOPCT.txt, and is 81,920 bytes in size.

[0004] The disclosed invention relates to an isolated antibody (Ab), such as a monoclonal antibody (mAb), that specifically binds to CC motif chemokine receptor 8 (CCR8), and a method for treating cancer in a subject, comprising administering the anti-CCR8 Ab to the subject as monotherapy or in combination with an anticancer agent such as an immune checkpoint inhibitor. [Background technology]

[0005] Human cancers encompass numerous genetic and epigenetic alterations that generate novel antigens potentially recognizable by the immune system (Chakravarthi et al., 2016). Leveraging the cancer-treating properties of the adaptive immune system makes immunotherapy unique among all cancer treatment modalities, due to its applicability to a wide range of cancers and its ability to induce sustained antitumor effects.

[0006] Considerable success has already been achieved in treating a variety of solid tumors and hematological malignancies by stimulating cytotoxic T cell activity with checkpoint inhibitors such as the anti-PD-1 antibody nivolumab [OPDIVO®] and the anti-CTLA-4 antibody ipilimumab [YERVOY®]. However, typically, less than 15% of patients with cancers for which this treatment is possible benefit from checkpoint inhibitor therapy in the long term (Haslam and Prasad, 2019), and checkpoint inhibitors have proven to be relatively ineffective in certain cancers, including breast and prostate cancer. The persistence of immunosuppressive mechanisms, particularly those mediated by regulatory T cells (Tregs), may contribute to the observed resistance to checkpoint inhibitor therapy in certain cancers or in certain patients (Fares et al., 2019, Han et al., 2019). As one means of overcoming this resistance, this application discloses a method of stimulating the immune system by reducing the immunosuppressive effect of Treg.

[0007] CD4+ Tumor-infiltrating CD4, a subset of T cells + CD25 + FOXP3 + Tregs are mediators of immunological self-tolerance. Deficiencies in genes involved in Treg development and function result in systemic autoimmunity in both mice (Fontenot et al., 2003, Khattri et al., 2003, Tivol et al., 1995) and humans (Yagi et al., 2004, Kuehn et al., 2014), highlighting the crucial and essential role of Tregs in maintaining immune homeostasis. Tregs suppress the immune system through multiple mechanisms, including downregulation of effector T cell induction and proliferation, secretion of chemokines and suppressive cytokines, and inhibition of dendritic cell maturation and function (Shitara and Nishikawa, 2018, Han et al., 2019). Mechanisms that promote self-tolerance utilized by Tregs can be incorporated into the tumor microenvironment to suppress the anti-tumor immune response. In fact, systemic depletion of Tregs in mice is sufficient to enable immune-mediated tumor reduction (Teng et al., 2010). Therefore, Tregs are thought to play a role in mediating peripheral immune tolerance to autoantigens, preventing autoimmune diseases, and suppressing antitumor immune responses.

[0008] As a result, reducing the activity or number of tumor-infiltrating Tregs has been identified as an attractive method for reversing immunosuppressive activity in the tumor microenvironment and enhancing anti-tumor immunity (Finotello and Trajanoski, 2017, Han et al., 2019). Various methods targeting Tregs in cancer immunotherapy, such as ADCC-mediated depletion of Tregs using antibodies (Abs) against antigens expressed on Tregs, including CD25 (Arce Vargas et al., 2017), CCR4 (Ishida et al., 2012, Hagemann et al., 2014), and CTLA-4 (Korman et al., 2017), as well as inhibition of the E3 ubiquitin ligase Siah2 (Scortegagna et al., 2020) and Yes-related proteins (YAP, Ni et al., 2018), have been and continue to be pursued. However, clinical attempts to specifically target or deplete Tregs derived from the tumor microenvironment have been unsuccessful. Diphtheria toxin fused with IL-2 (denileukin difuticotox) failed to effectively reduce Treg numbers in melanoma patients (Luke et al., (2016)), and despite the demonstration of anti-CTLA-4 mediated Treg depletion in mouse tumor models (Selby et al., 2013, Simpson et al., 2013), there is no clear evidence of Treg depletion by ipilimumab or tremelimumab (anti-human CTLA-4 Ab) in human cancer (Sharma et al., 2019a, Sharma et al., 2019b). Treg depletion was achieved using mogamulizumab, a non-fucosylated (nf) anti-CCR4 Ab, but not the conventional CD4 + Severe T cell depletion and CD8 + A moderate decrease in T cell count has also been observed (Kurose et al., 2015), limiting its usefulness in treating solid tumors. Therefore, there remains a need for a safe and effective Treg depletion agent that also preserves effector T cells (Teff) for an optimal antitumor response.

[0009] CCR8 has been recently identified as a potential specific marker for tumor-infiltrating Tregs (Plitas et al., 2016, De Simone et al., 2016, Wang et al., 2019) because CCR8 expression is selectively upregulated in these Tregs in multiple cancers including breast, colorectal, and lung, and as a central member of the IRF4-dependent "effector" Treg gene program (Alvisi et al., 2020). These CCR8 + Tregs are a highly activated suppressive subset of Tregs, with high amounts of CCR8 in these tumor types + Tregs are associated with poor prognosis (Wang et al., 2019, De Simone et al., 2016). Therefore, CCR8 could be a promising therapeutic target to deplete tumor-resident Tregs and enhance anti-tumor immunity. Targeting CCR8 may also have the advantage of depleting suppressive Tregs while not depleting cytolytic effector cells that drive anti-tumor immune responses. Furthermore, since CCR8 is rarely expressed on Tregs and Teffs in peripheral blood or other tissues, targeting CCR8 + may result in a minimal risk of toxicity.

[0010] CCR8 is within tumor FOXP3 hiCCR8 is a seven-transmembrane G protein-coupled chemokine receptor (GPCR) primarily expressed in Treg cells (Wang et al., 2019, Plitas et al., 2016, De Simone et al., 2016). The N-terminal and ECL2 regions of CCR8 are crucial for binding to CC motif chemokine ligand 1 (CCL1), a functional ligand of CCR8 produced by tumor myeloid cells and T cells. In addition to influencing the migration of Treg cells into the tumor microenvironment (TME), the CCL1:CCR8 interaction also enhances the immunosuppressive capacity of Treg cells by upregulating CCR8, FOXP3, IL-10, and other inhibitory factors (Vila-Caballer et al., 2019).

[0011] U.S. Patent No. 10,087,259, granted in recent years, claims a method of treating cancer by administering an anti-CCR8 agent to cancer patients such that tumor-infiltrating Treg cells are specifically depleted to a greater extent than normal tissue-infiltrating T cells in cancer patients. However, this patent does not exemplify any anti-CCR8 agents, nor does it demonstrate any method of treating cancer by administering anti-CCR8 agents.

[0012] U.S. Patent No. 10,550,191 claims a method for treating cancer, comprising administering an Ab against CCR8. Its examples demonstrate that a single Ab, a commercially available rat IgG2b anti-mouse CCR8 (anti-mCCR8) Ab (clone SA214G2; BioLegend, San Diego, CA), reduces the volume of various tumors in a mouse tumor model. No anti-human CCR8 (anti-hCCR8) Abs, nor any chimeric, humanized, or human Abs suitable for use in human therapy are disclosed.

[0013] International Publication No. 2018 / 112033 relates to a method for treating cancer by administering a drug that induces cytotoxicity in tumor-infiltrating Treg cells expressing specified gene products included in Table 1 or 2, thereby reducing the number or activity of tumor-infiltrating Treg cells in the subject. Although CCR8 is not a gene product included in Table 1 or 2, Example 8 demonstrates moderate levels of antitumor activity of anti-mCCR8 Ab in a mouse MC38 colon adenocarcinoma model.

[0014] International Publication No. 2019 / 157098 relates to an immunogenic composition comprising a recombinant Listeria strain and anti-CCR8 Ab, and a method for treating tumors in a subject, comprising administering the immunogenic composition to the subject. Similar to U.S. Patent No. 10,550,191, WO2019 / 157098 does not report the generation of any anti-CCR8 Ab, but instead demonstrates the use of commercial SA214G2 anti-mCCR8 Ab in combination with Listeria-based immunotherapy to treat transplanted colon cancer tumors in a mouse model.

[0015] Furthermore, in recent years, various groups have reported the generation of humanized anti-CCR8 Ab for use in depleting tumor-associated Tregs and treating cancer (see, for example, Depis et al., 2020, WO2020 / 138489, Harbour BioMed, 2020).

[0016] The inventions disclosed herein demonstrate that CCR8 expression is highly restricted to tumor Tregs derived from diverse tumor types. The invention includes the production of anti-CCR8 mAbs, specifically human, humanized, and chimeric anti-hCCR8 mAbs, and demonstrates that anti-CCR8-mediated Treg depletion in mouse tumor models requires Fc binding. The disclosure also describes the development of nf anti-hCCR8 mAbs that mediate tumor-specific Treg depletion in ex vivo human tumor culture systems. Anti-CCR8 mA treatment, either as monotherapy or in combination with checkpoint blockers, e.g., inhibition of the PD-1 / PD-L1 signaling pathway, can induce potent antitumor responses and provide clinical benefit to patients who do not respond to anti-PD-1 monotherapy. The combination of anti-CCR8-mediated Treg depletion and checkpoint blockers in their mechanisms of action increases tumor cell death, thereby offering a unique opportunity to treat a broad range of cancers. [Overview of the project] [Means for solving the problem]

[0017] The present invention provides an isolated Ab, preferably an mAb, that specifically binds to CCR8 expressed on the surface of cells, such as human CCR8 (hCCR8), and exhibits various functional properties, including desirable properties for therapeutic Ab. These properties include high affinity for tumor infiltration and activation of CD4. + FOXP3 high It binds to CCR8-expressing cells such as Treg cells; aside from Treg cells, it binds only to rare, scattered immune cells in the thymic medulla and dermis of the skin, and does not bind to many other tissues; ADCC-activated CD4 in tumor infiltration. + FOXP3 high It mediates the depletion of CCR8-expressing cells such as Tregs; it specifically mediates the depletion of tumor-infiltrating Tregs and CCR8 in normal tissues. +It does not mediate T cell depletion; it inhibits CCR8 / CCL1 signaling by inhibiting the binding of CCL1 and CCR8; when it binds to CCR8 on the cell surface, it does not cause internal migration of CCR8 in the presence or absence of cross-linking Ab; and it inhibits CCR8 in in vitro and human tumor ex vivo tissue samples. + These include mediating the specific depletion of Treg cells; and inhibiting the growth of tumor cells in a subject, preferably a human subject, when administered to the subject as monotherapy or in combination with another anticancer agent. In a preferred embodiment, the anti-CCR8 Ab is a modified mAb containing a modified heavy chain constant region, such as a low-fucosylated or non-fucosylated (nf) heavy chain constant region, which binds to the Fcγ receptor (FcγR) with higher affinity compared to the unmodified mAb and mediates enhanced ADCC.

[0018] Specifically, this disclosure provides an isolated Ab, preferably an mAb, or its antigen-binding moiety, which specifically binds to CCR8 expressed on the surface of cells and mediates the depletion of CCR8-expressing cells by ADCC. In certain embodiments, CCR8 is hCCR8 having the amino acid sequence described in SEQ ID NO: 1. In certain other embodiments, the Ab, e.g., an mAb, or its antigen-binding moiety comprises a heavy chain constant region of a human IgG1 or IgG3 isotype.

[0019] This disclosure also provides a modified anti-hCCR8 mAb or its antigen-binding moiety, which includes a modified heavy chain constant region that binds to the Fcγ receptor (FcγR) with higher affinity compared to an unmodified mAb or its antigen-binding moiety and mediates enhanced ADCC. In preferred embodiments, the modified mAb or its antigen-binding moiety includes a modified IgG1 heavy chain constant region exhibiting reduced fucosylation. In certain embodiments, the mAb or its antigen-binding moiety binds to the N-terminal peptide of hCCR8, where the epitope is sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21The mAb comprises at least one amino acid in the peptide having (SEQ ID NO: 2), and further comprises a sulfated tyr-15 and / or sulfated tyr-17 residue. In certain preferred embodiments, the mAb or its antigen-binding moiety is bound to the N-terminal peptide of hCCR8, where the epitope is sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It comprises at least one amino acid in the peptide having (SEQ ID NO: 109), and further comprises sulfated tyr-15 and sulfated tyr-17 residues.

[0020] In certain embodiments, the anti-CCR8 mAb or its antigen-binding moiety has the following characteristics: (a) EC of about 1 nM or less 50 or approximately 2nM or less EC 50 (b) It specifically binds to CCR8 expressed on the surface of cells; (c) It binds to rare, scattered immune cells in the thymic medulla and dermis of the skin, but does not bind to any of the human brain, cerebellum, heart, liver, lung, kidney, tonsil, spleen, thymus, colon, stomach, pancreas, adrenal gland, pituitary gland, skin, peripheral nerves, testes, or uterine tissue, or peripheral blood mononuclear cells (PBMCs); (d) It has an IC of approximately 5 nM or less. 50 (d) inhibits CCR8 / CCL1 signaling by inhibiting the binding of CCL1 to CCR8, and when it binds to CCR8 on the cell surface, the EC is approximately 10 pM or less. 50 or EC of approximately 60 pM or less. 50 (e) Mediates the depletion of cells, when administered to the target, mediates the depletion of tumor-infiltrating Tregs, but CCR8 in the spleen, blood, skin, or thymus +(f) substantially preserving T cells; (g) not causing internal translocation of CCR8 in the presence or absence of cross-linked Ab when bound to CCR8 on the cell surface; (h) inhibiting tumor cell growth in the subject when administered to the subject as monotherapy; and (h) inhibiting tumor cell growth in the subject when administered to the subject in combination with additional therapeutic agents such as immune checkpoint inhibitors to treat cancer, exhibiting at least one of these, for example, at least two, three, four, five, six, seven, or all of the following.

[0021] In particular, in certain preferred embodiments, the anti-CCR8 mAb or its antigen-binding moiety has at least the following characteristics: (a) EC of about 1 nM or less 50 or approximately 2nM or less EC 50 (b) Specifically binds to CCR8 expressed on the cell surface, and (b) IC2 is about 5 nM or less. 50 (c) inhibits the binding of CCL1 to CCR8 and thereby inhibits CCR8 / CCL1 signaling, and (c) when bound to CCR8 on the cell surface, the EC is approximately 10 pM or less. 50 or EC of approximately 60 pM or less. 50 (d) mediates the depletion of cells, and when administered to the target, mediates the depletion of tumor-infiltrating Tregs, but CCR8 in the spleen, blood, skin, or thymus. + (e) effectively preserves T cells, inhibits tumor cell growth in the subject when administered as monotherapy, and (f) inhibits tumor cell growth in the subject when administered in combination with additional therapeutic agents such as immune checkpoint inhibitors to treat cancer.

[0022] In other embodiments, the anti-CCR8 mAb or its antigen-binding moiety has at least the following characteristics: (a) EC of about 1 nM or less 50 or approximately 2nM or less EC 50(b) Specifically binds to CCR8 expressed on the cell surface, and when bound to CCR8 on the cell surface, the EC is approximately 10 pM or less. 50 or EC of approximately 60 pM or less. 50 (c) Mediates the depletion of cells, and when administered to the target, mediates the depletion of tumor-infiltrating Tregs, but CCR8 in the spleen, blood, skin, or thymus. + (d) It effectively preserves T cells and, when administered to a subject in combination with additional therapeutic agents such as immune checkpoint inhibitors to treat cancer, inhibits the growth of tumor cells in the subject.

[0023] These properties were studied in detail in certain Abs of the present invention, including, for example, those referred to herein as 14S15 and 4A19. mAb 4A19 is an nf humanized Ab, and mAb 14S15 is an nf chimeric Ab containing a mouse Fab fragment transplanted into a human Fc region. Some of the assays described herein were performed using mAbs including 14S15 and 4A19. The 14S15 mAb was then humanized by modifying its framework sequence to correspond to the nearest human germline sequence, generating an Ab referred to as 14S15h (see Example 10). The final replicate of the humanized mAb, produced after affinity maturation of the heavy chain variable domain, exhibited a binding affinity to hCCR8 comparable to, in fact slightly higher than, that of the original mouse or chimeric form of this Ab (1.4 nM for the starting mouse mAb versus 0.64 nM for the 14S15h Fab fragment). D Therefore, 14S15h is expected to exhibit the same or very similar functional characteristics as demonstrated in the chimeric 14S15 mAb. Thus, the above functional characteristics may exist, individually or in combination, along with the structural features of mAbs 14S15, 14S15h, and 4A19.

[0024] For example, in a particular embodiment of the present invention, Ab or its antigen-binding moiety may include one or more of the above characteristics (for example, at least two, three, four, five, or six of the above characteristics), and V may include a sequence of continuously linked amino acids having the sequence described in Sequence ID No. 4. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L Each of these may contain the CDR1, CDR2, and CDR3 domains. As another example, such an Ab or its antigen-binding moiety may contain the following CDR domains as defined by the Kabat method: V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 33. H V contains a sequence of continuously linked amino acids having the sequence described in CDR1, SEQ ID NO: 34. H V contains continuously linked amino acids having the sequence described in CDR2, SEQ ID NO: 35. H V contains a sequence of continuously linked amino acids having the sequence described in CDR3, SEQ ID NO: 36. L V contains a sequence of continuously linked amino acids having the sequence described in CDR1, SEQ ID NO: 37. L V contains a sequence of amino acids having the sequence described in CDR2 and SEQ ID NO: 38. L It may contain CDR3. As a further example, such an Ab or its antigen-binding moiety may contain a V containing sequentially linked amino acids having the sequence described in SEQ ID NO: 4. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L It may include. As another example, such Ab may include a heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 100, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 112. Ab may have reduced fucosylation in its heavy chain, as described elsewhere in this specification, or it may have a low-fucosylated or non-fucosylated heavy chain constant region.

[0025] For example, in certain other embodiments of the present invention, Ab or its antigen-binding moiety may include one or more of the above properties (for example, at least two, three, four, five, or six of the above properties), and V may include a sequence of linked amino acids having the sequence described in Sequence ID No. 115. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L Each of these may contain the CDR1, CDR2, and CDR3 domains. As another example, such an Ab or its antigen-binding moiety may contain the following CDR domains as defined by the Kabat method: V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 103. H V contains a sequence of continuously linked amino acids having the sequence described in CDR1, SEQ ID NO: 104. H V contains a sequence of continuously linked amino acids having the sequence described in CDR2, SEQ ID NO: 105. H V contains a sequence of continuously linked amino acids having the sequence described in CDR3, SEQ ID NO: 106. L V contains a sequence of amino acids having the sequence described in CDR1, SEQ ID NO: 107. L V contains a sequence of amino acids having the sequence described in CDR2 and SEQ ID NO: 108. L It may contain CDR3. As a further example, such an Ab or its antigen-binding moiety may contain a V containing sequentially linked amino acids having the sequence described in SEQ ID NO: 115. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L It may include. As another example, such an Ab may include a heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 117, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 118. Ab may have reduced fucosylation in its heavy chain, as described elsewhere in this specification, or it may have a low-fucosylated or non-fucosylated heavy chain constant region.

[0026] For example, in certain other embodiments of the present invention, Ab or its antigen-binding moiety may include one or more of the above properties (for example, at least two, three, four, five, or six of the above properties), and V may include a sequence of continuously linked amino acids having the sequence described in Sequence ID No. 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L Each of these may contain the CDR1, CDR2, and CDR3 domains. As another example, such an Ab or its antigen-binding moiety may contain the following CDR domains as defined by the Kabat method: V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 45. H V contains a sequence of continuously linked amino acids having the sequence described in CDR1, SEQ ID NO: 46. H V contains continuously linked amino acids having the sequence described in CDR2, SEQ ID NO: 47. H V contains a sequence of continuously linked amino acids having the sequence described in CDR3, SEQ ID NO: 48. L V contains a sequence of continuously linked amino acids having the sequence described in CDR1, SEQ ID NO: 49. L V contains a sequence of amino acids having the sequence described in CDR2 and SEQ ID NO: 50. L It may contain CDR3. As a further example, such an Ab or its antigen-binding moiety may contain a V containing sequentially linked amino acids having the sequence described in SEQ ID NO: 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L It may include. As another example, such an Ab may include a heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 114. Ab may have reduced fucosylation in its heavy chain, as described elsewhere in this specification, or it may have a low-fucosylated or non-fucosylated heavy chain constant region.

[0027] This disclosure specifically binds to hCCR8 expressed on the surface of cells and includes the following combination of heavy and light chain variable regions: (a) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 3 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 15 L , (b) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 4 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 16 L , (c) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 5 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 17 L , (d) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 6 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 18 L , (e) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 7 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 19 L , (f) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 8 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 20 L , (g) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 9 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 21 L , (h) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 10 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 22 L , (i) V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 11 H and V comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 23 L , (j) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 12 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 24. L , (k) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 13 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 25. L , (l) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 14 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 26. L ,or (m) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L The present invention further provides isolated Abs, preferably mAbs, or their antigen-binding moieties, each comprising the CDR1, CDR2, and CDR3 domains.

[0028] It specifically binds to hCCR8 expressed on the cell surface and follows the following combination of heavy and light chain variable regions: (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L , and (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L An isolated Ab, preferably an mAb, or its antigen-binding moiety, containing the CDR1, CDR2, and CDR3 domains in each of these is a specific example.

[0029] It specifically binds to hCCR8 expressed on the cell surface and follows the following combination of heavy and light chain variable regions: (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L In this context, an isolated Ab, preferably an mAb, or its antigen-binding moiety, containing the CDR1, CDR2, and CDR3 domains, is a specific example.

[0030] The sequences of CDRs can be defined in various ways, including by Kabat, Chothia, AbM, contact, and IMGT definitions. Unless otherwise explicitly indicated, CDRs in this disclosure are identified by the Kabat definition.

[0031] This disclosure also provides isolated nucleic acids encoding any of the anti-CCR8 mAbs or their antigen-binding moieties as described herein. This disclosure provides an expression vector comprising the isolated nucleic acid and a host cell comprising the expression vector. The host cell may be used in a method for preparing an anti-CCR8 mAb or its antigen-binding moiety, comprising expressing the mAb or its antigen-binding moiety in the host cell and isolating the mAb or its antigen-binding moiety from the host cell.

[0032] In certain embodiments, the Disclosure provides a method for treating a subject affected by cancer, comprising administering to the subject a therapeutically effective dose of, for example, an anti-CCR8 mAb or antigen-binding moiety described herein that mediates the depletion of CCR8-expressing cells, so that the subject is treated. In other embodiments, the Disclosure provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective dose of, for example, an anti-CCR8 mAb or antigen-binding moiety described herein that mediates the depletion of CCR8-expressing cells, so that the growth of tumor cells in the subject is inhibited. In certain embodiments of these methods, the anti-CCR8 mAb, when bound to CCR8 on the surface of cells, has an EC of about 10 pM or less. 50 This mediates cell depletion. In certain other embodiments, the anti-CCR8 mAb, when bound to CCR8 on the cell surface, has an IC50 of about 5 nM or less. 50 This inhibits the binding of CCL1 to CCR8 and thereby inhibits CCR8 / CCL1 signaling. In additional embodiments, the Disclosure provides anti-CCR8 mAbs or antigen-binding moieties described herein for use in methods for treating subjects affected by cancer or for use in methods for inhibiting the growth of tumor cells in subjects, for example, by mediating the depletion of CCR8-expressing cells, each method comprising administering a therapeutically effective amount of the anti-CCR8 mAb or antigen-binding moiety to a subject.

[0033] This disclosure further provides a method for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective dose of (a) an anti-CCR8 Ab, e.g., an mAb, or its antigen-binding moiety described herein, which mediates the depletion of, for example, CCR8-expressing cells, and (b) an additional therapeutic agent for treating cancer, wherein the additional therapeutic agent may be a compound that suppresses or increases the stimulation of the immune system. In certain preferred embodiments, the additional therapeutic agent is an antagonist Ab or its antigen-binding moiety that specifically binds to PD-1, PD-L1, or CTLA-4. The method may also be a method for inhibiting the growth of tumor cells in the subject.

[0034] When such methods involve the use of combinations of therapeutic agents, they can be referred to in various ways. For example, if a treatment method uses drugs (A) and (B) to treat cancer, this can be referred to as (i) drugs (A) and drugs (B) for use in a method of treating cancer, (ii) drug (A) used together with drug (B) in a method of treating cancer, or (iii) drug (B) used together with drug (A) in a method of treating cancer. Thus, the above combinations are (i) A method for treating a subject affected by cancer or a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective dose of a combination of (A) an anti-CCR8 mAb or its antigen-binding moiety that mediates the depletion of, for example, CCR8-expressing cells, and (B) an additional therapeutic agent for treating cancer, for use in such a method, (A) an anti-CCR8 mAb or its antigen-binding moiety that mediates the depletion of CCR8-expressing cells, and (B) an additional therapeutic agent for treating cancer, or (ii) A method for treating a subject having cancer or a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of a combination of (A) an anti-CCR8 mAb or its antigen-binding moiety that mediates the depletion of, for example, CCR8-expressing cells and (B) an additional therapeutic agent for treating cancer, wherein (B) (A) an anti-CCR8 mAb or its antigen-binding moiety that mediates the depletion of, for example, CCR8-expressing cells used together with the additional therapeutic agent for treating cancer, (iii) A method for treating a subject having cancer or a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of (B) an additional therapeutic agent for treating cancer and (A) a combination of an anti-CCR8 mAb or its antigen-binding moiety that mediates the depletion of CCR8-expressing cells, wherein (A) an anti-CCR8 mAb or its antigen-binding moiety that mediates the depletion of CCR8-expressing cells, and (B) an additional therapeutic agent for treating cancer used in combination with (A) an anti-CCR8 mAb or its antigen-binding moiety that mediates the depletion of CCR8-expressing cells It can be called that.

[0035] The Disclosure also provides a kit comprising (a) an isolated Ab, preferably an mAb, or its antigen-binding moiety, in a single or multiple dose in the range of a fixed dose of about 0.01 to about 20 mg / kg body weight or about 0.1 to about 2,000 mg, which specifically binds to CCR8 expressed on the surface of cells and mediates the depletion of CCR8-expressing cells by ADCC; (b) a mAb or its antigen-binding moiety, optionally in a single or multiple dose in the range of about 200 to about 1600 mg, which specifically binds to PD-1, PD-L1, or CTLA-4; and (c) instructions for using the isolated Ab or its moiety, which specifically binds to CCR8, and optionally the mAb or its moiety, which specifically binds to PD-1, PD-L1, or CTLA-4, in the therapeutic methods disclosed herein.

[0036] Other features and advantages of the present invention will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references cited throughout this application, including chemical papers, GenBank entries, patents, and patent applications, are expressly incorporated herein by reference. [Brief explanation of the drawing]

[0037] [Figure 1-1] Figures 1A–1C show the analysis of gene correlations between human CCR8 and FOXP3 in The Cancer Genome Atlas (TCGA). A: A network based on the mutual rank of gene correlations across all non-heme TCGA tumor RNA-seq identifies CCR8 as a Treg selection marker. B: Analysis of hepatocellular carcinoma (HCC) single-cell RNA-seq shows that CCR8 is selectively expressed on FOXP3-high lymphocytes in HCC tumor samples. C: Spearman correlation analysis performed on FOXP3+ T lymphocytes shows that CCR8 expression is associated with higher levels of FOXP3 expression. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 2-1]Figures 2A-2F show that CCR8 expression is concentrated on tumor Tregs compared to other Treg target molecules. A: Flow cytometry analysis of CCR8, CCR4, CTLA-4, and CD25 positivity in Tregs derived from tumors and blood (n=8-18) of cancer patients (colorectal, renal, pulmonary, and melanoma). B: Relative expression levels (MFI) of Treg targets on CD4+FOXP3+ Tregs derived from blood (n=6-10) and tumors (n=7-20) of cancer patients. C-E: Treg target frequencies on CD4+ Tconv derived from tumors (C) and blood (D) of cancer patients, and on CD8+ T cells derived from tumors (E) (n=7-17) and peripheral blood samples (F) (n=5-13). Not all markers were analyzed for all patients, and not all patients had matched blood samples. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. A-B: Two-way ANOVA followed by Bonferroni multiple comparison tests; C-F: One-way ANOVA Kruskal-Wallis test followed by Dunn multiple comparison tests. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 3-1] Figures 3A–3C show flow cytometry analysis demonstrating high expression of CCR8 on tumor-infiltrating Tregs. CCR8 expression levels were measured in different T cell populations. A: Percentage of tumor-infiltrating T lymphocyte subsets expressing CCR8. Tumor-infiltrating Tregs express CCR8 at a significantly higher frequency compared to normal CD4+FOXP3-(CD4 Tconv) T cells and CD8+ T cells (CD8). B: Mean fluorescence intensity (MFI) of PE conjugate anti-CCR8 Ab bound to tumor-infiltrating T lymphocyte subsets. Tumor Tregs also express higher levels of CCR8 per cell compared to CD4 Tconv and CD8 cells. C: Comparison of Treg CCR8 expression levels on CCR8+ Tregs in the blood and tumor tissue of cancer patients. CCR8 expression on Tregs in peripheral blood is lower compared to the significantly higher levels on tumor-infiltrating Tregs. [Figure 3-2]Same as above. [Figure 4-1] Figures 4A–4D show that CCR8 is differentially expressed in different Treg subpopulations. A: Percentage of cells in different T cell populations in PBMCs from healthy subjects expressing CCR8. CCR8 is predominantly expressed on peripheral Tregs. B: Percentage of cells in different Treg subpopulations in healthy subjects expressing CCR8. Within the Treg population, CCR8 is expressed more highly in the effector memory population (EM), less highly in central memory cells (CM), and only slightly in naive Tregs. C: Percentage of cells in normal CD4 Tconv cells expressing CCR8. CCR8 expression is minimal in all of these CD4 Tconv cell subpopulations. D: Mean fluorescence intensity (MFI) of anti-CCR8 Ab bound to CCR8+ Tregs from peripheral blood or tumor-infiltrating Tregs from cancer patients. In cancer patients, CCR8 expression per cell is higher on tumor-infiltrating Tregs than on peripheral blood-derived Tregs. [Figure 4-2] Same as above. [Figure 5] Figure 5 shows that CCR8 is expressed in the most immunosuppressive CD4+FOXP3high Treg population. Tumors from two melanoma patients and one renal cell carcinoma (RCC) patient were dissociated and stained for FOXP3 and CCR8 expression. CCR8 is primarily expressed in the CD4+FOXP3high population, which represents the most activated Tregs. [Figure 6-1] Figures 6A–6D show the percentages of CCR8+ and CCR8- T cells expressing CD25, CD39, and IL1R2. A: Percentage of CCR8+ and CCR8- cells expressing CD25; B: Percentage of CCR8+ and CCR8- cells expressing CD39; C: Percentage of CCR8+ and CCR8- cells expressing IL1R2; D: Percentage of stimulated CCR8+ and CCR8- cells expressing IL1R2. [Figure 6-2] Same as above. [Figure 7-1] Figures 7A–7D show the percentages of CCR4+ and CCR4- T cells expressing CD25, CD39, and IL1R2. A: Percentage of CCR4+ and CCR4- cells expressing CD25; B: Percentage of CCR4+ and CCR4- cells expressing CD39; C: Percentage of CCR4+ and CCR4- cells expressing IL1R2; D: Percentage of stimulated CCR4+ and CCR4- cells expressing IL1R2. [Figure 7-2] Same as above. [Figure 8] Figures 8A and 8B show the percentage of CCR8+ and CCR8- T cells expressing the activation marker HLA-DR (8A), and the percentage of CCR4+ and CCR4- T cells expressing this marker (8B). [Figure 9-1] Figures 9A-9C show the percentages of stimulated CCR8+ and CCR8- T cells expressing IFNγ, IL-2, and granzyme B. A: Percentage of stimulated CCR8+ and CCR8- cells expressing IFNγ; B: Percentage of stimulated CCR8+ and CCR8- cells expressing IL-2; C: Percentage of stimulated CCR8+ and CCR8- cells expressing granzyme B. [Figure 9-2] Same as above. [Figure 10-1] Figures 10A-10C show the percentages of stimulated CCR4+ and CCR4- T cells expressing IFNγ, IL-2, and granzyme B. A: Percentage of stimulated CCR4+ and CCR4- cells expressing IFNγ; B: Percentage of stimulated CCR4+ and CCR4- cells expressing IL-2; C: Percentage of stimulated CCR4+ and CCR4- cells expressing granzyme B. [Figure 10-2] Same as above. [Figure 11-1]Figures 11A and 11B show the binding of different anti-hCCR8 mAbs to hCCR8-expressing cell lines or activated human Tregs. A: Anti-hCCR8 mAbs were generated exhibiting a range of binding affinity (EC50 bins at nM) to hCCR8-transfected cell lines (293F, CHO, Raji) and activated human Tregs. B: EC50 values ​​(nM) of a selected subset of anti-CCR8 mAbs that bind to activated Tregs are shown. [Figure 11-2] Same as above. [Figure 12-1] Figures 12A and 12B show the crystal structures of the 4A19 Fab fragment bound to the hCCR8 N-terminal peptide. A: 4A19 Fab fragment bound to a single sulfated (at tyr-17) hCCR8 N-terminal peptide at a resolution of 2.03 Å. The epitope bound by the 4A19 mAb contains residues 15-21 with a sulfated tyrosine-17 residue at its center; B: 4A19 Fab fragment bound to a double sulfated (at tyr-15 and tyr-17) hCCR8 N-terminal peptide at a resolution of 1.80 Å. The epitope bound by the 4A19 mAb contains residues 12-22. [Figure 12-2] Same as above. [Figure 13] Figures 13A and 13B show the tissue cross-reactivity of different anti-hCCR8 mAbs applied to normal human PBMCs and normal human thymus at 1 μg / ml (top panel) and 3 μg / ml (top panel), respectively. A: Binding of mAb 18Y12 (left panel), 16B13 (center panel), and 4A19 (right panel) to PBMCs. mAb 18Y12 and 4A19 did not bind to PBMCs, while 16B13 showed high levels of binding to targets unlikely to be CCR8. B: Binding of mAb 18Y12 (left panel), 16B13 (center panel), and 4A19 (right panel) to thymic tissue. mAb 18Y12 showed little staining, while positive staining for mAb 4A19 was observed in rare, scattered immune cells in the thymic medulla. MAb 16B13 exhibits strong and widespread staining in a dominant cytoplasmic and / or perinuclear pattern in the majority of immune cells in the thymus. [Figure 14-1] Figures 14A–14C show that anti-hCCR8 mAbs block the binding of human CCL1 (hCCL1) to hCCR8. A: When evaluated by inhibition of Ca2+ flow, anti-hCCR8 mAbs exhibit varying abilities to block the binding of hCCL1 to hCCR8 on hCCR8-transfected CHO cells. B: The percentage of CCL1 signaling (CCL1-induced Ca2+ flow) blockade is shown for seven selected anti-hCCR8 mAbs. C: IC50 curves for Ca2+ flow blockade are shown for four selected anti-CCR8 mAbs. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15-1] Figures 15A and 15B show that anti-hCCR8 mAbs mediate CD16 crosslinking in CD16-expressing reporter cells, reflecting the ADCC potential of anti-CCR8 mAbs. CD16-expressing luciferase reporter cells were co-cultured with CCR8-expressing Raji cells or activated Tregs and treated with anti-CCR8 Abs (with either non-fucosylated (nf) or wild-type hIgG1 backbones). A: Various CD16 crosslinking capabilities were observed. B: CD16 crosslinking using activated Tregs as targets is depicted for a curated set of anti-hCCR8 mAbs with hIgG1-nf backbones. [Figure 15-2] Same as above. [Figure 16] Figure 16 shows that anti-CCR8 Ab mediates the killing of activated Tregs by allogeneic NK cells. Activated Tregs were co-cultured with pre-stimulated allogeneic NK cells and titrated anti-hCCR8 Ab. Cell death was measured by annexin V positivity in the Tregs. [Figure 17]Figures 17A and 17B demonstrate that, in vitro, anti-CCR8 mAbs mediate the killing of patient tumor Tregs by allogeneic NK cells. Enzymatically dissociated patient endometrial tumors were co-cultured with pre-stimulated allogeneic NK cells at 37°C for 24 hours. Anti-CCR8 Ab, 14S15, specifically depleted patient tumor Tregs (Figure 17A) but not normal CD4+ T cells (Figure 17B). In contrast, anti-CCR4 Ab (CCR4) mediated the depletion of both Tregs and normal CD4+ T cells (Figures 17A and 17B). [Figure 18-1] Figures 18A–18I show that anti-hCCR8-hIgG1-nf Ab mediates CCR8+ Treg depletion in an ex vivo patient tumor slice culture system without the addition of allogeneic NK cells. A–C: Representative results for depletion of peripheral blood Tregs (A), CD4+ Tconv (B), and CD8+ T cells (C) in vitro by treatment with 14S15-IgG1-nf or anti-hCR4-IgG1-nf. D–F: Representative plots from allogeneic NK cell killing assays of non-small cell lung cancer (NSCLC) tumors comparing 14S15-IgG1-nf (D), anti-hCCR4-IgG1-nf (E), and isotype (F)Ab. A–F: 14S15-IgG1-nf, black circles; anti-hCCR4-IgG1-nf, squares; isotype IgG1-nf, white circles. G: Co-culture of allogeneic NK and NSCLC tumors comparing 16B13-IgG1-nf with anti-hCCR8-inert and isotype control Ab. G: Isotype IgG1-nf, white circle; 14S15-IgG1-nf, black circle; 14S15-IgG1-inert, triangle. H and I: Results from ex vivo primary intact tumor sections from renal cell carcinoma (H) and gastric cancer (I) cultured for 24 hours in the presence of 16B13-IgG1-nf or IgG1-nf isotype control (3-5 technical replicates for each condition). H and I: Isotype IgG1-nf, white circle; 14S15-IgG1-nf, black circle. *P<0.05, ****P<0.0001. Standard one-way ANOVA with Tukey's multiple comparison test (G). Mann-Whitney test (two-sided) (G-I). [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 19] Figure 19 shows that anti-CCR8 mAb does not induce CCR8 internal translocation in activated Tregs, with or without crosslinking. Activated Tregs were incubated with anti-CCR8 mAb 4A19, a positive control mAb (anti-ICOS), and an isotype control, with or without anti-human Fc crosslinked mAbs. CCR8 expression on the Treg surface was evaluated at various time points. [Figure 20-1] Figures 20A-20D show the depletion of tumor Tregs by anti-CCR8 when digested patient tumors were co-cultured in vitro with allogeneic natural killer (NK) cells. A: mAb 4A19 (CCR8-nf) induced a more measurable depletion of patient tumor Tregs than the depletion induced by non-fucosylated anti-hCCR4 mAb (CCR4-nf). B: Conversely, anti-CCR4-nf mAb induced depletion of CD4+ Teff cells, while mAb 4A19 did not. C: Neither anti-CCR8 nor anti-CCR4 depleted CD8+ Teffs. In contrast to D:4A19(CCR8-nf), neither the control anti-keyhole limpet hemocyanin (KLH)-nf mAb (isotype) with an unrelated targeting arm nor the 4A19 mAb (CCR8-inert) with an inactive backbone depleted Treg cells. [Figure 20-2] Same as above. [Figure 21-1] Figures 21A–21D show that anti-CCR8 inhibits the growth of CT26 colon cancer in a mouse model. A: Treatment of CT26 colon cancer with anti-CCR8-mIgG2a, BioLegend rat anti-mCCR8 mAb, and anti-mCCR8 Ab with the mIgG2a isotype derived from clone SA214G2 significantly reduced tumor growth and increased the number of tumor-free mice. Individual tumor volumes are depicted. B: Change in mean tumor volume. C: Tumor Treg depletion was observed with respect to anti-CCR8 treatment, but the number of splenic Tregs was not affected by anti-CCR8 treatment (D). [Figure 21-2] Same as above. [Figure 22-1]Figures 22A-22F show the effects of anti-CCR8 on the T lymphocyte population in mice with CT26 colon adenocarcinoma, as analyzed by flow cytometry. A: Percentages of CD4+FOXP3+ Tregs (Treg), FOXP3-CD4+ effector cells (CD4eff), and CD8+ T (CD8T) cells expressing CCR8 in the spleen, blood, tumor Tregs, and skin. B: Percentages of double-negative CD4-CD8-(DN), single-positive CD4+CD8-(CD4 SP), single-positive CD4-CD8+(CD8 SP), and double-positive CD4+CD8+(DP) thymocytes expressing CCR8. C: Percentages of Foxp3+ Tregs in the spleen, blood, tumor, and skin after treatment with anti-CCR8-mIgG2a mAb and isotype controls. D: Percentage of DP, CD8 SP, CD4 SP, and DN thymocytes in the thymus after treatment with anti-CCR8-mIgG2a and isotype control. E: Percentage of CD4+ T cells in the skin after treatment with anti-CCR8-mIgG2a and isotype control. F: Percentage of CD8+ T cells in the skin after treatment with anti-CCR8-mIgG2a and isotype control. [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 23] Figures 23A and 23B show that anti-CCR8 inhibits the growth of MC38 colon cancer in a mouse model. A: Treatment of MC38 colon cancer with anti-CCR8-mIgG2a significantly reduced tumor growth and increased the number of tumor-free mice. B: Change in mean tumor volume. [Figure 24-1]Figures 24A–24D demonstrate that anti-mCCR8 Ab-induced Treg depletion in the MC38 colon cancer mouse model results in potent monotherapy efficacy and enhanced pharmacodynamic and pharmacokinetic responses. A single dose of anti-mCCR8 mAb leads to a dose-dependent decrease in tumor volume (A), Treg (% Foxp3+CD4+) depletion (B), and an increase in the percentage of tumor-infiltrating CD8+ T cells (C). Error bars indicate the standard deviation of the mean. D: Anti-mCCR8 mAb exhibits non-linear PK in the dosing range of 0.03–3 mg / kg. [Figure 24-2] Same as above. [Figure 25-1] Figures 25A-25C show the effect of the combination of mouse anti-mPD-1 Ab and anti-mCCR8 Ab on tumor growth compared to anti-PD-1 or anti-CCR8 Ab treatment alone, as measured by changes in tumor volume in the MB49 mouse bladder cancer model. A: Anti-CCR8-mIgG2a and anti-PD-1 induce moderate and low levels of tumor growth inhibition, respectively, but the combination shows synergistic efficacy in completely inhibiting tumor growth. B: Treatment with anti-CCR8-mIgG2a in or without anti-PD-1 significantly reduced the frequency of tumor Tregs but increased the frequency of antitumor CD8+ T cells (C). [Figure 25-2] Same as above. [Figure 26] Figure 26 shows the effect of the combination of mouse anti-mPD-1 Ab and mouse anti-mCCR8 Ab on tumor growth compared to anti-PD-1 or anti-CCR8 Ab treatment alone, as measured by changes in tumor volume in a 4T1 mouse breast cancer model. Anti-PD-1 showed no activity in inhibiting tumor growth, and tumor growth closely resembled that in mice treated with the negative control Ab combination, while anti-CCR8-mIgG2a induced a moderate level of tumor growth inhibition. Anti-CCR8 interacted synergistically with anti-PD-1 to almost completely inhibit tumor growth. [Figure 27]Figures 27A and 27B show that anti-mCCR8 Ab, including an inactive Fc constant region, exhibits antitumor activity in a SA1N fibrosarcoma mouse model. A: Treatment with anti-CCR8-mIgG2a potently reduced tumor growth, and all nine mice were tumor-free by day 25 after implantation. Blockade of CCR8 with Fc-inert Ab (anti-CCR8-mIgG1-D265A) partially reduced tumor growth. B: Depletion of tumor Tregs by anti-CCR8 treatment was achieved with anti-CCR8-mIgG2a treatment, but not with Fc-inert anti-CCR8-mIgG1-D265A. [Figure 28-1] Figures 28A–28F show that Fc receptor engagement is required for anti-mCCR8 Ab activity in the MC38 tumor model. Mean (A) and individual growth curves of implanted MC38 tumors in C57BL / 6 mice treated with 200 μg / mouse / treatment of anti-CCR8-mIgG2a (n=10) (B), anti-CCR8-mIgG1-D265A (n=10) (C), or IgG2a isotype control (n=10) (D) at 7, 10, and 14 days post-implantation. E: MC38 tumors were harvested at 15 days post-implantation, and Treg depletion was assessed by flow cytometry (n=5 per group). **P<0.01. One-way ANOVA Kruskal-Wallis test with Dunn's multiple comparison. F: Percentage of Ccr8+ / + and Ccr8- / - donor-derived Tregs in MC38 tumors and peripheral tissues 13 days after MC38 tumor implantation. **P<0.01. Two-way ANOVA, followed by Bonferroni multiple comparison test. [Figure 28-2] Same as above. [Figure 28-3] Same as above. [Figure 29-1]Figures 29A–29I show that anti-CCR8-mIgG2a induces a productive memory response in a heterogeneous re-exposure model. Mice implanted with CT26 tumors were randomized when the tumors reached 100–120 mm3. A–C: Growth curves and Treg depletion in CT26 tumors treated with 0.2 mg / mouse / treatment with (A) anti-CCR8-mIgG2a (n=8), (B) anti-CTLA4-mIgG2a (n=8), or (C) mIgG2a isotype control (n=8) at 1, 4, and 8 days after randomization. At 9 days after randomization, six tumors from each group were analyzed by flow cytometry to assess Treg depletion (D) and the frequency of AH1 tetramer+ CD8+ T cells (E). F: Frequency of AH1 tetramer+ CD8+ T cells in the blood 92 days after treatment. G: Frequency of AH1 tetramer + CD8+ effector memory T cells (TEM) in blood 5 days after exposure to LM-AH1A5. H and I: Intracellular cytokine staining for (H) IFNγ+ and (I) multifunctional IFNγ+ TNFα+ CD8+ T cells in the spleen after 5 hours of stimulation with AH1A5 peptide; not significant; *P<0.05, **P<0.01. One-way ANOVA Kruskal-Wallis test (D~I) with Dunn's multiple comparisons. [Figure 29-2] Same as above. [Figure 29-3] Same as above. [Figure 30] Figures 30A and 30B show the diverse expression of CCR8 in multiple human cancers. Immunohistochemistry (IHC) was performed on 17 cancer types / subtypes derived from two sets of samples (full-size tissue section sets and MTB sets). A: Full-size tissue sections showing whole-slide image analysis of CCR8+ cells in formalin-fixed paraffin-embedded (FFPE) slides of 6 tumor types / subtypes, with 14–24 samples per tumor type. B: Sets of multiple tumor blocks (MTBs) showing 16 tumor types / subtypes, including 20 cases / tumor type. Each MTB contained 5 cases with a single sign per FFPE block and 1 hyperplastic tonsil sample as a positive control. [Modes for carrying out the invention]

[0038] The present invention relates to an mAb that binds specifically and with high affinity to CCR8 expressed on the cell surface, and a method for treating cancer in a subject, comprising administering the anti-CCR8 Ab to the subject as monotherapy or in combination with anticancer agents such as immune checkpoint inhibitors, chemotherapeutic agents, and / or radiotherapy. The effect of CCR8-mediated Treg depletion on potent inhibition of tumor growth, both alone and in combination with PD-1 blockers, is demonstrated herein in several diverse preclinical mouse tumor models.

[0039] term To make this disclosure more easily understandable, several terms are defined at the outset. Where used in this application, unless otherwise expressly stated herein, each of the following terms shall have the meanings set forth below. Additional definitions are provided throughout this application.

[0040] "Administering," "administering," or "dosing" refers to the physical introduction of a composition containing a therapeutic agent into a target using any of the various methods and delivery systems known to those skilled in the art. The preferred route of administration for therapeutic agents such as anti-CCR8 and anti-PD-1 Ab is intravenous (IV) administration. Other routes of administration include subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), spinal, or other parenteral administration routes, such as by injection or infusion. When used herein, the term "parenteral administration" means a method of administration, usually by injection, other than enteral and topical administration, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the Ab of the present invention may be administered via non-parenteral routes such as topical, epidermal, or mucosal administration routes, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical. Administration may also be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0041] An "antibody" (Ab) is defined as a glycoprotein immunoglobulin (Ig) comprising at least two heavy (H) chains and two light (L) chains, or the antigen-binding portion thereof, that specifically bind to an antigen and are interconnected by disulfide bonds. Each H chain is a heavy chain variable region (V in this specification). H It includes the heavy chain constant region (abbreviated as C). The heavy chain constant region of IgG Ab consists of three constant domains, namely C H1 , C H2 , and C H3 Includes. Each light chain has a light chain variable region (V in this specification). L It includes the light chain constant region (abbreviated as C). The light chain constant region of IgG Ab is one constant domain, namely C L Includes. V H and V LThe region can be further subdivided into hyper-variable regions called complementary determination regions (CDRs), which are inserted between more conserved regions called framework regions (FRs). H and V L It contains three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: 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, including Kabat, Chothia, AbM, contact, and IMGT definitions, have been used to describe the CDR domains within Ab. The constant region of Ab can mediate the binding of Ig to various cells of the immune system (e.g., effector cells) and to host tissues or factors, including the first component (C1q) of the classical complement system.

[0042] As used herein, an Ab described in conventional usage as containing "one (a)" heavy chain and / or "one (a)" light chain means an Ab containing "at least one" of the described heavy and / or light chains, and therefore may encompass an Ab having two or more heavy and / or light chains. Specifically, an Ab described in this way may encompass a conventional Ab having two substantially identical heavy chains and two substantially identical light chains. The Ab chains may be substantially identical, but not entirely identical if they differ due to post-translational modifications, such as C-terminal cleavage and selective glycosylation patterns of lysine residues.

[0043] Ig may originate from any of the generally known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. “Isotype” refers to an Ab class or subclass (e.g., IgM, IgG1, or IgG4) encoded by a heavy chain constant region gene. The term “antibody” includes, by example, both naturally occurring and non-naturally occurring Abs, monoclonal and polyclonal Abs, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs, and single-stranded Abs. Non-human Abs may be partially or fully humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise explicitly stated and indicated by the context, the term “antibody” also includes antigen-binding fragments or moieties of any of the aforementioned Ig, including monovalent and bivalent fragments or moieties, and single-stranded Abs.

[0044] An "isolated" Ab refers to an Ab that substantially does not contain other Abs with different antigen specificities (for example, an isolated Ab that specifically binds to CCR8 substantially does not contain Abs that specifically bind to antigens other than CCR8, such as an Ab that binds to CCR4). However, an isolated Ab that specifically binds to human CCR8 (hCCR8) may cross-reactive to other antigens, such as CCR8 polypeptides from different species, such as mice and cynomolgus monkeys. Furthermore, in certain contexts, an isolated Ab may also mean an Ab that has been purified to substantially exclude other cellular material and / or chemicals. In contrast, an "isolated" nucleic acid refers to a nucleic acid composition of a substance that is significantly different from naturally occurring nucleic acids, i.e., has distinct chemical identity, properties, and utility. For example, isolated DNA, unlike natural DNA, is a free-standing portion of natural DNA and not a larger structural complex found in nature, i.e., an integrated portion of a chromosome. Furthermore, unlike native DNA, isolated DNA can, among other things, be used as PCR primers or hybridization probes to detect biomarker genes or mutations by measuring gene expression to diagnose diseases or predict the effectiveness of therapeutic drugs. In addition, in certain contexts, isolated nucleic acids may mean nucleic acids that have been purified using standard techniques well known in the art so as to be substantially free of other cellular components and other contaminants, such as other cellular nucleic acids and proteins.

[0045] The term "monoclonal" Ab (mAb) refers to a non-naturally occurring preparation of an Ab molecule with a single molecular composition, i.e., an Ab molecule whose primary sequence is essentially identical and which exhibits a single binding specificity and affinity for a particular epitope. mAbs are an example of isolated Abs. mAbs can be prepared by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0046] A "chimera" Ab refers to an Ab whose variable region originates from one species and whose steady region originates from another species; for example, an Ab whose variable region originates from mouse Ab and whose steady region originates from human Ab.

[0047] A “human” mAb (HuMAb) refers to an mAb having variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the Ab contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. The human Abs of this invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutagenesis in vivo). However, as used herein, the term “human” Ab is not intended to include Abs in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence. The terms “human” Ab and “fully human” Ab are used synonymously.

[0048] A "humanized" mAb refers to an mAb in which some, most, or all amino acids outside the CDR domain of a non-human mAb are replaced by corresponding amino acids derived from human immunoglobulins. In one embodiment of the humanized form of Ab, some, most, or all amino acids outside the CDR domain are replaced by amino acids derived from human immunoglobulins, while some, most, or all amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not suppress the Ab's ability to bind to a particular antigen. "Humanized" Abs retain antigen specificity similar to that of the original Ab.

[0049] An "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, and an anti-PD-1 Ab is an Ab that specifically binds to PD-1. As used herein, an "anti-PD-1 / anti-PD-L1" Ab is an Ab used to disrupt the PD-1 / PD-L1 signaling pathway, and may be an anti-PD-1 Ab or an anti-PD-L1 Ab.

[0050] The "antigen-binding portion" or "antigen-binding fragment" of Ab refers to one or more fragments of Ab, such as an mAb, that retain the ability to specifically bind to the antigen to which the complete Ab binds.

[0051] Antibody-dependent cell-mediated injury ("ADCC") refers to in vitro or in vivo cell-mediated cytotoxic activity in which nonspecific effector cells expressing Fc receptors (FcRs) on the surface of effector cells [e.g., natural killer (NK) cells, macrophages, neutrophils, and eosinophils] recognize the Fc region of an Ab bound to the surface antigen of target cells and actively lyse the target cells. In principle, any effector cell with activated FcRs can be induced to mediate ADCC. The therapeutic Ab of the present invention for use in human subjects is preferably an anti-hCCR8 Ab specifically modified to mediate enhanced ADCC activity against cells expressing CCR8. The ADCC activity of Ab can be measured as described, for example, in any of Examples 17-20.

[0052] In the present invention, "enhanced ADCC" or "enhanced ADCC activity" of a modified Ab refers to a level of ADCC activity greater than that induced by an unmodified Ab. A modified anti-CCR8 IgG1 Ab exhibiting enhanced ADCC is, for example, a modified form of Ab that induces greater ADCC than an Ab having its native IgG1 constant domain. Non-fucosylated (nf) mAbs are examples of modified Abs that induce enhanced ADCC via improved binding of IgG to activated FcγRIIIA. In certain embodiments, the level of enhanced ADCC activity is measured, for example, in an NK cell lysis assay, such as the EC of cell lysis in the NK cell lysis assay described in Example 17. 50 When measured by a decrease in EC, 50 This is a reduction to less than half, preferably less than one-tenth, and more preferably less than one-hundredth.

[0053] Cancer refers to a broad group of diseases characterized by the unregulated growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors, which may invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0054] "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 7-transmembrane GPCR, and this GPCR is primarily used in tumor FOXP3 hi It has been shown to be expressed in Treg cells. The term "CCR8," as used herein, includes human CCR8 (hCCR8), variants, isoforms, and species homologs of hCCR8, such as mouse CCR8 (mCCR8), and analogs having at least one epitope in common with hCCR8. The complete amino acid sequences of hCCR8 and mCCR8 can be found in GENBANK® accession numbers AAI07160.1 and NP_031746.1, respectively.

[0055] A "cell surface receptor" refers to a molecule or molecular complex expressed on the surface of a cell that can receive signals and transmit those signals across the cell's plasma membrane.

[0056] "Effector function" refers to the interaction between the Ab-Fc region and an FcR or ligand, or the resulting biochemical events. Examples of effector functions include FcγR-mediated effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), FcR binding, ADCC, and Ab-dependent cell-mediated phagocytosis (ADCP), as well as the downregulation of cell surface receptors (e.g., B cell receptors, BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an Ab-variable domain).

[0057] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind to IgG Ab include the FcγR family of receptors, which encompass allele variants and alternatively spliced ​​forms of the FcγR family of receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mouse; FcγRIA, FcγRIIA, and FcγRIIIA in human) and one inhibitory receptor (FcγRIIB). Table 1 summarizes the various properties of human FcγR. The majority of native effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, while NK cells selectively express one activating Fc receptor (FcγRIII in mouse and FcγRIIIA in human) but do not express the inhibitory FcγRIIB in mouse and human.

[0058] [Table 1]

[0059] The "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of the Ab heavy chain that mediates the binding of Ig to host tissues or factors, such as FcR or the first component (C1q) of the classical complement system located on various cells of the immune system (e.g., effector cells). Therefore, the Fc region is a polypeptide that contains the constant region of Ab excluding the first constant region Ig domain. In IgG, IgA, and IgD Ab isotypes, the Fc region is the second (C1q) of the two heavy chains of Ab. H2 ) and the third (C H3 It consists of two identical protein fragments derived from the constant domain of ), and the IgM and IgE Fc regions each polypeptide chain has three heavy chain constant domains (C H It contains domains 2-4). In IgG, the Fc region includes the Ig domains Cγ2 and Cγ3, as well as the hinge between Cγ1 and Cγ2. The boundary of the Fc region of immunoglobulin heavy chains may be variable, but the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position C226 or P230 to the C-terminus of the heavy chain, where the numbering follows the Kabat EU index. H2 The domain consists of approximately 231 to 340 amino acids, while C H3 The domain is C in the Fc region. H2 It is located at the C-terminal end of the domain, i.e., it extends from approximately amino acid 341 to approximately amino acid 447 of IgG. As used herein, the Fc region may be the natural sequence Fc or a mutant Fc.

[0060] "Fucosylation" and "non-fucosylation," as used herein, refer to the presence and absence of a core fucose residue in the N-linked glycan at position N297 of Ab, respectively. Unless otherwise indicated or made clear from the context, amino acid residue numbering in the Fc region of Ab follows EU numbering conventions (the EU index of Kabat et al., 1991).

[0061] "Immune response" refers to the biological response in vertebrates to foreign substances that protect the organism from these substances and the diseases they cause. The immune response is mediated by the action of immune system cells (e.g., T lymphocytes, B lymphocytes, NK cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including alpha, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0062] The term "immunotherapy" refers to the treatment of individuals who have a disease or are at risk of developing or experiencing a relapse of the disease, by means of inducing, enhancing, suppressing, or otherwise modifying the immune response.

[0063] The term "monotherapy" refers to a single type of treatment, such as the administration of a single drug to a patient to treat a disease or condition, or the use of radiotherapy or surgical procedures alone. The administration of a drug alone does not constitute monotherapy if another type of treatment for the disease or condition, such as the administration of additional drugs, is performed before or after it in the same course of treatment.

[0064] In contrast, “combination therapy” refers to, for example, the administration of two or more drugs to a patient to treat a disease or condition, or a treatment modality that combines drug administration with at least two other therapies, such as radiotherapy or surgical procedures. These two or more therapies do not need to be delivered to the patient simultaneously, but are part of the same course of treatment. In certain embodiments, different therapies are administered simultaneously. In other embodiments, the administration of one therapy overlaps with the administration of at least one other therapy. In further embodiments, different therapies are administered sequentially.

[0065] "Enhancing the immune response" means increasing the effectiveness or potency of an existing or induced immune response in a subject. This increase in effectiveness and potency can be achieved, for example, by reducing or overcoming mechanisms that suppress the endogenous host immune response, by stimulating mechanisms that enhance the endogenous host immune response, or by improving the immune response induced by an immunotherapy agent.

[0066] "Programmed death-1" (PD-1) refers to an immunosuppressive receptor belonging to the CD28 family that is predominantly expressed on already activated T cells in vivo and binds to two ligands, namely PD-L1 and PD-L2. The term "PD-1," as used herein, includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one epitope in common with hPD-1. The complete hPD-1 amino acid sequence can be found in GENBANK® accession number U64863.

[0067] "Programmed death ligand-1" (PD-L1) is one of two cell surface glycoprotein ligands to PD-1 (the other being PD-L2) that downregulate T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1," as used herein, includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs having at least one epitope common to hPD-L1. The complete hPD-L1 sequence can be found in GENBANK® accession number Q9NZQ7.

[0068] The term "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 human. The terms "subject" and "patient" are used interchangeably herein.

[0069] The “therapeutic effective dose” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of the drug or agent used alone or in combination with another therapeutic agent that protects a subject from the onset of the disease, or promotes disease regression in the subject, which is demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, prevention or reduction of functional or physical impairment resulting from the disease, or otherwise by improvement of disease symptoms. In addition, the terms “effective” and “effectiveness” in relation to a treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote disease regression in a patient, such as tumor reduction. Physiological safety refers to the tolerable level of toxicity or other harmful physiological effects (adverse effects) at the cellular, organ, and / or biological levels resulting from the administration of a drug. The effectiveness of a therapeutic agent can be evaluated using various methods known to those skilled in the art, for example, in human subjects during clinical trials, in animal model systems to predict effectiveness in humans, or by assaying the activity of the drug in in vitro assays.

[0070] As an example of tumor treatment, a therapeutically effective dose of an anticancer agent preferably inhibits cell growth or tumor growth by at least about 20%, preferably at least about 40%, more preferably at least about 60%, even more preferably at least about 80%, and even more preferably at least about 100% compared to an untreated subject. The ability of a drug or treatment to inhibit tumor growth can be evaluated in animal model systems that predict efficacy in human tumors, such as any of the CT26 colon adenocarcinoma, MC38 colon adenocarcinoma, SA1N fibrosarcoma, 4T1 breast cancer, and MB49 bladder cancer mouse tumor models. Alternatively, tumor growth inhibition can be measured by evaluating the ability of a drug or treatment to inhibit cell growth in vitro using assays known to those skilled in the art. In preferred embodiments of the present invention, tumor reduction may be observed and persist in human subjects for a period of at least about 30 days, more preferably at least about 60 days, and even more preferably at least about 6 months.

[0071] The therapeutically effective dose of a drug includes a “preventive effective dose,” which is any amount of the drug administered alone or in combination with another therapeutic agent to a person at risk of developing the disease (e.g., a person with a precancerous condition at risk of developing cancer) or at risk of suffering from a recurrence of the disease, thereby inhibiting the onset or recurrence of the disease (e.g., cancer). In preferred embodiments, the preventive effective dose completely prevents the onset or recurrence of the disease. To “inhibit” the onset or recurrence of the disease means either to reduce the likelihood of the onset or recurrence of the disease, or to completely prevent the onset or recurrence of the disease.

[0072] The term "treatment" or "therapy" for a subject means any type of intervention or process performed on the subject, including the administration of an active agent, with the aim of reversing, alleviating, improving, inhibiting, delaying, or preventing the onset, progression, onset, severity, or recurrence of a symptom, complication, condition, or biochemical sign associated with a disease.

[0073] Where used herein, the indefinite article “a” or “an” should be understood to refer to “one or more” of any components listed or enumerated.

[0074] When applied to a numerical value, the term "approximately" refers to a value that is reasonably close to the stated value, within the tolerance range determined by those skilled in the art, and that is, in part, dependent on the method by which the value is measured or determined, i.e., the limitations of the measuring system. For example, "approximately" may mean within a range of plus or minus 50%, preferably plus or minus 25%, and more preferably plus or minus 10% of the stated reference value. Where a specific value is provided in this application, the meaning of "approximately" should be understood, unless otherwise stated, to be within the tolerance range of that specific value according to the conventions of the art.

[0075] The terms “substantially identical” or “essentially identical” refer to a sufficiently high degree of similarity between two or more numerical values, compositions, or features such that a person skilled in the art can consider that, in relation to the property being measured, there is little or no biological and / or statistical significance in the differences between them. The differences between the numerical values ​​being measured may be, for example, less than about 50%, preferably less than about 25%, and more preferably less than about 10%.

[0076] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range described, and, where appropriate, its fractional part (e.g., one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0077] Various aspects of the present invention are described in further detail in the following subsections.

[0078] Specific expression of CCR8 in highly immunosuppressed tumor-infiltrating Tregs The various experiments reported herein demonstrate that CCR8 is specifically expressed by Tregs in human tumors, and unlike CCR4, a Treg depletion target currently undergoing clinical trials, CCR8 is selectively expressed in suppressor tumor Tregs and minimally expressed in pro-inflammatory Tef. An analysis of the correlation between human CCR8 and FOXP3 genes in The Cancer Genome Atlas (TCGA; National Cancer Institute, 2021) (Example 1) showed that CCR8 expression had the highest correlation with FOXP3 (a master transcription factor of Tregs) in most cancer types (Figure 1A). CCR8 is associated with tumor FOXP3 hi It is expressed in Tregs but is rarely observed in peripheral blood Tregs and Teffs. CCR8 is also expressed in FOXP3 in HCC tumor samples. hi It is selectively expressed in lymphocytes, but FOXP3 in patient tumors mid and FOXP3 neg CCR8 is not expressed in CD8 and CD4 Teff cells (Figure 1B). CCR8 expression is associated with higher levels of FOXP3 expression and typical Treg markers (IL2RA, IKZF2, BATF), while lower CCR8 expression is associated with cytotoxic T cell markers (GZMA, CD8A) (Figure 1C).

[0079] By comparing the expression profiles of various Treg-related molecules (Example 3), CCR8 expressed more tumor-associated FOXP3 than Tregs derived from patient-matched blood. + FOXP3 was highly expressed in Tregs, but there was little difference in the expression of CCR4, CTLA-4, and CD25 between tumor and blood-derived Tregs (Figure 2A). + The amount of CCR8 per cell in tumor Tregs is higher compared to peripheral blood Tregs (Figure 2B), and this is due to the CD4 levels in both tumors and peripheral blood. + Tconv (Figures 2C and D) and CD8 +Given the low frequency of CCR8 expression in T cells (Figure 2E and F), this suggests that CCR8 could be a highly selective therapeutic marker for targeting tumor Tregs with a low risk of damaging the anti-tumor Teff cell population.

[0080] Flow cytometry analysis of CCR8 expression levels in various subsets of human tumor-associated T lymphocytes (Example 4) showed that CCR8 is expressed by a high proportion of tumor-resident Tregs (median 82%) defined by FOXP3 (Figure 3A). In contrast, it is expressed by a much lower proportion of tumor-infiltrating CD4 + T cells and CD8 + T cells express CCR8 (median 12.65% and 4.55%, respectively, Figure 3B). While only a small percentage of CD4 Tconv cells express CCR8, the per-cell expression level of CCR8 is significantly higher in Tregs than in CD4 Tconv cells (median MFI 2106 vs. 132, P<0.0001), and CD8 + T cells express CCR8 at negligible levels (Figure 3B). Peripheral blood Tregs were also shown to express CCR8, but at significantly lower levels than tumor-infiltrating Tregs (Figure 3C).

[0081] Differences in CCR8 expression across various human Treg subpopulations were also analyzed by flow cytometry (Example 5). In PBMCs derived from healthy subjects, CCR8 was found to be predominantly expressed in peripheral blood Tregs, but this was only a small fraction of these peripheral Tregs (median 21%) compared to the median expression of approximately 82% of tumor-infiltrating Tregs (see Figure 3A) (Figure 4A). Therefore, anti-CCR8 Ab-mediated cell depletion is associated with a low proportion of CCR8 in the peripheral Treg population. +For Tregs, it is thought that there is no significant impact on the peripheral Treg compartment. This means that the risk of autoimmune toxicity associated with depleting tumor-associated Tregs by targeting CCR8 is lower compared to the indiscriminate depletion of non-tumor-associated Tregs. Within the Treg population, CCR8 is expressed more highly in the effector memory population and less highly in central memory cells, as well as in naive Tregs (Figure 4B) or conventional CD4 + CCR8 is hardly expressed in T cells (Figure 4C). In cancer patients, CCR8 expression per cell in tumor-infiltrating Tregs is higher than in peripheral blood-derived Tregs, as measured by MFI of bound anti-CCR8 Ab (Figure 4D).

[0082] In clinical comparisons with other Treg-targeted NF agents (CCR4 mogamulizumab and CTLA-4-Ipi-NF), CCR8 is superior to FOXP3. hi It is specifically expressed in the most activated immunosuppressive subset of tumor Tregs, associated with low survival rates (Plitas et al., 2016, Wang et al., 2019), and effectively eliminates Teffs expressing granzymes and other effector cytokines. Two melanoma tumors and one RCC tumor were dissociated and stained for FOXP3 and CCR8 expression (Example 6). Figure 5 shows that CCR8 is the most activated Treg, the most immunosuppressive CD4 + FOXP3 high This indicates that it is expressed in the Treg population. mid In melanoma samples where populations exist, the majority of CCR8 expression is FOXP3 high FOXP3 cells, which are T cells and whose CCR8 expression levels are predominantly effector cells expressing granzymes and other effector cytokines, are found in T cells. mid and FOXP3 neg The levels are significantly lower in tumor T cells. Most patients have FOXP3 + In RCC samples where T cells exhibit high levels of FOXP3, CCR8 expression overlaps with FOXP3 expression. high CD4+ T cells can be activated conventional T cells or resting Tregs (FOXP3). mid CD4 + Unlike T cells, it was shown to be a true Treg.

[0083] CCR8 + Tumor-infiltrating T cell population and CCR8 - The expression of functionally related molecules in tumor-infiltrating T cell populations was investigated (Example 7). CCR8 derived from patient tumor samples. + Cells were shown to co-express several proteins with potential immunosuppressive functions, such as CD25, IL1R2, and CD39 (Figure 6A-D), but CCR4 + No equivalent enrichment was observed in T cells (Figures 7A-D). When HLA-DR was used as an activation marker, CCR8 expression also correlated with higher levels of HLA-DR expression (Figure 8A), thus identifying activated Tregs, but CCR4 expression did not (Figure 8B). Therefore, CCR8 + Cell depletion can eliminate activated immunosuppressive Tregs expressing CD25, CD39, and IL1R2, but Tregs expressing these molecules cannot be eliminated by CCR4-targeted depletion. Furthermore, ex vivo stimulation of dissociated patient tumor cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin can eliminate CD4 + T cell CCR8 + Minimal production of IL-2, IFNγ, and granzyme B was induced in the fraction (Figure 9A-C). In contrast, CCR4 + CD4 + T cells are the primary producers of IFNγ and IL-2 (Figure 10A and B), and CCR8 + Not cells, but CCR4 + This suggests that targeted depletion of [the target] may be detrimental to antitumor immunity.

[0084] The data summarized above, revealed in Examples 1-7, shows that all CD4 in tumors + FOXP3 + It's not that T cells express CCR8, but rather that CCR8+ The fraction is FOXP3 + This suggests that it appears to be a more immunosuppressive subset of cells. Therefore, CCR8 expression is enriched in tumors and represents a subset of highly suppressive Tregs that can interfere with anti-tumor immunity. Tumor-specific Tregs, in particular, the most activated immunosuppressive CD4 + FOXP3 high The high specificity of CCR8 expression to intratumor Tregs makes CCR8 an ideal target for mediating the depletion of these highly immunosuppressive Tregs by ADCC using anti-CCR8 Ab.

[0085] Therefore, anti-hCCR8 mAbs were generated in the experiments described herein (Example 8) and screened to identify mAbs exhibiting several desirable properties in therapeutic mAbs for treating cancer (Examples 9-31). Surrogate mouse anti-mCCR8-mIgG2a mAbs potently inhibited tumor growth in several mouse tumor models, and non-fucosylated (nf) anti-hCCR8 mAbs drove pro-inflammatory CD4 iontophoresis, which drives anti-tumor immune responses. + and CD8 + This specification demonstrates that human tumor Tregs were depleted in ex vivo patient tumor samples and in vitro while preserving Teff cells (Examples 20 and 22). Depletion of tumor-associated Tregs reduces their immunosuppressive effects, thereby improving the overall immune response in fighting cancer. Furthermore, since CCR8 is rarely expressed in Tregs and Teffs in peripheral blood or other tissues, targeting Tregs carries minimal toxicity risk. In contrast, in vitro anti-hCCR4-IgG1-nf treatment depletes Treg cells and CD4 in the peripheral and tumor microenvironment. + This resulted in depletion of both Tconv cells (Examples 20 and 22). This is consistent with clinical evidence that mogamulizumab (anti-CCR4) depletes the Teff population in peripheral blood (Kurose et al., 2015).

[0086] Generation of anti-hCCR8 mAbs Chemokine receptors, including CCR8, have traditionally been "very difficult antigens to develop abs from" due to their low cell surface profile and the relatively difficult utilization of ab binding (WO2007 / 044756). Furthermore, abs generated against peptides corresponding to the extracellular domain of chemokine receptors often fail to recognize intact receptors in cells, possibly due to differences in secondary structure. Because of these differences, efforts to generate abs against chemokine receptors have a low success rate (WO2007 / 044756). CCR8 is a particularly difficult GPCR and has been described as a "very challenging" target for ab generation (Harbour BioMed, 2020).

[0087] Initial attempts to generate anti-hCCR8 mAbs by immunizing mice with hCCR8-expressing cells were unsuccessful. However, in this study (Example 8), we attempted to immunize various rodents using various hCCR8 antigens and various combinations of these antigens, and subsequently generated anti-hCCR8 mAbs.

[0088] Immunization of rodents Common C57Bl / 6 mice, transgenic mice of various strains expressing the human Ig repertoire, and specifically created CCR8 mice. - / -Humanized or human anti-CCR8 mAbs were generated by immunizing various rodents, including knockout mouse strains, rats, and hamsters, with immunogens containing various hCCR8 antigens, including hCCR8-overexpressing Chinese hamster ovaries (CHO), mouse BAF3, and human HEK 293F cells, or plasma membranes purified from these cells. These immunogens were boosted with chemosynthetic peptides derived from the hCCR8 N-terminus (residues 1-35 of hCCR8), which importantly contain single or double sulfated tyrosine residues at positions 15 and / or 17, conjugated with either bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Subsequently, some of the mAbs generated in non-transgenic mice were modified into humanized or chimeric derivatives.

[0089] Proteins often undergo post-translational modifications, some of which, such as glycosylation, have been well-studied and validated. In contrast, the sulfation of certain extracellular tyrosine residues has been largely unstudied, with only 32 molecules to date identified as containing sulfated tyrosine (Mehta et al., 2020). While further proteins containing sulfated tyrosine are expected to be discovered, sulfated tyrosine is estimated to be quite rare (Moore, 2003). Chemically, they are highly distinct from all other amino acids and therefore become a key focus in generating Abs with very high specificity and low off-target binding. Chemokine receptors, including CCR8, constitute one of several protein classes that have been well-studied for containing sulfated tyrosine at their N-terminus (Ludeman and Stone, 2014). In some of these receptors, tyrosine sulfation has been found to be crucial for ligand, i.e., chemokine, binding (Zhu et al., 2011). Therefore, Ab, which binds to sulfated tyrosine, is likely to disrupt the binding of chemokines to chemokine receptors, thereby preventing chemokine receptor activation. As discussed elsewhere in this specification, sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 (Sequence ID 109) contains at least one amino acid in a peptide, for example sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22The present invention comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the amino acids in the peptide having (SEQ ID NO: 109), and the Ab of the present invention is of particular interest in that amino acids Y15 and Y17 bind to an epitope that is sulfated. Example 11 shows that mAb 4A19 has about 1.6 nM K D This indicates that it binds to the N-terminus of double-sulfated CCR8 (see Table 5).

[0090] Reduced fucosylation, non-fucosylation, and low fucosylation of anti-CCR8 mAbs The interaction between Ab and FcγR can be enhanced by modifying the glycan moiety that binds to each Fc fragment at the N297 residue (EU numbering). In particular, the absence of core fucose residues potently enhances ADCC via improved binding of IgG to activated FcγRIIIA without altering antigen binding or complement-dependent cell-mediated cytotoxicity (CDC) (Natsume et al., 2009). The binding of nf Ab to CCR8 in human Tregs facilitates the binding of FcγR in NK cells and myelomonocytic Tregs. This FcγR binding drives NK cell activation, leading to enhanced Treg death by ADCC. There is compelling evidence that defucosylation of tumor-specific Ab resulted in enhanced therapeutic activity in mouse models in vivo (Nimmerjahn and Ravetch, 2005; Moessner et al., 2010).

[0091] Modification of Ab glycosylation can be achieved, for example, by expressing Ab in host cells with an altered glycosylation mechanism. Some of the anti-hCCR8 mAbs disclosed herein have reduced or eliminated fucosylation and exhibit enhanced ADCC particularly useful for the methods of the present invention. Therefore, the anti-hCCR8 mAbs disclosed herein can be produced in a form with reduced or eliminated fucosylation, for example by expressing the anti-hCCR8 mAb in cells with an altered glycosylation mechanism, resulting in enhanced ADCC particularly useful for the methods of the present invention. Cells with an altered glycosylation mechanism are described in the Art and can be used as host cells that express the recombinant Ab of this disclosure and thereby produce Ab, e.g., mAbs, with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase enzyme FUT8 [α-(1,6)fucosyltransferase, see U.S. Patent Application Publication No. 2004 / 0110704, Yamane-Ohnuki et al., 2004], and as a result, the Abs expressed in these cell lines lack fucose in their carbohydrates. European Patent No. 1176195 also describes a cell line having a functionally disrupted FUT8 gene and a cell line having little or no activity to add fucose to N-acetylglucosamine bound to the Fc region of Ab, e.g., rat myeloma cell line YB2 / 0 (ATCC CRL1662). Many other methods for producing Abs containing reduced fucosylation are described in the Art. The low-fucosylated or nf chimeric, humanized, or human anti-hCCR8 Ab disclosed herein lacks the FUT8 enzyme essential for fucosylation. - / - It was produced by expression in knockout cells (Thermo Fisher Scientific, San Diego, CA).

[0092] Although nf Ab exhibits significantly enhanced ADCC compared to fucosylated Ab, Ab preparations do not necessarily need to be completely free of fucosylated heavy chains to be useful in the methods of the present invention. The residual level of fucosylated heavy chains is not considered to significantly interfere with the ADCC activity of substantially nf heavy chain preparations. Ab produced in conventional CHO cells that have full ability to attach core fucose to N-glycans may nevertheless contain several percent to up to 15% nf Ab. Since nf Ab can exhibit approximately 10-fold higher affinity for CD16 and up to 30-100-fold enhancement of ADCC activity, even a slight increase in the proportion of nf Ab can dramatically increase the ADCC activity of a preparation. Any preparation containing more nf Ab than can be produced in normal CHO cells in culture, e.g., wild-type CHO cells with unaltered glycosylation mechanisms, may exhibit a certain level of enhanced ADCC. Such Ab preparations are referred to herein as preparations with reduced fucosylation. Depending on the intrinsic level of non-fucosylation obtained from normal CHO cells, e.g., wild-type CHO cells, reduced fucosylation preparations may contain 50%, 30%, 20%, 10%, or even less than 5% nf Ab. Reduced fucosylation can be functionally defined as a preparation exhibiting approximately twofold or greater ADCC enhancement compared to Ab prepared in normal CHO cells, without referring to any fixed percentage of nf species.

[0093] However, as used herein, unless otherwise indicated, the level of non-fucosylation is defined structurally. Specifically, a “non-fucosylated” (nf) or “defucosylated” (these terms are used synonymously) Ab preparation is an Ab preparation containing more than 95% nf Ab heavy chains, including 100%; “low fucosylated” refers to an Ab preparation in which 80–95% of the heavy chains lack fucose; and “low fucosylated or non-fucosylated” refers to an Ab preparation in which 80% or more of the heavy chains lack fucose.

[0094] The level of fucosylation in Ab preparations can be determined by methods known in the art, including but not limited to gel electrophoresis, liquid chromatography (LC), and mass spectrometry (MS). Unless otherwise indicated, for the purposes of this invention, the level of fucosylation in Ab preparations is determined by hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC). To determine the level of fucosylation in Ab preparations, the sample is denatured with PNGase F to cleave the N-linked glycans, and then analyzed for fucose content. LC / MS of the full-length Ab chain is an alternative method for detecting the level of fucosylation in Ab preparations, although mass spectrometry is inherently less quantitative.

[0095] Therapeutic anti-hCCR8 mAbs have a high probability of causing ADCC. Due to significant difficulties encountered in generating anti-CCR8 Ab in preliminary immunization experiments, immunization was ultimately performed using a wide variety of antigens, as described above and in Example 8. The generated hybridomas were screened as described in Example 9 to confirm the binding of Ab to CCR8 and the CCR8 N-terminal peptide. The selected mouse Ab was humanized as described in Example 10.

[0096] The present invention also provides a method for generating an Ab against CCR8, comprising an immunization schedule comprising one or more immunizations, wherein in at least one immunization, the administration of an hCCR8 antigen which is a KLH conjugate hCCR8 peptide containing residues Y15 and Y17 of hCCR8, and residues Y15 and Y17 are sulfotyrosine residues, and further comprising immunizing rodents at least once with one or more hCCR8 antigens in the immunization schedule. Preferably, both residues Y15 and Y17 are sulfotyrosine residues.

[0097] The hCCR8 peptide may consist of or comprise at least 25, 30, or 35 amino acids from the N-terminal-most part of hCCR8. The hCCR8 peptide may also consist of the 35 amino acids from the N-terminal side of hCCR8.

[0098] The rodent may be a mouse, rat, or hamster. Preferably, the rodent is a mouse.

[0099] Rodents may be immunized in at least one immunization using cells expressing hCCR8, or preferably a plasma membrane-enriched fraction isolated from cells expressing CCR8, either alone or in combination with a KLH-conjugated hCCR8 peptide. The immunization schedule may include immunizing rodents with hCCR8-expressing HEK 293F cells and a KLH-conjugated N-terminal hCCR8 peptide. The immunization schedule may also include immunizing rodents with a plasma membrane-enriched fraction of hCCR8-expressing BAF3 cells and a KLH-conjugated hCCR8 peptide.

[0100] In certain embodiments, the disclosure relates to an isolated Ab, preferably an mAb, or its antigen-binding moiety, which specifically binds to CCR8 expressed on the surface of cells and mediates the depletion of CCR8-expressing cells by ADCC. In certain embodiments, the CCR8 to which the mAb or its antigen-binding moiety binds is hCCR8, whose sequence is described in SEQ ID NO: 1. In other embodiments, the CCR8 is mCCR8, whose sequence is described in SEQ ID NO: 120.

[0101] Human IgG1 and IgG3 Ab isotypes can mediate ADCC by binding to activated Fcγ receptors, particularly the CD16(FcγRIIIa) receptor expressed by human NK cells and monocytes (see Table 1). Many commercially available therapeutic Abs have a human IgG1 isotype that can induce stronger ADCC and CDC compared to other human Ab isotypes. In addition, therapeutic IgG1 Abs have long-term stability in the blood, mediated by binding to the neonatal Fc receptor (FcRn). The activity of several therapeutic agents, including anti-CD20 rituximab [RITUXAN®] (Dall'Ozzo et al., 2004), anti-Her2 trastuzumab [HERCEPTIN®] (Gennari et al., 2004), anti-tumor necrosis factor-α (anti-TNF-α) infliximab [REMICADE®] (Louis et al., 2004), and anti-RhD (Miescher et al., 2004), is mediated, at least partially, by ADCC.

[0102] In certain embodiments of the disclosed invention, the anti-CCR8 mAb or its antigen-binding moiety comprises a heavy chain constant region of a human IgG1 or IgG3 isotype. In preferred embodiments, the anti-CCR8 mAb or its antigen-binding moiety is that of human IgG1.

[0103] The present invention also provides a modified anti-hCCR8 mAb or its antigen-binding moiety, comprising a modified heavy chain constant region that binds to FcγR with higher affinity compared to the corresponding unmodified mAb or its antigen-binding moiety and mediates enhanced ADCC. In certain embodiments, the modified anti-hCCR8 mAb or its antigen-binding moiety is used, for example, in NK cell lysis assays, such as the NK cell lysis assay described in Example 17, for example, to control EC of cell lysis. 50When measured by a decrease in ADCC activity, it mediates at least (a) approximately 2-fold enhanced ADCC activity, (a) approximately 5-fold enhanced ADCC activity, (c) approximately 10-fold enhanced ADCC activity, (d) approximately 30-fold enhanced ADCC activity, or (e) approximately 100-fold enhanced ADCC activity. In certain embodiments, the modified anti-hCCR8 mAb or its antigen-binding moiety includes a modified IgG1 heavy chain constant region exhibiting reduced fucosylation.

[0104] In further embodiments, the modified anti-hCCR8 mAb or its antigen-binding moiety comprises a modified IgG1 heavy chain constant region that is either low-fucosylated or unfucosylated. In certain other embodiments, the modified mAb or its antigen-binding moiety comprises a modified IgG1 heavy chain constant region that contains an enhanced ADCC-mediated mutation or multiple mutation. In further embodiments, the mutation or multiple mutation is selected from G236A, S239D, F243L, E333A, G236A / I332E, S239D / I332E, S267E / H268F, S267E / S324T, H268F / S324T, G236A / S239D / I332E, S239D / A330L / I332E, S267E / H268F / S324T, and G236A / S239D / A330L / I332E. In certain embodiments, the modified anti-hCCR8 mAb or its antigen-binding moiety comprises a modified IgG1 heavy chain constant region that is either low-fucosylated or unfucosylated, and further contains the mutation or multiple mutation listed above that mediates enhanced ADCC.

[0105] Functional screening of anti-hCCR8 mAbs that mediate ADCC Human, humanized, and chimeric anti-hCCR8 mAb clones were functionally screened to identify Abs that bind to hCCR8 with high affinity (Example 11), Abs that specifically bind to CCR8-expressing human cells (Example 14), Abs that block the binding of CCL1 ligand to CCR8 (Example 15), Abs that mediate ADCC in CCR8-expressing cells containing Tregs when bound to CCR8 on the surface of such cells (Examples 17, 19, and 20), Abs that do not induce internal migration of CCR8 into cells (Example 21), Abs that promote depletion of human tumor-associated Tregs in in vitro (Example 22) and ex vivo human tumor slice samples (Example 20), and Abs that promote depletion of CCR8 in normal tissues. + We identified therapeutic agents that exhibit desirable properties, including agents that specifically mediate tumor Treg depletion while preserving T cells (Example 24), and agents that suppress tumor growth in mice when administered as monotherapy or in combination with checkpoint inhibitors to preclinical mouse tumor models (Examples 23-29).

[0106] Clones, EC below nanomolar 50After initially demonstrating that the mAbs bind to CCR8 in human cells at a specific value and specifically to CCR8-expressing cells without cross-reactivity to diverse human tissues that do not express CCR8, they were selected for further characterization. The DNA encoding the variable regions in these mAbs was sequenced by next-generation sequencing, and clones were selected for diversity based on sequence homology and limited potential sequence tendencies, e.g., asparagine deamidation, methionine oxidation, and glycosylation sites. Based on their efficacy in mediating ADCC in CCR8-expressing cells, binding kinetics, and sequence family diversity, certain selected clone mAbs were further tested for desirable functions in therapeutic mAbs, including the ability to inhibit tumor growth in mouse tumor models, and subjected to sequence optimization to mitigate sequence tendencies, optimize binding affinity, and return to germline amino acids. The selected mAbs were also analyzed for biophysical properties through various means such as analytical size exclusion chromatography, capillary isoelectric focusing, hydrophobicity assessment, thermal stability, and aggregation ability to identify clones suitable for further development.

[0107] Characterization of the binding affinity of anti-CCR8 mAbs to CCR8 Some of the anti-CCR8 mAbs of the present invention bind to hCCR8 with high affinity. The mAbs typically have a dissociation constant (K) of 1 μM to 10 pM or less. D It specifically binds to its alloantigen with high affinity, as reflected by ). Any K greater than approximately 100 μM D In general, IgG Ab is considered to exhibit nonspecific binding. When used herein, IgG Ab that "specifically binds" to the antigen is defined as having a K content of about 100 nM or less, preferably about 10 nM or less, more preferably about 5 nM or less, and even more preferably between about 5 nM and 0.1 nM or less. DThis refers to an Ab that binds to an antigen and substantially identical antigens with high affinity, meaning it has high affinity to unrelated antigens. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity with that given antigen, for example, if it exhibits at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% sequence identity with that given antigen.

[0108] "K D The term "when used herein" means k off k on The ratio to (i.e., k off / k on It is intended to refer to the dissociation constant of a specific Ab-antigen interaction, obtained from and expressed as a molar concentration (e.g., nM). on The term "k" refers to the association rate or "binding rate" of the association interaction between Ab and its antigen, and "k off The term refers to the dissociation rate of the Ab-antigen complex. D The value can be determined using well-established methods in the art, such as surface plasmon resonance (SPR), coupled equilibrium exclusion [KinExA®, Sapidyne Instruments, Boise, ID], or biolayer interferometry (BLI, ForteBio, Fremont, CA). The K of a single Ab determined by various methods D The value can vary significantly, for example, by up to 1,000 times. Therefore, the K of various Ab values... D When comparing values, these K D It is important to determine the values ​​using the same method. Unless otherwise stated and indicated in the context, the K of Ab bonds disclosed herein D The values ​​were determined by SPR using the BIACORE® biosensor system (GE Healthcare, Chicago, IL).

[0109] The binding affinity of an Ab that binds to targets such as hCCR8 is the EC of the Ab concentration that achieves half of the maximum binding, relating to binding to CCR8-expressing cell lines. 50 It can also be determined by measuring the following: Studies of mAbs that bind to CHO,293F,Raji cell lines expressing CCR8 have shown that the EC2 is less than 1 nM. 50 This was observed (Figure 11A). When bound to activated Treg, these Abs exhibited a broader binding affinity, with less than half of the Abs having an EC of less than 1 nM. 50 The binding was observed at (Figure 11A). The mAbs of the selected set shown in Figure 11B had an EC of picomoles to nanomoles. 50 It exhibits the range of [this].

[0110] Therefore, in certain embodiments of the disclosed invention, the anti-CCR8 mAb or its antigen-binding moiety has an EC of about 10 nM or less, preferably about 5 nM or less, preferably about 2 nM or less, more preferably about 1.7 nM or less, more preferably about 1 nM or less, more preferably about 0.5 nM or less, and even more preferably about 0.1 nM or less 50 It specifically binds to human CCR8-expressing CHO cells. In certain preferred embodiments, the anti-hCCR8 mAb or its antigen-binding moiety contains approximately 0.1 nM EC 50 It binds to hCCR8-expressing CHO cells. In certain other preferred embodiments, the anti-hCCR8 mAb or its antigen-binding moiety contains about 1.7 nM EC 50 It binds to hCCR8-expressing CHO cells. In certain embodiments, the mAb has an EC of approximately 0.1 nM to approximately 10 nM. 50 They are joined together. In certain other embodiments, EC 50 The range is between approximately 0.1 nM and approximately 2 nM. In certain other embodiments, EC 50 The EC is between approximately 0.5 nM and approximately 5 nM. In a particular preferred embodiment, 50 The EC is between approximately 1 nM and approximately 2 nM. In other embodiments, the mAb or its antigen-binding moiety is between approximately 0.5 nM and approximately 1 nM. 50It binds to human hCCR8. In certain preferred embodiments, EC 50 The value is measured by the binding assay described in Example 11.

[0111] In certain other embodiments of the present invention, the anti-hCCR8 mAb or its antigen-binding moiety has an EC of about 50 nM or less, about 14 nM or less, about 10 nM or less, preferably about 5 nM or less, more preferably about 2 nM or less, more preferably about 0.5 nM or less, more preferably about 0.3 nM or less, even more preferably about 0.1 nM or less, and even more preferably about 0.03 nM or less. 50 It specifically binds to activated human Tregs. In certain preferred embodiments, the anti-hCCR8 mAb or its antigen-binding moiety contains approximately 1.7 nM EC 50 It binds to hCCR8-expressing CHO cells. In certain embodiments, the mAb has an EC of approximately 0.03 nM to approximately 10 nM. 50 They are joined together. In a particular preferred embodiment, EC 50 The EC is between approximately 0.1 nM and approximately 5 nM. In a more preferred embodiment, the mAb or its antigen-binding moiety has an EC between approximately 0.2 nM and approximately 2 nM. 50 It binds to human hCCR8. In certain preferred embodiments, EC 50 The value is measured by the binding assay described in Example 11.

[0112] The selected anti-hCCR8 mAb, namely the Fab fragment of 4A19, was shown by X-ray crystallography to bind to an epitope containing residues 15-21 in the N-terminal peptide of hCCR8, which has sulfated tyrosine-17 residues at the center of the epitope (Figure 12A) (Example 11). Therefore, in certain embodiments, the anti-hCCR8 mAb or its antigen-binding moiety described herein binds to the N-terminal epitope of human CCR8 as determined by X-ray crystallography, and the epitope is sequence Y 15 Y 16 Y 17 P 18 D19 I 20 F 21 It contains at least one amino acid in the peptide having (SEQ ID NO: 2). In certain preferred embodiments, the epitope peptide contains a sulfated tyrosine-17 residue. In certain embodiments, the epitope contains two, three, four, five, six, or all of the amino acids in the peptide having the sequence of SEQ ID NO: 2.

[0113] Binding of the bisulfated peptide (Y15 and Y17 are sulfated) to the 4A19 Fab fragment confirmed the identity and orientation of the epitope's center. This, in addition, allowed for the depiction of a more elongated linear epitope, including N-terminal residues 12–22 of hCCR8 (VTDYYYPDIFS, SEQ ID NO: 109) (Figure 12B). The elongation of the epitope from that revealed by the monosulfated peptide is likely a result of sulfo-Y15 defining a larger segment of the hCCR8 N-terminal peptide, allowing for the visualization of its interaction with 4A19 Ab. Since this Ab was generated using an immunization strategy involving multiple immunizations with the hCCR8 N-terminal peptide to enhance the immune response against the hCCR8 N-terminus, the epitope sequence observed in the structure with the bisulfated peptide may constitute the entire epitope to which mAb 4A19 binds. If other amino acid residues in the N-terminal peptide form an epitope, this would be expected to be visible in the crystal structure, but it is not actually observed (Figure 12B).

[0114] Therefore, in a particular preferred embodiment, the epitope peptide to which 4A19 is bound contains sulfated tyrosine-15 and tyrosine-17 residues. In a particular embodiment, amino acid Y of this peptide 15 and / or Y 17 It is sulfated. In a particular preferred embodiment, amino acid Y of this peptide 15 and Y 17 Both are sulfated.

[0115] This disclosure relates to an ADCC that can mediate and specifically bind to an epitope on hCCR8 described in SEQ ID NO: 1, and the epitope, when determined by X-ray crystallography, is approximately 15-21 amino acid residues (Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 It provides an Ab, such as an mAb, or its antigen-binding moiety, located in the N-terminal domain of hCCR8 within the peptide, including SEQ ID NO: 2). In a particular embodiment, the epitope is sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 It contains at least one amino acid in a peptide having (SEQ ID NO: 2). In other embodiments, the epitope is sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 The epitope contains two, three, four, five, six, or all of the amino acids in the peptide having (SEQ ID NO: 2). In a particular preferred embodiment, the epitope contains all seven amino acids having the sequence of SEQ ID NO: 2.

[0116] This disclosure also allows ADCC to mediate, and the sequence specifically binds to an epitope on hCCR8 described in SEQ ID NO: 1, and the epitope, when determined by X-ray crystallography, is approximately 12-22 amino acid residues (V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It provides an Ab, such as an mAb, or its antigen-binding moiety, located in the N-terminal domain of hCCR8 within the peptide (SEQ ID NO: 109). In a particular embodiment, the epitope is sequence V12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It contains at least one amino acid in a peptide having (SEQ ID NO: 109). In other embodiments, the epitope is sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 The epitope contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the amino acids in the peptide having (SEQ ID NO: 109). In a particular preferred embodiment, the epitope contains all 11 amino acids having the sequence of SEQ ID NO: 109. In a particular other embodiment, the epitope consists of the peptide having the sequence of SEQ ID NO: 109.

[0117] In certain aspects of the present invention, the anti-CCR8 mAb or its antigen-binding moiety is about 100 nM or less, preferably about 50 nM or less, preferably about 10 nM or less, more preferably about 5 nM or less, more preferably about 1.6 nM or less, more preferably about 1.0 nM or less, even more preferably about 0.5 nM or less, and even more preferably about 0.1 nM or less of K D The N-terminal peptide of hCCR8 contains sulfated tyr-15 and tyr-17 residues [for example, a peptide of N-terminal CCR8 residues 1-35 (CCR8-2 sulfo) that are sulfated at positions tyr-15 and tyr-17]. In certain preferred embodiments, the anti-hCCR8 mAb or its antigen-binding moiety contains approximately 1.6 nM K DIt binds to an N-terminal epitope peptide, for example, a peptide of N-terminal CCR8 residues 1-35 (CCR8-2 sulfo) that is sulfated at positions Tyr15 and Tyr17. In certain embodiments, the mAb is between approximately 100 nM and approximately 0.1 nM. D They are joined together. In a particular preferred embodiment, K D The K is between approximately 50 nM and approximately 0.5 nM. In a more preferred embodiment, the mAb or its antigen-binding moiety has a K between approximately 10 nM and approximately 1 nM. D The binding occurs. In a more preferred embodiment, the mAb or its antigen-binding moiety has a K between approximately 2 nM and approximately 1 nM. D They are joined together. In a particular preferred embodiment, K D The value is measured by the SPR method described in Example 11.

[0118] Sulfation of the tyrosine residues (tyr-15 and tyr-17) at the N-terminus of hCCR8 is required for binding to 4A19 Ab (see Example 11). When only one tyrosine residue, i.e., tyr-15, is sulfoylated, the K2 level is more than 10 times higher than that of the bisulfated peptide, and approximately 1,000 times higher. D As demonstrated, the 4A19 Fab fragment does not bind relatively closely to the N-terminal peptide, and when only tyr-17 is sulfated, there is a reduction in affinity to approximately one-tenth (Table 5). Therefore, the present invention provides an anti-hCCR8 mAb or its antigen-binding moiety that binds to the N-terminal peptide of hCCR8 (CCR8-sulfoY17) containing a single sulfated residue, i.e., tyr-17, at a concentration of about 100 nM or less, preferably about 50 nM or less, preferably about 25 nM or less, more preferably about 10 nM or less, and even more preferably about 1.0 nM or less. In certain preferred embodiments, the anti-hCCR8 mAb or its antigen-binding moiety has a concentration of about 20 nM of K D It binds to a single sulfated epitope peptide. In certain embodiments, the mAb is between approximately 100 nM and approximately 1 nM. D They are joined together. In a particular preferred embodiment, K DThe K is between approximately 50 nM and approximately 10 nM. In a more preferred embodiment, the K of the mAb or its antigen-binding moiety is between approximately 30 nM and approximately 20 nM. D They are joined together. In a particular preferred embodiment, K D The value is measured by the SPR method described in Example 11.

[0119] Tissue cross-reactivity of anti-hCCR8 mAbs Since therapeutic anti-CCR8 mAbs can be used to deplete target cells expressing CCR8, it is important that the mAb specifically binds to the intended target cells, i.e., tumor-infiltrating Tregs, and not to other cell types essential in the body where depletion could induce toxic or undesirable side effects. Therefore, candidate mAbs were tested for binding to a wide variety of normal human tissue types (Example 14). Anti-hCCR8 mAbs 18Y12 and 4A19 were shown to bind primarily to sparsely distributed immune cells in the thymic medulla and dermis of the skin, but binding was not observed in many other tissues examined. Another mAb, 16B13, was observed to bind nonspecifically to PBMCs containing immune cells in lymphoid organs and lymphoid-rich tissues, as well as to various human tissues and many other tissues where immune cells are present. Staining showed a cytoplasmic pattern. Abs such as 16B13, which nonspecifically bind to targets other than CCR8 on the cell surface, are not suitable for therapeutic use targeting CCR8-expressing cells due to depletion, despite other desirable functional properties they may exhibit.

[0120] Accordingly, this disclosure provides an anti-CCR8 mAb or its antigen-binding moiety that specifically binds to CCR8-expressing cells such as tumor Tregs, as well as to rare, scattered immune cells in the thymic medulla and dermis of the skin, but does not bind to any of the diverse human tissues, including the cerebrum, cerebellum, heart, liver, lungs, kidneys, tonsils, spleen, thymus, colon, stomach, pancreas, adrenal gland, pituitary gland, skin, peripheral nerves, testes, or uterine tissue, or to any PBMCs. For example, an anti-CCR8 mAb or its antigen-binding moiety specifically binds to tumor-infiltrating Tregs but does not bind to PBMCs, and may not show cytoplasmic staining in fixed PBMCs, for example. The non-binding of Ab to the cells and tissues listed above can be demonstrated, for example, by performing standard staining using the relevant Ab on a fixed tissue sample, for example, by the method described in Example 14.

[0121] Inhibition of CCR8 / ligand signaling by anti-CCR8 mAbs Since the binding of CCL1 to CCR8 in CHO cells induces calcium (Ca) flow, Examples 15 and 16 investigated the blocking of binding of hCCL1 and mCCL1 to hCCR8 and mCCR8, respectively, as well as the blocking of CCR8 / CCL1 signaling by anti-CCR8 mAbs, by performing Ca flow assays on CCR8-expressing CHO cell lines. CCL1 is the only ligand known to bind to CCR8. Multiple studies have shown that the binding of CCL1 to CCR8 can improve Treg suppression in in vitro assays and suppress autoimmune inflammatory responses in mouse models (Barsheshet et al., 2017). Blocking anti-CCR8 mAbs in a SA1N fibrosarcoma model also demonstrated partial antitumor efficacy (Figure 28A). Therefore, in certain embodiments of the present invention, the anti-CCR8 Ab or its antigen-binding moiety inhibits CCR8 / CCL1 signaling by inhibiting the binding of CCL1 to CCR8, such as hCCR8 or mCCR8. In preferred embodiments, inhibition of Ca flow is measured as described in Examples 15 and 16 for hCCR8 and mCCR8, respectively.

[0122] In certain embodiments, the anti-hCCR8 mAb is about 10 nM or less, about 5 nM or less, preferably about 1 nM or less, more preferably about 0.5 nM or less, more preferably about 0.1 nM or less, and even more preferably about 0.01 nM or less IC 50 This inhibits CCR8 / CCL1 signaling. In a particular preferred embodiment, an anti-hCCR8 mAb has an IC5 of approximately 0.5 nM. 50 This inhibits CCR8 / CCL1 signaling. In certain embodiments, anti-hCCR8 Ab has an IC50 between approximately 0.01 nM and approximately 10 nM. 50 This inhibits CCR8 / CCL1 signaling. In certain other embodiments, anti-hCCR8 Ab has an IC50 between approximately 0.05 nM and approximately 5 nM. 50 This inhibits CCR8 / CCL1 signaling. In certain preferred embodiments, anti-hCCR8 Ab has an IC50 between approximately 0.1 nM and approximately 1 nM. 50 This inhibits CCR8 / CCL1 signaling. In a more preferred embodiment, anti-hCCR8 Ab has an IC50 between approximately 0.2 nM and approximately 0.5 nM. 50 This inhibits CCR8 / CCL1 signaling. 50 The values ​​are based on the assay described in Example 15.

[0123] ADCC-mediated death of CCR8-expressing cells by anti-CCR8 mAbs The capacity of anti-hCCR8 and anti-mCCR8 mAbs to induce ADCC-mediated death of CCR8-expressing cells was indirectly evaluated by measuring their ability to induce crosslinking in human or mouse reporter cells expressing the Fc receptor. In certain embodiments, the mAb of the present invention or its antigen-binding moiety, when measured by a CD16 crosslinking assay, showed a certain EC 50 This mediates the depletion of CCR8-expressing cells. In preferred embodiments, ADCC activity is measured by crosslinking assays described in Examples 17 and 18 for hCCR8 and mCCR8, respectively.

[0124] In certain embodiments, the anti-hCCR8 mAb or its antigen-binding moiety, when measured by a CD16 crosslinking assay, has an EC of about 100 pM or less, preferably about 30 pM or less, preferably about 10 pM or less, preferably about 3 pM or less, more preferably about 1 pM or less, more preferably about 0.5 pM or less, more preferably about 0.1 pM or less, or even more preferably about 0.05 pM or less. 50 This mediates the depletion of CCR8-expressing cells. In certain preferred embodiments, the anti-hCCR8 mAb is present at approximately 0.7 pM EC 50 This mediates the depletion of CCR8-expressing cells. In certain embodiments, anti-hCCR8 Ab is used to reduce EC between about 0.05 pM and about 50 pM, preferably between about 0.1 pM and about 10 nM, more preferably between about 0.3 nM and about 7 nM, and even more preferably between about 0.6 nM and about 3 nM. 50 This mediates the depletion of CCR8-expressing cells. These ECs 50 The values ​​are based on the CD16 crosslinking assay described in Example 17.

[0125] Direct killing of activated Tregs and patient tumor Tregs was demonstrated in Examples 19 and 20. In certain embodiments, the anti-hCCR8 mAb or its antigen-binding moiety, as measured by an apoptosis assay, had an EC of about 500 pM or less, preferably about 100 pM or less, preferably about 30 pM or less, more preferably about 15 pM or less, even more preferably about 5 pM or less, or even more preferably about 1 pM or less. 50 This mediates the depletion of activated Tregs. In certain preferred embodiments, the anti-hCCR8 mAb is present at approximately 13 pM EC 50 This mediates the depletion of CCR8-expressing cells. In certain embodiments, anti-hCCR8 mAbs are used to induce EC between about 1 pM and about 500 pM, preferably between about 5 pM and about 100 pM, more preferably between about 10 pM and about 50 pM. 50 This mediates the depletion of CCR8-expressing cells. These ECs 50The values ​​are based on the apoptosis assay described in Example 19.

[0126] CCR8 does not undergo internal migration due to anti-CCR8 mAbs. Medications specific to certain cell surface receptors induce internal translocation of the receptor via receptor-mediated endocytosis, which can be essential for the targeted delivery of certain drugs, toxins, or enzymes for therapeutic purposes. As shown in Figure 19, anti-inducible T cell costimulator (ICOS) mAbs do not induce internal translocation of the ICOS receptor in the absence of cross-linked mAbs, but induce significant ICOS internal translocation in the presence of cross-linked mAbs. In contrast, anti-CCR8 mAbs do not induce internal translocation of CCR8, either in the presence or absence of cross-linked mAbs. Internal translocation of CCR8 by anti-CCR8 mAbs may reduce its ability to mediate Treg depletion due to the removal of the receptor from the Treg cell surface. Therefore, the lack of CCR8 internal translocation by anti-CCR8 mAbs further ensures the therapeutic efficacy of these mAbs. CCR8 internal translocation can be measured by standard techniques used in the art, e.g., the experimental protocol in Example 21.

[0127] Therefore, the present invention provides an anti-CCR8 mAb or its antigen-binding moiety that, when bound to CCR8 on the cell surface, does not cause internal translocation of CCR8, both in the presence or absence of a cross-linked Ab. In a particular preferred embodiment, the cell expressing CCR8 on the cell surface is a Treg.

[0128] Depletion of human tumor Tregs in in vitro and ex vivo patient tumor samples The ability of anti-hCCR8 mAbs to mediate tumor Treg depletion was evaluated in an in vitro system in which warm-immersed patient tumors were co-cultured with allogeneic NK cells (Example 22). The anti-hCCR8 mAb 4A19 was shown to induce tumor Treg depletion without reducing the Teff population. In this in vitro assay system, mAb 4A19 was more effective than anti-CCR4-nf mAbs in Treg depletion (Figure 20A), and CD4 +Anti-CCR4-nf Ab did not induce depletion of effector T cells, but it did cause measurable depletion of these cells (Figure 20B). Both anti-CCR8 and anti-CCR4 are CD8 + Effector T cells were not depleted (Figure 20C).

[0129] The ability of anti-hCCR8 mAbs to induce tumor Treg depletion was also evaluated in an ex vivo assay system using sliced ​​patient tumors (Example 20). mAb 16B13-IgG1-nf was shown to mediate Treg depletion without the addition of allogeneic NK cells (Figures 18H and 18I).

[0130] In certain embodiments, the anti-CCR8 mAb of the present invention or its antigen-binding moiety is used in vitro to bind CD4 + or CD8 + This induces tumor Treg depletion without reducing the effector T cell population. In a preferred embodiment, in vitro tumor Treg depletion is measured using the assay described in Example 22.

[0131] In certain other embodiments, the anti-CCR8 mAb of the present invention or its antigen-binding moiety induces tumor Treg depletion in ex vivo patient tumor tissue samples. In certain preferred embodiments, tumor Treg depletion in ex vivo patient tumor tissue samples is measured using the assay described in Example 20.

[0132] Anti-mCCR8-mediated specific depletion of tumor Tregs, rather than CCR8 T cells, in normal tissue samples. In addition to tumor Tregs, CCR8 is also expressed in a small subset of thymic T cells and in cutaneous commensal T cells, a rare population found in the skin. In the CT26 mouse syngeneic tumor model, which exhibits a CCR8 expression profile very similar to that of humans, the anti-mCCR8 depletion mAb, anti-CCR8-mIgG2a, is found to reduce CCR8 expression in tumors. + Tregs were selectively depleted, but CCR8 was not found in the skin, thymus, spleen, or blood.+ It did not deplete T cells (Example 24). The highly specific depletion activity of anti-CCR8-mIgG2a in tumors rather than other CCR8-expressing organs is due to the relatively low frequency of FcγRIV-expressing cells near Ab-binding target cells, or the presence of CCR8 in the skin, thymus, spleen, and blood. + This is thought to be due to a lower CCR8 surface density in T cells. This suggests that human or humanized IgG1-nf anti-CCR8 mAbs, such as 4A19 or 14S15, which have an optimized affinity for activated FcγR, may be effective in human patients with low CD16 expression in the skin. + I strongly support the view that this could enable powerful tumor Treg depletion without the accidental depletion of T cells.

[0133] In certain embodiments of the disclosed invention, an anti-hCCR8 mAb or its antigen-binding moiety is used to target CCR8 in the skin, thymus, spleen, and blood. + This method specifically induces tumor Treg depletion without depleting T cells. In certain preferred embodiments, tumor Treg depletion in vivo is measured using the assay described in Example 24.

[0134] Anti-CCR8 mAb that binds to the same CCR8 epitope as reference Ab. The present invention also binds to the same hCCR8 epitope as reference Ab, and reference Ab is (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 3 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 15. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 5 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 17.L , (d) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L , (e) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 7 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 19. L , (f) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 8 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 20. L , (g) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 9 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 21. L , (h) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 10 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 22. L , (i) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 11 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 23. L , (j) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 12 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 24. L , (k) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 13 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 25. L , (l) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 14 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 26.L ,or (m) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L The present invention provides an isolated Ab, preferably an mAb, or its antigen-binding moiety, which includes the above.

[0135] In a particular preferred embodiment, the reference Ab is bound to the same hCCR8 epitope as reference Ab, (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 1144 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L ,or (c) VH containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 6, and VL containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 18. An isolated Ab, preferably an mAb, or its antigen-binding moiety, is provided.

[0136] In a particular preferred embodiment, V is bound to the same hCCR8 epitope as reference Ab, and reference Ab contains a sequence of linked amino acids having the sequence described in SEQ ID NO: 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L An isolated Ab, preferably an mAb, or its antigen-binding moiety, is provided.

[0137] Anti-CCR8 mAb cross-competes with reference Ab for binding to CCR8. Isolated Abs, preferably mAbs, or their antigen-binding moieties, that specifically bind to hCCR8 expressed on the cell surface and cross-compete with a reference Ab or its reference antigen-binding moiety for binding to hCCR8 are also included within the scope of the disclosed invention. 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, sterically interfering with the binding of the second Ab to that particular epitope region, and conversely, that the second Ab binds to substantially the same epitope region of the antigen as the first Ab, sterically interfering with the binding of the first Ab to that epitope region. Thus, the ability of a test Ab to competitively inhibit the binding of, for example, mAb 14S15 or 4A19 to hCCR8 demonstrates that the test Ab binds to substantially the same epitope region of hCCR8 as mAb 14S15 or 4A19.

[0138] A first Ab is considered to bind to "substantially the same epitope" as the second Ab if it reduces the binding of the second Ab to the antigen by at least about 40%. Preferably, the first Ab reduces the binding of the second Ab to the antigen by more than about 50% (e.g., at least about 60% or at least about 70%). In a more preferred embodiment, the first Ab reduces the binding of the second Ab to the antigen by more than about 70% (e.g., at least about 80%, at least about 90%, or about 100%). The order of the first and second Abs may be reversed, i.e., the "second" Ab may bind to the surface first, and then the "first" may come into contact with the surface in the presence of the "second" Ab. Regardless of the order in which the Abs are added to the immobilized antigen, if a competitive reduction in binding to the antigen is observed, the Abs are considered to "cross-compete".

[0139] Cross-competitive Abs are expected to have very similar functional properties to a reference Ab for binding to the epitope region of substantially the same antigen, such as the CCR8 receptor. The higher the degree of cross-competition, the more similar the functional properties will be. For example, two cross-competitive Abs are expected to have essentially the same functional properties if each inhibits the other's binding to the epitope by at least about 80%. This similarity in function is due to the fact that the cross-competitive Abs have a dissociation constant (K). D If the affinities for binding to the epitope are similar when measured by ), then an even closer bond is expected.

[0140] Cross-competitive anti-antigens (Abs) can be readily identified using either recombinant antigen molecules or cell surface-expressed antigen molecules in standard antigen-binding assays, including BIACORE® analysis, ELISA assays, or flow cytometry, based on their detectably competitive ability. For example, a simple competition assay to identify whether test Ab competes with mAb 4A19 for binding to hCCR8 may involve (1) measuring the binding of 4A19, used at a saturated concentration, to a BIACORE® chip (or other suitable medium for SPR analysis) on which hCCR8 is immobilized, and (2) measuring the binding of 4A19 to an hCCR8-coated BIACORE® chip (or other suitable medium) on which test Ab is pre-bound. The binding of 4A19 to the hCCR8-1 coated surface is compared in the presence and absence of test Ab. A significant reduction (e.g., over 40%) in the binding of 4A19 in the presence of test Ab indicates that both Abs recognize substantially the same epitope to compete for binding to the hCCR8 target. The percentage of the binding of the first Ab to the antigen inhibited by the second Ab can be calculated as [1 - (detected binding of the first Ab in the presence of the second Ab) / (detected binding of the first Ab in the absence of the second Ab)] × 100. To determine whether the Abs cross-compete, the competitive binding assay is repeated, except that the binding of test Ab to the hCCR8 coated chip is measured in the presence of 4A19.

[0141] Any of the anti-CCR8 Abs disclosed herein may serve as a reference Ab in cross-competition assays. In certain embodiments, the reference Ab is: (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 3 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 15. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 5 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 17. L , (d) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L , (e) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 7 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 19. L , (f) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 8 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 20. L , (g) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 9 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 21. L , (h) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 10 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 22. L , (i) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 11 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 23. L , (j) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 12 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 24. L , (k) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 13 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 25. L , (l) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 14 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 26. L ,or (m) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L Includes.

[0142] In a particular preferred embodiment, reference Ab is (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 1144 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L ,or (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L Includes.

[0143] In a particular preferred embodiment, reference Ab is V, which contains a sequence of linked amino acids having the sequence described in Sequence ID No. 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L Includes.

[0144] Structurally defined anti-CCR8 mAb This disclosure also specifically binds to hCCR8 expressed on the surface of cells, (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 3 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 15. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 5 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 17. L , (d) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L , (e) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 7 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 19. L , (f) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 8 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 20. L , (g) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 9 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 21. L, (h) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 10 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 22. L , (i) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 11 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 23. L , (j) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 12 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 24. L , (k) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 13 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 25. L , (l) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 14 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 26. L ,or (m) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L The present invention provides an isolated Ab, preferably an mAb, or its antigen-binding moiety, each containing the CDR1, CDR2, and CDR3 domains.

[0145] It specifically binds to hCCR8 expressed on the surface of cells, (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 1144 HV containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L ,or (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L In each of these, an isolated Ab, preferably an mAb, or its antigen-binding moiety, is preferred, comprising the CDR1, CDR2, and CDR3 domains.

[0146] V contains a sequence of continuously linked amino acids that specifically binds to hCCR8 expressed on the cell surface and has the sequence described in SEQ ID NO: 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L In each of these, an isolated Ab, preferably an mAb, or its antigen-binding moiety, is preferred, comprising the CDR1, CDR2, and CDR3 domains.

[0147] Various methods have been developed to describe the CDR domain within Ab. The methods of Kabat and his collaborators (Wu and Kabat, 1970; Kabat et al., 1983) were based on the assumption that CDRs contain the most variable positions in Ab and are therefore identifiable even when aligned with a fairly limited number of Ab sequences available at the time. Based on this alignment, Kabat et al. introduced a residue numbering scheme in the hypervariable region to determine which positions represent the start and end points of each CDR (http: / / bioinf.org.uk / abs / simkab.html).

[0148] In addition to the widely used definition of Kabat, other definitions are used, 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 IMGT (Lefranc et al., 2003, http: / / www.imgt.org), which attempt to address the shortcomings of the Kabat definition. Kabat's definition, despite being developed in an era when structural information on Ab was not available, remains the most commonly used method for CDR domain prediction.

[0149] Unless otherwise explicitly stated or indicated by the context, the CDRs disclosed herein are identified using the Kabat definition. The amino acid sequences of the six CDR domains defined using the Kabat method, as well as the V of mAb 16B13, 14S15, 14S15h, 18Y12, 4A19, 2M18, 15C17, 13T20, 10R3, 8D55, 1V11, 11K16, and 12F27. H , V L The amino acid sequences of the heavy chain and light chain are shown in Table 10.

[0150] This invention relates to the following CDR domains as defined by the Kabat method: (a) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 27, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 28, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 29, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 30, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 31, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 32. (b) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 33, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 34, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 35, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 36, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 37, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 38. (c) Heavy chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 39, heavy chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 40, heavy chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 41, light chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 42, light chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 43, and light chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 44. (d) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 45, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 46, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 47, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 48, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 49, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 50. (e) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 51, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 52, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 53, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 54, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 55, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 56. (f) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 57, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 58, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 59, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 60, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 61, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 62. (g) Heavy chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 63, heavy chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 64, heavy chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 65, light chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 66, light chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 67, and light chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 68. (h) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 69, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 70, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 71, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 72, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 73, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 74. (i) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 75, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 76, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 77, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 78, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 79, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 80. (j) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 81, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 82, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 83, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 84, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 85, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 86. (k) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 87, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 88, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 89, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 90, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 91, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 92. (l) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 93, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 94, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 95, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 96, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 97, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 98, or (m) Heavy chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 103, heavy chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 104, heavy chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 105, light chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 106, light chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 107, and light chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 108 The present invention provides an isolated Ab, preferably an mAb, containing [the specified substance].

[0151] A preferred isolated Ab, preferably an mAb, has the following CDR domain as defined by the Kabat method: (a) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 33, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 34, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 35, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 36, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 37, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 38. (b) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 103, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 104, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 105, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 106, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 107, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 108, or (c) Heavy chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 45, heavy chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 46, heavy chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 47, light chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 48, light chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 49, and light chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 50 Includes.

[0152] In a particular preferred embodiment, the isolated Ab, preferably an mAb, comprises the following CDR domains as defined by the Kabat method: heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 45; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 46; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 47; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 48; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 49; and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 50.

[0153] The disclosed invention also relates to an isolated Ab, preferably an mAb, or its antigen-binding moiety, which specifically binds to hCCR8 expressed on the surface of a cell, wherein the isolated Ab or its antigen-binding moiety (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 3 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 15. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 5 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 17. L , (d) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L , (e) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 7 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 19. L , (f) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 8 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 20. L , (g) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 9 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 21. L , (h) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 10 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 22. L , (i) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 11 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 23. L , (j) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 12 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 24. L , (k) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 13 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 25. L , (l) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 14 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 26. L ,or (m) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L It comprises an isolated Ab, preferably an mAb, or its antigen-binding moiety.

[0154] A preferred isolated Ab, preferably an mAb, or its antigen-binding moiety specifically binds to hCCR8 expressed on the cell surface. (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L , (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L ,or (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L Includes.

[0155] A preferred isolated Ab, preferably an mAb, or its antigen-binding moiety specifically binds to hCCR8 expressed on the cell surface and contains a V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L Includes.

[0156] V has an amino acid sequence that is highly similar to or homologous to any of the above anti-CCR8 Ab amino acid sequences.H and V L Anti-CCR8 Abs that include the region and retain the functional properties of these Abs are also suitable for use in this method. For example, a suitable Ab is a V containing continuously linked amino acids, each having a sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NOs. 6 and / or 18, respectively. H and / or V L Examples include mAbs containing regions. In further embodiments, for example, V H and / or V L The amino acid sequences exhibit at least 85%, 90%, 95%, or 99% identity with the sequences described in Sequence ID No. 6 and / or 18, respectively. As used herein, the sequence identity percentage between two amino acid sequences is a function of the number of identical positions shared by the sequences to the length of the sequences being compared, taking into account the number of any gaps introduced to maximize the degree of sequence identity between the two sequences and the length of each such gap (i.e., identity % = number of identical positions / total number of positions being compared × 100). The comparison of sequences and the determination of the identity percentage between two sequences can be achieved using mathematical algorithms well known to those skilled in the art.

[0157] V has an amino acid sequence that is highly similar to or homologous to any of the above anti-CCR8 Ab amino acid sequences. H and V L If anti-CCR8 Abs are disclosed that include regions and retain the functional properties of these Abs, they shall be 100% identical in at least one, two, three, four, five, or six CDRs, as well as the associated complete V H and / or V L The sequence may have at least 85%, 90%, 95%, or 99% identity.

[0158] For example, an isolated Ab, preferably an mAb, or its antigen-binding moiety, that specifically binds to hCCR8 expressed on the surface of cells. (a) V containing a sequence of continuously linked amino acids having at least 85%, 90%, 95%, or 99% the same sequence as described in Sequence ID No. 4 H V containing a sequence of continuously linked amino acids having at least 85%, 90%, 95%, or 99% the same sequence as described in Sequence ID No. 16. L And V H This includes at least one, two, or all three (sequences 33-35) of the CDRs defined with respect to sequence number 4, and V H This may include at least one, two, or all three (sequences 36-38) of the CDRs defined with respect to sequence number 16, V H and V L , (b) V containing a sequence of continuously linked amino acids having at least 85%, 90%, 95%, or 99% the same sequence as described in Sequence ID No. 115 H V containing a sequence of continuously linked amino acids having at least 85%, 90%, 95%, or 99% the same sequence as described in Sequence ID No. 116. L And V H This includes at least one, two, or all three (sequences 103-105) of the CDRs defined with respect to sequence number 115, and V L This may include at least one, two, or all three (sequences 106-108) of the CDRs defined with respect to sequence number 116, V H and V L ,or (c) V containing a sequence of continuously linked amino acids having at least 85%, 90%, 95%, or 99% the same sequence as described in Sequence ID No. 6 H V containing a sequence of continuously linked amino acids having at least 85%, 90%, 95%, or 99% the same sequence as described in Sequence ID No. 18. L And V H This includes at least one, two, or all three (sequences 45-47) of the CDRs defined with respect to sequence number 6, and V LThis may include at least one, two, or all three (sequences 48-50) of the CDRs defined with respect to sequence number 18, V H and V L It may include.

[0159] Similarly, in any of the other isolated Abs mentioned herein, Ab is, reference V H V containing a sequence that is at least 85%, 90%, 95%, or 99% identical to the sequence. H , and reference V H V containing a sequence that is at least 85%, 90%, 95%, or 99% identical to the sequence. L And V H Reference V H The sequence includes at least one, two, or all three of the CDRs defined for the sequence, V L Reference V L V may include at least one, two, or all three of the CDRs defined for the array. H and V L It may include.

[0160] The present invention relates to an isolated Ab, preferably an mAb, or an antigen-binding moiety thereof, which may specifically bind to hCCR8 expressed on the surface of cells, wherein the isolated Ab or its antigen-binding moiety is (a) A heavy chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 99, and a light chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 111, (b) A heavy chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 100, and a light chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 112. (c) A heavy chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 101, and a light chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 113. (d) A heavy chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 114. (e) A heavy chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 117, and a light chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 118, or (f) A heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 110, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 119. It further includes an isolated Ab, preferably an mAb, or its antigen-binding moiety, which contains the above.

[0161] An isolated Ab, preferably an mAb, or its antigen-binding moiety, which may specifically bind to hCCR8 expressed on the surface of cells, wherein the isolated Ab or its antigen-binding moiety is (a) A heavy chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 100, and a light chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 112. (b) A heavy chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 117, and a light chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 118, or (c) A heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 114. An isolated Ab, preferably an mAb, or its antigen-binding moiety, containing an isolated Ab, is preferred.

[0162] An isolated Ab, preferably an mAb, or its antigen-binding moiety, which may specifically bind to hCCR8 expressed on the surface of cells, wherein the isolated Ab or its antigen-binding moiety is (a) A heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 114. An isolated Ab, preferably an mAb, or its antigen-binding moiety, containing an isolated Ab, is preferred.

[0163] In certain embodiments, the isolated anti-CCR8 Ab or its antigen-binding moiety of the present invention is a human Ab or a fragment thereof. In other embodiments, it is a humanized Ab or a fragment thereof. In further embodiments, it is a chimeric Ab or a fragment thereof. In other embodiments, the isolated anti-CCR8 Ab or its antigen-binding moiety is a mouse Ab or a fragment thereof. For administration to human subjects, the Ab is preferably a chimeric Ab, more preferably a humanized or human Ab. Such chimeric, humanized, human, or mouse mAbs can be prepared and isolated by methods well known in the art.

[0164] The anti-CCR8 Ab disclosed herein includes not only full-length Ab but also antigen-binding fragments capable of mediating ADCC.

[0165] Anti-CCR8 immunoconjugate In another aspect, the present invention relates to any isolated anti-hCCR8 Ab or its antigen-binding moiety disclosed herein, which is conjugated to a cell-lysating agent such as a cytotoxin or a radioisotope. Such conjugates are referred to herein as “immunoconjugates.” Cytotoxins can be conjugated with the Ab of the present invention using linker techniques available in the art. Methods for preparing radioactive immunoconjugates have also been demonstrated in the art.

[0166] bispecific molecules In another embodiment, the present invention relates to a bispecific molecule comprising one of the isolated anti-hCCR8 mAbs or their antigen-binding moieties disclosed herein, linked to a binding domain having different binding specificity from the anti-hCCR8 mAb or its antigen-binding moiety. The binding domain may be a functional molecule (e.g., another Ab, the antigen-binding moiety of an Ab, or a ligand for a receptor), and as a result, the resulting bispecific molecule binds to at least two different binding sites or target molecules.

[0167] Nucleic acid encoding anti-hCCR8 mAb and its use for expressing Ab Another aspect of this disclosure relates to nucleic acids encoding any of the isolated anti-hCCR8 Abs of the present invention. This disclosure provides isolated nucleic acids encoding any of the anti-CCR8 mAbs or their antigen-binding moieties described herein.

[0168] "Isolated" nucleic acids refer to nucleic acid compositions of substances that are significantly different from naturally occurring nucleic acids, i.e., possess distinct chemical identity, properties, and utility. For example, isolated DNA, unlike natural DNA, is an independent portion of natural DNA and not part of the larger structural complex found in nature, i.e., an integrated portion of a chromosome. Furthermore, unlike natural DNA, isolated DNA can, among other things, be used as PCR primers or hybridization probes to detect biomarker genes or mutations by measuring gene expression to diagnose diseases or predict the effectiveness of therapeutic drugs. Isolated nucleic acids can also be purified using standard techniques well known in the art so as to be substantially free of other cellular components and other contaminants, such as other cellular nucleic acids and proteins.

[0169] The nucleic acids of the present invention can be obtained using standard molecular biological techniques. In the case of Ab expressed by a hybridoma (for example, a hybridoma prepared from a transgenic mouse possessing the human Ig gene as described in Example 8), the cDNA encoding the light and heavy chains or variable regions of Ab produced by the hybridoma can be obtained by standard PCR amplification techniques. H and V L Once segment-coding DNA fragments are obtained, these fragments can be further manipulated using standard recombinant DNA techniques to convert, for example, variable region DNA into full-length Ab strand genes, Fab fragment genes, or scFv genes. In the case of Ab obtained from an Ig gene library (e.g., using phage display techniques), the nucleic acid encoding Ab can be recovered from the library.

[0170] The nucleic acid of the present invention may be, for example, RNA, cDNA, or DNA such as genomic DNA. In preferred embodiments, the nucleic acid is cDNA.

[0171] This disclosure also provides an expression vector comprising an isolated nucleic acid encoding an anti-CCR8 mAb or its antigen-binding moiety. This disclosure further provides host cells comprising the expression vector. Eukaryotic cells, most preferably mammalian host cells, are preferred as host cells for expressing the Ab. This is because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to construct and secrete properly folded immunologically active Abs. 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.

[0172] Host cells may be used in a method for preparing an anti-CCR8 mAb or its antigen-binding moiety, comprising expressing the mAb or its antigen-binding moiety in host cells and isolating the mAb or its antigen-binding moiety from the host cells. Host cells may be used ex vivo or in vivo. The DNA encoding the Ab heavy and light strands may be inserted into separate expression vectors, or more typically, both may be inserted into the same vector. H and V L The segment is V H The segment is C in the vector H It is operationally linked to the segment(s), V κ The segment is C in the vector L These variable regions can be used to produce full-length mAbs of any isotype by inserting the DNA encoding them into an expression vector already encoding the heavy and light chain constant regions of the desired isotype, so as to be operationally linked to the segments. Cell lines lacking the α-(1,6) fucosyltransferase (FUT8) enzyme can be used to produce mAbs lacking fucose in their carbohydrate (see, for example, U.S. Patent Application Publication No. 2004 / 0110704, Yamane-Ohnuki et al., 2004, and European Patent No. 1176195).

[0173] Another aspect of the present invention relates to a transgenic mouse comprising a human Ig heavy and light chain transgene, expressing any of the anti-CCR8 HuMAbs disclosed herein. The present invention also includes a hybridoma prepared from the mouse, which produces a HuMAb.

[0174] Treatment anti-CCR8 methods In this study, since the anti-hCCR8 mAb generated does not bind to mCCR8, the efficacy of anti-CCR8 as an anticancer drug was tested in a mouse tumor model using a mouse anti-mCCR8 mAb as a surrogate for human or humanized nf IgG1 anti-hCCR8 mAb. The selection of an appropriate AB isotype was determined by the abundance of FcγRIV in mouse tumors. + It has been demonstrated that it is primarily driven by cell interaction (Nimmerjahn et al., 2010) and can be used to enhance ADCC / ADCP-mediated CCR8-mediated tumor Treg depletion in mice (Simpson et al., 2013). An anti-mCCR8 mAb with the mouse IgG2a isotype, referred to as anti-CCR8-mIgG2a, was used as a surrogate for humanized or human anti-hCCR8 mAbs that could be used as human therapeutic agents. Anti-CCR8-mIgG2a is derived from a commercially available rat anti-mCCR8 mAb marketed by BioLegend as clone SA214G2, modified to change the rat IgG2b isotype to the mouse IgG2a isotype. Similar to the human anti-hCCR8 mAbs A419 and 14S15, anti-CCR8-mIgG2a is an N-terminal binding agent, and its mIgG2a scaffold achieves maximum FcγR binding for driving the ADCC response similar to the human nf structure.

[0175] Anti-CCR8-mIgG2a was demonstrated to have high ADCC activity comparable to some of the high ADCC activity of the tested anti-hCCR8 mAbs (Example 17) (Example 18), and to block the binding of mCCL1 to mCCR8-expressing cells (Example 16), but with lower potency than some of the tested anti-hCCR8 mAbs (Example 15). Anti-hCCR8 mAbs also exhibit potent ADCC activity (Examples 19 and 20). Anti-CCR8-mIgG2a also binds to mCCR8-expressing cells with lower affinity than some of the anti-hCCR8 mAbs of this disclosure (Example 13). This suggests that while anti-CCR8-mIgG2a is a suitable surrogate of anti-hCCR8 mAb for direct studies of tumor growth inhibition in mouse models, anti-hCCR8 Ab may be even more potent in inhibiting tumor growth in human subjects, considering its higher affinity binding to allogeneic hCCR8 targets, higher activity in blocking the binding of CCL1 ligand to CCR8, and higher ADCC capacity.

[0176] The antitumor activity of anti-CCR8-mIgG2a mouse surrogate mAbs, both alone and in combination with anti-PD-1, was determined using various syngeneic mouse tumor models. Anti-CCR8-mIgG2a mAb-mediated Treg depletion and subsequent pro-inflammatory responses (increases in CD8, CD4, interferon-γ, granzyme B, and Ki67) induced robust tumor growth inhibition with high percentages of complete tumor clearance in immunogenic mouse tumor models, including the CT26 and MC38 colon adenocarcinoma models and the SA1N fibrosarcoma model (see Examples 23-26 and 29), and in combination with anti-PD-1 in the immunotherapy-resistant models MB49 and 4T1. In contrast, treatment with CCR8-mIgG1-D265A, a variant Ab containing an inactive Fc-inactive mIgG1-D265A heavy chain (Baudino et al., 2008), had the least effect on tumor reduction (Examples 29 and 30). To determine whether a dose-dependent relationship exists between Treg depletion and tumor efficacy in anti-CCR8 Ab treatment, single-dose monotherapy studies were conducted in the MC38 model, and a dose-dependent pharmacokinetic / pharmacodynamic / efficacy relationship was observed (Example 26). In addition, increased titrable CCL1 ligand changes were observed in the tumor supernatant upon depletion (data not shown), which may serve as a surrogate biomarker.

[0177] In MB49 mouse bladder cancer, anti-CCR8-mIgG2a showed only partial antitumor activity, but this was stronger than the activity of anti-PD-1 mAb (Example 27). Similarly, in the 4T1 mouse mammary cancer model, anti-CCR8-mIgG2a also showed only partial antitumor activity, but this tumor was completely resistant to treatment with anti-PD-1 mAb (Example 28). In both tumor models, anti-CCR8-mIgG2a enhanced the anti-PD-1 induced immune response, resulting in a significant increase in the potency of the antitumor response. The combination of the two Abs is synergistic in that it potently inhibits tumor growth in these refractory tumor models. Herein, Abs are considered to interact synergistically if the antitumor efficacy of the combination of these Abs is greater than the sum of the antitumor efficacy exhibited individually by each Ab.

[0178] Tumor Treg-specific depletion in human tumor explantation models was also demonstrated with treatment using 14S15, 16B13, and 4A19 mAbs (Example 20).

[0179] Cancer treatment using anti-CCR8 mAb monotherapy The data provided herein has high affinity (EC 50 = approximately 0.1~2nM, Example 11) The production of anti-hCCR8 mAbs that bind to hCCR8 on the surface of activated Tregs induces potent ADCC-mediated Treg death in an in vitro system in which primary activated human Tregs are co-cultured with allogeneic activated NK cells (EC 50 = approximately 10-60 pM, Example 19), CCR8 in human tumor explants and mouse models + These mAbs demonstrate specific mediation of tumor-infiltrating Treg depletion (Examples 20, 22, and 24). These mAbs exhibit robust inhibition of tumor growth in diverse immunogenic and immunotherapy-resistant mouse tumor models (Examples 23, 25, 27-29), and the data strongly support the clinical evaluation of CCR8 depletion as a novel immunotherapy for cancer, either as monotherapy or in combination with immune checkpoint blockers.

[0180] Accordingly, as supported by the data provided in the examples, the present disclosure provides a method for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of any one of the Treg-depleting anti-CCR8 Abs disclosed herein, e.g., mAbs, immunoconjugates, or bispecific molecules, or a pharmaceutical composition comprising any one of the said Abs, e.g., anti-CCR8 mAbs, immunoconjugates, or bispecific molecules, so that the subject is treated.

[0181] The disclosure also provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of any one of the Treg-depleting anti-CCR8 Abs disclosed herein, e.g., mAbs, immunoconjugates, or bispecific molecules, or a pharmaceutical composition comprising any one of the anti-CCR8 Abs, e.g., mAbs, immunoconjugates, or bispecific molecules, such that the growth of tumor cells in the subject is inhibited.

[0182] Cancer treatment using anti-CCR8 Ab in combination with other anticancer drugs This disclosure provides a method for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of (a) a pharmaceutical composition comprising any one of the Treg-depleting anti-CCR8 Ab, immunoconjugate, or bispecific molecule disclosed herein, or any one of the anti-CCR8 Ab, immunoconjugate, or bispecific molecule, and (b) an additional therapeutic agent for treating cancer, which may be a compound that suppresses or increases the stimulation of the immune system, so that the subject is treated.

[0183] The disclosure also provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of (a) one of the Treg-depleting anti-CCR8 Ab, immunoconjugate, or bispecific molecule disclosed herein, or a pharmaceutical composition comprising one of the anti-CCR8 Ab, immunoconjugate, or bispecific molecule, and (b) an additional therapeutic agent for treating cancer. In certain preferred embodiments, the additional therapeutic agent is a compound that suppresses or increases the inhibition of the immune system so as to inhibit the growth of tumor cells in the subject.

[0184] In any other preferred embodiment of this method, the subject is a human patient.

[0185] Treg-mediated immunosuppression may be a major barrier to the optimal antitumor immune response in cancer immunotherapy. Many molecules targeted by cancer immunotherapies, such as PD-1, CTLA-4, LAG3, TIM3, and TIGIT, are upregulated in Tregs (Kumar et al., 2018). Consequently, these T cell-based immunotherapies may also amplify the Treg response. For example, PD-1 blockade may enhance Treg suppression and increase Treg proliferation (Kamada et al., 2019), and there is clinical evidence of Treg enlargement after anti-CTLA-4 therapy (Kavanagh et al., 2008). Therefore, Treg depletion agents not only improve the antitumor response as monotherapy but also enhance the activity of other immunotherapies as disclosed herein (see Examples 27 and 28). Accordingly, the present disclosure provides a method for enhancing a therapeutic agent-induced antitumor immune response in a subject with cancer, wherein the therapeutic agent is an immunotherapy agent such as cancer immunotherapy, for example, an antibody that specifically binds to PD-1, CTLA-4, LAG3, TIM3, or TIGIT.

[0186] In a mouse tumor model, potent antitumor activity was observed in mice treated with both anti-CCR8-mIgG2a and anti-PD-1 (Examples 27 and 28). Similar CD8 activity was observed in anti-CCR8 monotherapy and combination therapy. + Despite the increased T cell frequency, anti-PD-1 may be required to enhance effector functionality. In addition, anti-CCR8 treatment may enhance persistent antigen-specific CD8 + It was demonstrated that it induces T cell memory (Example 31). These findings have important clinical implications for the treatment of advanced cancers that often develop resistance to anti-PD1 / PD-L1 therapy (Jenkins et al., 2018) and exhibit high recurrence rates (Mahvi et al., 2018). Overall, these studies highlight the specificity and targetability of CCR8 in tumor Tregs and support the evaluation of anti-CCR8 depletion agents in the treatment of advanced solid tumors, either as monotherapy or in combination with other immunotherapies such as anti-PD-1, anti-PD-L1, or anti-CTLA-4.

[0187] In certain embodiments of the disclosed method, the additional therapeutic agent is a compound that suppresses the inhibition of the immune system. For example, the additional therapeutic agent may be a small molecule compound, a macrocyclic peptide, a fusion protein, or an Ab, such as an mAb. In further embodiments, additional therapeutic agents are antagonists such as programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activator gene-3 (LAG-3), B and T lymphocyte attenuator (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), killer immunoglobulin-like receptor (KIR), killer cell lectin-like receptor G1 (KLRG-1), adenosine A2a receptor (A2aR), T cell immune receptor having Ig and ITIM domains (TIGIT), V domain Ig-containing T cell activation inhibitor (VISTA), proto-oncogene tyrosine protein kinase MER (MerTK), natural killer cell receptor 2B4 (CD244), or antagonist mAbs that specifically bind to CD160.

[0188] In certain preferred embodiments, the additional therapeutic agent is an antagonist Ab or its antigen-binding moiety that specifically binds to PD-1. In further embodiments, the Ab that specifically binds to PD-1 is selected from nivolumab, pembrolizumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, retifanlimab, and pimivalimab, for example, selected from nivolumab, pembrolizumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, and, for example, tripalimab, and, for example, selected from nivolumab and pembrolizumab.

[0189] In other preferred embodiments, the additional therapeutic agent is an antagonist Ab that specifically binds to PD-L1 or its antigen-binding moiety. In further embodiments, the Ab that specifically binds to PD-L1 is selected from atezolizumab, durvalumab, avelumab, emvafolimab, BMS-936559, CK-301, CS-1001, SHR-1316, CBT-502, BGB-A333, and KN035, for example, selected from atezolizumab, durvalumab, avelumab, emvafolimab, BMS-936559, CK-301, CS-1001, SHR-1316, CBT-502, BGB-A333, for example, selected from atezolizumab, durvalumab, and avelumab.

[0190] In other preferred embodiments, the additional therapeutic agent is an antagonist Ab or its antigen-binding moiety that specifically binds to CTLA-4. In further embodiments, the Ab that specifically binds to CTLA-4 is ipilimumab or tremelimumab, selected from ipilimumab, for example.

[0191] This disclosure further provides a method for enhancing a therapeutically induced antitumor immune response in a subject with cancer, comprising administering to the subject a therapeutically effective amount of one of the Treg-depleting anti-CCR8 Ab, immunoconjugate, or bispecific molecule disclosed herein, or a pharmaceutical composition comprising one of the anti-CCR8 Ab, immunoconjugate, or bispecific molecule, such that the subject experiences a stronger immune response against cancer compared to the immune response induced by the therapeutic agent alone. In certain preferred embodiments of this method, the therapeutic agent is a checkpoint inhibitor, such as an anti-PD-1, anti-PD-L1, or anti-CTLA-4 mAb. In certain preferred embodiments of this method, the therapeutic agent is nivolumab, an anti-PD-1 Ab. In other preferred embodiments, the therapeutic agent is pembrolizumab, an anti-PD-1 Ab. In certain preferred embodiments, the therapeutic agent is atezolizumab, an anti-PD-L1 Ab. In other preferred embodiments, the therapeutic agent is durvalumab, an anti-PD-L1 Ab. In a more preferred embodiment, the therapeutic agent is avelumab, an anti-PD-L1 Ab. In a particular preferred embodiment of this method, the therapeutic agent is ipilimumab, an anti-CTLA-4 Ab. In a particular other embodiment, the therapeutic agent is radiotherapy.

[0192] Cancers treatable by the methods disclosed herein Cancer immunotherapy, which relies on using the virtually limitless flexibility of the immune system to attack and destroy cancer cells, is applicable to a very wide range of cancers (see, e.g., Yao et al., 2013; Callahan et al., 2016; Pianko et al., 2017; Farkona et al., 2016; Kamta et al., 2017). For example, nivolumab, an anti-PD-1 absorber, has been shown to be effective in treating many different types of cancer (see, e.g., Brahmer et al., 2015; Guo et al., 2017; Pianko et al., 2017; see International Publication No. 2013 / 173223), and is currently undergoing clinical trials in several solid tumors and hematological malignancies. Therefore, the methods of this disclosure, which utilize CCR8-mediated depletion of tumor-infiltrating Tregs, either as monotherapy or in combination with other immunotherapies such as immune checkpoint inhibitors, are applicable to the treatment of a wide variety of solid and liquid tumors.

[0193] A wide range of treatable cancers The antibodies (Abs) used in the cancer treatment methods disclosed herein do not directly target cancer cells, but rather target and enhance the immune system by depleting immunosuppressive Tregs. Therefore, when combined with immune checkpoint inhibitors, which facilitate the immune system's attack and destruction of cancer cells, these antibodies are applicable to a wide range of cancer treatments. The efficacy of nivolumab in the treatment of diverse cancers has been demonstrated by its approval for the treatment of advanced melanoma, advanced non-small cell lung cancer, metastatic renal cell carcinoma, classical Hodgkin lymphoma, advanced squamous cell carcinoma of the head and neck, metastatic urothelial carcinoma, MSI-H or dMMR metastatic colorectal cancer, hepatocellular carcinoma, small cell lung cancer, and esophageal squamous cell carcinoma (Drugs.com - Opdivo Approval History: https: / / www.drugs.com / history / opdivo.html), and clinical trials are underway for many other cancers. Similarly, anti-PD-L1 drugs [e.g., atezolizumab (TECENTRIQ®), durvalumab [IMFINZI®], and avelumab [BAVENCIO®]] are gaining approval for a variety of indications. Therefore, a wide variety of different cancers can be treated using anti-CCR8 agents, and, as appropriate, combinations of anti-CCR8 and anti-PD-1 / PD-L1 agents. Given the high efficacy demonstrated by this combination therapy, it becomes possible to focus on cancers that suffer from significant unmet medical needs.

[0194] In certain embodiments, the cancer therapies of this disclosure may be used to treat cancers that are solid tumors. For example, in certain embodiments, these solid tumors include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, head and neck cancer, breast cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, small intestine cancer, liver cancer, hepatocellular carcinoma (HCC), pancreatic cancer (PAC), kidney cancer, renal cell carcinoma (RCC), bladder cancer, urethral cancer, ureteral cancer, colorectal cancer (CRC), colon cancer, anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, paranasal sinus natural killer, melanoma, skin cancer, bone cancer, and cervical cancer. The cancers are selected from uterine cancer, endometrial carcinoma, fallopian tube carcinoma, ovarian cancer, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, testicular cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, penile cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, solid tumors in children, environment-induced cancer, virus-associated cancer, cancer of viral origin, advanced cancer, unresectable cancer, metastatic cancer, refractory cancer, recurrent cancer, and any combination thereof. In certain embodiments, the cancer is advanced, unresectable, metastatic, refractory, and / or recurrent cancer.

[0195] Based on the demonstration of effective treatment of various cancers with anti-CCR8 in mouse models (Examples 23-29), certain tumor types are expected to be particularly suitable for treatment with anti-CCR8 Ab. Therefore, in certain embodiments, solid tumors are cancers selected from colon adenocarcinoma, bladder cancer, breast cancer, and fibrosarcoma. The finding that anti-CCR8 is effective in tumor reduction in mouse models where anti-PD-1 shows little efficacy, e.g., M49 bladder (Example 27) and 4T1 breast cancer model (Example 28), suggests that anti-CCR8 may be very broadly applicable in cancer treatment and may be broadly more effective than anti-PD-1. Furthermore, combinations of anti-CCR8 with checkpoint blockers, e.g., anti-PD-1, anti-PD-L1, or anti-CTLA-4, may be even more broadly applicable to the treatment of diverse cancers.

[0196] CCR8 + To analyze the various gene expressions of Treg, single-cell RNA sequencing was performed on human tumors (Example 32). Based on the relatively high expression of CCR8 and CD8A, and their high CCR8 / CD8A ratios, the following cancer types were identified as particularly suitable for treatment with anti-CCR8 Ab: 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). Therefore, in certain embodiments, solid tumors are cancers selected from HNSC, LUAD, STAD, LUSC, PAAD, READ, ESCA, BRCA, COAD, CESC, follicular lymphoma, acute lymphoblastic leukemia, and lymphoma as cancer types that are expected to be particularly suitable for treatment with anti-CCR8 Ab.

[0197] In 17 tumor types or subtypes, CCR8 expression, as assessed by IHC in formalin-fixed paraffin-embedded (FFPE) tissue samples, was found to be high in head and neck squamous cell carcinoma (HNSCC; also referred herein to as head and neck squamous cell carcinoma [SCCHN]) and lowest in glioblastoma multiforme (GBM; Example 33). Based on the fact that tumors expressing high levels of CCR8 are more responsive to treatment with anti-CCR8 Ab, this tumor profiling data supports prioritizing HNSCC, cervical 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 for anti-CCR8 treatment. Thus, in certain embodiments, the solid tumor is a cancer selected from HNSCC, cervical cancer, CRC, NSCLC-SCC, NSCLC-ADC, pancreatic cancer, gastric cancer, bladder cancer, and breast cancer.

[0198] In certain embodiments, this treatment method may be used to treat cancers that are hematological malignancies. Hematological malignancies include humoral tumors that originate from either of two major blood cell lineages [i.e., myeloid cell lineages (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphocyte cell lineages (producing B cells, T cells, NK cells, and plasma cells)], such as all types of leukemia, lymphoma, and myeloma. Hematological malignancies that can be treated with this therapy include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal immunoglobulinemia of unknown significance (MGUS), advanced, metastatic, refractory, and / or recurrent hematological malignancies, and any combination of the aforementioned hematological malignancies.

[0199] TARGET (Therapeutically Applicable Research to Generate Effective Treatments, https: / / ocg.cancer.gov / programs / target) analysis revealed that among the hematological malignancies examined, follicular lymphoma, acute lymphoblastic leukemia, and lymphoma showed the highest relative expression of CCR8, and therefore should be prioritized for treatment with anti-CCR8 mAbs (Example 32). Therefore, in certain embodiments of this therapy, the hematological malignancies are follicular lymphoma, acute lymphoblastic leukemia, and lymphoma.

[0200] In certain other embodiments, hematological malignancies include acute, chronic, lymphocytic (lymphoblastic) and / or myeloid leukemias, e.g., ALL, AML, CLL, and CML; lymphomas, e.g., HL, NHL, of which about 85% are B-cell lymphomas, e.g., diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphoma [mucosa-associated lymphoma]. [MALT lymphoma, nodal marginal zone B-cell lymphoma, and perisplenic zone B-cell lymphoma], Burkitt lymphoma, lymphoplasmacytoid lymphoma [LPL; also known as Waldenström macroglobulinemia (WM)], hairy cell lymphoma, and primary central nervous system (CNS) lymphoma, T-cell lymphomas such as NHL, e.g., progenitor T-lymphoblastic lymphoma / leukemia, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, e.g., cutaneous lymphoma T-cell lymphoma (CTLC, i.e., mycosis fungoides, Sézary syndrome, etc.), adult T-cell lymphoma / leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma nasal type, enteropathy-associated enteric T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), and unspecified peripheral T-cell lymphoma, acute myeloid lymphoma, lymphoplasmacytic lymphoma, monocyticoid B-cell lymphoma, vascular central lymphoma, enteric T-cell lymphoma, mediastinal primary B-cell large cell lymphoma, post-transplant lymphoma Cancers selected from: proliferative diseases, histiocytic lymphoma, primary exudative lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, and precursor B lymphoblastic lymphoma; myeloma, e.g., multiple myeloma, smoldering myeloma (also known as low-grade myeloma), monoclonal immunoglobulinemia of unknown significance (MGUS), solitary plasmacytoma, IgG myeloma, light chain myeloma, non-secretory myeloma, and amyloidosis; and any combination of the aforementioned hematological malignancies.

[0201] In further embodiments, the hematological malignancies are selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), T-cell lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal immunoglobulinemia of unknown significance (MGUS), advanced, metastatic, refractory, and / or recurrent hematological malignancies, and any combination of the said hematological malignancies.

[0202] This method is also applicable to the treatment of progressive, metastatic, refractory, and / or recurrent hematological malignancies.

[0203] Regarding clinical trials of anti-CCR8 in cancer treatment (Example 34), specific solid tumors were selected based on the following results from the various preclinical studies described above: demonstration of the efficacy of anti-CCR8 in mouse tumor models (Examples 23-29); RNA expression of CCR8 and CD8A in 33 tumor types represented by the Cancer Genome Atlas (National Cancer Institute, 2021); identification of tumor types with relatively high CCR8 expression and enrichment of CD8A expression (Example 32); and levels of CCR8 expression in 17 tumor types or subtypes as measured by IHC (Example 33). The selected tumor types are NSCLC, SCCHN, MSS-CRC, gastric / gastroesophageal (GE) junction cancer, and cervical cancer [squamous cell carcinoma (SCC) or adenocarcinoma]. While available data on progressive tumors are limited, this selection is supported by high CCR8 expression levels or the presence of Tregs, which correlate with poor prognosis in NSCLC, CEC, gastric cancer, and cervical cancer (Shah et al., 2011; Yi et al., 2018; Zhao et al., 2016; Tao et al., 2012; Xu et al., 2020). Reports on the prognosis of SCCHN are mixed, likely due in part to the various methods and molecular markers used to detect the presence of Tregs (Saleh et al., 2020). Accordingly, in certain embodiments of the methods for treating cancer of this disclosure, the solid tumor is a cancer selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer. In certain preferred embodiments, the solid tumor is NSCLC. In other preferred embodiments, the solid tumor is SCCHN. In another preferred embodiment, the solid tumor is MSS-CRC. In a further preferred embodiment, the solid tumor is gastric / GE junction cancer. In yet another preferred embodiment, the solid tumor is cervical cancer.

[0204] Medical use of anti-CCR8 Ab and anti-PD-1 / anti-PD-L1 Ab This disclosure also provides isolated anti-CCR8 Ab (preferably an mAb or its antigen-binding moiety) for use in methods for treating subjects affected by cancer. This disclosure further provides isolated anti-CCR8 Ab, preferably an mAb or its antigen-binding moiety, and a checkpoint inhibitor, e.g., isolated anti-PD-1 / anti-PD-L1 Ab, preferably an mAb or its antigen-binding moiety, for use in combination in methods for treating subjects affected by cancer, including dual Treg depletion and blockade of checkpoint pathways (e.g., PD-1 / PD-L1 signaling pathways). This anti-CCR8 Ab may be used as a monotherapy or in combination with a checkpoint inhibitor (e.g., anti-PD-1 / anti-PD-L1 Ab) for all treatments of cancers disclosed herein.

[0205] One aspect of the invention of this disclosure encompasses the use of the isolated anti-CCR8 Ab or its antigen-binding moiety for the preparation of pharmaceuticals for treating subjects affected by cancer. This anti-CCR8 Ab may be used alone or in combination with a checkpoint inhibitor (e.g., isolated anti-PD-1 / anti-PD-L1 Ab or its antigen-binding moiety) for the preparation of pharmaceuticals for treating cancer patients. Any such use of anti-CCR8 Ab and anti-PD-1 / anti-PD-L1 Ab for the preparation of pharmaceuticals is broadly applicable to all cancers disclosed herein.

[0206] This disclosure also provides anti-CCR8 Ab or its antigen-binding moiety, combined with a checkpoint inhibitor (e.g., isolated anti-PD-1 / anti-PD-L1 Ab or its antigen-binding moiety), for use in methods of treating cancer, corresponding to all embodiments of treatment methods employing this combination of therapeutic agents described herein.

[0207] Anti-CCR8 Ab suitable for use in therapeutic applications of the present disclosure Suitable anti-CCR8 Abs for use in the methods of this disclosure are isolated Abs (preferably mAbs or their antigen-binding moieties) that specifically bind with high specificity and affinity to CCR8 expressed on the surface of cells and mediate the depletion of CCR8-expressing cells by ADCC. Such Abs exhibit one or more properties important for therapeutic efficacy. Specifically, the isolated Ab or its antigen-binding moiety exhibits at least one of the following properties: (a) EC of about 20 nM or less, preferably about 2 nM or less, preferably about 1 nM or less 50 It specifically binds to CCR8 expressed on the surface of cells; (b) It specifically binds to sparse and scattered immune cells in the thymic medulla and dermis of the skin, but does not bind to, for example, human cerebrum, cerebellum, heart, liver, lung, kidney, tonsil, spleen, thymus, colon, stomach, pancreas, adrenal gland, pituitary gland, skin, peripheral nerves, testicular or uterine tissue, or PBMCs. For example, an anti-CCR8 mAb or its antigen-binding moiety may specifically bind to tumor-infiltrating Tregs, but not to PBMCs, and does not show cytoplasmic staining in, for example, fixed PBMCs. The non-binding of the Ab to the list of cells and tissues enumerated above can be established, for example, by performing standard staining with the relevant Ab on, for example, fixed tissue samples, in the manner described in Example 14; (c) ICs with a minimum impedance of approximately 5 nM 50 This inhibits the binding of CCL1 to CCR8, thereby inhibiting CCR8 / CCL1 signaling; (d) When bound to CCR8 on the cell surface, the EC is about 100 pM or less, preferably about 60 pM or less, preferably about 40 pM or less, more preferably about 13 pM or less, more preferably 10 pM or less. 50 And this mediates the depletion of these cells; (e) When bound to CCR8 on the cell surface, it does not cause internal translocation of CCR8, whether in the presence or absence of cross-linked Ab; (f) When administered as monotherapy to the target, it inhibits the growth of tumor cells in that target; Furthermore (g) When administered to a subject in combination with an additional therapeutic agent for treating cancer, the additional therapeutic agent may be an immune checkpoint inhibitor, which may be anti-PD-1, anti-PD-L1, or anti-CTLA-4 Ab.

[0208] In certain embodiments, the isolated Ab or its antigen-binding moiety exhibits at least two or three, preferably four, five, or six of the properties described above. In more preferred embodiments, the isolated Ab or its binding moiety exhibits all of the properties described above. For example, in certain preferred embodiments, the isolated Ab or its antigen-binding moiety exhibits: (a) EC of about 20 nM or less, preferably about 2 nM or less, preferably about 1 nM or less 50 It then specifically binds to CCR8 expressed on the surface of cells; (b) When bound to CCR8 on the cell surface, the EC is about 100 pM or less, about 60 pM or less, preferably about 40 pM or less, more preferably about 13 pM or less, more preferably 10 pM or less. 50 And this mediates the depletion of these cells; (c) When administered as monotherapy to the subject, it inhibits the growth of tumor cells in that subject; and (d) When administered to a subject in combination with additional therapeutic agents for treating cancer, the additional therapeutic agent may inhibit the growth of tumor cells in that subject, and the additional therapeutic agent may be an immune checkpoint inhibitor.

[0209] In other preferred embodiments, the isolated Ab or its antigen-binding moiety is (a) EC of approximately 2nM or less 50 It then specifically binds to CCR8 expressed on the surface of cells; (b) When bound to CCR8 on the cell surface, the EC is approximately 40 pM or less. 50 And this mediates the depletion of these cells; and (c) When administered to a subject in combination with additional therapeutic agents for treating cancer, the additional therapeutic agent may be an anti-PD-1, anti-PD-L1, or anti-CTLA-4 Ab.

[0210] In certain embodiments, an isolated Ab, such as an mAb, exhibiting one or more of the functional properties described above, and up to all of them, or exhibiting its antigen-binding moiety, further comprises a K of approximately 10 nM or less. D Then, it binds to an epitope located in the N-terminal domain of hCCR8, and this epitope is sequence Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 The peptide comprises (SEQ ID NO: 2) and sulfated tyr-15 and / or tyr-17 residues. In certain other embodiments, an isolated Ab (e.g., mAb) or its antigen-binding moiety exhibiting one or more, and up to all, of the above-described functional properties is further expressed as K10 nM or less. D Then, it binds to an epitope located in the N-terminal domain of human CCR8, and this epitope is sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 This product contains a peptide having (SEQ ID NO: 109) and sulfated tyr-15 and tyr-17 residues.

[0211] In certain embodiments, an isolated Ab, preferably an mAb, or its antigen-binding moiety may have the above properties and / or contain a V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. LEach of these may include the CDR1 domain, CDR2 domain, and CDR3 domain. For example, such an Ab or its antigen-binding moiety may include the following CDRs as defined by the Kabat method: heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 33; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 34; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 35; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 36; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 37; light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 38. As another example, such an Ab or its antigen-binding moiety may include a V containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 4. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L This may include. As a further example, such an Ab may include a heavy chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 100, and a light chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 112. Ab may have reduced fucosylation of its heavy chain, or it may have a low-fucosylated or unfucosylated heavy chain constant region as described elsewhere in this specification.

[0212] In certain embodiments, an isolated Ab, preferably an mAb, or its antigen-binding moiety may have the above properties and / or contain a V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 115. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. LEach of these may include a CDR1 domain, a CDR2 domain, and a CDR3 domain. For example, such an Ab or its antigen-binding moiety may include the following CDRs as defined by the Kabat method: heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 103; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 104; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 105; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 106; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 107; light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 108. As another example, such an Ab or its antigen-binding moiety may include a V containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 115. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L This may include. As a further example, such an Ab may include a heavy chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 117, and a light chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 118. Ab may have reduced fucosylation of its heavy chain, or it may have a low-fucosylated or unfucosylated heavy chain constant region as described elsewhere in this Spec.

[0213] In certain embodiments, an isolated Ab, preferably an mAb, or its antigen-binding moiety may have the above properties and / or contain a V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. LEach of these may include a CDR1 domain, a CDR2 domain, and a CDR3 domain. For example, such an Ab or its antigen-binding moiety may include: heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 45; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 46; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 47; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 48; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 49; light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 50. In another example, such an Ab or its antigen-binding moiety may include a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L This may include. As a further example, such an Ab may include a heavy chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 114. Ab may have reduced fucosylation of its heavy chain, or it may have a low-fucosylated or unfucosylated heavy chain constant region as described elsewhere in this specification.

[0214] Combination therapy Human CCR8 + Treg also co-expresses PD-1 at high levels (data not shown). Treatment with anti-PD-1 mAbs reduces PD-1 +Treg activation may explain hyperprogression or primary resistance in gastric cancer patients treated with nivolumab (Kamada et al., 2019). Therefore, Treg depletion is likely to improve the overall response rate to anti-PD-1 therapy. This combination is also supported by preclinical mouse studies in which the anti-PD-1 synergistic effect of anti-CCR8 was observed in MB49 and 4T-1 tumor models (Examples 27 and 28).

[0215] While the efficacy of combination therapy with anti-CCR8 Ab and checkpoint inhibitors has been demonstrated herein using anti-PD-1 Ab, several other costimulatory and inhibitory receptors that modulate the T cell response have been identified. Examples of stimulatory receptors include inducible T cell costimulatory molecules (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, lucocorticoid-inducible TNFR-related protein (GITR), and herpesvirus entry mediator (HVEM). Examples of inhibitory receptors include PD-1 / PD-L1, as well as cytotoxic T lymphocyte-associated protein 4 (CTLA-4), B and T lymphocyte attenuators (BTLA), T cell immunoglobulins, and mucins. Examples include domain-3 (TIM-3), lymphocyte-activating gene-3 (LAG-3), killer immunoglobulin-like receptor (KIR), adenosine A2a receptor (A2aR), killer cell lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), CD160, T cell immune receptors with Ig and ITIM domains (TIGIT), and the receptor for the V-domain Ig suppressor of T cell activation (VISTA) (Mellman et al., 2011; Pardoll, 2012; Baitsch et al., 2012). These receptors and their ligands provide therapeutic targets designed to stimulate or prevent the suppression of the immune response, thereby attacking tumor cells (Weber, 2010; Mellman et al., 2011; Pardoll, 2012). Stimulatory receptors or receptor ligands are targets for agonists, and inhibitory receptors or receptor ligands are targets for blocking agents. Since many immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by Ab or readily modulated by recombinant ligands or receptors. One or more of the costimulatory and inhibitory receptors and ligands that modulate T cell responses other than PD-1 / PD-L1 may provide targets for synergistic effects with anti-CCR8 Ab disclosed herein regarding the inhibition of tumor growth.For example, in one particular embodiment, anti-CCR8 Ab is combined with anti-CTLA-4 Ab. In other embodiments, anti-CCR8 Ab is combined with anti-LAG-3 Ab.

[0216] Radiotherapy has been associated with increased Treg infiltration after treatment in mouse models (Muroyama et al., 2017) and humans (unpublished data). Increased Treg infiltration may lead to undesirable suppression of antitumor immunity, and CCR8-mediated depletion may enhance the effectiveness of radiotherapy.

[0217] This disclosure provides an anti-CCR8 Ab that is effective in enhancing the immune response by enhancing the antitumor effect of treatments such as checkpoint inhibition or radiotherapy, and exhibits at least one, some, or all of the following desired properties: (a) EC of about 1 nM or less 50 (b) It specifically binds to hCCR8 expressed on the surface of cells; (c) It specifically binds to sparse and scattered immune cells in the thymic medulla and dermis of the skin, but does not bind to, for example, human cerebrum, cerebellum, heart, liver, lung, kidney, tonsil, spleen, thymus, colon, stomach, pancreas, adrenal gland, pituitary gland, skin, peripheral nerves, testicular or uterine tissue, or PBMCs. For example, an anti-CCR8 mAb or its antigen-binding moiety may specifically bind to tumor-infiltrating Tregs, but not to PBMCs, and does not show cytoplasmic staining in, for example, fixed PBMCs. The non-binding of the Ab to the list of cells and tissues enumerated above can be established, for example, by performing standard staining with the relevant Ab on fixed tissue samples (for example, by the method described in Example 14); (c) IC2000 or less of about 5 nM 50 (d) When CCL1 binds to CCR8, it inhibits CCR8 / CCL1 signaling; (d) When it binds to CCR8 on the cell surface, the EC is below approximately 50 pM. 50Therefore, it mediates the depletion of these cells; (e) when bound to CCR8 on the cell surface, it does not cause internal translocation of CCR8 in the presence or absence of cross-linked Ab; (f) when administered to a subject as monotherapy, it inhibits the growth of the subject's tumor cells; and (g) when administered to a subject in combination with an additional therapeutic agent to treat cancer, it inhibits the growth of the subject's tumor cells, the additional therapeutic agent may be anti-PD-1, anti-PD-L1, or anti-CTLA-4 Ab.

[0218] Certain anti-CCR8 mAbs that may be used in the therapies, compositions, or kits described herein are those that bind specifically to hCCR8 on the cell surface with high affinity and have an EC of approximately 10 pM or less. 50 Examples include mAbs that mediate cell depletion and exhibit at least two other, preferably all, of the above-mentioned properties. In certain embodiments, an isolated Ab, preferably an mAb, or its antigen-binding moiety may have at least one of the above-mentioned properties and / or contain a sequence of linked amino acids having the sequence described in SEQ ID NO: 4. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L Each of these may include the CDR1 domain, CDR2 domain, and CDR3 domain. For example, such an Ab or its antigen-binding moiety may include the following CDR as defined by the Kabat method: V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 33 H V containing continuously linked amino acids having the sequence described in CDR1;SEQ ID NO: 34 H V containing continuously linked amino acids having the sequence described in CDR2;SEQ ID NO: 35 H V containing continuously linked amino acids having the sequence described in CDR3;SEQ ID NO: 36 L V containing continuously linked amino acids having the sequence described in CDR1;SEQ ID NO: 37 L V containing continuously linked amino acids having the sequence described in CDR2 and SEQ ID NO: 38 LCDR3. In another example, such an Ab or its antigen-binding moiety contains a V with a sequence of linked amino acids having the sequence described in SEQ ID NO: 4. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L This may include. In further examples, such an Ab may include a heavy chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 100, and a light chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 112. The Ab may have reduced fucosylation of its heavy chain, or it may have a low-fucosylated or unfucosylated heavy chain constant region as described elsewhere in this specification.

[0219] In a particular embodiment, the isolated Ab, preferably an mAb, or its antigen-binding moiety may have at least one of the above characteristics and / or contain a V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 115. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L Each of these may include the CDR1 domain, CDR2 domain, and CDR3 domain. For example, such an Ab or its antigen-binding moiety may include the following CDR as defined by the Kabat method: V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 103 H V containing continuously linked amino acids having the sequence described in CDR1;SEQ ID NO: 104 H V containing continuously linked amino acids having the sequence described in CDR2;SEQ ID NO: 105 H V containing continuously linked amino acids having the sequence described in CDR3;SEQ ID NO: 106 L V containing continuously linked amino acids having the sequence described in CDR1;SEQ ID NO: 107 L V containing continuously linked amino acids having the sequence described in CDR2; and SEQ ID NO: 108 L CDR3. As another example, such an Ab or its antigen-binding moiety contains a V containing sequentially linked amino acids having the sequence described in SEQ ID NO: 115. HV containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L This may include. As a further example, such an Ab may include a heavy chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 117, and a light chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 118. Ab may have reduced fucosylation of its heavy chain, or it may have a low-fucosylated or unfucosylated heavy chain constant region as described elsewhere in this Spec.

[0220] In a particular embodiment, the isolated Ab, preferably an mAb, or its antigen-binding moiety may have at least one of the above characteristics and / or contain a V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L Each of these may include the CDR1 domain, CDR2 domain, and CDR3 domain. For example, such an Ab or its antigen-binding moiety may include the following CDR as defined by the Kabat method: V containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 45 H V containing continuously linked amino acids having the sequence described in CDR1;SEQ ID NO: 46 H V containing continuously linked amino acids having the sequence described in CDR2;SEQ ID NO: 47 H V containing continuously linked amino acids having the sequence described in CDR3;SEQ ID NO: 48 L V containing continuously linked amino acids having the sequence described in CDR1;SEQ ID NO: 49 L V containing continuously linked amino acids having the sequence described in CDR2; and SEQ ID NO: 50 L CDR3. In another example, such an Ab or its antigen-binding moiety contains a V with a sequence of linked amino acids having the sequence described in SEQ ID NO: 6. H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. LThis may include. In further examples, such Ab or its antigen-binding moiety may include a heavy chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 114. Ab may have reduced fucosylation of its heavy chain, or it may have a low-fucosylated or unfucosylated heavy chain constant region as described elsewhere in this specification.

[0221] Anti-PD-1 / anti-PD-L1 Ab suitable for use in the therapeutics of this disclosure Suitable anti-PD-1 Abs for use in the cancer treatment methods, compositions, or kits disclosed herein include isolated Abs, preferably mAbs or their antigen-binding moieties, that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and / or PD-L2 to PD-1, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. Similarly, suitable anti-PD-L1 Abs for use in these methods are isolated Abs, preferably mAbs or their antigen-binding moieties, that bind to PD-L1 with high specificity and affinity, block the binding of PD-L1 to PD-1 and CD80(B7-1), and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the therapeutic methods disclosed herein, the anti-PD-1 or anti-PD-L1 Ab comprises an antigen-binding moiety or fragment that binds to the PD-1 receptor or PD-L1 ligand, respectively, and exhibits functional properties similar to a complete Ab in the inhibition of receptor-ligand binding and reversal of inhibition of T cell activity, thereby upregulating the immune response.

[0222] Anti-PD-1 mAb mAbs that specifically bind to PD-1 with high affinity are disclosed in U.S. Patent No. 8,008,449. Other anti-PD-1 mAbs are described, for example, in U.S. Patents No. 7,488,802, No. 8,168,757, No. 8,354,509, and No. 9,205,148. The anti-PD-1 mAbs disclosed in U.S. Patent No. 8,008,449 have been demonstrated to exhibit some or all of the following characteristics: (a) K50 nM or less as determined by the SPR[BIACORE®] biosensor system D (b) binds to human PD-1; (c) substantially does not bind to human CD28, CTLA-4, or ICOS; (d) increases T cell proliferation, interferon-γ production, and IL-2 secretion in mixed lymphocyte reaction (MLR) assays; (e) binds to human PD-1 and cynomolgus monkey PD-1; (f) inhibits the binding of PD-L1 and PD-L2 to PD-1; (g) CD4 + CD25 - (a) relieves inhibition of T cell proliferation and interferon-γ production caused by Treg cells; (b) stimulates antigen-specific memory responses; (h) stimulates Ab responses; and (i) inhibits tumor cell growth in vivo. Anti-PD-1 Abs usable in the treatment methods, compositions, or kits of this disclosure include mAbs that bind specifically to human PD-1 with high affinity and exhibit at least five, preferably all, of the above characteristics. For example, anti-PD-1 Abs suitable for use in the therapeutics disclosed herein include (a) mAbs with a K content of about 10 nM to 0.1 nM as determined by SPR [BIACORE®]. D (b) binds to human PD-1; (c) increases T cell proliferation, interferon-γ production, and IL-2 secretion in MLR assays; (d) inhibits the binding of PD-L1 and PD-L2 to PD-1; (d) CD4 + CD25 - (a) reverses Treg-induced inhibition of T cell proliferation and interferon-γ production; (b) stimulates antigen-specific memory responses; and (f) inhibits tumor cell growth in vivo.

[0223] Other anti-PD-1 mAbs include, for example, U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Patent Application Publication No. 2016 / 0272708, and International Publication Nos. 2008 / 156712, 2012 / 145493, 2014 / 179664, 2014 / 194302, 2014 / 206107, 2015 / 035606, 2015 / 085847, 2015 / 112800, 2015 / 112900, and 2016 / 1 This is described in Brochures No. 06159, 2016 / 197367, 2017 / 020291, 2017 / 020858, 2017 / 024465, 2017 / 024515, 2017 / 025016, 2017 / 025051, 2017 / 040790, 2017 / 106061, 2017 / 123557, 2017 / 132827, and 2017 / 133540, each of which disclosures are incorporated herein by reference in their entirety.

[0224] In certain embodiments, the anti-PD-1 mAb is nivolumab [OPDIVO®; formerly named 5C4, BMS-936558, MDX-1106, or ONO-4538], pembrolizumab [KEYTRUDA®; formerly named lambrolizumab and MK-3475; see International Publication No. 2008 / 156712A1], PDR001 (see International Publication No. 2015 / 112900), MEDI-0680 (formerly named AMP-514; see International Publication No. 2012 / 145493), REGN-2810 (see International Publication No. 2015 / 112800), JS001 (Liu and See Wu, 2017), BGB-A317 (see International Publication No. 2015 / 035606 brochure and U.S. Patent Application Publication No. 2015 / 0079109 specification), INCSHR1210 (SHR-1210; see International Publication No. 2015 / 085847 brochure; see Liu and Wu, 2017), TSR-042 (ANB011; see International Publication No. 2014 / 179664 brochure), GLS-010 (WBP3055; see Liu and Wu, The group is selected from the following: (see 2017), AM-0001 (see International Publication No. 2017 / 123557), STI-1110 (see International Publication No. 2014 / 194302), AGEN2034 (see International Publication No. 2017 / 040790), and MGD013 (see International Publication No. 2017 / 106061).

[0225] In certain preferred embodiments of any of the therapies described herein, including the administration of an anti-PD-1 Ab, the anti-PD-1 Ab is nivolumab [OPDIVO®], which has already been approved by the US Food and Drug Administration (FDA) for the treatment of several different cancers. Nivolumab is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor Ab that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of antitumor T cell function (described as mAb C5 in U.S. Patent No. 8,008,449; Wang et al., 2014). In other preferred embodiments, the anti-PD-1 Ab is pembrolizumab [KEYTRUDA®; described as h409A11 in U.S. Patent No. 8,354,509; a humanized monoclonal IgG4 Ab against PD-1], which has also been approved for several cancer indications.

[0226] Examples of anti-PD-1 Abs usable in the methods, compositions, or kits of this disclosure include isolated Abs, preferably mAbs, that specifically bind to human PD-1 (hPD-1) and cross-compete with any one of the anti-PD-1s described herein for binding to human PD-1, e.g., nivolumab (5C4; see, e.g., U.S. Patent No. 8,008,449; see, International Publication No. 2013 / 173223) and pembrolizumab. The antigen, in this case the Ab that cross-competes with the reference Ab for binding to human PD-1, e.g., nivolumab or pembrolizumab, can be readily identified by standard PD-1 binding assays such as BIACORE® analysis, ELISA assay, or flow cytometry (see, e.g., International Publication No. 2013 / 173223). In certain embodiments, the anti-PD-1 Ab binds to the same epitope as any of the anti-PD-1 Abs described herein, for example, nivolumab or pembrolizumab.

[0227] Anti-PD-1 Abs usable in the methods of the present invention include antigen-binding moieties, such as Fab, F(ab')2, Fd or Fv fragments, sdAb, scFv, di-scFv or bi-scFv, diabodies, minibodies, or isolated CDRs (see Hollinger and Hudson, 2005; Olafsen and Wu, 2010 for further details).

[0228] In certain embodiments, the isolated anti-PD-1 Ab or its antigen-binding moiety includes a heavy chain constant region that is a human IgG1, IgG2, IgG3, or IgG4 isotype. In certain preferred embodiments, the anti-PD-1 Ab or its antigen-binding moiety includes a heavy chain constant region that is a human IgG4 isotype. In other embodiments, the anti-PD-1 Ab or its antigen-binding moiety is a human IgG1 isotype. In certain other embodiments, the IgG4 heavy chain constant region of anti-PD-1 or its antigen-binding moiety includes an S228P mutation (numbered using the Kabat system; Kabat et al., 1983), in which a serine residue in the hinge region is replaced with a proline residue normally found at the corresponding position in IgG1 isotype Ab. This mutation present in nivolumab prevents Fab arm exchange with endogenous IgG4 Ab while maintaining a low affinity for the activated Fc receptor associated with wild-type IgG4 Ab (Wang et al., 2014). In yet another embodiment, Ab includes a light chain steady region which is a human kappa or lambda steady region.

[0229] In other embodiments of this method, the anti-PD-1 Ab or its antigen-binding moiety is an mAb or its antigen-binding moiety. For administration to human subjects, the anti-PD-1 Ab is preferably a chimeric Ab, more preferably a humanized Ab or human Ab. Such chimeric mAbs, humanized mAbs, or human mAbs can be prepared and isolated by methods known in the art, as described, for example, in U.S. Patent No. 8,008,449.

[0230] Anti-PD-L1 mAb Since anti-PD-1 and anti-PD-L1 target the same signaling pathway and have been shown in clinical trials to exhibit comparable efficacy in various cancers (see, for example, Brahmer et al., 2012; see International Publication No. 2013 / 173223), anti-PD-L1 Ab may be substituted for anti-PD-1 Ab in the combination therapies disclosed herein.

[0231] Suitable anti-PD-L1 mAbs for use in the methods, compositions, or kits of this disclosure are isolated mAbs that bind to PD-L1 with high specificity and affinity, block the binding of PD-L1 to PD-1 and CD80, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. mAbs that specifically bind to PD-L1 with high affinity are disclosed in U.S. Patent No. 7,943,743. Other anti-PD-L1 mAbs are described, for example, in U.S. Patents Nos. 8,217,149, 8,779,108, 9,175,082, and 9,624,298, as well as in International Publication No. 2012 / 145493. The anti-PD-1 HuMAb disclosed in U.S. Patent No. 7,943,743 has been demonstrated to exhibit one or more of the following characteristics: (a) a K content of approximately 50 mM or less as determined by SPR [BIACORE®]. D(b) binds to human PD-1; (c) increases T cell proliferation, interferon-γ production, and IL-2 secretion in MLR assays; (d) stimulates the Ab response; (e) inhibits the binding of PD-L1 to PD-1; and (e) reverses the inhibitory effect of Treg on T cell effector cells and / or dendritic cells. Examples of anti-PD-L1 Abs for use in the therapies disclosed herein include isolated Abs (preferably mAbs) that bind specifically to human PD-L1 with high affinity and exhibit at least one, in some embodiments at least three, preferably all, of the above-described properties. For example, anti-PD-L1 Abs suitable for use in this method include (a) having about 50 mM to 0.1 mM K as determined by surface plasmon resonance [BIACORE®]. D It binds to human PD-1; (b) increases T cell proliferation, interferon-γ production, and IL-2 secretion in MLR assays; (c) inhibits the binding of PD-L1 to PD-1 and CD80; and (d) reverses the inhibitory effect of Treg on T cell effector cells and / or dendritic cells.

[0232] A suitable anti-PD-L1 agent for use in this method is BMS-936559 (formerly MDX-1105; named 12A4 in U.S. Patent No. 7,943,743). Other suitable anti-PD-L1 agents include atezolizumab [TECENTRIQ®; already known as RG7446 and MPDL3280A; named YW243.55S70 in U.S. Patent No. 8,217,149; Herbst et al.]. See also 2014], durvalumab [IMFINZI®; already known as MEDI-4736; named 2.14H9OPT in U.S. Patent No. 8,779,108], avelumab [BAVENCIO®; already known as MSB-0010718C; named A09-246-2 in U.S. Patent No. 9,624,298], STI-A1014 (named H6 in U.S. Patent No. 9,175,082), CX-072 (see International Publication No. 2016 / 149201), KN035 (see Zhang et al., 2017), LY3300054 (see International Publication No. 2017 / 034916, for example), and CK-301 (Gorelik et al., (See 2017)

[0233] In certain preferred embodiments of any of the therapies described herein, including the administration of an anti-PD-L1 Ab, the anti-PD-L1 Ab is atezolizumab [TECENTRIQ®]. In other preferred embodiments, the anti-PD-L1 Ab is durvalumab [IMFINZI®]. In even more preferred embodiments, the anti-PD-L1 Ab is avelumab [BAVENCIO®].

[0234] Suitable anti-PD-L1 Abs for use in the methods, compositions, or kits of this disclosure include isolated Abs that specifically bind to human PD-L1 and cross-compete with any one of the anti-PD-L1 Abs described herein with respect to binding to human PD-L1, such as BMS-936559 (12A4; see, e.g., U.S. Patent No. 7,943,743; see International Publication No. 2013 / 173223), atezolizumab, durvalumab, avelumab, or STI-A1014. The ability of an Ab to cross-compete with a reference Ab with respect to binding to human PD-L1 suggests that such an Ab is expected to bind to the same epitope region of PD-L1 as the reference Ab and, by binding to substantially the same epitope region of PD-L1, will have functional properties very similar to the reference Ab. In some embodiments, the anti-PD-L1 Ab binds to the same epitope as any of the anti-PD-L1 Abs described herein, for example, atezolizumab, durvalumab, avelumab, or STI-A1014. Cross-competing Abs can be readily identified to those skilled in the art based on their ability to cross-compete with a reference Ab (e.g., atezolizumab or avelumab) in standard PD-L1 binding assays known to those skilled in the art [e.g., BIACORE® analysis, ELISA assay, or flow cytometry] (see, for example, International Publication No. 2013 / 173223).

[0235] In certain preferred embodiments, the isolated anti-PD-L1 Ab for use in this method is an mAb. In other embodiments, particularly for administration to human subjects, this Ab is preferably a chimeric Ab, and more preferably a humanized Ab or human Ab. Chimeric Abs, humanized Abs, and human Abs can be prepared and isolated by methods known to those skilled in the art, for example, as described in U.S. Patent No. 7,943,743.

[0236] In certain embodiments, the anti-PD-L1 Ab or its antigen-binding moiety includes a heavy chain constant region that is a human IgG1, IgG2, IgG3, or IgG4 isotype. In certain other embodiments, the anti-PD-L1 Ab or its antigen-binding moiety is human IgG1 of the IgG4 isotype. In further embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-L1 Ab or its antigen-binding moiety includes an S228P mutation. In other embodiments, this Ab includes a light chain constant region that is a human kappa or lambda constant region.

[0237] The anti-PD-L1 Ab of the present invention also includes antigen-binding moieties of the above Ab, such as Fab, F(ab')2, Fd, Fv, and scFv, di-scFv or bi-scFv, and scFv-Fc fragments, nanobodies, diabodies, triabodies, tetrabodies, and isolated CDRs, which bind to PD-L1 and exhibit functional properties similar to the complete Ab in inhibiting receptor binding and upregulating the immune system.

[0238] Pharmaceutical compositions and dosage regimens mAbs used in any of the therapeutic methods disclosed and described herein may consist of a composition, for example, a pharmaceutical composition comprising an Ab and a pharmaceutically acceptable carrier. The present invention also provides compositions comprising any of the immunoconjugates or bispecific molecules of this disclosure and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic and absorption retardant, and others. Preferably, carriers for compositions comprising an Ab are suitable for intravenous (IV), intramuscular, subcutaneous (SC), parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0239] The SC injection options are based on Halozyme Therapeutics' ENHANZE® drug delivery technology, which involves co-formulations of an Ab, such as an mAb, and recombinant human hyaluronidase enzyme (rHuPH20), removing conventional limitations on the amount of biologics and drugs that can be delivered subcutaneously due to the extracellular matrix (U.S. Patent No. 7,767,429). It may be possible to co-formulate two Abs used in combination therapy into a single composition for SC administration.

[0240] The pharmaceutical composition of the present invention may comprise one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants, such as preservatives, humectants, emulsifiers, and dispersants.

[0241] The dosage regimen is adjusted to produce the optimal desired response, e.g., maximum therapeutic response and / or minimum side effects. In the case of administration including combination therapy with anti-CCR8, anti-PD-1, or anti-PD-L1 Ab, or their antigen-binding moieties, the dosage may range from about 0.01 to about 20 mg / kg of body weight, preferably from about 0.1 to about 10 mg / kg of body weight. For example, the dosage may be about 0.1, 0.3, 1, 2, 3, 5, or 10 mg / kg of body weight, more preferably about 0.3, 1, 3, or 10 mg / kg of body weight. Alternatively, instead of a body weight-based dose, a fixed or constant dose of Ab or its antigen-binding moiety may be administered, for example, about 0.1 to about 2,000 mg, preferably about 1 to about 1,000 mg, for example, about 0.3, 1, 3, 5, 10, 30, 60, 100, 150, 200, 240, 300, 400, 500, 600, 800, or 1,000 mg. Constant dosing (relative to body weight) is appealing due to its ease of preparation, low risk of medication error, and the observation that the two dosing approaches work similarly for most biologics (Wang et al., 2009).

[0242] The administration schedule is typically designed to achieve exposure that results in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of the Ab. Exemplary treatment regimens involve administration once weekly, once every two weeks, once every three weeks, once every four weeks, once a month, once every three to six months, or once over a longer period. In certain preferred embodiments, anti-CCR8, anti-PD-1, or anti-PD-L1 Ab, or their antigen-binding moieties, are administered to the subject every two weeks. In other preferred embodiments, Ab or its antigen-binding moiety is administered every three or four weeks. Dosage and schedule may be modified during treatment. The first-in-human (FIH) starting constant dose of 0.3 mg (4 μg / kg) IV Q2W of 4A19 was derived using data from a combination of pharmacology-based and toxicology-based approaches, with the aim of ensuring adequate safety while minimizing the risk of participant exposure and cytokine release to potentially sub-efficacious doses.

[0243] In the clinical trial described in Example 34, the dosage used in the first-in-human (FIH) clinical trial was determined using data from a combination of pharmacology-based and toxicology-based approaches. In the dose escalation phase of the anti-CCR8 monotherapy group, a constant dose of 0.3, 1, 3, 10, 30, 100, 300, or 800 mg of 4A19 was administered intravenously (IV) every two weeks (Q2W). In the combination therapy group, the same dose of 4A19 was administered in combination with nivolumab, which was administered IV at an FDA-approved constant dose of 480 mg every four weeks (Q4W), during the dose escalation phase. To facilitate dose expansion, single-treatment groups and randomized cohorts will be opened up from the escalation phase to include various tumor types and dose levels, and treatment as monotherapy or combination therapy will be continued until disease progression, unacceptable toxicity, withdrawal of consent, completion of 26 cycles of study treatment (104 weeks), or termination of the study, whichever comes first.

[0244] Accordingly, in certain embodiments of the therapies of this disclosure, anti-CCR8 Ab is administered to the subject as monotherapy in a constant dose of about 0.3 to about 800 mg at Q2W, or in combination with an immune checkpoint inhibitor, such as anti-PD-1 or anti-PD-L1 Ab. More specifically, in certain embodiments, anti-CCR8 Ab is administered at Q2W in doses of 0.3, 1, 3, 10, 30, 100, 300, or 800 mg. In certain preferred embodiments, anti-CCR8 Ab is mAb 4A19. In other preferred embodiments, anti-CCR8 Ab is mAb 14S5 or 14S15h. In certain embodiments, anti-CCR8 Ab is administered to the subject at a constant dose of 3 mg at Q2W. In certain other embodiments, anti-CCR8 Ab is administered at 10 mg at Q2W. In other embodiments, anti-CCR8 Ab is administered at a constant dose of 30 mg at Q2W. In yet another embodiment, anti-CR8 Ab is administered to the subject at a fixed dose of 100 mg during Q2W.

[0245] In combination therapy, in certain embodiments, the immune checkpoint inhibitor is an anti-PD-1, anti-PD-L1, or anti-CTLA-4 Ab. In certain preferred embodiments, the anti-PD-1 Ab is nivolumab. In preferred embodiments, nivolumab is administered to the subject at a fixed dose of 480 mg during Q4W.

[0246] When used in combination, doses less than the therapeutic dose of one or both Abs may be used, for example, doses of anti-CCR8, anti-PD-1, and / or anti-PD-L1 Ab, or doses of their antigen-binding moieties. As used herein, “sub-therapeutic” doses or dosages of therapeutic Abs, etc., refer to doses lower than typical or approved monotherapy doses. For example, a dose of nivolumab lower than the 3 mg / kg every two weeks initially approved by the FDA, for example, 1.0 mg / kg or less every two, three, or four weeks, is considered a sub-therapeutic dose. Subsequently, nivolumab has been approved by the FDA at 240 mg every two weeks or 480 mg every four weeks. Therefore, for example, a dose of nivolumab lower than the approved 480 mg every four weeks, for example, 120 mg or less every two, three, or four weeks, is considered a sub-therapeutic dose. RO data from 15 subjects receiving nivolumab at doses of 0.3 mg / kg to 10 mg / kg suggest that PD-1 occupancy appears non-dose-dependent within this dose range. Across all doses, the mean occupancy was 85% (range, 70%–98%), and the mean plateau occupancy was 72% (range, 59%–81%) (Brahmer et al., 2010). Therefore, an administration of 0.3 mg / kg may provide sufficient exposure to yield significant biological activity.

[0247] In contrast, as used herein, a “sub-effective dose” or administration of a therapeutic agent such as therapeutic ab refers to a dose that is lower than the dose required for significant biological activity and therefore does not produce any significant therapeutic effect when administered as monotherapy or in combination therapy.

[0248] The synergistic interactions observed in mouse tumor models between anti-CCR8 Ab and anti-PD-1 / anti-PD-L1 Ab, or their antigen-binding moieties, may allow one or both of these therapeutic agents to be administered to cancer patients in sub-therapeutic doses. In certain embodiments of the combination therapy of this disclosure, anti-CCR8 Ab or its antigen-binding moiety is administered to the cancer patient in a sub-therapeutic dose. In other embodiments, anti-PD-1 / anti-PD-L1 Ab or their antigen-binding moieties are administered to the patient in a sub-therapeutic dose. In further embodiments, anti-PD-1 / anti-PD-L1 Ab and anti-CCR8 Ab, or their antigen-binding moieties, are each administered to the patient in sub-therapeutic doses.

[0249] Administering doses of one or both Abs below such therapeutic doses may reduce adverse events compared to the use of higher doses of the individual Abs in monotherapy. Therefore, the success of the combination therapy methods of this disclosure can be evaluated not only by the improved efficacy of the Ab combination compared to monotherapy with these Abs, but also by the improved safety (i.e., reduced incidence of adverse events) resulting from the use of a combination of drugs at lower doses compared to monotherapy doses.

[0250] In certain embodiments of any of the methods disclosed herein, anti-CCR8 Ab, anti-PD-1 Ab, and / or anti-PD-L1 Ab are formulated for intravenous (IV) administration or for subcutaneous (SC) injection. In certain embodiments, anti-CCR8 Ab or its antigen-binding moiety and anti-PD-1 / anti-PD-L1 Ab or its antigen-binding moiety are administered sequentially to the subject. “Sequential” administration means that one of the anti-CCR8 Ab and anti-PD-1 / anti-PD-L1 Ab is administered before the other. Either Ab may be administered first; that is, in certain embodiments, anti-PD-1 / anti-PD-L1 Ab is administered before anti-CCR8 Ab, and in other embodiments, anti-CCR8 Ab is administered before anti-PD-1 / anti-PD-L1 Ab. In certain embodiments, each Ab is administered by IV infusion, for example, by infusion over a period of about 60 minutes. In other embodiments, at least one Ab is administered by SC injection.

[0251] In certain embodiments of sequential IV administration, for the convenience of the patient, anti-CCR8 Ab and anti-PD-1 / anti-PD-L1 Ab, or a combination thereof, are administered to each other within 30 minutes. Typically, when both anti-CCR8 Ab and anti-PD-1 / anti-PD-L1 Ab are delivered via IV on the same day, separate infusion bags and filters are used for each infusion. After the infusion of the first Ab, the line for this Ab is quickly flushed with saline, and then the infusion of the second Ab is started. In other embodiments, the two Abs are administered to each other within 1, 2, 4, 8, 24, or 48 hours.

[0252] The delivery of at least one agent (Ab) via sequential administration reduces the time required for healthcare workers to administer the drug, thus shortening the time for drug administration. For example, the use of sequential injections has reduced the time required for IV administration, typically about 30-60 minutes, to about 5 minutes. In certain embodiments of sequential SC administration, anti-CCR8 Ab and anti-PD-1 / anti-PD-L1 Ab, or a combination thereof, are administered within 10 minutes of each other.

[0253] Because checkpoint inhibitors (Abs) are known to evoke highly durable responses, partly due to the memory component of the immune system (see, for example, International Publication No. 2013 / 173223; Lipson et al., 2013; Wolchok et al., 2013), the activity of administered anti-PD-1 / anti-PD-L1 Ab may persist for weeks, months, or even years. In certain embodiments, this combination therapy with sequential administration involves the administration of anti-CCR8 Ab to patients already treated with anti-PD-1 / anti-PD-L1 Ab. In further embodiments, anti-CCR8 Ab is administered to patients already treated with anti-PD-1 / anti-PD-L1 Ab whose cancer has progressed. In other embodiments, this combination therapy with sequential administration may involve the administration of anti-PD-1 / anti-PD-L1 Ab to patients already treated with anti-CCR8 Ab, and then administration to patients whose cancer has progressed after treatment with anti-CCR8 Ab.

[0254] In certain other embodiments, the anti-PD-1 / anti-PD-L1 mAb and the anti-CCR8 mAb are administered simultaneously either as a single composition in a pharmaceutically acceptable formulation for co-administration, or as separate compositions in which each Ab is incorporated into a pharmaceutically acceptable composition. In certain preferred embodiments, the anti-PD-1 / anti-PD-1 mAb and the anti-CCR8 mAb are administered simultaneously as separate compositions in which each Ab is incorporated into a pharmaceutically acceptable composition. In other embodiments, the anti-PD-1 / anti-PD-L1 mAb and the anti-CCR8 mAb are administered simultaneously as a single composition in a pharmaceutically acceptable formulation.

[0255] This disclosure provides isolated Abs, preferably mAbs, that specifically bind to hCCR8 expressed on the surface of cells, wherein the isolated Ab comprises a heavy chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 114, and is used in a method for treating patients with cancer, the cancer being selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer.

[0256] In certain embodiments, the Disclosure also provides an isolated Ab (preferably an mAb) that specifically binds to hCCR8 expressed on the surface of a cell, wherein the isolated Ab comprises a heavy chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 114, and is used in a manner for treating a patient with cancer in combination with another therapeutic agent, wherein the cancer is selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer, and the other therapeutic agent is an anti-PD-1, anti-PD-L1, or anti-CTLA-4 Ab.

[0257] In certain other embodiments, the Disclosure further provides an isolated Ab (preferably an mAb) that specifically binds to hCCR8 expressed on the surface of cells, wherein the isolated Ab comprises a heavy chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 114, and is used in a manner for treating a patient with cancer in combination with another therapeutic agent, wherein the cancer is selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer, and the other therapeutic agent is nivolumab, wherein the anti-CCR8 Ab is administered to the subject at a dose of 1 to 30 mg once every two weeks, and nivolumab is administered to the subject at a dose of 240 mg once every two weeks or at a dose of 480 mg once every four weeks.

[0258] The disclosure also provides a method for treating a subject with cancer, comprising administering to the subject a therapeutically effective dose of a low-fucosylated or non-fucosylated anti-CCR8 mAb comprising a heavy chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 114, wherein the cancer is selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer.

[0259] In certain embodiments, the Disclosure also provides a method for treating a subject having cancer, comprising administering to the subject a therapeutically effective dose of (a) a low-fucosylated or non-fucosylated anti-CCR8 mAb comprising a heavy chain having a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain having a sequence of linked amino acids having the sequence described in SEQ ID NO: 114, and (b) another therapeutic agent, the cancer being selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer, and the other therapeutic agent being anti-PD-1, anti-PD-L1, or anti-CTLA-4 Ab.

[0260] In certain other embodiments, the Disclosure further provides a method for treating a subject having cancer, comprising administering to the subject a therapeutically effective dose of (a) a low-fucosylated or non-fucosylated anti-CCR8 mAb comprising a heavy chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 114, and (b) another therapeutic agent, the cancer being selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer, and the other therapeutic agent being nivolumab, wherein the anti-CCR8 Ab is administered to the subject at a dose of 1 to 30 mg once every two weeks, and the nivolumab is administered to the subject at a dose of 240 mg once every two weeks or at a dose of 480 mg once every four weeks.

[0261] The disclosure further provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective dose of a low-fucosylated or non-fucosylated anti-CCR8 mAb comprising a heavy chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain containing a sequence of linked amino acids having the sequence described in SEQ ID NO: 114, such that the growth of tumor cells in the subject is inhibited, the cancer being selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer.

[0262] In certain embodiments, the Disclosure also provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a low-fucosylated or non-fucosylated anti-CCR8 mAb comprising (a) a heavy chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 114; and (b) a therapeutically effective dose of another therapeutic agent, such that the growth of tumor cells in the subject is inhibited, the cancer being selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer, and the other therapeutic agent being anti-PD-1, anti-PD-L1, or anti-CTLA-4 Ab.

[0263] In certain other embodiments, the Disclosure further provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a low-fucosylated or non-fucosylated anti-CCR8 mAb comprising (a) a heavy chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 102 and a light chain comprising a sequence of linked amino acids having the sequence described in SEQ ID NO: 114; and (b) a therapeutically effective dose of another therapeutic agent, such that the growth of tumor cells in the subject is inhibited, wherein the cancer is selected from NSCLC, SCCHN, MSS-CRC, gastric / GE junction cancer, and cervical cancer, and the other therapeutic agent is nivolumab, and the anti-CCR8 Ab is administered to the subject at a dose of 1 to 30 mg once every two weeks, and the nivolumab is administered to the subject at a dose of 240 mg once every two weeks, or at a dose of 480 mg once every four weeks.

[0264] kit Similarly, kits comprising anti-CCR8 Ab are included within the scope of the present invention. A kit typically includes a label indicating the intended use of the contents of the kit and instructions for use. The term "label" includes any written or recorded material provided on or with the kit, or otherwise accompanying the kit. Accordingly, this disclosure relates to a kit for treating a subject with cancer, comprising: (a) a single or multiple dose of an Ab, e.g., mAb or its antigen-binding moiety, in the range of about 0.01 to about 20 mg / kg body weight, e.g., about 0.1 or 1 mg / kg, or a constant dose of about 0.1 to about 2,000 mg, e.g., about 3, 10, 30 or 100 mg, which specifically binds to CCR8 and mediates the depletion of CCR8-expressing cells by ADCC; and (b) a substance that, as appropriate, specifically binds to PD-1, PD-L1, or CTLA-4. (c) a kit comprising instructions for using an Ab, for example, an mAb or its antigen-binding portion, in a single or multiple dose in the range of about 0.1 to about 20 mg / kg body weight, for example, about 2 to about 10 mg / kg, or a constant dose of about 200 to about 1600 mg, for example, about 240 or 480 mg; and (c) a kit comprising instructions for using an Ab or portion thereof that specifically binds to CCR8, and, as appropriate, an Ab or portion thereof that specifically binds to PD-1, PD-L1, or CTLA-4, in any of the therapies disclosed herein.

[0265] The Disclosure further provides a kit for treating a subject with cancer, comprising: (a) a single or multiple dose of an Ab, e.g., an mAb or its antigen-binding moiety, in the range of about 0.01 to about 20 mg / kg body weight, e.g., about 0.1 or 1 mg / kg, or a constant dose of about 0.1 to about 2,000 mg, e.g., about 3, 10, 30, or 100 mg; (b) a single or multiple dose of an anti-PD-1 / anti-PD-L1 mAb or its antigen-binding moiety, e.g., about 0.1 to about 20 mg / kg body weight, e.g., about 2 or 3 mg / kg, or a constant dose of about 200 to about 1,600 mg, e.g., about 240 or 480 mg; and (c) instructions for using the anti-CCR8 mAb and the anti-PD-1 / anti-PD-L1 mAb in any of the combination therapies disclosed herein.

[0266] In certain embodiments, the kit contains one or more fixed doses of anti-CCR8 Ab in the range of about 0.1 to about 2,000 mg, preferably about 0.3 to about 1,000 mg, for example, about 0.3, 1, 3, 5, 10, 30, 60, 100, 150, 200, 240, 300, 400, 500, 600, 800, or 1,000 mg, instead of a body weight-based dose. In certain embodiments, the kit contains 0.3, 1, 3, 10, 30, 100, 300, and 800 mg of anti-CCR8 Ab for administration once every two weeks. In certain other embodiments, the kit contains approximately 100 to approximately 600 mg of anti-PD-1 / PD-L1 Ab, for example, 240 mg of nivolumab for a dose every two weeks or 480 mg of nivolumab for a dose every four weeks. In certain preferred embodiments, the anti-CCR8 Ab is mAb 4A19. In other preferred embodiments, the anti-CCR8 Ab is mAb 14S15 or 14S15h. In certain preferred embodiments, the anti-PD-1 Ab is nivolumab. In other preferred embodiments, the anti-PD-1 Ab is pembrolizumab.

[0267] In certain embodiments, Ab may be co-packaged in unit dosage forms. In certain preferred embodiments for treating human patients, the kit comprises anti-human PD-1 Ab disclosed herein, for example, nivolumab or pembrolizumab.

[0268] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations. The contents of all references cited throughout this application are expressly incorporated herein by reference. This disclosure relates, for example, to the following: [Section 1] A monoclonal antibody or its antigen-binding moiety that specifically binds to CC motif chemokine receptor 8 (CCR8) expressed on the surface of cells and mediates the depletion of CCR8-expressing cells by antibody-dependent cell-mediated cytotoxicity (ADCC). [Section 2] A monoclonal antibody or its antigen-binding moiety as described in item 1, wherein the sequence specifically binds to human CCR8 (hCCR8) as described in Sequence ID No. 1. [Section 3] A monoclonal antibody or its antigen-binding moiety according to item 1 or 2, comprising a heavy chain constant region of a human IgG1 or IgG3 isotype. [Section 4] A modified anti-hCCR8 monoclonal antibody or its antigen-binding moiety, comprising a modified heavy chain constant region that binds to the Fcγ receptor (FcγR) with higher affinity than the monoclonal antibody or its antigen-binding moiety described in any one of the preceding items and mediates enhanced ADCC, wherein, optionally, in the NK cell lysis assay described in Example 17, cell lysis EC 50 When measured by the decrease, at least, (a) ADCC activity increased by 2 times, (a) 5-fold enhanced ADCC activity, or (c) 10-fold enhanced ADCC activity A modified anti-hCCR8 monoclonal antibody or its antigen-binding moiety that mediates the transmission of the virus. [Section 5] A modified anti-hCCR8 monoclonal antibody or its antigen-binding moiety according to claim 4, comprising a modified IgG1 heavy chain constant region exhibiting reduced fucosylation, and possibly comprising a modified IgG1 heavy chain constant region that is low in fucosylation or not fucosylated. [Section 6] A modified anti-hCCR8 monoclonal antibody or its antigen-binding moiety according to item 4 or 5, comprising a modified IgG1 heavy chain constant region containing an enhanced ADCC-mediated mutation or multiple mutation, wherein the mutation or multiple mutation may be selected from G236A, S239D, F243L, E333A, G236A / I332E, S239D / I332E, S267E / H268F, S267E / S324T, H268F / S324T, G236A / S239D / I332E, S239D / A330L / I332E, S267E / H268F / S324T, and G236A / S239D / A330L / I332E. [Section 7] (i) When measured by the binding assay described in Example 11, (a) Approximately 10 nM or less, (b) Approximately 5 nM or less, (c) Approximately 1.7 nM or less, (d) Approximately 1 nM or less, (e) Approximately 0.5 nM or less, (f) Approximately 0.1 nM or less, (g) Approximately 0.1 nM, (h) Approximately 1.7nM, (i) Between approximately 0.1 nM and approximately 10 nM, (j) Between approximately 0.1 nM and approximately 2 nM, (k) Between approximately 0.5 nM and approximately 5 nM, (l) Between approximately 1 nM and approximately 2 nM, (m) Between approximately 0.5 nM and 1 nM EC 50 It specifically binds to human CCR8-expressing Chinese hamster ovary (CHO) cells, and / or (ii) When measured by the binding assay described in Example 11, (a) Approximately 50 nM or less, (b) Approximately 14 nM or less, (c) Approximately 5 nM or less, (d) Approximately 2 nM or less, (e) Approximately 0.5 nM or less, (f) Approximately 0.3 nM or less, (g) Approximately 0.1 nM or less, (h) Approximately 0.03 nM or less, (i) Approximately 1.7nM, (j) Between approximately 0.03 nM and approximately 10 nM, (k) Between approximately 0.1 nM and approximately 5 nM, (l) Between approximately 0.2nM and approximately 2nM EC 50 It specifically binds to activated regulatory T cells (Treg), and / or (iii) When measured by surface plasmon resonance (SPR) as described in Example 11, (a) Approximately 100 nM or less, (b) Approximately 50 nM or less, (c) Approximately 10 nM or less, (d) Approximately 5 nM or less, (e) Approximately 1.6 nM, (f) Approximately 1.0 nM or less, (g) Approximately 0.5 nM or less, (h) Approximately 0.1 nM or less, (i) Between approximately 100 nM and approximately 0.1 nM, (j) Between approximately 50 nM and approximately 0.5 nM, (k) Between approximately 10 nM and approximately lnM, (l) Between approximately 2nM and approximately 1nM K D It binds to the N-terminal peptide of human CCR8 containing sulfated tyrosine-15 and tyrosine-17 residues, and / or (iv) When measured by the SPR described in Example 11, (a) Approximately 100 nM or less, (b) Approximately 50 nM or less, (c) Approximately 25 nM or less, (d) Approximately 10 nM or less, (f) Approximately 1.0 nM or less, (e) Approximately 20 nM, (i) Between approximately 100 nM and approximately 1 nM, (j) Between approximately 50 nM and approximately 10 nM, (k) Between approximately 30 nM and approximately 20 nM K D It binds to a single sulfated residue, namely tyrosine-15, in the N-terminal peptide of human CCR8. A monoclonal antibody or its antigen-binding portion as described in any one of the preceding paragraphs. [Section 8] A monoclonal antibody or its antigen-binding moiety according to any one of the preceding paragraphs, which specifically binds to rare, scattered immune cells in the medulla of the thymus and the dermis of the skin, but does not bind to any of the human cerebrum, cerebellum, heart, liver, lungs, kidneys, tonsils, spleen, thymus, colon, stomach, pancreas, adrenal gland, pituitary gland, skin, peripheral nerves, testes, or uterine tissue, or peripheral blood mononuclear cells (PBMCs). [Section 9] A monoclonal antibody or its antigen-binding moiety according to any one of the preceding items, which, when measured by inhibition of calcium flow as described in Example 15, further inhibits the binding of CC motif chemokine ligand 1 (CCL1) to CCR8 and inhibits CCR8 / CCL1 signaling. [Section 10] (i) (a) Approximately 10 nM or less, (b) Approximately 5 nM or less, (c) Approximately 1 nM or less, (d) Approximately 0.5 nM or less, (e) Approximately 0.1 nM or less, (f) Approximately 0.01 nM or less, (g) Between approximately 0.01 nM and approximately 10 nM, (h) Between approximately 0.05 nM and approximately 5 nM, (i) Between approximately 0.1 nM and approximately 1 nM, (j) Approx. 0.46nM IC 50 This inhibits CCR8 / CCL1 signaling, and / or (ii) When measured by the CD16 crosslinking assay described in Example 17, (a) Approximately 100 pM or less, (b) Approximately 30 pM or less, (c) Approximately 10 pM or less, (d) Approximately 3 pM or less, (e) Approximately 1 pM or less, (e) Approximately 0.5 pM or less, (f) Approximately 0.1 pM or less, (g) Approximately 0.05 pM or less, (h) approx. 0.7pM, (i) Between approximately 0.05 pM and approximately 50 pM, (j) Between approximately 0.1 pM and approximately 10 nM, (k) Between approximately 0.3 nM and approximately 7 nM, (l) Between approximately 0.6 nM and approximately 3 nM EC 50 This mediates the depletion of CCR8-expressing cells, and / or (iii) When measured by the apoptosis assay described in Example 19, (a) Approximately 500 pM or less, (b) Approximately 100 pM or less, (c) Approximately 30 pM or less, (d) Approximately 15 pM or less, (e) Approximately 5 pM or less, (f) Approximately 1 pM or less, (g) about 13pM, (h) Between approximately 1 pM and approximately 500 pM, (i) Between approximately 5 pM and approximately 100 pM, (j) Between approximately 10 pM and 50 pM EC 50 This mediates the depletion of activated Tregs. The monoclonal antibody or its antigen-binding portion as described in the preceding paragraph. [Section 11] When determined by X-ray crystallography, it binds to an epitope located in the N-terminal domain of human CCR8, and the epitope is 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: 109) comprises at least one amino acid in the peptide, (i) Array V 12 T 13 D 14 Y 15 Y 16 Y17 P 18 D 19 I 20 F 21 S 22 The peptide containing (SEQ ID NO: 109) may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the amino acids. (ii) Sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It may also contain all 11 amino acids in the peptide having (SEQ ID NO: 109), (iii) Amino acid Y 15 and Y 17 Both may be sulfated. A monoclonal antibody or its antigen-binding portion as described in any one of the preceding paragraphs. [Section 12] It can mediate ADCC and specifically bind to an epitope on the human CC motif chemokine receptor 8 (hCCR8) whose sequence is described in Sequence ID No. 1. The epitope, when determined by X-ray crystallography, is approximately 12-22 amino acid residues (V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 Located in the N-terminal domain of hCCR8 within the peptide (SEQ ID NO: 109), (i) Epitope is sequence V 12 T 13 D 14Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It may contain at least one amino acid in the peptide having (SEQ ID NO: 109), (ii) Epitope is sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 The peptide containing (SEQ ID NO: 109) may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the amino acids. (iii) Epitope is sequence V 12 T 13 D 14 Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 It may contain 11 amino acids in the peptide having (SEQ ID NO: 109), or (iv) Epitope is sequence V 12 T 13 D 14 Y 15 Y 16 Y17 P 18 D 19 I 20 F 21 S 22 It may consist of all 11 amino acids in the peptide having (SEQ ID NO: 109), A monoclonal antibody or its antigen-binding portion. [Section 13] (i) When bound to CCR8 on the cell surface, it does not cause internal migration of CCR8 in the presence or absence of the cross-linking antibody, and / or (ii) Promoting the depletion of human tumor-associated Tregs in vitro, and / or (iii) CCR8 in non-tumor tissue, which may be skin, thymus, spleen, or blood. + To specifically induce tumor Treg depletion without depleting T cells, and / or (iv) When administered to a subject as monotherapy, it inhibits the growth of tumor cells in the subject, and / or (v) When administered to a subject in combination with additional therapeutic agents for treating cancer, inhibits the growth of tumor cells in the subject. A monoclonal antibody or its antigen-binding portion as described in any one of the preceding paragraphs. [Section 14] The following characteristics: (a) EC of about 2 nM or less 50 It specifically binds to CCR8 expressed on the surface of cells. (b) Specifically binds to rare, scattered immune cells in the thymic medulla and dermis of the skin, but does not bind to any of the human cerebrum, cerebellum, heart, liver, lungs, kidneys, tonsils, spleen, thymus, colon, stomach, pancreas, adrenal glands, pituitary gland, skin, peripheral nerves, testes, or uterine tissue, or peripheral blood mononuclear cells (PBMCs). (c) ICs of approximately 5 nM or less 50 This inhibits the binding of CCL1 and CCR8, thereby inhibiting CCR8 / CCL1 signaling. (d) When bound to CCR8 on the cell surface, EC is approximately 10 pM or less. 50 to mediate cell depletion, (e) When bound to CCR8 on the cell surface, it does not cause internal migration of CCR8 in the presence or absence of the cross-linking antibody. (f) Promote the depletion of human tumor-associated Tregs in vitro using the assay described in Example 22. (g) Using the assay described in Example 20, promote the depletion of human tumor-associated Tregs in ex vivo human tumor slice samples. (h) CCR8 in non-tumor tissue + To preserve T cells while specifically mediating the depletion of tumor Tregs, (i) When administered to the target as monotherapy, it inhibits the growth of tumor cells in the target, and (j) When administered to a subject in combination with additional therapeutic agents for treating cancer, inhibiting the growth of tumor cells in the subject. It exhibits at least one, two, three, four, five, or six of the following: A monoclonal antibody or its antigen-binding moiety according to any one of the preceding items, which may exhibit all of these characteristics. [Section 15] Approximately 10 nM or less K D It then further binds to an epitope located in the N-terminal domain of human CCR8, and the epitope is sequence V 12 T 13 D 14Y 15 Y 16 Y 17 P 18 D 19 I 20 F 21 S 22 A monoclonal antibody or its antigen-binding moiety according to item 34 or 35, comprising a peptide having (SEQ ID NO: 109) and sulfated tyrosine-15 and tyrosine-17 residues. [Section 16] A modified anti-hCCR8 monoclonal antibody or its antigen-binding moiety, comprising a modified heavy chain constant region that binds to the Fcγ receptor (FcγR) with higher affinity than the monoclonal antibody or its antigen-binding moiety described in item 14 or 15, and which is at least 2, 5, or 10-fold enhanced in ADCC-mediated modified heavy chain constant region. [Section 17] The reference antibody binds to the same epitope, and the reference antibody (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 3 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 15. L 、 (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L 、 (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 5 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 17. L 、 (d) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L 、 (e) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 7 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 19. L 、 (f) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 8 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 20. L 、 (g) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 9 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 21. L 、 (h) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 10 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 22. L 、 (i) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 11 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 23.L 、 (j) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 12 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 24. L 、 (k) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 13 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 25. L 、 (l) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 14 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 26. L ,or (m) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L A monoclonal antibody or its antigen-binding moiety as described in any one of the preceding items, including the monoclonal antibody or its antigen-binding moiety. [Section 18] Regarding binding to hCCR8, it cross-competes with the reference antibody, and the reference antibody... (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 3 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 15. L 、 (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L 、 (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 5 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 17. L 、 (d) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L 、 (e) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 7 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 19. L 、 (f) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 8 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 20. L 、 (g) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 9 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 21. L 、 (h) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 10 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 22.L 、 (i) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 11 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 23. L 、 (j) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 12 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 24. L 、 (k) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 13 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 25. L 、 (l) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 14 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 26. L ,or (m) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L A monoclonal antibody or its antigen-binding moiety as described in any one of the preceding items, including the monoclonal antibody or its antigen-binding moiety. [Section 19] (a) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 3 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 15. L 、 (b) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 4 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 16. L 、 (c) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 5 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 17. L 、 (d) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 6 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 18. L 、 (e) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 7 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 19. L 、 (f) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 8 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 20. L 、 (g) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 9 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 21. L 、 (h) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 10 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 22. L 、 (i) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 11 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 23. L 、 (j) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 12 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 24. L 、 (k) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 13 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 25. L 、 (l) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 14 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 26. L ,or (m) V containing a sequence of linked amino acids having the sequence described in Sequence ID No. 115 H V containing a sequence of amino acids having the sequence described in Sequence ID No. 116. L A monoclonal antibody or its antigen-binding moiety according to any one of the preceding items, each comprising the CDR1, CDR2, and CDR3 domains. [Section 20] The following CDR domains are defined by the Kabat protocol: (a) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 27, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 28, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 29, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 30, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 31, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 32. (b) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 33, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 34, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 35, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 36, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 37, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 38. (c) Heavy chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 39, heavy chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 40, heavy chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 41, light chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 42, light chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 43, and light chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 44. (d) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 45, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 46, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 47, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 48, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 49, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 50. (e) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 51, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 52, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 53, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 54, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 55, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 56. (f) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 57, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 58, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 59, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 60, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 61, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 62. (g) Heavy chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 63, heavy chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 64, heavy chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 65, light chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 66, light chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 67, and light chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 68. (h) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 69, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 70, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 71, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 72, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 73, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 74. (i) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 75, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 76, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 77, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 78, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 79, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 80. (j) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 81, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 82, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 83, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 84, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 85, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 86. (k) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having t...

Claims

1. When measured by the binding assay described in Example 11, the EC was 20 nM ± 25% or less. ５０ A modified version of the anti-hCCR8 monoclonal antibody or its antigen-binding moiety, which specifically binds to the human C-C motif chemokine receptor 8 (hCCR8) described in Sequence ID No. 1, expressed on the surface of human activated regulatory T cells (Treg), and mediates antibody-dependent cell-mediated cell-mediated cytotoxicity (ADCC) killing of activated human Treg cells, comprising an anti-hCCR8 monoclonal antibody or its antigen-binding moiety, wherein the modified anti-hCCR8 monoclonal antibody has a higher affinity for the Fcγ receptor compared to the unmodified anti-hCCR8 antibody and mediates enhanced ADCC, The monoclonal antibody or modified monoclonal antibody or its antigen-binding moiety contains the following CDR domain as defined by the Kabat method: (a) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 33, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 34, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 35, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 36, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 37, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO:

38. (b) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 39, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 40, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 41, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 42, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 43, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO:

44. (c) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 45, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 46, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 47, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 48, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 49, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO:

50. (d) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 69, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 70, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 71, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 72, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 73, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO:

74. (e) Heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 75, heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 76, heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 77, light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 78, light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 79, and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 80, or (f) Heavy chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 103, heavy chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 104, heavy chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 105, light chain variable region CDR1 containing continuously linked amino acids having the sequence described in SEQ ID NO: 106, light chain variable region CDR2 containing continuously linked amino acids having the sequence described in SEQ ID NO: 107, and light chain variable region CDR3 containing continuously linked amino acids having the sequence described in SEQ ID NO: 108 Monoclonal antibodies or modified monoclonal antibodies, or their antigen-binding moieties, including [the specified substance].

2. (i) When measured by the binding assay described in Example 11, (a) 10 nM ± 25% or less, (b) 5 nM ± 25% or less, (c) 1.7 nM ± 25% or less, (d) 1 nM ± 25% or less, (e) 0.5 nM ± 25% or less, (f) 0.1 nM ± 25% or less, (g) 0.1 nM ± 25%, (h) 1.7nM±25%, (i) Between 0.1 nM ± 25% and 10 nM ± 25%, (j) Between 0.1 nM ± 25% and 2 nM ± 25%, (k) Between 0.5 nM ± 25% and 5 nM ± 25%, (l) Between 1 nM ± 25% and 2 nM ± 25%, or (m) Between 0.5 nM ± 25% and 1 nM ± 25% EC ５０ It specifically binds to human CCR8-expressing Chinese hamster ovary cells, and / or (ii) When measured by the binding assay described in Example 11, (a) 14 nM ± 25% or less, (b) 5 nM ± 25% or less, (c) 2nM ± 25% or less, (d) 0.5 nM ± 25% or less, (e) 0.3 nM ± 25% or less, (f) 0.1 nM ± 25% or less, (g) 0.03 nM ± 25% or less, (h) 1.7nM±25%, (i) Between 0.03 nM ± 25% and 10 nM ± 25%, (j) Between 0.1 nM ± 25% and 5 nM ± 25%, or (k) Between 0.2 nM ± 25% and 2 nM ± 25% EC ５０ It specifically binds to activated regulatory T cells (Treg), and / or (iii) When measured by surface plasmon resonance (SPR) as described in Example 11, (a) 100 nM ± 25% or less, (b) 50 nM ± 25% or less, (c) 10 nM ± 25% or less, (d) 5 nM ± 25% or less, (e) 1.6 nM ± 25%, (f) 1.0 nM ± 25% or less, (g) 0.5 nM ± 25% or less, (h) 0.1 nM ± 25% or less, (i) Between 100 nM ± 25% and 0.1 nM ± 25%, (j) Between 50 nM ± 25% and 0.5 nM ± 25%, (k) Between 10 nM ± 25% and 1 nM ± 25%, or (l) Between 2 nM ± 25% and 1 nM ± 25% K Ｄ It binds to the N-terminal peptide of human CCR8 containing sulfated tyrosine-15 and tyrosine-17 residues, and / or (iv) When measured by the SPR described in Example 11, (a) 100 nM ± 25% or less, (b) 50 nM ± 25% or less, (c) 25 nM ± 25% or less, (d) 10 nM ± 25% or less, (f) 1.0 nM ± 25% or less, (e) 20 nM ± 25%, (i) Between 100 nM ± 25% and 1 nM ± 25%, (j) Between 50 nM ± 25% and 10 nM ± 25%, or (k) Between 30 nM ± 25% and 20 nM ± 25% K Ｄ It binds to a single sulfated residue, namely tyrosine-15, in the N-terminal peptide of human CCR8. The monoclonal antibody or modified monoclonal antibody according to claim 1, or the antigen-binding portion thereof.

3. A monoclonal antibody or modified monoclonal antibody or its antigen-binding moiety according to claim 1 or 2, which specifically binds to rare, scattered immune cells in the thymic medulla and dermis of the skin, but does not bind to any of the human cerebrum, cerebellum, heart, liver, lungs, kidneys, tonsils, spleen, thymus, colon, stomach, pancreas, adrenal gland, pituitary gland, skin, peripheral nerves, testes, or uterine tissue, or peripheral blood mononuclear cells (PBMCs).

4. (i) When measured by inhibition of calcium flow as described in Example 15, the binding of C-C motif chemokine ligand 1 (CCL1) to CCR8 is inhibited, (a) 10 nM ± 25% or less, (b) 5 nM ± 25% or less, (c) 1 nM ± 25% or less, (d) 0.5 nM ± 25% or less, (e) 0.1 nM ± 25% or less, (f) 0.01 nM ± 25% or less, (g) Between 0.01 nM ± 25% and 10 nM ± 25%, (h) Between 0.05 nM ± 25% and 5 nM ± 25%, (i) Between 0.1 nM ± 25% and 1 nM ± 25%, or (j) 0.46nM±25% IC ５０ This inhibits CCR8 / CCL1 signaling, and / or (ii) When measured by the CD16 crosslinking assay described in Example 17, (a) 100 pM ± 25% or less, (b) 30 pM ± 25% or less, (c) 10 pM ± 25% or less, (d) 3 pM ± 25% or less, (e) 1 pM ± 25% or less, (e) 0.5 pM ± 25% or less, (f) 0.1 pM ± 25% or less, (g) 0.05 pM ± 25% or less, (h) 0.7 pM ± 25%, (i) Between 0.05 pM ± 25% and 50 pM ± 25%, (j) Between 0.1 pM ± 25% and 10 nM ± 25%, (k) Between 0.3 nM ± 25% and 7 nM ± 25%, or (l) Between 0.6 nM ± 25% and 3 nM ± 25% EC ５０ This mediates the depletion of CCR8-expressing cells, and / or (iii) When measured by the apoptosis assay described in Example 19, (a) 500 pM ± 25% or less, (b) 100 pM ± 25% or less, (c) 30 pM ± 25% or less, (d) 15 pM ± 25% or less, (e) 5 pM ± 25% or less, (f) 1 pM ± 25% or less, (g) 13 pM ± 25%, (h) Between 1 pM ± 25% and 500 pM ± 25%, (i) Between 5 pM ± 25% and 100 pM ± 25%, or (j) Between 10 pM ± 25% and 50 pM ± 25% EC ５０ This mediates the depletion of activated Treg, A monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof, as described in any one of claims 1 to 3.

5. When determined by X-ray crystallography, it binds to an epitope located in the N-terminal domain of human CCR8. (a) The epitope contains at least one amino acid in the peptide having the sequence Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ (SEQ ID NO: 2) and contains a sulfated Y15 or sulfated Y17 residue (b) Epitope is sequence Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ A peptide containing two, three, four, five, six, or all of the amino acids in (SEQ ID NO: 2), and containing a sulfated Y15 or sulfated Y17 residue, (c) Epitope is sequence Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ The peptide containing (SEQ ID NO: 2) includes all seven amino acids and contains a sulfated Y15 or sulfated Y17 residue, (d) The epitope consists of all seven amino acids in a peptide having the sequence Y15 Y16 Y17 P18 D19 I20 F21 (SEQ ID NO: 2), and includes a sulfated Y15 or a sulfated Y17 residue. (e) The epitope is sequence V １２ T １３ D １４ Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ S ２２ A peptide having (SEQ ID NO: 109) comprising at least one amino acid, comprising sulfated Y15 and sulfated Y17 residues, (f) Epitope is sequence V １２ T １３ D １４ Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ S ２２ A peptide containing two, three, four, five, six, seven, eight, nine, ten, or all of the amino acids in (SEQ ID NO: 109), and containing sulfated Y15 and sulfated Y17 residues, (g) Epitope is sequence V １２ T １３ D １４ Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ S ２２ A peptide containing all 11 amino acids, including sulfated Y15 and sulfated Y17 residues, or (h) Epitope is sequence V １２ T １３ D １４ Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ S ２２ The peptide having (SEQ ID NO: 109) consists of all 11 amino acids, and includes sulfated Y15 and sulfated Y17 residues, A monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof, as described in any one of claims 1 to 4.

6. A modified anti-hCCR8 monoclonal antibody or its antigen-binding moiety capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC), comprising a modified heavy chain constant region that binds to the Fcγ receptor with higher affinity than an unmodified anti-hCCR8 monoclonal antibody or its antigen-binding moiety and mediates enhanced ADCC, wherein the modified anti-hCCR8 monoclonal antibody or its antigen-binding moiety specifically binds to an epitope on the human C-C motif chemokine receptor 8 (hCCR8) located in the N-terminal domain of human CCR8, and the epitope is (a) Amino acid Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ (SEQ ID NO: 2) contains amino acid Y １５ or Y １７ It is sulfated, or (b) Amino acid Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ consisting of (SEQ ID NO: 2), and amino acid Y １５ or Y １７ is sulfated, or (c) Array V １２ T １３ D １４ Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ S ２２ contains all 11 amino acids in the peptide having (SEQ ID NO: 109), and the amino acids Y １５ and Y １７ are sulfated, or (d) Array V １２ T １３ D １４ Y １５ Y １６ Y １７ P １８ D １９ I ２０ F ２１ S ２２ It consists of all 11 amino acids in the peptide having (SEQ ID NO: 109), and amino acid Y １５ and Y １７ is sulfated Monoclonal antibodies or modified monoclonal antibodies or their antigen-binding moieties.

7. A monoclonal antibody according to claim 5 or 6, or its antigen-binding moiety, which binds to an epitope located in the N-terminal domain of human CCR8 at a KD of 10 nM ± 25% or less, Epitope, (a) A peptide having a sequence comprising Y15 Y16 Y17 P18 D19 I20 F21 (SEQ ID NO: 2) and a sulfated Y15 or Y17 residue, or (b) A sequenced peptide comprising 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: 109) and sulfated Y 15 and Y 17 residues. A monoclonal antibody or its antigen-binding moiety, including the above.

8. A monoclonal antibody or a modified monoclonal antibody or the antigen-binding portion thereof, in the NK cell lysis assay described in Example 17, the EC of cell lysis. ５０ A monoclonal antibody or modified monoclonal antibody or its antigen-binding moiety according to any one of claims 1 to 7, which mediates ADCC activity that is at least 2-fold, 5-fold, or 10-fold enhanced compared to an unmodified monoclonal antibody or its antigen-binding moiety, as measured by a decrease in ADCC activity.

9. (a) Promoting the depletion of human tumor-associated Tregs in vitro, and / or (b) CCR8 in non-tumor tissue ＋ To specifically induce tumor Treg depletion without depleting T cells, and / or (c) When administered to the subject as monotherapy, it inhibits the growth of tumor cells in the subject, and / or (d) When administered to a subject in combination with additional therapeutic agents for treating cancer, inhibits the growth of tumor cells in the subject. A monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof according to any one of claims 1 to 8.

10. The following characteristics: (a) When measured by the antibody binding assay described in Example 11, the EC is 2 nM ± 25% or less. ５０ It specifically binds to CCR8 expressed on the surface of cells. (b) It specifically binds to rare, scattered immune cells in the thymic medulla and dermis of the skin, but does not bind to any of the human cerebrum, cerebellum, heart, liver, lungs, kidneys, tonsils, spleen, thymus, colon, stomach, pancreas, adrenal glands, pituitary gland, skin, peripheral nerves, testes, or uterine tissue, or peripheral blood mononuclear cells (PBMCs). (c) ICs with a 5 nM ± 25% or less ５０ This inhibits the binding of CCL1 and CCR8, thereby inhibiting CCR8 / CCL1 signaling. (d) When bound to CCR8 on the cell surface, EC of 10 pM ± 25% or less ５０ mediates the depletion of cells, (e) When bound to CCR8 on the cell surface, it does not cause internal migration of CCR8, both in the presence and absence of the cross-linking antibody. (f) To promote the depletion of human tumor-associated Tregs in vitro, (g) To promote the depletion of human tumor-related Treg in ex vivo human tumor slice samples, (h) CCR8 in non-tumor tissue ＋ To preserve T cells while specifically mediating the depletion of tumor Tregs, (i) When administered to the target as monotherapy, it inhibits the growth of tumor cells in the target, and (j) When administered to a subject in combination with additional therapeutic agents for treating cancer, inhibiting the growth of tumor cells in the subject. Exhibiting at least one, two, three, four, five, or six, or all of the following: A monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof according to any one of claims 1 to 9.

11. V binds to the same hCCR8 epitope as the reference antibody and / or cross-competes with the reference antibody for binding to hCCR8, and the reference antibody contains a sequence of linked amino acids having the sequence described in SEQ ID NO:

6. Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

18. Ｌ A monoclonal antibody or modified monoclonal antibody or antigen-binding moiety thereof according to any one of claims 1 to 10, comprising:

12. (a) V containing a sequence of amino acids having the sequence described in Sequence ID No. 4 Ｈ V containing a sequence of amino acids having the sequence described in Sequence ID No.

16. Ｌ , (b) V containing a sequence of amino acids having the sequence described in Sequence ID No. 5 Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

17. Ｌ , (c) V containing a sequence of amino acids having the sequence described in Sequence ID No. 6 Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

18. Ｌ , (d) V containing a sequence of amino acids having the sequence described in Sequence ID No. 10 Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

22. Ｌ , (e) V containing a sequence of amino acids having the sequence described in Sequence ID No. 11 Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

23. Ｌ ,or (f) V containing a sequence of continuously linked amino acids having the sequence described in Sequence ID No. 115 Ｈ V containing a sequence of amino acids having the sequence described in Sequence ID No.

116. Ｌ A monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof according to any one of claims 1 to 11, comprising the CDR1, CDR2, and CDR3 domains in each of the above.

13. A monoclonal antibody or a modified monoclonal antibody or its antigen-binding moiety according to any one of claims 1 to 12, which, when bound to CCR8 on the surface of a cell, does not cause internal migration of CCR8, whether in the presence or absence of a cross-linking antibody.

14. The following CDR domains are defined by the Kabat method: heavy chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 45; heavy chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 46; heavy chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 47; light chain variable region CDR1 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 48; light chain variable region CDR2 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO: 49; and light chain variable region CDR3 containing a sequence of continuously linked amino acids having the sequence described in SEQ ID NO:

50. A monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof according to any one of claims 1 to 13.

15. V contains a sequence of continuously linked amino acids having a sequence that is at least 80% identical to the sequence described in Sequence ID No.

6. Ｈ V containing a sequence that is at least 80% identical to the sequence described in Sequence ID No. 18, and V Ｌ V includes and has the sequence described in SEQ ID NO: 6 and SEQ ID NO: 18, respectively. Ｈ and V Ｌ Maintaining the functional properties of the antibody including the region, Alternatively, V containing a sequence of continuously linked amino acids having the sequence described in Sequence ID No.

6. Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

18. Ｌ including, Alternatively, the material may include a heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO:

114. The monoclonal antibody or modified monoclonal antibody according to claim 14, or the antigen-binding portion thereof.

16. (a) V containing a sequence of amino acids having the sequence described in Sequence ID No. 4 Ｈ V containing a sequence of amino acids having the sequence described in Sequence ID No.

16. Ｌ , (b) V containing a sequence of amino acids having the sequence described in Sequence ID No. 5 Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

17. Ｌ , (c) V containing a sequence of amino acids having the sequence described in Sequence ID No. 6 Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

18. Ｌ , (d) V containing a sequence of amino acids having the sequence described in Sequence ID No. 10 Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

22. Ｌ , (e) V containing a sequence of amino acids having the sequence described in Sequence ID No. 11 Ｈ V, and V containing a sequence of amino acids having the sequence described in Sequence ID No.

23. Ｌ ,or (f) V containing a sequence of continuously linked amino acids having the sequence described in Sequence ID No. 115 Ｈ V containing a sequence of amino acids having the sequence described in Sequence ID No.

116. Ｌ Includes or, (g) A heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 100, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO:

112. (h) A heavy chain comprising continuously linked amino acids having the sequence described in SEQ ID NO: 101, and a light chain comprising continuously linked amino acids having the sequence described in SEQ ID NO:

113. (i) A heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 114, or (j) A heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 117, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO:

118. A monoclonal antibody or modified monoclonal antibody or antigen-binding moiety thereof according to any one of claims 1 to 15, comprising:

17. An anti-hCCR8 monoclonal antibody or modified monoclonal antibody or antigen-binding moiety thereof according to any one of claims 1 to 16, comprising a chimeric antibody, a humanized antibody, a human antibody, a human IgG1 isotype, a human IgG3 isotype, or a modified IgG1 heavy chain constant region mediating an enhanced ADCC, or a fragment mediating the ADCC of any of the antibodies.

18. (i) low-fucosylated, or non-fucosylated, and / or (ii) containing enhanced ADCC-mediated mutations or multiple mutations A monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof according to claim 17, comprising a modified IgG1 heavy chain constant region.

19. A mutation or multiple mutation of IgG1 is selected from G236A; S239D; F243L; E333A; G236A / I332E; S239D / I332E; S267E / H268F; S267E / S324T; H268F / S324T; and G236A / S239D / I332E. The modified anti-hCCR8 monoclonal antibody or its antigen-binding moiety according to claim 18.

20. A modified monoclonal antibody comprising a heavy chain constant region that is low-fucosylated or non-fucosylated, which specifically binds to human C-C motif chemokine receptor 8 (hCCR8) described in SEQ ID NO: 1, which is expressed on the surface of cells, and mediates the depletion of CCR8-expressing cells by antibody-dependent cell-mediated cytotoxicity (ADCC), comprising a heavy chain containing continuously linked amino acids having the sequence described in SEQ ID NO: 102, and a light chain containing continuously linked amino acids having the sequence described in SEQ ID NO:

114.

21. An immunoconjugate comprising a monoclonal antibody or a modified monoclonal antibody according to any one of claims 1 to 20, or an antigen-binding moiety thereof, which is linked to a cell lysating agent.

22. The immunoconjugate according to claim 21, wherein the cell lysing agent is a cytotoxic or a radioisotope.

23. A bispecific molecule comprising a monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof according to any one of claims 1 to 20, wherein the bispecific molecule is linked to a binding domain having a different binding specificity from the monoclonal antibody or modified monoclonal antibody or its antigen-binding moiety.

24. (a) A monoclonal antibody or a modified monoclonal antibody according to any one of claims 1 to 20, or an antigen-binding moiety thereof (b) The immunoconjugate according to claim 21 or 22, or (c) The bispecific molecule described in claim 23, Pharmacologically acceptable carriers and A composition containing the following:

25. A monoclonal antibody or a modified monoclonal antibody according to any one of claims 1 to 20, or an isolated nucleic acid encoding an antigen-binding portion thereof.

26. An expression vector comprising the nucleic acid described in claim 25.

27. A host cell comprising the expression vector described in claim 26.

28. The host cell according to claim 27, comprising a modified glycosylation mechanism.

29. The host cell according to claim 28, wherein the modified glycosylation mechanism lacks the α-(1,6) fucosyltransferase (FUT8) enzyme, and the antibody produced in the cell is low-fucosylated or non-fucosylated.

30. A host cell according to any one of claims 27 to 29, which is a eukaryotic cell.

31. The host cell according to claim 30, wherein the eukaryotic cell is a mammalian cell.

32. A method for preparing an anti-CCR8 monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof, comprising expressing the antibody or its antigen-binding moiety in a host cell according to any one of claims 27 to 31, and isolating the antibody or its antigen-binding moiety from the host cell.

33. A pharmaceutical composition for treating cancer in a subject suffering from cancer, comprising the composition described in claim 24.

34. A pharmaceutical composition for inhibiting the growth of tumor cells in a target, comprising the composition described in claim 24.

35. The pharmaceutical composition according to claim 33 or 34, for use in combination with an additional therapeutic agent for treating a therapeutically effective amount of cancer.

36. The pharmaceutical composition according to claim 35, wherein the additional therapeutic agent is a compound that suppresses or increases the stimulation of the immune system, an immune checkpoint inhibitor, a chemotherapeutic agent, radiotherapy, a small molecule compound, a macrocyclic peptide, a fusion protein, and / or an antibody.

37. The pharmaceutical composition according to any one of claims 33 to 36, wherein the additional therapeutic agent is an antagonist or an agonist.

38. (a) The antagonist is an antagonist monoclonal antibody that specifically binds to programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activator gene-3 (LAG-3), B and T lymphocyte attenuator (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), killer immunoglobulin-like receptor (KIR), killer cell lectin-like receptor G1 (KLRG-1), adenosine A2a receptor (A2aR), T cell immune receptor having Ig and ITIM domains (TIGIT), V domain Ig-containing T cell activation inhibitor (VISTA), oncogene tyrosine protein kinase MER (MerTK), natural killer cell receptor 2B4 (CD244), or CD160, or (b) The agonist is an agonist monoclonal antibody that specifically binds to inducible T cell costimulatory factor (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-induced TNFR-related protein (GITR), or herpesvirus entry mediator (HVEM). The pharmaceutical composition according to claim 37.

39. (a) The antagonist is an antagonist antibody that specifically binds to PD-1 or its antigen-binding portion, or (b) An antibody that specifically binds to PD-1 is selected from nivolumab, pembrolizumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, retifanlimab, and pimivarimab, or (c) The antagonist is an antagonist antibody that specifically binds to PD-L1 or its antigen-binding portion, or (d) An antibody that specifically binds to PD-L1 is selected from atezolizumab, durvalumab, avelumab, emvafolimab, BMS-936559, CK-301, CS-1001, SHR-1316, CBT-502, and BGB-A333, or (e) The antagonist is an antagonist antibody that specifically binds to CTLA-4 or its antigen-binding portion, or (f) The antibody that specifically binds to CTLA-4 is ipilimumab or tremelimumab. The pharmaceutical composition according to claim 38.

40. The pharmaceutical composition according to any one of claims 33 to 39, wherein the cancer is a solid tumor or a hematological malignancy, or the tumor cells are cells of a solid tumor or a hematological malignancy.

41. A solid tumor, (a) 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, anal cancer, endometrial cancer, prostate cancer, fibrosarcoma, neuroblastoma, glioma, glioblastoma, germ cell tumor, pediatric sarcoma, sinus natural killer cancer, melanoma, skin cancer, bone cancer, cervical cancer, uterine cancer, endometrial cancer Cancers of the cervix, fallopian tubes, ovarian cancer, cervical cancer, vaginal cancer, vulvar cancer, testicular cancer, endocrine cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcomas, penile cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumors, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, solid tumors in children, environmentally induced cancers, virus-associated cancers, cancers of viral origin, advanced cancers, unresectable cancers, metastatic cancers, refractory cancers, recurrent cancers, and any combination thereof selected from these, or (b) A cancer selected from non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colorectal cancer (CRC), gastric cancer, gastroesophageal (GE) junction cancer, and cervical cancer, or (c) Cancer selected from head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), gastric adenocarcinoma (STAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), rectal adenocarcinoma (READ), esophageal cancer (ESCA), invasive breast cancer (BRCA), colon adenocarcinoma (COAD), and cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), or (d) A cancer selected from squamous cell carcinoma of the head and neck (SCCHN), cervical cancer, colorectal cancer (CRC), non-small cell lung cancer-squamous cell carcinoma (NSLC-SCC), NSCLC-adenocarcinoma (NSLC-ADC), pancreatic cancer, gastric cancer, bladder cancer, and breast cancer, or (e) Cancer selected from colon adenocarcinoma, bladder cancer, breast cancer, and fibrosarcoma The pharmaceutical composition according to claim 40.

42. Hematological malignancies, (a) acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), T-cell lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal immunoglobulinemia of unknown significance (MGUS), advanced, metastatic, refractory, and / or recurrent hematological malignancies, and any combination of the said hematological malignancies, or (b) Acute, chronic, lymphocytic (lymphoblastic) and / or myeloid leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphoma [mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, and splenic lymphoma] Perivisceral zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytoid lymphoma [LPL; also known as Waldenström macroglobulinemia (WM)], hairy cell lymphoma, and primary central nervous system (CNS) lymphoma, T-cell lymphomas such as NHL, progenitor T-lymphoblastic lymphoma / leukemia, T-lymphoblastic lymphoma / leukemia (T-Lby / T-ALL), peripheral T-cell lymphoma, cutaneous T-cell lymphoma (CTLC), bacteria Sarcoma assisi, Sézary syndrome, adult T-cell lymphoma / leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma nasal type, enteropathy-associated enteric T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), and unspecified peripheral T-cell lymphoma, acute myeloid lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, vascular lymphoma, enteric T-cell lymphoma, mediastinal primary B-cell large cell lymphoma, post-transplant lymphoma Lymphocyte proliferative disorders, histiocytic lymphoma, primary exudative lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, and precursor B lymphoblastic lymphoma; myeloma, multiple myeloma, smoldering myeloma (low-grade myeloma), monoclonal immunoglobulinemia of unknown significance (MGUS), solitary plasmacytoma, IgG myeloma, light chain myeloma, non-secretory myeloma, and amyloidosis; and any combination of the aforementioned hematological malignancies. The pharmaceutical composition according to claim 40.

43. A pharmaceutical composition for enhancing the antitumor immune response induced by a therapeutic agent in a subject suffering from cancer, comprising the composition described in claim 24.

44. The pharmaceutical composition according to claim 43, wherein the therapeutic agent is an immune checkpoint inhibitor, a chemotherapeutic agent, or radiotherapy.

45. The pharmaceutical composition according to claim 44, wherein the immune checkpoint inhibitor is an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody.

46. A pharmaceutical composition for treating cancer in a subject, comprising the modified monoclonal antibody described in Claim 20.

47. The pharmaceutical composition according to claim 46, for use in combination with nivolumab.

48. A pharmaceutical composition according to any one of claims 33 to 47, wherein the target is a human.

49. A kit for treating cancer in subjects suffering from cancer, (a) A single or multiple dose in the range of 0.01±25% to 20±25% mg / kg body weight, comprising an anti-CCR8 monoclonal antibody or a modified monoclonal antibody or an antigen-binding moiety thereof according to any one of claims 1 to 20, (b) Instructions for treating cancer in the subject and A kit that includes this.

50. A monoclonal antibody or its antigen-binding moiety that specifically binds to PD-1, PD-L1, or CTLA-4, administered in a single or multiple dose in the range of 0.1±25% to 20±25% mg / kg body weight, or in a constant dose of 200±25% to 1600±25% mg. Instructions for treating cancer with a monoclonal antibody or its portion that specifically binds to PD-1, PD-L1, or CTLA-4 in combination with the aforementioned anti-CCR8 monoclonal antibody or modified monoclonal antibody or its antigen-binding portion, and The kit according to claim 49, further comprising:

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