Anti-CCR8 Antibodies and Uses Thereof

Abstract

Described herein is an anti-CCR8 antibody and its antigen-binding fragment.Described herein is also a nucleic acid encoding anti-CCR8 antibody and its antigen-binding fragment, a composition comprising anti-CCR8 antibody and its antigen-binding fragment, and a method for producing and using anti-CCR8 antibody and its antigen-binding fragment to treat or prevent cancer in a subject in need of cancer treatment or prevention.The anti-CCR8 antibody and its antigen-binding fragment specifically binds to chemokine (CC motif) receptor 8 (CCR8).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. Section 119(e) of International Patent Application No. PCT / CN2021 / 113913, filed on August 20, 2021, now withdrawn. The disclosure of the prior application is deemed to be part of the disclosure of this application and is incorporated by reference in its entirety into the disclosure of this application.

[0002] FIELD OF THEINVENTION The present invention relates to an isolated anti-chemokine (CC motif) receptor 8 (CCR8) monoclonal antibody or antigen-binding fragment thereof, nucleic acid and expression vector encoding the antibody, recombinant cells containing the vector, and compositions comprising the antibody. Methods of making the antibody and methods of using the antibody to treat diseases, including cancer and / or associated complications, are also provided.

[0003] Reference to Electronically Submitted Sequence Listing This application contains a Sequence Listing that has been submitted electronically via EFS-Web as a Sequence Listing in ASCII format having a size of 49 kb, with a filename of "065798.6WO1 Sequence Listing" and a creation date of August 6, 2021. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated by reference in its entirety herein. [Background technology]

[0004] 2. Background of the Invention CCR8 is a chemokine receptor that mediates cell migration down a CCL1 or CCL18 gradient (Islam et al., JEM210(10):1889-1898(2013)). Recently, CCR8 has been identified as a highly specific cell surface marker of tumor-infiltrating regulatory T cells (TITRs) in human cancers, with significantly higher expression on Tregs present in tumors compared to circulating Tregs and no or very low expression on other T cell populations (cytotoxic or effector T cells, respectively). Furthermore, CCR8 is predominantly expressed on highly immunosuppressive Tregs that express FoxP3high, CD25high, TIGIT+, LAG3+ and release high IL-10 and TGF-β. Depleting CCR8+ Tregs would reduce immunosuppressive cytokines and modulate the tumor-induced microenvironment to restore antitumor immunity. Interestingly, in patients with breast or pancreatic cancer, high CCR8+ Treg numbers correlated with more advanced disease stage and possibly reduced overall survival. Therefore, CCR8 is an ideal target for cancer immunotherapy to treat and potentially cure CCR8-positive cancers. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Islam et al., JEM210(10):1889-1898(2013) Summary of the Invention

[0006] Brief Summary of the Invention In one general aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to chemokine (CC motif) receptor 8 (CCR8). 1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (2) SEQ ID NOs: 13, 2, 14, 4, 28, and 6, respectively; (3) SEQ ID NOs: 13, 2, 15, 4, 5, and 6, respectively; (4) SEQ ID NOs: 16, 17, 18, 29, 30, and 6, respectively; (5) SEQ ID NOs: 19, 20, 21, 4, 5, and 6, respectively; (6) SEQ ID NOs: 22, 23, 24, 31, 5, and 32, respectively; or (7) SEQ ID NOs: 25, 26, 27, 33, 34, and 35, respectively; and a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having a polypeptide sequence of An isolated monoclonal antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment thereof specifically binds to chemokine (CC motif) receptor 8 (CCR8), preferably human CCR8.

[0007] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7, 36, 38, 40, 42, 44, or 46, or a light chain variable region having a polypeptide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8, 37, 39, 41, 43, 45, or 47.

[0008] In one embodiment, the isolated anti-CCR8 monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7 and a light chain variable region having the polypeptide sequence of SEQ ID NO:8; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:36 and a light chain variable region having the polypeptide sequence of SEQ ID NO:37; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:38 and a light chain variable region having the polypeptide sequence of SEQ ID NO:39; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 40, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 41; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:42 and a light chain variable region having the polypeptide sequence of SEQ ID NO:43; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 44 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 45; or (7) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 46 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 47. Includes.

[0009] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof binds to CCR8 and induces effector-mediated tumor cell lysis through antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or mediates the activity of a conjugated drug, and / or can form a bispecific antibody with another monoclonal antibody or antigen-binding fragment thereof that has a cancer-killing effect.

[0010] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric or human or humanized.

[0011] In certain embodiments, the humanized monoclonal antibody or antigen-binding fragment thereof comprises: (1) SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively; (2) SEQ ID NOs: 1, 2, 49, 4, 5, and 6, respectively; (3) SEQ ID NOs: 1, 2, 49, 50, 51, and 6, respectively; (4) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; or (5) SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively. and heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having a polypeptide sequence of:

[0012] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9, 52, 53, 54, 55, 56, 57, 58, 59, or 60, or a light chain variable region having a polypeptide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10, 61, 62, or 63.

[0013] In one embodiment, the isolated anti-CCR8 monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:52 and a light chain variable region having the polypeptide sequence of SEQ ID NO:63; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:52 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:53 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:54 and a light chain variable region having the polypeptide sequence of SEQ ID NO:63; or (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:59 and a light chain variable region having the polypeptide sequence of SEQ ID NO:62; Includes.

[0014] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof specifically binds to cynomolgus CCR8.

[0015] Also provided is an isolated bispecific antibody or antigen-binding fragment thereof comprising a monoclonal antibody or antigen-binding fragment thereof of the invention.

[0016] Also provided is an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention.

[0017] Also provided is a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention.

[0018] Host cells comprising vectors containing isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof, or bispecific antibodies or antigen-binding fragments thereof of the invention are also provided.

[0019] In certain embodiments, a pharmaceutical composition is provided comprising an isolated monoclonal antibody or antigen-binding fragment thereof or an isolated bispecific antibody or antigen-binding fragment thereof of the invention and a pharma- ceutically acceptable carrier.

[0020] Also provided is a method for specifically targeting CC motif chemokine receptor 8 (CCR8) on the surface of cancer cells in a subject in need of specific targeting of CCR8 on the surface of cancer cells, comprising administering a pharmaceutical composition of the invention to the subject.

[0021] Also provided is a method for treating cancer in a subject in need of cancer treatment, comprising administering the pharmaceutical composition of the present invention to the subject.Cancer can be, for example, a solid tumor, preferably a solid tumor with infiltrating T cells, more preferably a solid tumor with infiltrating Treg cells, more preferably a solid tumor with highly suppressive Treg cells expressing CCR8, most preferably a solid tumor with infiltrating highly suppressive Treg cells overexpressing CCR8 with natural killer (NK) cell infiltration.Examples of cancer can be selected from, for example, lung cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, bladder cancer, liver cancer, kidney cancer, and melanoma, but are not limited thereto.In an embodiment, the subject can, for example, contain CCR8-expressing Treg cells.

[0022] Also provided is a method of producing a monoclonal antibody or antigen-binding fragment thereof or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell containing a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof under conditions to produce the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof from the cell or culture.

[0023] Also provided is a method for producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising combining the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable carrier to obtain a pharmaceutical composition.

[0024] Also provided is a method for determining the level of CCR8 in a subject. The method includes (a) obtaining a sample from a subject; (b) contacting the sample with an anti-CCR8 monoclonal antibody or an antigen-binding fragment thereof of the present invention; and (c) determining the level of CCR8 in the subject. In some embodiments, the sample is a tissue sample. The tissue sample can be, for example, a cancer tissue sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample comprises Treg cells.

[0025] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings. [Brief description of the drawings]

[0026] [Figure 1] Figures 1A-1C show that anti-CCR8 monoclonal antibodies specifically bind to CCR8-expressing cells with an EC50 of less than nM. Figure 1A shows the binding of the parental antibody to CCR8.CHO cells. Figure 1B shows the binding of the parental antibody to the parental cells. Figure 1C is a table summarizing the EC50s measured in Figures 1A and 1B.

[0027] [Diagram 2] Figure 2 shows that anti-CCR8 monoclonal antibodies recognize the N-terminal epitope as well as hCCR8 loop 1 involved in protein conformation, but not hCCR4. Top: Diagram of the chimeric construct (black: hCCR8, grey: hCCR4); Bottom: Binding profile associated with the construct.

[0028] [Figure 3A-B]Figures 3A-3C show that anti-CCR8 monoclonal antibodies can bind to human CCR8 protein and cross-react with mouse and / or cynomolgus monkey CCR8 protein. Figure 3A shows anti-CCR8 monoclonal antibodies that bind to human, mouse and cynomolgus monkey CCR8 protein. Figure 3B shows the EC50 of HFB11-21 and HFB11-19. Figure 3C shows the EC50 of HFB11-21 and HFB11-2. [Figure 3C] Figures 3A-3C show that anti-CCR8 monoclonal antibodies can bind to human CCR8 protein and cross-react with mouse and / or cynomolgus monkey CCR8 protein. Figure 3A shows anti-CCR8 monoclonal antibodies that bind to human, mouse and cynomolgus monkey CCR8 protein. Figure 3B shows the EC50 of HFB11-21 and HFB11-19. Figure 3C shows the EC50 of HFB11-21 and HFB11-2.

[0029] [Figure 4] 4A-4B show the different CCL1 blocking profiles for anti-CCR8 monoclonal antibodies (FIG. 4A) and the associated IC50 table (FIG. 4B). Cynomolgus cross-reactive antibodies do not block hCCL1, but rather strong hCCR8 binding antibodies block hCCL1.

[0030] [Figure 5A-F] 5A-5G show that anti-CCR8 monoclonal antibodies inhibit intracellular Ca2+ flux. FIG. 5A shows Ca2+ changes in the presence of buffer. FIG. 5B shows Ca2+ changes in the presence of 1 nM CCL1. FIG. 5C shows Ca2+ changes in the presence of 1 nM CCL1+HFB11-3. FIG. 5D shows Ca2+ changes in the presence of 1 nM CCL1+HFB11-10. FIG. 5E shows Ca2+ changes in the presence of buffer+10 nM CCL1. FIG. 5F shows Ca2+ changes in the presence of 10 nM CCL1+HFB11-3. FIG. 5G shows Ca2+ changes in the presence of 10 nM CCL1+HFB11-10. [Figure 5G]5A-5G show that anti-CCR8 monoclonal antibodies inhibit intracellular Ca2+ flux. FIG. 5A shows Ca2+ changes in the presence of buffer. FIG. 5B shows Ca2+ changes in the presence of 1 nM CCL1. FIG. 5C shows Ca2+ changes in the presence of 1 nM CCL1+HFB11-3. FIG. 5D shows Ca2+ changes in the presence of 1 nM CCL1+HFB11-10. FIG. 5E shows Ca2+ changes in the presence of buffer+10 nM CCL1. FIG. 5F shows Ca2+ changes in the presence of 10 nM CCL1+HFB11-3. FIG. 5G shows Ca2+ changes in the presence of 10 nM CCL1+HFB11-10.

[0031] [Figure 6] Figures 6A-6B show that anti-CCR8 antibodies strongly engage CD16 in ADCC reporter bioassays and mediate ADCC through CD16 modified cells. Figure 6A shows the RLU signal after 5 minutes of incubation with luciferase substrate. Figure 6B shows a table of EC50 and Emax values ​​for all antibodies.

[0032] [Figure 7] Figures 7A-7B show that humanized anti-CCR8 antibodies in an ADCC enhancement format specifically bind to CCR8-expressing cells. Figure 7A shows the MFI signal. Figure 7B shows a table of EC50 values ​​for all antibodies.

[0033] [Figure 8A] Figures 8A-8B show that humanized anti-CCR8 antibodies block CCL1 binding to CCR8-expressing cells. Figure 8A shows the percentage of CCL1 blocking. Figure 8B shows a table of IC50 values ​​for all antibodies. [Figure 8B] Figures 8A-8B show that humanized anti-CCR8 antibodies block CCL1 binding to CCR8-expressing cells. Figure 8A shows the percentage of CCL1 blocking. Figure 8B shows a table of IC50 values ​​for all antibodies.

[0034] [Figure 9] FIG. 9 shows that the humanized HFB11-10Hz37 anti-CCR8 antibody engages both CD16 F and V variants in an ADCC reporter bioassay.

[0035] [Figure 10A] Figures 10A-10B show that humanized anti-CCR8 antibodies mediate ADCC on CCR8-expressing cells. Figure 10A shows the percentage of specific lysis. Figure 10B shows a table of EC50 values ​​for all antibodies. [Figure 10B] Figures 10A-10B show that humanized anti-CCR8 antibodies mediate ADCC on CCR8-expressing cells. Figure 10A shows the percentage of specific lysis. Figure 10B shows a table of EC50 values ​​for all antibodies.

[0036] [Figure 11A-B] Figures 11A-11G show in vitro characterization of anti-CCR8 mAb HFB101110. Figure 11A shows binding of HFB101110 to high copy (CHOK1-hCCR8, approximately 30,000 receptors / cell, left) or low copy (M300.19-hCCR8, approximately 2,000 receptors / cell, right) cells as measured by flow cytometry. Figure 11B shows ADCC activity of HFB101110 on M300.19-hCCR8 cells. Figure 11C shows binding of HFB101110 to the related chemokine receptor CCR4 assessed by flow cytometry. Figure 11D shows a domain swap experiment to identify the region of CCR8 recognized by HFB101110. Figure 11E shows blocking of hCCL1 binding to hCCR8 by HFB101110 as measured by flow cytometry. Figure 11F shows HFB101110-mediated blockade of chemotaxis of CCR8+ cells induced by recombinant hCCL1 as measured by transwell migration assay. Figure 11G shows HFB101110-mediated blockade of calcium flux induced by addition of hCCL1 to CCR8+ cells. [Figure 11C-E]Figures 11A-11G show in vitro characterization of anti-CCR8 mAb HFB101110. Figure 11A shows binding of HFB101110 to high copy (CHOK1-hCCR8, approximately 30,000 receptors / cell, left) or low copy (M300.19-hCCR8, approximately 2,000 receptors / cell, right) cells as measured by flow cytometry. Figure 11B shows ADCC activity of HFB101110 on M300.19-hCCR8 cells. Figure 11C shows binding of HFB101110 to the related chemokine receptor CCR4 assessed by flow cytometry. Figure 11D shows a domain swap experiment to identify the region of CCR8 recognized by HFB101110. Figure 11E shows blocking of hCCL1 binding to hCCR8 by HFB101110 as measured by flow cytometry. Figure 11F shows HFB101110-mediated blockade of chemotaxis of CCR8+ cells induced by recombinant hCCL1 as measured by transwell migration assay. Figure 11G shows HFB101110-mediated blockade of calcium flux induced by addition of hCCL1 to CCR8+ cells. [Fig. 11F-G]Figures 11A-11G show in vitro characterization of anti-CCR8 mAb HFB101110. Figure 11A shows binding of HFB101110 to high copy (CHOK1-hCCR8, approximately 30,000 receptors / cell, left) or low copy (M300.19-hCCR8, approximately 2,000 receptors / cell, right) cells as measured by flow cytometry. Figure 11B shows ADCC activity of HFB101110 on M300.19-hCCR8 cells. Figure 11C shows binding of HFB101110 to the related chemokine receptor CCR4 assessed by flow cytometry. Figure 11D shows a domain swap experiment to identify the region of CCR8 recognized by HFB101110. Figure 11E shows blocking of hCCL1 binding to hCCR8 by HFB101110 as measured by flow cytometry. Figure 11F shows HFB101110-mediated blockade of chemotaxis of CCR8+ cells induced by recombinant hCCL1 as measured by transwell migration assay. Figure 11G shows HFB101110-mediated blockade of calcium flux induced by addition of hCCL1 to CCR8+ cells.

[0037] [Figure 12] FIG. 12 shows that humanized HFB11-10Hz37 anti-CCR8 antibody mediates ADCP of CCR8+ expressing cells.

[0038] [Figure 13] Figures 13A-13C show that humanized anti-CCR8 antibodies mediate anti-tumor activity against MC38 cells in hCCR8-KI mice. Figure 13A shows tumor volume after treatment with MG053 isotype. Figure 13B shows tumor volume after treatment with Hz variant. Figure 13C shows comparison of tumor volume after treatment with MG053 isotype or Hz variant.

[0039] [Figure 14]Figures 14A-14F show that humanized anti-CCR8 antibody therapy reprograms the tumor microenvironment in vivo. Figure 14A shows the percentage of Tregs among CD4+ after treatment with isotype or HFB11-10Hz37. Figure 14B shows the percentage of CD4+ T effectors among CD4+ after treatment with isotype or HFB11-10Hz37. Figure 14C shows the percentage of CD8+ among CD3+ after treatment with isotype or HFB11-10Hz37. Figure 14D shows the CD8+ / Treg ratio after treatment with isotype or HFB11-10Hz37. Figure 14E shows the percentage of natural killer among CD45+ after treatment with isotype or HFB11-10Hz37. Figure 14F shows the percentage of CCR8+ among Tregs after treatment with isotype or HFB11-10Hz37.

[0040] [Figure 15] Figures 15A-15B show CCR8 expression in T cell populations from human primary tumors from renal cell carcinoma (RCC) and lung cancer patients. Each point represents data from an individual patient (Figure 15A). Figure 15B shows histograms overlaid with CCR8 expression in CD8+ (CD8+CD3+), Teff (FoxP 3-CD4+CD3+), and Treg (FoxP 3+CD4+CD3+) from TILs of RCC patients.

[0041] [Figure 16] FIG. 16 shows CCR8 expression in T cell populations from the circulation in healthy and malignant PBMCs.

[0042] [Figure 17A-C]Figures 17A-17E show ex vivo ADCC activity mediated by humanized HFB11-10 antibody on primary human TILs from RCC patients (n=13). Figure 17A shows a schematic of sample collection and ADCC assay. Figure 17B shows depletion of different T cell subsets. Each point represents data from an individual patient. Figure 17C shows dot plots showing Treg populations gated as CD3+CD4+Foxp 3+TIGIT+ cells from isotype groups versus the group treated with 50 nM HFB11-10Hz antibody in TILs from a representative RCC patient. Figure 17D shows dose response of Treg depletion activity of HFB101110. Figure 17E shows depletion of Tregs in ADCC assay as a function of the amount of exogenous NK cells added. [Fig. 17D-E] Figures 17A-17E show ex vivo ADCC activity mediated by humanized HFB11-10 antibody on primary human TILs from RCC patients (n=13). Figure 17A shows a schematic of sample collection and ADCC assay. Figure 17B shows depletion of different T cell subsets. Each point represents data from an individual patient. Figure 17C shows dot plots showing Treg populations gated as CD3+CD4+Foxp 3+TIGIT+ cells from isotype groups versus the group treated with 50 nM HFB11-10Hz antibody in TILs from a representative RCC patient. Figure 17D shows dose response of Treg depletion activity of HFB101110. Figure 17E shows depletion of Tregs in ADCC assay as a function of the amount of exogenous NK cells added.

[0043] [Figure 18A-B] Figures 18A-18C show safety and pharmacokinetic studies. Figure 18A shows a schematic of a single-dose PK study in cynomolgus monkeys. Figure 18B shows the serum PK profile of HFB101110 in cynomolgus monkeys. Figure 18C shows an in vitro cytokine release study from human PBMCs using a soluble antibody format. [Figure 18C]Figures 18A-18C show safety and pharmacokinetic studies. Figure 18A shows a schematic of a single-dose PK study in cynomolgus monkeys. Figure 18B shows the serum PK profile of HFB101110 in cynomolgus monkeys. Figure 18C shows an in vitro cytokine release study from human PBMCs using a soluble antibody format. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Detailed Description of the Invention Various publications, articles, and patents are cited or described throughout the background and specification, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like included in the present specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items form part of the prior art with respect to the invention disclosed or claimed.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Other terms used herein have the meanings defined herein.

[0046] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0047] Unless otherwise specified, any numerical values, such as concentrations or concentration ranges described herein, should be understood to be modified in all cases by the term "about". Thus, numerical values ​​typically include ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within that range, integers and portions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0048] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to every element of the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0049] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variations thereof, are intended to mean the inclusion of a recited integer or group of integers, but not the exclusion of any other integer or group of integers, and are non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, and may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or device. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present), B is false (or absent), A is false (or absent), B is true (or present), and both A and B are true (or present).

[0050] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within the meaning and thus meet the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more options is also understood to be within the meaning and thus meet the requirements of the term "and / or".

[0051] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout this specification and the claims, indicates that any integer or group of integers listed is inclusive, but that no additional integers or groups of integers may be added to the specified method, structure, or composition.

[0052] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, refers to the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP § 2111.03.

[0053] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0054] The words "right", "left", "lower" and "upper" designate directions in the drawings to which reference is made.

[0055] It should also be understood that terms such as "about," "approximately," "generally," and "substantially," as used herein when referring to dimensions or characteristics of preferred inventive components, indicate that the described dimensions / characteristics are not precise boundaries or parameters, but do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0056] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-CCR8 antibodies and the polynucleotides encoding them, CCR8 polypeptides and the CCR8 polynucleotides encoding them) refers to two or more sequences or subsequences that are the same, or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection.

[0057] For sequence comparison, typically, one sequence acts as the reference sequence with which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence based on designated program parameters.

[0058] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 1981;2:482, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 1970;48:443, by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 1988;85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or by visual inspection (generally as described in Current Protocols in Molecular Biology, FMAusubel et al. (eds.), Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John This can be done by the methods described in Wiley & Sons, Inc., 1995 (see Supplement (Ausubel)).

[0059] Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 1990;215:403-410 and Altschul et al., Nucleic Acids Res. 1997;25:3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0060] Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value, the cumulative score becomes 0 or less due to the accumulation of one or more negative scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 1989;89:10915).

[0061] In addition to calculating percent sequence identity, BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 1993;90:5873-5787).One measure of similarity provided by BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance.For example, a nucleic acid is considered to be similar to a reference sequence when the minimum sum probability in the comparison between test nucleic acid and reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0062] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions.Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0063] As used herein, the term "isolated" means that a biological component (e.g., a nucleic acid, peptide, or protein) is substantially separated from, produced separately from, or purified from other biological components of the organism in which it naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins may be part of a composition, and remain isolated when the composition is not part of the original environment of the nucleic acid, peptide, or protein. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0064] As used herein, the term "polynucleotide" is interchangeably referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions that include RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contains one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA, and thus "polynucleotide" includes chemically, enzymatically or metabolically modified forms of polynucleotides that are typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also includes relatively short nucleic acid strands that are often referred to as oligonucleotides.

[0065] As used herein, the term "vector" is a replicon into which another nucleic acid segment may be operatively inserted so as to bring about the replication or expression of the segment.

[0066] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the invention. A "host cell" can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a "host cell" is a cell that has been transfected with a nucleic acid molecule of the invention. In another embodiment, a "host cell" is the progeny or potential progeny of such a transfected cell. The progeny of a cell may or may not be identical to the parent cell, for example, due to mutations or environmental influences that may occur in subsequent generations, or due to integration of the nucleic acid molecule into the host cell genome.

[0067] The term "expression" as used herein refers to the biosynthesis of a gene product. This term includes the transcription of a gene into RNA. This term also includes the translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed antibody may be in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or may be anchored to a cell membrane.

[0068] As used herein, the term "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, for example, by formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art, are also included in the definition.

[0069] The peptide sequences described herein are written according to the usual convention with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomeric forms of amino acids are known, it is the L-form of the amino acid that is represented unless otherwise specified.

[0070] antibody

[0071] The present invention generally relates to isolated anti-chemokine (CC motif) receptor 8 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, recombinant cells expressing the antibodies, and compositions comprising the antibodies. Methods of making the antibodies and methods of using the antibodies to treat diseases such as cancer are also disclosed. The antibodies of the present invention have one or more desirable functional properties, including but not limited to high affinity binding to CCR8, high specificity to CCR8, the ability to stimulate antibody-dependent cellular phagocytosis (ADCP) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing CCR8, and the ability to inhibit tumor growth in subjects and animal models when administered alone or in combination with other anti-cancer therapies.

[0072] In a general aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to chemokine (CC motif) receptor 8 (CCR8).

[0073] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules, including human, humanized, composite and chimeric antibodies, as well as antibody fragments that are monoclonal or polyclonal. In general, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. Antibody structure is well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM) depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subdivided as isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Thus, the antibodies of the present invention can be of any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3 or IgG4. The antibody light chains of vertebrate species can be assigned to one of two distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Thus, the antibodies of the present invention can contain a kappa light chain constant domain or a lambda light chain constant domain. According to certain embodiments, the antibody of the present invention comprises a heavy chain constant region and / or a light chain constant region from a rat or human antibody. In addition to the heavy chain constant domain and the light chain constant domain, the antibody comprises an antigen-binding region composed of a light chain variable region and a heavy chain variable region, each of which comprises three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are also referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are also referred to as HCDR1, HCDR2, and HCDR3.

[0074] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CCR8 is substantially free of antibodies that do not bind to CCR8). Moreover, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0075] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts. The monoclonal antibody of the present invention can be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or by recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic non-human animals, such as transgenic mice or rats, whose genomes comprise human heavy chain transgenes and light chain transgenes.

[0076] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), single domain antibody (sdab), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment can bind to the same antigen that the parent antibody or parent antibody fragment binds. According to certain embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain. According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab').

[0077] As used herein, the term "single chain antibody" refers to a conventional single chain antibody in the art that contains a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art that contains a heavy chain variable region and a heavy chain constant region, or contains only a heavy chain variable region.

[0078] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0079] As used herein, the terms "humanized antibody" and / or "humanized antigen-binding domain" refer to a non-human antibody that has been modified to increase its sequence homology with that of a human antibody such that the antigen-binding properties of the antibody are retained but its antigenicity in the human body is reduced.

[0080] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0081] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, where a plurality of first immunoglobulin variable domain sequences has binding specificity for a first epitope and a plurality of second immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody comprises a third, fourth or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0082] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises an scFv or fragment thereof having binding specificity for a first epitope and an scFv or fragment thereof having binding specificity for a second epitope. In one embodiment, the first epitope is located on CCR8 and the second epitope is located on PD-1, PD-L1, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD3, and / or other tumor-associated immunosuppressants or surface antigens.

[0083] As used herein, an antibody that "specifically binds to CCR8" is one that is greater than or equal to 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less, 1×10 -9 M or less, 5 x 10 -10 M or less, or 1 x 10-10 It refers to an antibody and / or antigen binding domain that binds to CCR8, preferably human CCR8, with a KD of M or less. In one embodiment, the antibody and / or antigen binding domain binds to cynomolgus CCR8. The term "KD" refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods in the art in light of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, for example, by using a biosensor system such as a Biacore® system, or by using biolayer interferometry technology such as an Octet RED 96 system.

[0084] The smaller the KD value of an antibody, the higher the affinity with which the antibody binds to a target antigen.

[0085] As used herein, "IC 50 The term IC refers to the half-maximal inhibitory concentration of a monoclonal or bispecific antibody or antigen-binding fragment thereof of the invention. 50 is a measure of the potency of the monoclonal or bispecific antibody or antigen-binding fragment thereof of the invention to inhibit binding of CCL1 to CCR8 in cells or to inhibit the function of CCR8. In one embodiment, the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof is about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 Has a KD of less than M.

[0086] As used herein, "EC 50 The term "half maximal effective concentration" refers to the monoclonal or bispecific antibody or antigen-binding fragment thereof of the invention.50 refers to the concentration of a monoclonal or bispecific antibody or antigen-binding fragment thereof to induce a biological response (i.e., cell death) halfway between baseline and maximum over a specified exposure time. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof has an EC of less than about 1 μM, about 1000 nM to about 100 nM, about 100 nM to about 10 nM, about 10 nM to about 1 nM, about 1000 pM to about 500 pM, about 500 pM to about 200 pM, less than about 200 pM, about 200 pM to about 150 pM, about 200 pM to about 100 pM, about 100 pM to about 10 pM, or about 10 pM to about 1 pM. 50 has.

[0087] According to a particular aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, the monoclonal antibody or antigen-binding fragment or antigen-binding domain comprising: (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (2) SEQ ID NOs: 13, 2, 14, 4, 28, and 6, respectively; (3) SEQ ID NOs: 13, 2, 15, 4, 5, and 6, respectively; (4) SEQ ID NOs: 16, 17, 18, 29, 30, and 6, respectively; (5) SEQ ID NOs: 19, 20, 21, 4, 5, and 6, respectively; (6) SEQ ID NOs: 22, 23, 24, 31, 5, and 32, respectively; or (7) SEQ ID NOs: 25, 26, 27, 33, 34, and 35, respectively; and a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having a polypeptide sequence of The antibody or antigen-binding fragment thereof or antigen-binding domain thereof relates to an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to chemokine (CC motif) receptor 8 (CCR8), preferably human CCR8.

[0088] According to another particular embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, 36, 38, 40, 42, 44, or 46, or a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, 37, 39, 41, 43, 45, or 47. According to a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8, respectively.

[0089] According to certain embodiments, the isolated anti-CCR8 monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7 and a light chain variable region having the polypeptide sequence of SEQ ID NO:8; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:36 and a light chain variable region having the polypeptide sequence of SEQ ID NO:37; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:38 and a light chain variable region having the polypeptide sequence of SEQ ID NO:39; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 40, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 41; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:42 and a light chain variable region having the polypeptide sequence of SEQ ID NO:43; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 44 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 45; or (7) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 46 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 47. Includes.

[0090] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8.

[0091] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 13, 2, 14, 4, 28, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 36, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 37.

[0092] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 13, 2, 15, 4, 5, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 38 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 39.

[0093] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 16, 17, 18, 29, 30, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 40, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 41.

[0094] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 19, 20, 21, 4, 5, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 42, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 43.

[0095] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 22, 23, 24, 31, 5, and 32, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 44 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 45.

[0096] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 25, 26, 27, 33, 34, and 35, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 46, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 47.

[0097] According to another particular aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the present invention, wherein said monoclonal antibody or bispecific antibody or antigen-binding fragment thereof is chimeric.

[0098] According to another particular aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the present invention, wherein said monoclonal antibody or bispecific antibody or antigen-binding fragment thereof is human or humanized.

[0099] According to another particular aspect, the present invention comprises (1) SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively; (2) SEQ ID NOs: 1, 2, 49, 4, 5, and 6, respectively; (3) SEQ ID NOs: 1, 2, 49, 50, 51, and 6, respectively; (4) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; or (5) SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively. The present invention relates to a humanized monoclonal antibody or antigen-binding fragment thereof, comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having a polypeptide sequence of:

[0100] According to another particular embodiment, the present invention relates to a humanized monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having a polypeptide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9, 52, 53, 54, 55, 56, 57, 58, 59, or 60, or a light chain variable region having a polypeptide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10, 61, 62, or 63.

[0101] According to another particular embodiment, the humanized anti-CCR8 monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:52 and a light chain variable region having the polypeptide sequence of SEQ ID NO:63; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:52 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:53 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:54 and a light chain variable region having the polypeptide sequence of SEQ ID NO:63; or (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:59 and a light chain variable region having the polypeptide sequence of SEQ ID NO:62; Includes.

[0102] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10.

[0103] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 1, 2, 49, 4, 5, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 52, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 63. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 52 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 63.

[0104] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 1, 2, 49, 50, 51, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 52, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 52 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10.

[0105] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 1, 2, 49, 50, 51, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 53, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 53, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10.

[0106] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 54, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 63. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 54 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 63.

[0107] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively. According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 59, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 62. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 59, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 62.

[0108] In another general aspect, the present invention relates to an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof and / or bispecific antibody or antigen-binding fragment thereof of the present invention. It will be understood by those skilled in the art that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, it will be understood by those skilled in the art that the nucleic acid sequence encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0109] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof of the present invention. Any vector known to the skilled artisan in view of the present disclosure can be used, such as a plasmid, cosmid, phage vector or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any element for establishing the conventional function of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive promoter, an inducible promoter, or a repressible promoter. Several expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein for the production of an antibody or antigen-binding fragment thereof in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate the recombinant expression vector according to embodiments of the present invention.

[0110] In another general aspect, the present invention relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof of the present invention. Any host cell known to the skilled artisan in view of the present disclosure can be used for recombinant expression of an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is an E. coli TG1 or BL21 cell (e.g., for expression of scFv or Fab antibodies), a CHO-DG44 or CHO-K1 cell, or a HEK293 cell (e.g., for expression of full-length IgG antibodies). According to certain embodiments, the recombinant expression vector is transformed into the host cell by conventional methods such as chemical transfection, heat shock, or electroporation, and stably integrated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0111] In another general aspect, the invention relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof under conditions to produce the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof of the invention, and recovering the monoclonal antibody and / or bispecific antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed monoclonal antibody and / or bispecific antibody or antigen-binding fragment thereof can be harvested from the cells and purified according to conventional techniques as known in the art and described herein.

[0112] Pharmaceutical Compositions

[0113] In another general aspect, the invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, an isolated polynucleotide and / or an isolated polypeptide of the invention and a pharma- ceutical acceptable carrier.

[0114] The term "pharmaceutical composition" as used herein means a product comprising the isolated polynucleotide of the present invention, the isolated polypeptide of the present invention, the anti-CCR8 monoclonal antibody or antigen-binding fragment thereof, and / or the bispecific antibody of the present invention together with a pharmaceutically acceptable carrier. The polynucleotide, polypeptide, anti-CCR8 monoclonal antibody or antigen-binding fragment thereof, and / or the bispecific antibody of the present invention, and compositions comprising them, are also useful for the manufacture of medicaments for the therapeutic applications referred to herein.

[0115] As used herein, the term "carrier" refers to any pharmaceutical excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, pharmaceutical excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy of the composition according to the present invention or the biological activity of the composition according to the present invention. According to certain embodiments, any pharmaceutical acceptable carrier suitable for use in antibody pharmaceutical compositions in light of the present disclosure can be used in the present invention.

[0116] Formulation of pharmacologic active ingredients with pharmacologic acceptable carriers is known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st Edition (2005), and later editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity adjusting agents, preservatives, stabilizers, and chelating agents. One or more pharmacologic acceptable carriers can be used to formulate the pharmaceutical composition of the present invention.

[0117] In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation.Preferred examples of liquid formulations are aqueous formulations, i.e. formulations that contain water.Liquid formulations can include solutions, suspensions, emulsions, microemulsions, gels, etc.Aqueous formulations typically contain at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w water.

[0118] In one embodiment, the pharmaceutical composition can be formulated as an injectable that can be injected, for example, via an injection device (e.g., a syringe or an infusion pump). The injection can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0119] In another embodiment, the pharmaceutical composition is a solid formulation, for example a freeze-dried or spray-dried composition, which can be used as is or the physician or patient adds solvent and / or diluent to them before use.Solid dosage forms can include tablets, for example compressed tablets, and / or coated tablets, and capsules (for example hard or soft gelatin capsules).The pharmaceutical composition can be in the form of, for example, sachets, dragees, powders, granules, lozenges, or powders for reconstitution.

[0120] The dosage form may be of immediate release type, in which case it may include a water soluble or dispersible carrier, or it may be of delayed, sustained or modified release type, in which case it may include a water insoluble polymer that modulates the dissolution rate of the dosage form in the gastrointestinal tract or under the skin.

[0121] In other embodiments, the pharmaceutical compositions may be delivered intranasally, bucally, or sublingually.

[0122] The pH of the aqueous formulation may be from pH 3 to pH 10. In one embodiment of the present invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the present invention, the pH of the formulation is from about 3.0 to about 7.0.

[0123] In another embodiment of the invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffers may be present individually or aggregated at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these particular buffers constitute alternative embodiments of the invention.

[0124] In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservatives may be present individually or aggregated in a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific preservatives constitute alternative embodiments of the invention.

[0125] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonicity agent. Non-limiting examples of isotonicity agents include salts (e.g., sodium chloride), amino acids (e.g., glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), alditols (e.g., glycerol, 1,2-propanediol propylene glycol), 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (e.g., PEG400), and mixtures thereof. Other examples of isotonicity agents include sugars. Non-limiting examples of sugars can be monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans (e.g., including fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose). Another example of an isotonicity agent is a sugar alcohol, the term "sugar alcohol" being defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. The isotonicity agents may be present individually or aggregated in a concentration of about 0.01 mg / ml to about 50 mg / ml, e.g., about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific isotonicity agents constitute alternative embodiments of the invention.

[0126] In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agents may be present individually or aggregated at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific chelating agents constitute alternative embodiments of the invention.

[0127] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0128] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, said stabilizer being carboxy- / hydroxycellulose and its derivatives (e.g., HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (e.g., PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (e.g., sodium chloride), sulfur-containing substances such as monothioglycerol, or thioglycolic acid. The stabilizers may be present individually or aggregated in a concentration of about 0.01 mg / ml to about 50 mg / ml, for example about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these particular stabilizers constitute alternative embodiments of the invention.

[0129] In a further embodiment of the invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecule or ion composed of a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactant may be selected, for example, from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactants may be present individually or aggregated, at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific surfactants constitute alternative embodiments of the invention.

[0130] In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitors may be present individually or aggregated at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific protease inhibitors constitute alternative embodiments of the invention.

[0131] In another general aspect, the invention relates to a method for producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising combining the monoclonal antibody or antigen-binding fragment thereof and / or the bispecific antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable carrier to obtain a pharmaceutical composition.

[0132] How to use

[0133] In another general aspect, the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an anti-CCR8 monoclonal and / or bispecific antibody or antigen-binding fragment thereof of the present invention. The cancer can be selected from, for example, but not limited to, lung cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, renal cancer, and melanoma.

[0134] In some embodiments, the present invention relates to a method for inducing tumor-infiltrating regulatory T cell (TITC) depletion in a subject in need of such induction, comprising administering to the subject a pharmaceutical composition comprising the anti-CCR8 monoclonal and / or bispecific antibody or antigen-binding fragment thereof of the present invention. In various embodiments, other T cells, including CD4+ T cells and CD8+ T cells, are not affected by the induced cell depletion. In other embodiments, the method further induces reprogramming of the tumor microenvironment. In various embodiments, inducing TITC depletion comprises inducing natural killer (NK)-mediated killing of CCR-8-expressing cancer cells. In other embodiments, inducing NK-mediated killing comprises inhibiting TITC-induced immunosuppression.

[0135] In another general aspect, the present invention relates to a method of targeting CCR8 on the surface of a cancer cell in a subject to achieve cell death, comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof that specifically binds to CCR8, or a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof of the present invention. Binding of an anti-CCR8 monoclonal or bispecific antibody or antigen-binding fragment to CCR8 can mediate antibody-dependent cellular phagocytosis (ADCP) and / or antibody-dependent cellular cytotoxicity (ADCC) or other effects that result in the death of the target cancer cell. The monoclonal or bispecific antibody or antigen-binding fragment thereof can, for example, serve to recruit a conjugated drug and / or form a bispecific antibody with another monoclonal antibody to mediate the death of the target cancer cell.

[0136] The functional activity of the antibody and its antigen-binding fragments that bind to CCR8 can be characterized by methods known in the art and described herein.Methods for characterizing the antibody and its antigen-binding fragments that bind to CCR8 include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and OctetRed analysis, and detection of the binding of the antibody and its antigen-binding fragments to CCR8 on cells (either cells transfected with CCR8 or cells that naturally express CCR8) by FACS.According to certain embodiments, the methods for characterizing the antibody and its antigen-binding fragments that bind to CCR8 include those described below.

[0137] In another general aspect, the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof that specifically binds to CCR8 or a pharmaceutical composition of the present invention. The cancer may be, for example, a solid tumor, preferably a solid tumor with infiltrating T cells, more preferably a solid tumor with infiltrating Treg cells, more preferably a solid tumor with highly suppressive Treg cells that express CCR8, most preferably a solid tumor with infiltrating highly suppressive Treg cells that overexpress CCR8 with natural killer (NK) cell infiltration. The cancer may be, for example, selected from, but not limited to, lung cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, bladder cancer, liver cancer, kidney cancer, and melanoma. According to certain embodiments, the subject may, for example, comprise CCR8-expressing Treg.

[0138] As used herein with respect to an anti-CCR8 antibody or its antigen-binding fragment, a therapeutically effective amount refers to the amount of an anti-CCR8 antibody or its antigen-binding fragment that modulates the immune response in a subject in need thereof.Also, as used herein with respect to an anti-CCR8 antibody or its antigen-binding fragment, a therapeutically effective amount refers to the amount of an anti-CCR8 antibody or its antigen-binding fragment that results in the treatment of a disease, disorder, or condition, preventing or delaying the progression of the disease, disorder, or condition, or reducing or completely alleviating the symptoms associated with the disease, disorder, or condition.

[0139] According to certain embodiments, the disease, disorder or condition to be treated is cancer.Cancer can be, for example, solid tumor, preferably solid tumor with infiltrating T cells, more preferably solid tumor with infiltrating Treg cells, more preferably solid tumor with highly suppressive Treg cells expressing CCR8, most preferably solid tumor with highly suppressive Treg cells overexpressing CCR8 with natural killer (NK) cell infiltration.Cancer can be, for example, selected from, but not limited to, lung cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, bladder cancer, liver cancer, kidney cancer, and melanoma.

[0140] According to certain embodiments, a therapeutically effective amount refers to an amount of therapy sufficient to achieve one, two, three, four or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder or condition being treated, or symptoms associated therewith; (ii) reducing the duration of the disease, disorder or condition being treated, or symptoms associated therewith; (iii) preventing the progression of the disease, disorder or condition being treated, or symptoms associated therewith; (iv) causing regression of the disease, disorder or condition being treated, or symptoms associated therewith; (v) preventing the onset or development of the disease, disorder or condition being treated, or symptoms associated therewith. (vi) preventing the recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reducing hospitalization of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (viii) shortening the length of hospitalization of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (ix) increasing the survival of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibiting or reducing the disease, disorder or condition being treated, or symptoms associated therewith in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another therapy.

[0141] The therapeutically effective amount or dosage may vary depending on various factors, such as the disease, disorder or condition being treated, the means of administration, the target site, the physiological condition of the subject (including, for example, age, weight, health), whether the subject is a human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0142] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject.For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

[0143] As used herein, the terms "treat", "treating", and "treatment" are all intended to refer to an improvement or reversal of at least one measurable physical parameter associated with cancer, which may, but is not necessarily, discernible in the subject. The terms "treat", "treating", and "treatment" can also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to alleviating, preventing the onset or development, or shortening the duration of one or more symptoms associated with a disease, disorder, or condition, such as a tumor or, more preferably, a cancer. In certain embodiments, "treat", "treating", and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to increasing the survival of a subject having a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

[0144] According to certain embodiments, compositions are provided for use in the treatment of cancer. For cancer treatment, the compositions provided can be used in combination with other treatments, including but not limited to chemotherapy, anti-CD20 mAb, anti-EGFR mAb, anti-HER-2 mAb, anti-CD19 mAb, anti-CD33 mAb, anti-CD47 mAb, anti-CD73 mAb, anti-PD-1 mAb, anti-PD-L1 mAb, anti-CTLA mAb, anti-TNFR2 mAb, anti-OX40 mAb, other immuno-oncology drugs, anti-angiogenic agents, radiation therapy, antibody-drug conjugates (ADC), targeted therapy, other anti-cancer drugs, and / or treatments targeting immune-modulating targets, including but not limited to PD1 and PD-L1. Antibodies to CCR8 can be used to construct bispecific antibodies with partner mAbs to PD-1, PD-L1, CTLA-4, CTLA, TNFR2, OX40, EGFR, HER-2, CD19, CD20, CD33, CD73, CD47, and / or CD3. Two antibodies that recognize two different epitopes on CCR8 can also be used to construct bispecific antibodies to treat cancers / tumors expressing CCR8.

[0145] As used herein, the term "in combination" in the context of administration of two or more therapies to a subject refers to the use of two or more therapies. The use of the term "in combination" does not restrict the order in which the therapies are administered to a subject. For example, a first treatment (e.g., a composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second treatment to a subject.

[0146] In another general aspect, the invention relates to a method for determining the level of CCR8 in a subject, the method comprising: (a) obtaining a sample from a subject; (b) contacting the sample with a monoclonal antibody or antigen-binding fragment thereof of the invention; and (c) determining the level of CCR8 in the subject.

[0147] As used herein, "sample" refers to a biological sample isolated from a subject, and may include, but is not limited to, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy (e.g., cancer tissue), lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluid. "Blood sample" refers to whole blood or any fraction thereof, including blood cells, serum, and plasma. The sample may, for example, include Treg cells.

[0148] In an embodiment, the level of CCR8 in a subject can be determined using an assay selected from, but not limited to, Western blot assay, immunohistochemistry (IHC) and ELISA. Relative protein level can be determined by using Western blot analysis and IHC, and absolute protein level can be determined by using ELISA. When determining the relative level of CCR8, the level of CCR8 can be determined between at least two samples, for example, between samples from the same subject at different time points, between samples from different tissues of the same subject, and / or between samples from different subjects. Alternatively, when determining the absolute level of CCR8 by ELISA or the like, the absolute level of CCR8 in a sample can be determined by making an ELISA standard before testing the sample. Those skilled in the art will understand which analytical technique to use to determine the level of CCR8 in a sample from a subject using the antibody of the present invention or its antigen-binding fragment.

[0149] Using the method of determining the level of CCR8 in a sample from a subject, abnormal (elevated, decreased or insufficient) CCR8 level in disease can be diagnosed and appropriate treatment decision can be made.Such disease can be cancer.Furthermore, by monitoring the level of CCR8 in a subject, the risk of developing the above-mentioned disease can be determined based on the knowledge of the level of CCR8 in a particular disease and / or during the progression of a particular disease. Embodiment

[0150] The present invention also provides the following non-limiting embodiments.

[0151] Embodiment 1 is an isolated monoclonal antibody or antigen-binding fragment thereof, comprising: (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (2) SEQ ID NOs: 13, 2, 14, 4, 28, and 6, respectively; (3) SEQ ID NOs: 13, 2, 15, 4, 5, and 6, respectively; (4) SEQ ID NOs: 16, 17, 18, 29, 30, and 6, respectively; (5) SEQ ID NOs: 19, 20, 21, 4, 5, and 6, respectively; (6) SEQ ID NOs: 22, 23, 24, 31, 5, and 32, respectively; or (7) SEQ ID NOs: 25, 26, 27, 33, 34, and 35, respectively; and a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having a polypeptide sequence of The antibody or antigen-binding fragment thereof is an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CCR8, preferably human CCR8.

[0152] Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 1, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 7, 36, 38, 40, 42, 44, or 46, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 8, 37, 39, 41, 43, 45, or 47.

[0153] Embodiment 3 is the isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 1 or 2, (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7 and a light chain variable region having the polypeptide sequence of SEQ ID NO:8; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:36 and a light chain variable region having the polypeptide sequence of SEQ ID NO:37; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:38 and a light chain variable region having the polypeptide sequence of SEQ ID NO:39; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 40, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 41; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:42 and a light chain variable region having the polypeptide sequence of SEQ ID NO:43; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 44 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 45; or (7) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 46 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 47. The antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof comprising:

[0154] Embodiment 4 is the isolated monoclonal antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 3, wherein the antibody or antigen-binding fragment thereof is chimeric and / or human or humanized.

[0155] Embodiment 5 is the isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 4, comprising: The isolated monoclonal antibody or antigen-binding fragment thereof comprises: (1) SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively; (2) SEQ ID NOs: 1, 2, 49, 4, 5, and 6, respectively; (3) SEQ ID NOs: 1, 2, 49, 50, 51, and 6, respectively; (4) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; or (5) SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively. and a polypeptide sequence of:

[0156] Embodiment 6 is the isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 5, The isolated monoclonal antibody or antigen-binding fragment thereof is an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 9, 52, 53, 54, 55, 56, 57, 58, 59, or 60, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 10, 61, 62, or 63.

[0157] Embodiment 7 is the isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 6, comprising: The isolated monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:52 and a light chain variable region having the polypeptide sequence of SEQ ID NO:63; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:52 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:53 and a light chain variable region having the polypeptide sequence of SEQ ID NO:10; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:54 and a light chain variable region having the polypeptide sequence of SEQ ID NO:63; or (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:59 and a light chain variable region having the polypeptide sequence of SEQ ID NO:62; The present invention is an isolated monoclonal antibody or antigen-binding fragment thereof comprising:

[0158] Embodiment 8 is the isolated monoclonal antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 7, wherein the isolated antibody or antigen-binding fragment thereof is capable of forming a bispecific antibody with another mAb or antigen-binding fragment thereof that has the following functions: antibody-dependent cellular cytotoxicity (ADCC); effector-mediated tumor cell lysis through antibody-dependent cellular phagocytosis (ADCP); and / or induces mobilization of a conjugated drug; and / or has a cancer-killing effect.

[0159] Embodiment 9 is an isolated monoclonal antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 8, wherein the monoclonal antibody or antigen-binding fragment thereof specifically binds to cynomolgus CCR8.

[0160] Embodiment 10 is an isolated bispecific antibody or antigen-binding fragment thereof comprising the monoclonal antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 9.

[0161] Embodiment 11 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-9.

[0162] Embodiment 12 is an isolated nucleic acid encoding the bispecific antibody or antigen-binding fragment thereof of embodiment 10.

[0163] Embodiment 13 is a vector comprising the isolated nucleic acid of embodiment 11 or 12.

[0164] Embodiment 14 is a host cell comprising the vector of embodiment 13.

[0165] Embodiment 15 is a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment of any one of Embodiments 1 to 9, or the bispecific antibody or antigen-binding fragment thereof of Embodiment 10, and a pharma- ceutical acceptable carrier.

[0166] Embodiment 16 is a method of targeting CCR8 on the surface of cancer cells and / or treating cancer, comprising administering the pharmaceutical composition of embodiment 15 to a subject.

[0167] Embodiment 17 is the method of embodiment 16, wherein the cancer is, for example, a solid tumor, preferably a solid tumor with infiltrating T cells, more preferably a solid tumor with infiltrating Treg cells, more preferably a solid tumor with highly suppressive Treg cells that express CCR8, most preferably a solid tumor with infiltrating highly suppressive Treg cells that overexpress CCR8 accompanied by natural killer (NK) cell infiltration.

[0168] Embodiment 18 is the method of embodiment 16 or 17, wherein the cancer is selected from the group consisting of lung cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, renal cancer, and melanoma.

[0169] Embodiment 19 is the method of any one of embodiments 16 to 18, wherein the subject comprises CCR8-expressing Treg cells.

[0170] Embodiment 20 is a method for producing the monoclonal antibody or antigen-binding fragment of any one of Embodiments 1 to 9, or the bispecific antibody or antigen-binding fragment thereof of Embodiment 10, comprising culturing a cell containing a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, under conditions for producing the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof from the cell or culture.

[0171] Embodiment 21 is a method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 9, or the bispecific antibody or antigen-binding fragment thereof of Embodiment 10, comprising combining the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable carrier to obtain a pharmaceutical composition.

[0172] Embodiment 22 is a method for determining a level of CCR8 in a subject, the method comprising: a. obtaining a sample from a subject; b. contacting the sample with the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-9; c. determining the level of CCR8 in the subject; The method includes:

[0173] Embodiment 23 is the method of embodiment 22, wherein the sample is a tissue sample or a blood sample, optionally wherein the tissue sample is a cancer tissue sample.

[0174] Embodiment 24 is the method of embodiment 22, wherein the sample comprises Treg cells. EXAMPLES

[0175] Example 1: Antibody Preparation

[0176] Immunity:

[0177] Anti-CCR antibodies were developed by immunization of Balb / c mice with four DNA injections followed by two to four boosts with stable M300.19 cells expressing the hCCR8 receptor. Mice showing high specific titers (>10,000) were processed for single B cell sorting on the CelliGo™ platform.

[0178] B Cell Sorting with the CelliGo Platform:

[0179] Splenocytes from immunized mice were activated in vitro with X-vivo 15 medium (supplemented with 1% penicillin / streptomycin, 2x L-glutamine, 10% low IgG serum, 100ng / mL IL-2 and 2.5μg / mL R848) at 37℃ and 5% CO2 for 5 days. B cells were enriched using a Pan B isolation kit (Miltenyi Biotec; Bergisch Gladbach, North Rhine-Westphalia, Germany), labeled with Celltrace™ Violet reagent (Thermofisher; Waltham, MA), and then sorted using the HiFiBio Celligo platform (HiFiBio; Cambridge, MA). A cell-based screening assay was developed using HiFiBio microfluidic technology to detect and sort specific B cells that bind to target cells in droplets.

[0180] VH / VL were amplified from single B cells in droplets by RT-PCR and PCR, and sequences were analyzed using Absolution HiFibio software (HiFibio).

[0181] Chimeric antibodies were produced in a human IgG1 scaffold and screened by fluorescence-activated cell sorting (FACS) for specific binding on CCR8 high copy cells (stable CCR8 CHO-K1 cell line, Perkin Elmer; Waltham, MA) compared to parental cells.

[0182] Among the 25 specific anti-hCCR8 antibodies, we identified a panel of antibodies with variable biological properties (Figures 1-6).

[0183] The antibody sequences are provided below.

[0184] [Table 1-1] [Table 1-2]

[0185] [Table 2]

[0186] [Table 3]

[0187] [Table 4]

[0188] Example 2: Anti-CCR8 monoclonal antibodies specifically bind to CCR8-expressing cells

[0189] Briefly, purified antibodies (ranging from 30 mM in PBS, 3-fold dilutions) were added to 1 × 10 5The cells were incubated with 1000 s of stable CCR8-expressing CHOK1 cells (Perkin Elmer; Waltham, MA) for 30 min at 4 °C. After washing with MACS buffer (Miltenyi; Bergisch Gladbach, Germany), secondary anti-human AF647 antibody (Jackson ImmunoResearch; West Grove, PA) was incubated at 12 nM in PBS for 30 min. After washing with MACS buffer, the cell pellet was resuspended in 50 μl PBS and samples were analyzed on an iQue cytometer (Sartorious; Göttingen, Germany) using Forecyt software (Intellicyt; Ann Arbor, MI). The average fluorescence in the AF647 channel from single cells is shown in Figure 1.

[0190] No signal was detected in parental CHOK1 cells, therefore the tested antibodies were specific for hCCR8 (Figure 1B). EC values ​​obtained from the titration curves shown in Figure 1A 50 Values ​​ranged from 0.5 to 9 nM (Figure 1C).

[0191] Example 3: Anti-CCR8 monoclonal antibodies recognized the N-terminus as well as loop 1 involved in protein conformation

[0192] To map the epitope of CCR8, constructs were generated expressing chimeric proteins containing portions of human CCR8 (Uniprot / Swiss-Prot:P51685 (SEQ ID NO:11)) fused to the closest chemokine receptor CCR4 (UniProt / Swiss-Prot:P51679 (SEQ ID NO:12)). The resulting proteins contain either the N-terminal domain (amino acids 1-40 of SEQ ID NO:12), extracellular domain 1 ((ECL1) amino acids 100-112 of SEQ ID NO:12), extracellular domain 2 ((ECL2) amino acids 177-205 of SEQ ID NO:12), or extracellular domain 3 ((ECL3) amino acids 269-286 of SEQ ID NO:12) from human CCR4, with the remaining sequence being from human CCR8 (SEQ ID NO:11). Constructs were made in pcDNA3.1 vectors containing a cleavable intracellular GFP tag, and constructs were transiently expressed on the surface of ExpiHEK293 cells. Purified antibodies (50 nM) were added to 1 × 10 cells transiently transfected with either human, mouse, or cynomolgus CCR8-GFP protein. 5 ExpiHEK293 cells were incubated for 30 min at 4 °C. After washing, secondary anti-human AF647 antibody (Jackson ImmunoResearch; West Grove, PA) was incubated for 30 min at 12 nM in PBS. After washing, cell pellets were resuspended in 50 μL of PBS and samples were analyzed on an iQue cytometer (Sartorious; Göttingen, Germany) using Forecyt software (Intellicyt; Ann Arbor, MI). The average fluorescence intensity of specific signals is represented among GFP+ cells in Figure 2.

[0193] All antibodies were specific for human CCR8 and did not cross-react with its closest family member, CCR4. The antibodies showed a similar profile against CCR8 / CCR4 mutants. When the N-terminal domain was replaced by CCR4, the anti-CCR8 antibodies lost reactivity. The binding activity was strongly reduced upon replacement of the extracellular domain 1 (ECL1). However, the majority of the antibodies did not cross-react with cynomolgus CCR8 (Figure 3), and the ECL1 is 100% identical to that of human CCR8, suggesting that the ECL1 loop played an important role in the protein conformation but may not be the epitope for the antibody.

[0194] Substitutions in ECL2 and 3 had no effect, determining that the generated anti-CCR8 antibody contains an epitope located in the N-terminal domain.

[0195] Example 4: Anti-CCR8 monoclonal antibodies are specific for the human CCR8 protein and can cross-react with mouse and / or cynomolgus monkey CCR8 receptors.

[0196] Briefly, purified antibodies (at a saturating concentration of 50 nM, FIG. 3A, or at concentrations ranging from 100 nM, 3-fold dilutions, FIG. 3B-3C) were transiently transfected with 1×10 CCR8 antibodies transiently transfected with either human (SEQ ID NO: 11; Uniprot / Swiss-Prot: P51685), mouse CCR8 (SEQ ID NO: 64; Uniprot / Swiss-Prot: P56484), or cynomolgus CCR8 (SEQ ID NO: 65; Uniprot / Swiss-Prot: G7NYJ2) fused to GFP protein via a cleavable linker. 5The cells were incubated with ExpiHEK 293 cells for 30 min at 4 °C. After washing, secondary anti-human AF647 antibody (Jackson ImmunoResearch; West Grove, PA) was incubated at 12 nM in PBS for 30 min. After washing, the cell pellet was resuspended in 50 µL of PBS and the samples were analyzed by flow cytometry on an iQue cytometer (Sartorious, Göttingen, Germany) using Forecyt software (Intellicyt, Ann Arbor, MI). The average fluorescence intensity for specific signals among GFP+ cells is represented in Figures 3A-3C. All antibodies tested at saturating concentrations are shown in Figure 3A. Titration curves of cross-reactive antibodies against mouse CCR8 and cynomolgus CCR8 are shown in Figures 3B and 3C, respectively.

[0197] Different cross-reactivity profiles were observed among the anti-CCR8 antibodies: one major group, including HFB11-3, HFB11-5, HFB11-8 and HFB11-10, was specific only to the human protein and did not cross-react with other species. HFB11-2 was cross-reactive to mouse CCR8 protein; HFB11-19 recognized cynomolgus monkey CCR8 protein; HFB11-21 was able to bind to both mouse and cynomolgus monkey CCR8 proteins. These antibodies appear to recognize different epitopes between the N-terminal domains. The titration curves of mouse CCR8 antibodies showed subnanomolar EC 50 demonstrated similar binding properties for HFB11-2 with an EC 50 On the other hand, this antibody demonstrated higher binding properties to the cynomolgus monkey CCR8 receptor and exhibited EC 50 HFB11-19 was a more potent binder to cynomolgus monkey CCR8 and was 15.8 nM. 50 is 1.8 nM (Figure 3C).

[0198] Example 5: Anti-CCR8 antibodies block CCL1 binding to CCR8-expressing cells

[0199] The ability to block CCL1 binding to human CCR8-expressing cells was evaluated. Anti-CCR8 antibodies at several concentrations (dilution range starting from 100 nM) were added to stable CCR8-expressing cells (CHOK1, Perkin Elmer) for 30 min at 4 °C. After washing in MACS buffer, 30 nM hCCL1-AF647 (Almac) was added and incubated for 45 min at 4 °C. After washing in MACS buffer, pelleted cells were resuspended in 50 μL and then read by flow cytometry using an iQue cytometer (Sartorious, Gottingen, Germany) and Forecyt software (Intellicyt, Ann Arbor, MI). Maximum mean fluorescence intensity (MFI) was obtained without antibody to reflect 100% of CCL1 binding. The MFI of the AF647 channel was used to calculate %CCL1 blocking (formula = 100-(MFI サンプル *100 / MFI CCL1単独 )). Figure 4 shows results demonstrating that the 5 / 7 anti-CCR8 antibody blocks CCL1 binding to CCR8.

[0200] HFB11-19 and HFB-21, both of which were cynomolgus monkey cross-reactive, were unable to block hCCL1 binding to cell surface-expressed hCCR8, whereas all other antibodies efficiently blocked hCCL1 and exhibited IC 50 The values ​​ranged from approximately 0.4 to 1.4 nM (Figure 4B), suggesting that HFB11-19 and HFB11-21 share a distinct epitope compared to other antibodies on the N-terminal domain.

[0201] Example 6: Anti-CCR8 antibody inhibits intracellular Ca 2+ Inhibits flux

[0202] CCR8 low copy cells (M300.19 mouse pre-B cells) were treated with 100 nM HFB11-10 antibody (gray arrows, Figure 5D and 5G), HFB11-3 antibody (gray arrows, Figure 5D) or buffer (Figure 5E) at time 0 s, followed by incubation for 90 s and addition of 1 nM CCL1 (clear arrows, Figure 5B-5D) or 10 nM CCL1 (clear arrows, Figure 5E-5G). A negative control of buffer only is shown in Figure 5A (clear arrow). Figures 5C and 5D show that both antibodies suppressed CCL1-induced Ca in CCR8-M300.19 cells. 2+ We demonstrate that CCL1 inhibits spiking and shows complete signal inhibition at 1 nM CCL1. When the CCL1 concentration was increased to 10 nM, partial inhibition of 61 and 77.5% was observed for HFB11-3 and HFB11-10, respectively (Figures 5F and 5G). We demonstrate that anti-CCR8 antibodies that blocked CCL1 binding also block chemokine-induced intracellular signaling.

[0203] Example 7: Anti-CCR8 antibodies were able to engage CD16 in an antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay

[0204] CCR8-expressing CHOK1 cells (Perkin Elmer) were used as target cells in the ADCC reporter bioassay (Promega). Briefly, anti-CCR8 antibodies (range from 30 nM, 3-fold dilution), target cells (25000 cells / well) were co-cultured with Jurkat engineered cells expressing CD16 and luciferase reporter genes at E / T=3:1. After 6 h incubation at 37°C, 5% CO2, Bio-glo reagent was added and bioluminescence was read using a Tecan plate reader after 5 min incubation at room temperature. RLU signals are shown in the graph (Figure 6A).

[0205] All anti-CCR8 antibodies were able to engage CD16 with variable efficacy. The non-blocking antibodies HFB11-19 and HFB11-21 showed lower potency (lower E max and E.C.50 HFB11-2, HFB11-3, HFB11-5, HFB11-8 and HFB11-10 showed high potency and EC 50 Values ​​ranged from 0.1 nM to 0.3 nM, with Emax between >25,000 and >40,000 RLU (Figure 6B).

[0206] Example 8: Humanized anti-CCR8 antibodies strongly bind to CCR8-expressing cells

[0207] Purified antibody (produced in an ADCC-enhanced format) was added to 1 × 10 5 hCCR8-CHOK1 stable cells or parental CHOK1 cells (ranging from 50 nM in MACS buffer, 3-fold dilution) were incubated for 30 min at 4 °C. After washing, secondary anti-human AF647 antibody was incubated at 12 nM in MACS buffer for 30 min. After washing, cell pellets were resuspended in 50 μL of MACS buffer and samples were analyzed by cytometry on an iQue cytometer (Sartorious, Gottingen, Germany) using Forecyt software (Intellicyt, Ann Arbor, MI). The average fluorescence in the AF647 channel among gated single cells is shown in Figure 7.

[0208] Titration curves show that all humanized anti-CCR8 antibody candidates have very low ECs of 0.40, 0.30, 0.37, 0.21, 0.58 and 0.10 nM for humanized variants Hz25, Hz28, Hz29, Hz35, Hz37 and Hz4, respectively, without any isotype antibody background. 50 We demonstrated that the antibody was able to bind to CCR8-expressing cells at 100 ng / mL (Figures 7A and 7B).

[0209] The humanized antibody sequence is provided below:

[0210] [Table 5]

[0211] [Table 6]

[0212] [Table 7]

[0213] [Table 8]

[0214] Example 9: Humanized anti-CCR8 antibodies blocked CCL1 binding to CCR8

[0215] 1×10 5 Stable CHOK1.hCCR8 cells (Perkin Elmer) were incubated with a range of humanized anti-CCR8 antibodies (produced in ADCC-enhanced format) or isotype antibodies (concentration range from 33 nM, 3-fold dilutions) for 30 min at 4° C. After centrifugation at 300 g for 3 min, cells were resuspended with 30 nM human CCL1 ligand labeled with AF647 (50 μL) (Almac) and incubated for 45 min at 4° C. After two washes, cells were analyzed by flow cytometry using an iQue cytometer (Sartorious, Gottingen, Germany) with Forecyt software (Intellicyt, Ann Arbor, MI). % blocking was calculated as described in Example 5 and is shown in FIG. 8.

[0216] Humanized anti-CCR8 antibodies in an ADCC-enhanced format inhibited human CCL1 binding to CCR8-expressing cells with potent IC values ​​of 0.09, 1.09, 0.39, 0.68, 0.49 and 0.81 nM for HFB11-10Hz4, HFB11-10Hz25, HFB11-10Hz28, HFB11-10Hz29, HFB11-10Hz35 and HFB11-10Hz37 antibodies, respectively. 50 We were able to block the effect (Figure 8).

[0217] Example 10: Humanized HFB11-10Hz37 anti-CCR8 antibody engaged CD16F and V allotypes in ADCC reporter assays

[0218] Stable hCCR8-CHOK1 cells (Perkin Elmer) were used as target cells and co-cultured with engineered stable Jurkat cells expressing CD16 (FF or VV phenotype) and the reporter NFAT gene from the ADCC reporter bioassay kit at a 3:1 E / T ratio (Promega). A series of HFB11-10Hz37 antibodies was also added to the cultures starting at 30 nM (3-fold serial dilutions). After 6 h of incubation at 37°C and 5% CO2, BioGlo luciferase substrate was added as recommended by the supplier and the luminescence signal was read after 5 min of incubation using a Tecan plate reader. The bioluminescence signal is the reporter as RLU in the graph (Figure 9).

[0219] The humanized anti-hCCR8 antibody was able to engage both CD16F and V allotypes in the ADCC reporter assay to mediate target cell killing. As expected, it showed higher potency at the high affinity Fcγ receptor (VV allotype) with an EC of 0.014 nM. 50 and increased to 0.080 nM for the low affinity Fcγ receptor (FF allotype) (Figure 9).

[0220] Example 11: Humanized anti-CCR8 antibody-mediated ADCC on cells expressing low copies of CCR8

[0221] NK cells were isolated from healthy human PBMCs using an NK cell isolation kit (Miltenyi). Two million NK cells per well in a 24-well plate were activated in IL2 RPMI medium at 37°C, 5% CO2 for approximately 30 hours.

[0222] Cells expressing low copy numbers of CCR8 (stably transfected M300.19 mouse pre-B cells) were labeled with Celltrace™ FarRed (Invitrogen; Waltham, MA) and then co-cultured with NK cells (E / T ratio 2:1) and a range of HFB11-10 antibody concentrations (humanized and non-humanized in ADCC-enhanced format) in U-bottom 96-well plates for 16 hours at 37°C. Dead cells were labeled with Nucgreen and fluorescence was read using a cytoflex cytometer (Beckman Coulter). The percentage of dead target cells was assessed as double positive cells, and the percentage of specific target cell lysis was assessed as (% dead target cells with antibody - % dead target cells without antibody) x 100 / (100 - % dead target cells without antibody). Spontaneous cell death was assessed in co-culture with NK cells without antibody.

[0223] Figure 10 shows the specific ADCC NK killing mediated by humanized anti-CCR8 antibodies. Humanized variant and parental antibodies (ADCC enhanced format) showed high potency in mediating ADCC NK killing of CCR8-expressing low copy cells in the assay (E:T ratio 3:1). EC 50 was low, ranging from 0.003 nM (for Hz29, Hz35, parental Ab) to 0.007 nM for the Hz25 antibody (average from n=3 donors). All humanized antibodies showed very potent activity in ADCC assays using primary NK cells.

[0224] Example 12: In vitro characterization of humanized anti-CCR8 antibody HFB101110

[0225] The defucosylated humanized anti-CCR8 antibody HFB101110, related to HFB11-10, was further studied. In the following examples, the anti-CCR8 antibodies HFB101110 and HFB11-10 can be used alternatively.

[0226] Anti-CCR8 mAb HFB101110 was evaluated in more detail, as shown in Figures 11A-11G. The antibody bound strongly to CCR8 on both high-copy (CHOK1-hCCR8, approx. 30,000 receptors / cell, left) and low-copy (M300.19-hCCR8, approx. 2,000 receptors / cell, right) cells as measured by flow cytometry, and inhibited EC 50 were confirmed to be 0.23 nM and 0.26 nM, respectively (FIG. 11A). A parent antibody against HFB101110 formatted as a hIgG1 antibody with normal glycosylation and DE mutations in the Fc region to enhance ADCC activity (HFB101110-hIgG1-DE) was used as a positive control for defucosylated HFB101110. HFB101110 was able to bind to both high and low copy hCCR8 expressing cells with similar sub-nM EC50 values.

[0227] Anti-CCR8 mAb HFB101110 induced ADCC activity against M300.19-hCCR8 cells. Exogenous primary NK cells isolated from PBMCs were added at an effector:target ratio of 3:1, and dead cells were quantified by flow cytometry using NucGreen staining after 16 h of co-culture. The average values ​​of experiments using NK cells from three different donors are shown. HFB101110-hIgG1-DE and another anti-hCCR8 DE format hIgG1 antibody were used as comparisons (Figure 11B).

[0228] Binding of HFB101110 to the related chemokine receptor CCR4 was assessed by flow cytometry, with the anti-CCR4 antibody mogamulizumab used as a positive control. HFB101110 showed no binding to hCCR4, indicating specific recognition of hCCR8 by this antibody (Figure 11C).

[0229] To identify the region of CCR8 recognized by HFB101110, we performed domain swap experiments. A portion of hCCR8 was replaced with the homologous domain of the related chemokine receptor CCR4, the resulting chimeric protein was expressed in 293 cells, and the binding of HFB101110 was assessed by flow cytometry (Figure 11D). Based on these experiments, it was determined that HFB101110 recognizes the N-terminal extracellular domain of hCCR8.

[0230] Blockade of hCCL1 binding to hCCR8 by HFB101110 was measured by flow cytometry as shown in Figure 11E. HFB101110-mediated blockade of recombinant hCCL1-induced CCR8+ cell chemotaxis was measured by transwell migration assay. CCL1 was present at a concentration of 30 nM with varying amounts of antibody present. HFB101110-hIgG1-DE as well as another humanized variant were used as comparisons (Figure 11F).

[0231] As shown in Figure 11G, HFB101110-mediated blockade of calcium flux induced by addition of hCCL1 to CCR8+ cells was assessed. CCR8+ cells were added with 10 nM hCCL1 in the presence or absence of 10 ug / mL HFB101110 at the times indicated by the arrows.

[0232] Example 13: Humanized anti-CCR8 antibodies demonstrated antibody-dependent cellular phagocytosis (ADCP) activity

[0233] Primary macrophages were isolated from human PBMCs after CD14+ bead isolation (Miltenyi). 2M cells / well were seeded in 6-well plates containing serum-free RPMI 1640 medium for 1 h at 37°C, 5% CO2. The medium was then replaced with activation medium (RPMI 1640, 10% FBS, 1% penicillin / streptomycin, 50ng / mL M-CSF) and refreshed every 2 days for 1 week. IL13 was added to the activation medium for the last 2 days of activation. Target cells (stable hCCCR8.M300.19 cells) were labeled with Celltrace™ violet (Invitrogen) (20 min incubation at 37°C, 5% CO2). After washing with complete medium, CCR8 cells were incubated with humanized anti-CCR8 antibody at saturating concentration (100 nM) for 15 min at 37 °C, 5% CO2, and then co-cultured with activated macrophages for 4 h. Macrophages were stained with anti-CD11b-APC antibody (Biolegend) and then analyzed by flow cytometry using a Cytoflex cytometer (Beckman Coulter). Phagocytic cells were identified as double positive cells using FlowJo software.

[0234] As shown in Figure 12, approximately 15% of the cells were phagocytosed or in the process of phagocytosis by macrophages after 4 hours of incubation (Figure 12). The humanized anti-CCR8 antibody HFB11-10Hz37 specifically mediated CCR8+ cell phagocytosis through the ADCP mechanism using primary macrophage cells.

[0235] Example 14: Humanized anti-CCR8 antibody-mediated anti-tumor activity against MC38 cells in hCCR8-KI mice

[0236] Mice and tumor cell lines:

[0237] Female 8-week-old C57BL / 6 mice were purchased from Biocytogen Jiangsu Co., Ltd. (Jiangxi, China). MC38 mouse colon carcinoma cell line was used for in vivo tumor efficacy studies (Biocytogen Jiangsu Co., Ltd.). MC38 cell line was passaged twice before storage, thawed, and passaged twice before implantation for all tumor experiments described. Cells were determined to be free of mycoplasma.

[0238] Studies were conducted according to the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines and in accordance with protocols reviewed and approved by Biocytogen's Institutional Animal Care and Use Committee (IACUC).

[0239] Inject MC38 tumor cells (5 × 10) in 0.1 mL of PBS into the right anterior flank of hCCR8 KI C57BL / 6 mice (8 weeks old, 15–21 g). 5 One week after tumor implantation, 10 mg / kg of humanized HFB11-10 or isotype antibody was intraperitoneally injected every other week for 3 weeks (n=8 / group). Tumor volume (mm 3 (See FIG. 13A for a schematic of an in vivo efficacy study in hCCR8 knock-in mice.) For this study, a non-defucosylated version of HFB101110 formatted as an mIgG2a antibody was used to promote engagement with mouse Fc receptors.

[0240] Figures 13B-13D show the evaluation of antitumor activity by administration of humanized anti-CCR8 antibodies (Figures 13C-13D) or isotype mIgG2a (Figures 13B-13D) using colorectal cancer-derived MC38 cells in hCCR8-KI mice. Starting from day 9 after the start of treatment, tumor growth was significantly reduced in the anti-human CCR8 treatment group compared to the isotype control (p<0.01, **). Furthermore, tumor regression was observed from day 11 after tumor implantation in 3 out of 8 mice. Taken together, these results demonstrated that anti-CCR8 antibodies significantly reduced tumor growth.

[0241] Example 15: Humanized anti-CCR8 antibody therapy reprogrammed the tumor microenvironment

[0242] To better clarify the mechanism of action, we investigated the changes in the MC38 tumor microenvironment (TME) induced by anti-CCR8 antibody therapy. As shown in Figure 5. 14A-14F, anti-CCR8 antibody treatment altered the tumor-infiltrating lymphocyte (TIL) composition in treated mice.

[0243] Anti-CCR8 antibody administration was observed to significantly increase the percentage of tumor-infiltrating CD8+ (*, p-value=0.0262, FIG. 14C). Furthermore, a significant increase in the CD8 / Treg ratio was observed (*, p-value=0.0240, FIG. 14D), further suggesting a tilt of the immune balance in favor of anti-tumor immunity. Treatment also slightly increased the percentage of NK cells and CD4+ effector T cells in the TME (FIGS. 14E and 14B).

[0244] Furthermore, while anti-CCR8 antibody treatment did not significantly reduce Foxp 3+CD4+ at the final endpoint (Figure 14A), it did significantly reduce the CCR8+ Treg population (Figure 14F), demonstrating the target-specific effect of this agent. The 58% reduction in CCR8+ Tregs observed compared to the isotype group reflected target occupancy by the humanized antibodies used in the FAC panel competing for the same epitope.

[0245] Example 16: CCR8 expression in T cell populations from human primary tumors from renal cell carcinoma (RCC) and lung cancer patients

[0246] Fresh tumor-infiltrating lymphocytes were digested, dissociated into single cell suspensions, counted, and frozen prior to analysis. Briefly, a gentle MACS tumor dissociation kit (Miltenyi) was used first. Tumors were cut into small pieces, incubated with an enzyme mix, and tissue was digested using gentle MACS (Miltenyi). Samples were then centrifuged at 300g for 5 min and washed twice with 10 mL cold medium. Samples were then filtered at 30 μM and resuspended in cold freezing medium (90% FBS and 10% DMSO) prior to further analysis. One aliquot was thawed and incubated with an antibody panel for CCR8 phenotyping. Samples were first stained with live / dead dye and FcBlock. Extracellular staining with anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD56, anti-TIGIT antibodies (Biolegend) and anti-CCR8 (BD) was then performed. Finally, samples were permeabilized and stained with FoxP3 for intracellular staining. Samples were analyzed by flow cytometry using FLOWJo software.

[0247] CCR8 expression was observed on Tregs from 55-95% of renal cell carcinoma patients (n=13), with a mean of 72.8±12.8% (Figure 15A). Conversely, CCR8 expression on FoxP3-CD4+ T effector cells was less than 20% (mean 7.9±5.6%) and close to zero for CD8+ T cells (mean of 1.2±0.8%, Figure 14A). In the histogram shown in Figure 15B, cytometry data is reported for donor #726, where 96% of Tregs were depleted. CCR8 was confirmed to be a desired target for depletion of Tregs without affecting other T cell populations within RCC tumors.

[0248] Example 17: CCR8 expression in T cell populations from the circulation in healthy and malignant PBMCs.

[0249] CCR8 phenotyping was performed as described in Example 15. Briefly, PBMCs from healthy (n=4) or malignant (n=5) donors were isolated from fresh blood. 1 / 3 volume of Ficoll was added to the blood samples, followed by centrifugation at 2000 rpm at room temperature for 30 min. The white rings containing immune cells were collected and washed twice with PBS by centrifugation at 1250 rpm at room temperature for 10 min before FACS analysis. The percentage of CCR8+ in the T cell population is shown in Figure 16.

[0250] CCR8 expression observed in the circulation of healthy or malignant patients was very low in all T cell populations (<4%) (Figure 16). There is no difference between Treg or Teff and CD8+ T cells. This analysis confirms the specificity of the target restricted to the tumor site, no side effects are expected in the circulation, and the risk of toxicity and autoimmunity is limited.

[0251] Example 18: Humanized anti-CCR8 antibody-mediated ADCC NK killing activity on primary human tumor infiltrating lymphocytes (TILS) from RCC patients

[0252] Figure 17A shows a schematic of sample collection and ADCC assays performed for ex vivo killing of primary tumor infiltrating lymphocytes mediated by HFB101110. Single cell suspensions of tumor infiltrating lymphocytes (TILs) from RCC patients (n=13) were thawed and viability was assessed using AO / PI fluorescence in a countess II device. TILs were split into 96-well U-bottom plates at 100 μL / well for ADCC NK killing assays. 50 μL of antibody was added to the TILs followed by the addition of allogeneic primary NK cells at an E / T ratio of 20:1.

[0253] NK cells from healthy donors were pre-expanded using the Miltenyi NK Isolation and Expansion Kit. Aliquots were thawed and incubated for 10 min until TIL preparation. 7The NK cells were stored at 37°C in TIL medium at 100 cells / mL. The amount of NK cells required for the experiment was calculated based on the number of target cells (Tregs) previously evaluated during the phenotyping experiment. The final volume was adjusted to 200 mL, followed by co-culture for 24 hours at 37°C, 5% CO2. FACS staining was performed to identify immune cell populations as described in Example 15. Samples were analyzed by flow cytometry using FlowJo software: B cells were gated as Alive+ / CD45+ / CD19+; CD8+ cells were gated as Alive+ / CD45+ / CD3+ / CD8+; Teff cells were gated as Alive+ / CD45+ / CD3+ / CD4+ / FoxP3-, and Tregs were gated as Alive+ / CD45+ / CD3+ / CD4+ / FoxP3+ / TIGIT+. Statistical analysis was performed using one-way ANOVA test (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05).

[0254] A strong Treg depletion (as % relative to CD3+) was observed (Figure 17 B) compared to the isotype group (50%-90%, mean 71.3 ± 13%, p value < 0.0001). This population disappeared in the dot plot of one representative patient in Figure 17 C. As shown in Figure 17 B, this depletion was specific for FoxP3+CD4+ T cells (Tregs), since no depletion was observed in other cell populations. Conversely, a significant increase in CD8+ cells was observed (CD8+ relative to CD3+ cells), mean +9.8 ± 9%, **p = 0.0067. Moreover, the B cell population was significantly increased in the treatment group at +16.7 ± 19.6% (p < 0.0317). Anti-CCR8 antibodies were able to modulate the TME by specific Treg depletion ex vivo and restore antitumor immunity by increasing CD8+ T and B cells in TILs from RCC patients.

[0255] As further shown in Figure 17D, Treg depletion was assessed in a dose-response experiment after overnight co-culture of primary tumor infiltrating lymphocytes (TILs) with primary NK cells at an effector:target ratio of 20:1. The remaining cells were quantified by flow cytometry. Treg depletion in ADCC assays as a function of the amount of exogenous NK cells added was also assessed (Figure 17E). Two of the nine samples profiled showed substantial Treg depletion even in the absence of exogenous NK cells, suggesting that endogenous NK cells present in the tumor samples can mediate ADCC.

[0256] Example 19: Safety and Pharmacokinetic Evaluation

[0257] Pharmacokinetic and safety studies of HFB101110 were conducted in cynomolgus monkeys (see FIG. 18A for outline of single-dose PK study).

[0258] The serum PK profile of HFB101110 in cynomolgus monkeys revealed that no anti-drug antibodies were detected after 15 days (see FIG. 18B), and in vitro cytokine release studies from human PBMCs using a soluble antibody format did not observe significant cytokine release (see FIG. 18C).

[0259] References

[0260] Villarreal, DO, L'Huillier, A., Armington, S., Mottershead, C., Filippova, EV, Coder, BD, Petit, RG & Princiotta, MF (2018) Targeting CCR8 induces protective antitumor immunity and enhances vaccine-induced responses in colon cancer,Cancer Res.78,5340-5348.

[0261] Van Damme,H.,Dombrecht,B.,Kiss,M.,Roose,H.,Allen,E.,Van Overmeire,E.,Kancheva,D.,Martens,L.,Murgaski,A.,Bardet,PMR,Blancke,G.,Jans,M.,Bolli,E.,Martins,MS,Elkrim,Y.,Dooley,J.,Boon,L .,Schwarze,JK,Tacke,F.,Movahedi,K.,Vandamme,N.,Neyns,B.,Ocak,S.,Scheyltjens,I.,Vereecke,L.,Nana,FA,Merchiers,P.,Laoui,D.&Van Ginderachter,JA(2021)Therapeutic depletion of CCR8(+)tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy,J Immunother Cancer.9,e001749。

[0262] Campbell,JR,McDonald,BR,Mesko,PB,Siemers,NO,Singh,PB,Selby,M.,Sproul,TW,Korman,AJ,Vlach,LM,Houser,J.,Sambanthamoorthy,S.,Lu,K.,Hatcher,SV,Lohre,J.,Jain,R.R.F.Y. Anti-CCR8 Antibody Depletes Regulatory T Cells in Human Tumor Models,Cancer Res.81,2983-2994。

[0263] Whiteside, SK, Grant, FM, Gyori, DS, Conti, AG, Imianowski, CJ, Kuo, P., Nasrallah, R., Sadiyah, F., Lira, SA. , Tacke, F., Eil, RL, Burton, OT, Dooley, J., Liston, A., Okkenhaug, K., Yang, J. & Roychoudhuri, R. (2021) CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function,Immunology.163,512-520.

[0264] Bhatt, D., Kang, B., Sawant, D., Zheng, L., Perez, K., Huang, Z., Sekirov, L., Wolak, D., Huang, JY, Liu, X., DeVoss, J., Manzanillo, PS, Pierce, N., Zhang, Z., Symons, A. & Ouyang, W. (2021) STARTRAC analyzes of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets, J Exp Med.218.e20201329.

[0265] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood therefore that the invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the invention as defined herein.

Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: (1) SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively; (2) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (3) SEQ ID NOs: 13, 2, 14, 4, 28, and 6, respectively; (4) SEQ ID NOs: 13, 2, 15, 4, 5, and 6, respectively; (5) SEQ ID NOs: 16, 17, 18, 29, 30, and 6, respectively; (6) SEQ ID NOs: 19, 20, 21, 4, 5, and 6, respectively; (7) SEQ ID NOs: 22, 23, 24, 31, 5, and 32, respectively; (8) SEQ ID NOs: 25, 26, 27, 33, 34, and 35, respectively; (9) SEQ ID NOs: 1, 2, 49, 4, 5, and 6, respectively; or (10) SEQ ID NOs: 1, 2, 49, 50, 51, and 6, respectively; and a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having a polypeptide sequence of: the antibody or antigen-binding fragment thereof specifically binds to chemokine (C-C motif) receptor 8 (CCR8), preferably human CCR8; An isolated monoclonal antibody or antigen-binding fragment thereof.

2. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the polypeptide sequences of SEQ ID NOs: 1, 48, 49, 50, 51, and 6, respectively; An isolated monoclonal antibody or antigen-binding fragment thereof.

3. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10; The isolated monoclonal antibody or antigen-binding fragment thereof of claim 2.

4. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO:9 and a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO:

10. An isolated monoclonal antibody or antigen-binding fragment thereof.

5. a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 7, 9, 36, 38, 40, 42, 44, 46, 52, 53, 54, 55, 56, 57, 58, 59, or 60, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 8, 10, 37, 39, 41, 43, 45, 47, 61, 62, or 63; The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1.

6. (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 36 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 37; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 38 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 39; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 40 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 41; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 42 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 43; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 44 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 45; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 46 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 47; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10; (9) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 52 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 63; (10) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 52 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10; (11) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 53 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 54 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 63; or (13) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 59 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

62. Including, The monoclonal antibody or antigen-binding fragment thereof according to claim 5.

7. the antibody or antigen-binding fragment thereof is capable of forming a bispecific antibody with another mAb or antigen-binding fragment thereof that induces effector-mediated tumor cell lysis through antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); and / or induces the mobilization of a conjugated drug; and / or has cancer-killing effects; The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. the monoclonal antibody or antigen-binding fragment thereof specifically binds to cynomolgus monkey CCR8; The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

9. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. Bispecific antibodies or antigen-binding fragments thereof.

10. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

11. 10. An isolated nucleic acid encoding the bispecific antibody or antigen-binding fragment thereof of claim 9.

12. A vector comprising the isolated nucleic acid of claim 10.

13. A vector comprising the isolated nucleic acid described in claim 11.

14. A host cell comprising the vector of claim 12.

15. A host cell comprising the vector described in claim 13.

16. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or a bispecific antibody or antigen-binding fragment thereof comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

17. 17. The pharmaceutical composition of claim 16 for targeting CCR8 on the surface of cancer cells and / or treating cancer in a subject in need thereof.

18. The cancer is, for example, a solid tumor, preferably a solid tumor with infiltrating T cells, more preferably a solid tumor with infiltrating Treg cells, more preferably a solid tumor with highly suppressive Treg cells that express CCR8, and most preferably a solid tumor with infiltrating highly suppressive Treg cells that overexpress CCR8 accompanied by natural killer (NK) cell infiltration; 18. The pharmaceutical composition of claim 17.

19. the cancer is selected from the group consisting of lung cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, renal cancer, and melanoma; 18. The pharmaceutical composition of claim 17.

20. the subject comprises CCR8-expressing Treg cells.

18. The pharmaceutical composition of claim 17.

21. 10. A method for producing the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6, or a bispecific antibody or antigen-binding fragment thereof comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the method comprising culturing cells comprising nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof under conditions to produce the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof from the cells or culture.

22. 10. A method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6, or a bispecific antibody or antigen-binding fragment thereof comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6, comprising combining the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

23. 1. A method for determining the level of CCR8 in a subject, the method comprising: a. contacting a sample with the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the sample is isolated from the subject. What you can get and b. determining the level of CCR8 in said subject; A method comprising:

24. 24. The method of claim 23, wherein the sample is a tissue sample or a blood sample, optionally wherein the tissue sample is a cancer tissue sample.

25. 24. The method of claim 23, wherein the sample comprises Treg cells.

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