CCR8 antigen-binding unit and uses thereof

Abstract

This application describes antibodies against CCR8 with enhanced binding, ligand blocking, and antibody-dependent cytotoxicity, and also describes methods of using formulations containing said CCR8 antibodies to target tumor-infiltrating lymphocytes in cancer patients to treat cancer.

Description

[Technical field]

[0001] The present disclosure relates to the field of antibody-based therapeutics. More specifically, the present disclosure relates to anti-CCR8 compositions and methods of using them to treat diseases. [Background technology]

[0002] Despite the promise of immunotherapy in cancer treatment, nearly 80% of patients do not respond to checkpoint inhibitor (CPI) therapy. Regulatory T cells (Tregs), which suppress immune responses in the tumor microenvironment through multiple inhibitory mechanisms, are thought to play a key role in the lack of CPI efficacy. Therefore, targeting and depleting Tregs would promote more effective antitumor immunity. CCR8 is a chemokine receptor selectively expressed on activated human tumor-resident Tregs, and it has been shown that these intratumoral CCR8+ Tregs can promote immune suppression and cause poor prognosis. Therefore, CCR8 can be targeted for cancer immunotherapy by selectively depleting intratumoral immunosuppressive Treg cells. Antibodies targeting human CCR8 can cause Treg depletion in tumors and increase tumor sensitivity to CPI therapy, such as anti-PD-1 treatment. DISCLOSURE OF THEINVENTION [Means for solving the problem]

[0003] Disclosed herein are antigen-binding units that include a light chain complementarity determining region (CDR) (referring to a light chain variable region) and a heavy chain CDR (referring to a heavy chain variable region). In some embodiments, the antigen-binding unit binds to CCR8 and prevents binding of CCR8 to CC motif chemokine ligand 1 (CCL1) expressed on the surface of immune cells.

[0004] In some embodiments, the antigen-binding unit, when linked to a fragment crystallizable (Fc) region, particularly a mutated Fc variant, enhances antibody-dependent cellular cytotoxicity (ADCC), thereby killing CCR8-expressing Treg cells.

[0005] In some embodiments, the antigen binding unit comprises at least one mutation at a post-translational modification (PTM) site. In some embodiments, the at least one mutation is at least one of a N28G mutation, a N28Q mutation, a N28I mutation, a N28S mutation, a G29A mutation, a G29I mutation, and a G29V mutation. In some embodiments, the mutation is a N28G mutation in the light chain CDR1.

[0006] In some embodiments, the heavy chain CDR comprises HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 each comprise a sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (including any range (e.g., 60% to 85%)) to a sequence selected from SEQ ID NOs: 13 to 15, 19 to 22, 71 to 73, 77, 78, 81, 83 to 85, 89, 91 to 93, 97, 98, and 112 to 118.

[0007] In some embodiments, the heavy chain CDRs comprise HC-CDR1, HC-CDR2, and HC-CDR3, each of which comprises a sequence selected from SEQ ID NOs: 13-15, 19-22, 71-73, 77, 78, 81, 83-85, 89, 91-93, 97, 98, and 112-118.

[0008] In some embodiments, the heavy chain CDRs comprise HC-CDR1, HC-CDR2, and HC-CDR3, each of which comprises a sequence selected from SEQ ID NOs: 13-15, 19-22, 89, 114, and 115.

[0009] In some embodiments, the heavy chain CDRs comprise HC-CDR1, HC-CDR2, and HC-CDR3, each of which comprises a sequence selected from SEQ ID NOs: 83-85 and 116-118.

[0010] In some embodiments, HC-CDR1 comprises a sequence selected from SEQ ID NOs: 13, 71, 83, 115, and 117, HC-CDR2 comprises a sequence selected from SEQ ID NOs: 14, 72, 77, 84, 89, 91, 92, 97, 114, 116, and 118, and HC-CDR3 comprises a sequence selected from SEQ ID NOs: 15, 19-22, 73, 78, 81, 85, 93, 98, 112, and 113.

[0011] In some embodiments, HC-CDR1 comprises a sequence selected from SEQ ID NOs: 13 and 115, HC-CDR2 comprises a sequence selected from SEQ ID NOs: 14, 89, and 114, and HC-CDR3 comprises a sequence selected from SEQ ID NOs: 15 and 19-22.

[0012] In some embodiments, HC-CDR1 comprises a sequence selected from SEQ ID NOs: 83 and 117, HC-CDR2 comprises a sequence selected from SEQ ID NOs: 84, 116, and 118, and HC-CDR3 comprises the sequence of SEQ ID NO: 85.

[0013] In some embodiments, the light chain CDR comprises LC-CDR1, LC-CDR2, and LC-CDR3, and each of the LC-CDR1, LC-CDR2, and LC-CDR3 comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (including any range (e.g., 60% to 85%)) to a sequence selected from SEQ ID NOs: 16 to 18, 23, 24, 25, 74 to 76, 79, 80, 82, 86 to 88, 90, 94 to 96, and 99 to 111.

[0014] In some embodiments, the light chain CDRs comprise LC-CDR1, LC-CDR2, and LC-CDR3, each of which comprises a sequence selected from SEQ ID NOs: 16-18, 23, 24, 25, 74-76, 79, 80, 82, 86-88, 90, 94-96, and 99-111.

[0015] In some embodiments, the light chain CDRs comprise LC-CDR1, LC-CDR2, and LC-CDR3, each of which comprises a sequence selected from SEQ ID NOs: 16-18, 23, 24, 25, 90, and 102-106.

[0016] In some embodiments, the light chain CDRs comprise LC-CDR1, LC-CDR2, and LC-CDR3, each of which comprises a sequence selected from SEQ ID NOs: 86-88.

[0017] In some embodiments, LC-CDR1 comprises a sequence selected from SEQ ID NOs: 16, 74, 79, 86, 90, 94, 99, and 102-109, LC-CDR2 comprises a sequence selected from SEQ ID NOs: 17, 75, 80, 87, 95, 100, 110, and 111, and LC-CDR3 comprises a sequence selected from SEQ ID NOs: 18, 23-25, 76, 82, 88, 96, and 101.

[0018] In some embodiments, LC-CDR1 comprises a sequence selected from SEQ ID NOs: 16, 90, and 102-106, LC-CDR2 comprises a sequence of SEQ ID NO: 17, and LC-CDR3 comprises a sequence selected from SEQ ID NOs: 18 and 23-25.

[0019] In some embodiments, LC-CDR1 comprises the sequence of SEQ ID NO:86, LC-CDR2 comprises the sequence of SEQ ID NO:87, and LC-CDR3 comprises the sequence of SEQ ID NO:88.

[0020] In some embodiments, the heavy chain CDRs comprise HC-CDR1, HC-CDR2, and HC-CDR3, wherein said HC-CDR1, HC-CDR2, and HC-CDR3 are selected from the following groups: SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:19; SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20; SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21; SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:22; SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; SEQ ID NO:71, SEQ ID NO:77, and SEQ ID NO:78; SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:81; SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO: No. 85; SEQ ID NO:13, SEQ ID NO:89, and SEQ ID NO:15; SEQ ID NO:13, SEQ ID NO:91, and SEQ ID NO:15; SEQ ID NO:71, SEQ ID NO:92, and SEQ ID NO:93; SEQ ID NO:13, SEQ ID NO:97, and SEQ ID NO:98; SEQ ID NO:13, SEQ ID NO:97, and SEQ ID NO:112; SEQ ID NO:13, SEQ ID NO:97, and SEQ ID NO:113; SEQ ID NO:13, SEQ ID NO:114, and SEQ ID NO:15; SEQ ID NO:115, SEQ ID NO:114, and SEQ ID NO:15; SEQ ID NO:83, SEQ ID NO:116, and SEQ ID NO:85; SEQ ID NO:117, SEQ ID NO:116, and SEQ ID NO:85; and SEQ ID NO:83, SEQ ID NO:118, and SEQ ID NO:85.

[0021] In some embodiments, the light chain CDRs comprise LC-CDR1, LC-CDR2, and LC-CDR3, wherein said LC-CDR1, LC-CDR2, and LC-CDR3 are selected from the following groups: SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:23; SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:24; SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:25; SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76; SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:76; SEQ ID NO:79, SEQ ID NO:75, and SEQ ID NO:82; SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88; SEQ ID NO:90, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:94, SEQ ID NO: SEQ ID NO:95, and SEQ ID NO:96; SEQ ID NO:79, SEQ ID NO:75, and SEQ ID NO:76; SEQ ID NO:99, SEQ ID NO:100, and SEQ ID NO:101; SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:103, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:104, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:105, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:106, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:107, SEQ ID NO:100, and SEQ ID NO:101; SEQ ID NO:108, SEQ ID NO:100, and SEQ ID NO:101; SEQ ID NO:109, SEQ ID NO:100, and SEQ ID NO:101; and SEQ ID NO:99, SEQ ID NO:111, and SEQ ID NO:101.

[0022] In some embodiments, the heavy chain CDRs comprise HC-CDR1, HC-CDR2, and HC-CDR3, and the light chain CDRs comprise LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 are selected from the following groups: SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively; SEQ ID NOs: 13, 14, 19, 16, 17, and 18; SEQ ID NOs: 13, 14, 20, 16, 17, and 18; SEQ ID NOs: 13, 14, 21, 16, 17, and 18; SEQ ID NOs: 13, 14, 22, 16, 17, and 18; SEQ ID NOs: 13, 14, 15, 16, 17, and 23; SEQ ID NOs: 13, 14, 15, 16, 17, and 24; SEQ ID NOs: 13, 14, 15, 16, 17, and 25; SEQ ID NOs: Nos. 13, 14, 19, 16, 17, and 23; SEQ ID NOs. 13, 14, 20, 16, 17, and 23; SEQ ID NOs. 13, 14, 21, 16, 17, and 23; SEQ ID NOs. 13, 14, 22, 16, 17, and 23; SEQ ID NOs. 13, 89, 15, 90, 17, and 18; SEQ ID NOs. 13, 89, 15, 102, 17, and 18; SEQ ID NOs. 13, 89, 15, 103, 17, and 18; SEQ ID NOs. The present invention includes amino acid sequences selected from the group consisting of SEQ ID NOs: 13, 89, 15, 104, 17, and 18; SEQ ID NOs: 13, 89, 15, 105, 17, and 18; SEQ ID NOs: 13, 89, 15, 106, 17, and 18; SEQ ID NOs: 13, 114, 15, 16, 17, and 18; and SEQ ID NOs: 115, 114, 15, 16, 17, and 18.

[0023] In some embodiments, the heavy chain CDRs comprise HC-CDR1, HC-CDR2, and HC-CDR3, and the light chain CDRs comprise LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 comprise amino acid sequences selected from the following group: SEQ ID NOs: 83, 84, 85, 86, 87, and 88; SEQ ID NOs: 83, 116, 85, 86, 87, and 88; SEQ ID NOs: 117, 116, 85, 86, 87, and 88; and SEQ ID NOs: 83, 118, 85, 86, 87, and 88.

[0024] In some embodiments, the antigen binding unit comprises a heavy chain CDR, wherein the heavy chain CDR comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (including any range between these endpoints, e.g., 60% to 85%) to a sequence selected from SEQ ID NOs: 1, 3, 4, 5, 6, 7, 11, 12, 26, 28, 30, 32, 34, 36, 37, 39, 41, 54-59, 61, and 66-70.

[0025] In some embodiments, the antigen binding unit comprises a heavy chain CDR, and the heavy chain CDR comprises a sequence selected from SEQ ID NOs: 1, 3, 4, 5, 6, 7, 11, 12, 26, 28, 30, 32, 34, 36, 37, 39, 41, 54-59, 61, and 66-70.

[0026] In some embodiments, the antigen binding unit comprises a light chain CDR, wherein the light chain CDR comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (including any range between these endpoints, e.g., 60% to 85%) to a sequence selected from SEQ ID NOs: 2, 8, 9, 10, 27, 29, 31, 33, 35, 38, 40, 42 to 53, 60, and 62 to 65).

[0027] In some embodiments, the antigen binding unit comprises a light chain CDR, and the light chain CDR comprises a sequence selected from SEQ ID NOs: 2, 8, 9, 10, 27, 29, 31, 33, 35, 38, 40, 42-53, 60, and 62-65.

[0028] In some embodiments, the antigen binding unit comprises a heavy chain CDR and a light chain CDR, wherein the heavy chain CDR and the light chain CDR comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 2; SEQ ID NOs: 4 and 2; SEQ ID NOs: 5 and 2; SEQ ID NOs: 6 and 2; SEQ ID NOs: 7 and 2; SEQ ID NOs: 1 and 8; SEQ ID NOs: 1 and 9; SEQ ID NOs: 1 and 10; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 8; SEQ ID NOs: 5 and 8; SEQ ID NOs: 6 and 8; SEQ ID NOs: 11 and 8; SEQ ID NOs: 12 and 8; SEQ ID NOs: 34 and 35; SEQ ID NOs: 34 and 43; SEQ ID NOs: 34 and 44; SEQ ID NOs: 34 and 45; SEQ ID NOs: 34 and 46; SEQ ID NOs: 34 and 47; SEQ ID NOs: 34 and 48; SEQ ID NOs: 56 and 2; SEQ ID NOs: 57 and 2; SEQ ID NOs: 58 and 2; SEQ ID NOs: 59 and 2; SEQ ID NOs: 56 and 60; SEQ ID NOs: 57 and 60; SEQ ID NOs: 58 and 60; and SEQ ID NOs: 58 and 60. In some embodiments, the heavy chain CDRs and light chain CDRs each comprise an amino acid sequence selected from the following group: SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 2; SEQ ID NOs: 4 and 2; SEQ ID NOs: 5 and 2; SEQ ID NOs: 6 and 2; SEQ ID NOs: 7 and 2; SEQ ID NOs: 1 and 8; SEQ ID NOs: 1 and 9.

[0029] In some embodiments, the antigen binding unit comprises a heavy chain CDR and a light chain CDR, wherein the heavy chain CDR and the light chain CDR respectively comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 and 33; SEQ ID NOs: 61 and 62; SEQ ID NOs: 61 and 63; SEQ ID NOs: 61 and 64; SEQ ID NOs: 61 and 65; SEQ ID NOs: 66 and 62; SEQ ID NOs: 67 and 62; SEQ ID NOs: 68 and 62; SEQ ID NOs: 69 and 62; and SEQ ID NOs: 70 and 62.

[0030] In some embodiments, the antigen binding unit comprises an IgG1 framework, with or without mutations in the Fc region.

[0031] In some embodiments, the antigen-binding unit is a monoclonal antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antigen-binding unit is an scFv, Fab', single-chain Fab (scFab'), Fd or F(ab')2, sFc, Fv, or ccFv. In some embodiments, the antigen-binding unit competes for binding to the epitope recognized by the antigen-binding unit.

[0032] In other embodiments, a pharmaceutical composition is provided comprising any one of the antigen binding units disclosed herein and a pharma- ceutically acceptable excipient.

[0033] In other embodiments, an isolated nucleic acid encoding any one of the antigen binding units disclosed herein is provided.

[0034] In other embodiments, a vector is provided that comprises a nucleic acid sequence encoding any one of the antigen binding units disclosed herein.

[0035] In other embodiments, a host cell is provided that expresses any one of the antigen binding units disclosed herein.

[0036] In other embodiments, a host cell is provided that contains a nucleic acid encoding any one of the antigen binding units disclosed herein.

[0037] In other embodiments, a method of producing any one of the antigen binding units disclosed herein is provided, the method comprising culturing any of the host cells disclosed herein under conditions suitable for expressing the antigen binding unit, and isolating the antigen binding unit expressed by the host cell.

[0038] In other embodiments, a method of eradicating an immune cell population is provided. The method comprises contacting an immune cell with any of the antigen binding units disclosed herein. In some aspects, the immune cell is a regulatory T cell (Treg). In other embodiments, the immune cell is a tumor-resident Treg.

[0039] In other embodiments, a method of treating cancer in a subject in need thereof is provided. The method comprises administering to the subject in need thereof an effective amount of an antigen binding unit as described herein. In embodiments, the method comprises repeating the administering step over a period of time, such as until the subject is cancer-free. In some embodiments, the cancer is a hematological cancer or a solid tumor. In some embodiments, treating the cancer comprises reducing tumor volume.

[0040] In other embodiments, a method of treating cancer in a subject in need of treatment is provided. The method comprises administering to the subject in need an effective amount of a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and any of the antigen binding units disclosed herein. In some embodiments, the method comprises repeating the administering step over a period of time, such as until the subject is cancer-free. In some embodiments, the cancer is a hematological cancer or a solid tumor. In some embodiments, treating the cancer comprises reducing tumor volume. [Brief description of the drawings]

[0041] [Figure 1] FIG. 1 shows a multiple sequence alignment of the amino acid sequences of human, cynomolgus monkey, mouse and rat CCR8. [Figure 2A-2C] 2A to 2C are diagrams showing that antibodies from which post-translational modification (PTM) sites have been removed bind to HEK293-human CCR8, HEK293 cells, and HEK293-cynomolgus CCR8. [Figure 3A-3B] 3A-3B show that antibodies with deleted post-translational modification (PTM) sites block the binding of human CCL1 to CHO-K1-human CCR8 cells. [Figure 4A-4B] 4A-4B show binding of the indicated humanized antibodies to ExpiCHO-S-CCR1 and ExpiCHO-S-CCR4 cells. [Figure 5A-5B] 5A to 5B show antibody-dependent cellular cytotoxicity (ADCC) activity of humanized antibodies against the CHO-K1-human CCR8 cell line. [Figure 6] FIG. 6 shows that anti-CCR8 inhibited CCL1-induced β-arrestin recruitment. [Figure 7] FIG. 7 shows the ability of humanized anti-CCR8 antibodies to inhibit CCL1-induced cell migration. [Figure 8A-8B] 8A-8B show FACS binding of periplasmic extracts (PPE) to CHO-K1-human CCR8 cells. [Figure 9A-9B] 9A-9B show the ability of affinity matured antibodies to inhibit CCL1-induced β-arrestin recruitment. [Figure 10] FIG. 10 shows that mature anti-CCR8 antibodies block CCL1-induced cell migration. [Figure 11A-11B] 11A to 11B show that Fc variants of humanized 149 antibody and humanized 348 antibody bind to HuT78 cells. [Figure 12A-12B] 12A to 12B show the ADCC activity of V variant CD16a / NFAT-Jurkat cells against CHO-K1-human CCR8 cells. [Figure 13A-13B] 13A to 13B show the ADCC activity of F variant CD16a / NFAT-Jurkat cells against CHO-K1-human CCR8 cells. [Figure 14A-14B] 14A to 14B show the ADCC activity of V variant CD16a / NFAT-Jurkat cells against HuT78 cells. [Figure 15A-15B] 15A to 15B are diagrams showing the ADCC activity of F variant CD16a / NFAT-Jurkat cells against HuT78 cells. [Figure 16A-16B]16A to 16B show the ADCC activity of human PBMC cells against CHO-K1-human CCR8 cells. [Figure 17A-17B] 17A-17B show the ability of affinity matured antibodies to inhibit CCL1-induced β-arrestin recruitment. [Figure 18] 18 shows that major immune cell populations in peripheral blood do not express CCR8. PBMCs from a kidney cancer patient (patient number 200003119) were analyzed by flow cytometry. [Figure 19] 19 shows that CCR8 is expressed only by Tregs from hDTCs. Kidney hDTCs from patient number 200003119 were analyzed by flow cytometry. [Figures 20A-20N] 20A-20N are diagrams showing that CCR8 is expressed by Tregs from hDTCs of different cancer types. Two CCR8 subsets (subset 1: CCR8 low (lo) and subset 2: CCR8 high (hi)) were observed. [Figure 21] FIG. 21 shows that treatment with Hu149-11G1m significantly reduced tumor volume. [Figure 22] FIG. 22 is a graph of tumor volume versus days after tumor implantation for the different treatment groups (control, Hu149-11G1m, anti-PD-1, and combo) in a study using hCCR8 knock-in transgenic mice inoculated with M38 tumors. [Figure 23] FIG. 23 is a Kaplan-Meier survival plot of % survival of animals versus days after tumor inoculation in different treatment groups (control, Hu149-11G1m, anti-PD-1, and combo) in a study using hCCR8 knock-in transgenic mice inoculated with M38 tumors. [Figure 24] FIG. 24 is a graph of tumor volume versus days after tumor implantation in different treatment groups (control, Hu149-11G1m, anti-PD-1, and combo) in a study using hCCR8 knock-in transgenic mice inoculated with E0771 tumors. [Diagram 25]FIG. 25 is a Kaplan-Meier survival plot of % survival of animals versus days after tumor inoculation in different treatment groups (control, Hu149-11G1m, anti-PD-1, and combo) in a study using hCCR8 knock-in transgenic mice inoculated with E0771 tumors. [Figure 26] FIG. 26 shows flow cytometry analysis of immune cell populations within E0771 tumors 7 days after initiation of treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. The following claims are intended to define the scope of the invention, and methods and structures within the scope of these claims, and their equivalents, are intended to be covered thereby.

[0043] I. Definition For convenience, certain terms used in the specification, examples, and claims are collected here. Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0044] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other specified or intervening value in that range is included, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges are independently included in the smaller ranges and are also encompassed within said ranges, subject to any limit expressly excluded in the stated range. Where the stated range includes one or both of the limits, ranges excluding one or both of those limits are also included.

[0045] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0046] The word "about" immediately preceding a numerical value means a range of plus or minus 10% of that value, unless the context of this disclosure indicates otherwise or is inconsistent with such an interpretation, e.g., "about 50" means 45 to 55, and "about 25,000" means 22,500 to 27,500. For example, in a list of numerical values ​​such as "about 49", "about 50", and "about 55", "about 50" means a range of less than half the interval between the previous value and the next value, e.g., a range of greater than 49.5 to less than 52.5. Furthermore, the phrases "less than about a value" or "greater than about a value" should be understood in light of the definition of the term "about" provided herein.

[0047] The compositions of the present disclosure can comprise, consist essentially of, or consist of the disclosed components.

[0048] All percentages, parts, and ratios are based on the total weight of the topical composition unless otherwise specified, and all measurements are made at about 25°C.

[0049] The phrase "pharmacologically acceptable" is used to refer to compounds, salts, compositions, dosage forms, and the like that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and / or other mammals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments, "pharmacologically acceptable" means approved by a regulatory agency of the U.S. Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeia for use in mammals (e.g., animals), especially humans.

[0050] The term "treatment" is used herein, for example, with respect to methods of treating cancer, and generally includes administration of a compound or composition that reduces the frequency or delays the onset of symptoms of a condition (such as cancer) in a subject, compared to a subject to whom the compound or composition is not administered. This can include reversing, alleviating, or arresting the symptoms, clinical signs, and underlying pathology of the condition (such as regression of tumor growth) in a manner that improves or stabilizes the subject's condition.

[0051] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to polymers of amino acids of any length. The polymers may be linear, cyclic or branched, may contain modified amino acids and may be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified, for example, by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation or ubiquitination, or other manipulations, such as conjugation with a labeling moiety. As used herein, the term "amino acid" refers to any naturally occurring and / or unnatural or synthetic amino acid, including glycine and D- or L-form optical isomers, amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide. Preferably, the polypeptide has an amino acid sequence that is essentially identical to that of a polypeptide encoded by the sequence, or a portion thereof, which portion consists of at least 10-20 amino acids, or at least 20-30 amino acids, or at least 30-50 amino acids, or is immunologically distinguishable from the polypeptide encoded by the sequence. The term also includes polypeptides expressed from a specified nucleic acid sequence.

[0052] The term "antigen-binding unit" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds ("immunoreacts") with an antigen. The term "antigen-binding unit" also includes immunoglobulin molecules from various species, including invertebrates and vertebrates. Structurally, the simplest natural antibody (e.g., IgG) is composed of four polypeptide chains, two heavy (H) and two light (L) chains interlinked by disulfide bonds. Immunoglobulins represent a large family of molecules that includes several types of molecules, such as IgD, IgG, IgA, IgM, and IgE. The term "immunoglobulin molecule" includes, for example, hybrid or engineered antibodies, and fragments thereof. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a natural antibody. These fragments are collectively referred to as "antigen-binding units." The term "antigen-binding unit" also includes a molecular structure comprising a polypeptide chain having a specific shape that fits and recognizes an epitope, and one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope.

[0053] An antigen-binding unit "specifically binds" or is "immunoreactive" with an antigen if it binds to the antigen with greater affinity or avidity than it binds to one or more other reference antigens, including polypeptides or other substances.

[0054] As used herein, "antigen" refers to a substance that is specifically recognized and bound by an antigen-binding unit. Antigens can include peptides, proteins, glycoproteins, polysaccharides, lipids, portions thereof, and combinations thereof. Non-limiting exemplary antigens include CCR8 from human, mouse, and other homologs thereof.

[0055] The term "biological sample" encompasses a variety of sample types obtained from a living organism and can be used in diagnostic or monitoring assays. The term includes blood and other liquid samples from an organism, solid tissue samples such as a biopsy specimen or tissue cultures or cells and their progeny obtained therefrom. The term includes samples that have been manipulated in any way after procurement, such as by treatment with reagents, solubilization, or enrichment for particular components. The term includes clinical samples, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.

[0056] A "chimeric" protein contains at least one fusion polypeptide that contains regions that are in a different sequence position than occurs in nature. These regions may be normally present in separate proteins and are brought together in the fusion polypeptide, or they may be normally present in the same protein but are arranged in a new configuration in the fusion polypeptide. Chimeric proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0057] A genetic "database" refers to a set of stored data representing a collection of sequences, including nucleotide sequences and peptide sequences, which sequences represent a collection of biological reference material.

[0058] "Domain" refers to a portion of a protein that is physically or functionally distinct from other portions of the protein or peptide. Physically defined domains include amino acid sequences that are particularly hydrophobic or hydrophilic, such as membrane-associated or cytoplasmic-associated sequences. Domains may also be defined by internal homology, for example resulting from gene duplication. Functionally defined domains have a distinct biological function. For example, the ligand-binding domain of a receptor is the domain that binds to a ligand. An antigen-binding domain refers to the portion of an antigen-binding unit or antibody that binds to an antigen. A functionally defined domain need not be encoded by a contiguous amino acid sequence. A functionally defined domain may include one or more physically defined domains. For example, receptors are commonly divided into an extracellular ligand-binding domain, a transmembrane domain, and an intracellular effector domain.

[0059] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA (also called a "transcript") is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide are collectively referred to as the gene product. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0060] "Cell line" or "cell culture" refers to a bacterial, plant, insect, or higher eukaryotic cell grown or maintained in vitro. The progeny of a cell may not be completely identical (morphologically, genotypically, or phenotypically) to the parent cell.

[0061] A "fusion gene" is a gene that is constructed by linking at least two heterologous polynucleotides.

[0062] The terms "gene" or "gene fragment" are used interchangeably herein. They refer to a polynucleotide that contains at least one open reading frame that can encode a particular protein after being transcribed and translated. A gene or gene fragment can be either genomic, cDNA, or synthetic, so long as the polynucleotide contains at least one open reading frame and covers the entire coding region or a segment thereof.

[0063] "Heterologous" means derived from a genotypically distinct entity from the other entity to which it is being compared. For example, a promoter removed from its native coding sequence and operably linked to a coding sequence other than the native coding sequence is a heterologous promoter. The term "heterologous" as applied to a polynucleotide, polypeptide means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from the other entity to which it is being compared. For example, a heterologous polynucleotide or antigen may be derived from a different species of origin, a different cell type, and the same type of cell from a different individual.

[0064] "Host cell" includes an individual cell or cell culture that can be or has been a recipient of a subject vector. A host cell includes the progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. A host cell includes a cell that has been transfected in vivo with a vector. "Host cell" may refer to a prokaryotic, eukaryotic cell, or cell line cultured as a single-cell entity that can be or has been used as a recipient for a recombinant vector or other transfer polynucleotide, and includes the progeny of the original transfected cell. The progeny of a single cell may not necessarily be completely identical in morphology or genomic or total DNA complement to the original parent cell due to natural, accidental, or deliberate mutations.

[0065] The term "isolated" as used herein means that the polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof is separated from cellular and other components that normally accompany the polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof in nature. As will be apparent to one of skill in the art, a non-natural polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not need to be "isolated" to be distinguished from its natural counterpart. Furthermore, an "enriched," "isolated," or "diluted" polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof is distinguishable from its natural counterpart in that the concentration or number of molecules per volume is greater than "enriched" or less than "isolated" compared to its natural counterpart. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or can be measured relative to a second, potentially interfering substance present in the source mixture. In some embodiments, increased enrichment is preferred. Thus, for example, 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, and 1000-fold enrichment is even more preferred. An agent can also be provided in isolated form by an artificial construction process, such as chemical synthesis or recombinant expression.

[0066] "Linked" and "fused" or "fusion" are used interchangeably herein. These terms refer to the joining of two or more chemical elements or components by any means, including chemical conjugation or recombinant means. "In-frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a continuous, longer ORF in a manner that maintains the correct reading frame of the original ORFs. The resulting recombinant fusion protein is thus a single protein that contains two or more segments that correspond to the polypeptides encoded by the original ORFs (these segments are not normally joined in this manner in nature). In this way, the reading frame is continuous throughout the fusion segments, but the segments may be physically or spatially separated, for example, by an in-frame linker sequence (e.g., "Flexon").

[0067] "Operably linked" or "operably linked" refers to a juxtaposition in a relationship that allows components to function in their intended manner. For example, a promoter sequence is operably linked to a coding sequence if it promotes transcription of the coding sequence. For example, a peptide sequence (e.g., Fc) is linked to another peptide sequence (e.g., an antigen binding unit) in a manner that allows for a functional structure (e.g., an antibody).

[0068] The terms "polynucleotide", "nucleic acid", "nucleotide" and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, gene sites (locuses) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, oligonucleotides, or synthetic DNA. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component.

[0069] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures, that result in a construct that differs from polynucleotides found in nature.

[0070] In the context of a polypeptide, a "sequence" is the order of amino acids in a polypeptide from the amino terminus to the carboxyl terminus, where residues adjacent to each other in a sequence are contiguous in the primary structure of the polypeptide. A sequence may be the linear sequence of a portion of a polypeptide that is known to include additional residues in one or both directions.

[0071] A "vector" is a nucleic acid molecule, preferably self-replicating, that transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily to insert DNA or RNA into a cell, replicating vectors that function primarily to replicate DNA or RNA, and expression vectors that function to transcribe and / or translate DNA or RNA. Also included are vectors that provide one or more of the above functions. An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, is transcribed and translated into a polypeptide. An "expression system" usually refers to a suitable host cell that comprises an expression vector capable of producing a desired expression product.

[0072] Terms such as "treatment / treating / treat" are generally used herein to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in the sense of completely or partially preventing a disease or its symptoms, and / or therapeutic, in the sense of partially or completely stabilizing or curing a disease and / or side effects resulting from said disease. As used herein, "treatment" includes any treatment of a disease in a mammal, such as a mouse, rat, rabbit, pig, primate (including humans and other apes, and especially humans), and further includes (a) preventing a disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed, (b) suppressing a disease symptom, (c) halting the progression of the disease, (d) alleviating a disease symptom, (e) causing regression of a disease or condition, or a combination thereof.

[0073] The terms "recipient," "individual," "subject," "host," and "patient" may be used interchangeably herein and refer to any mammalian subject, particularly humans, in need of diagnosis, treatment, or therapy.

[0074] The terms "cancer," "neoplasm," "tumor," and "carcinoma" are used interchangeably herein to refer to cells that exhibit relatively autonomous growth, resulting in an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. In general, cells of interest for detection or treatment in this application include precancerous (e.g., benign), malignant, premetastatic, metastatic, and nonmetastatic cells. The term "normal," as used in the context of "normal cells," refers to cells of an untransformed phenotype or cells that exhibit the morphology of nontransformed cells of the tissue type being examined. A "cancerous phenotype" generally refers to any of a variety of biological phenomena that are characteristic of cancer cells, and these phenomena may vary depending on the type of cancer. A cancerous phenotype is typically identified by abnormalities, such as, for example, cell growth or proliferation (e.g., uncontrolled growth or proliferation), cell cycle regulation, cell motility, cell-cell interactions, or metastasis.

[0075] Less than the complete scope of the disclosure may be claimed for any reason by reserving the right to exclude or qualify individual members of such groups, including subranges or combinations of subranges within groups that may be claimed according to ranges or in a similar manner. Further, less than the complete scope of the disclosure may be claimed for any reason by reserving the right to exclude or qualify individual substituents, analogs, compounds, ligands, structures, or groups thereof, or any members of a claimed group.

[0076] Throughout this disclosure, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications are incorporated by reference in their entireties into this disclosure to more fully describe the state of the art as known to those skilled in the art as of the date of this disclosure. In the event of a conflict between the cited patents, patent applications, and publications and this disclosure, the disclosure shall control.

[0077] II. Antigen-binding units, compositions, and methods In one embodiment, the disclosure provides an antigen binding unit comprising a light chain complementarity determining region (CDR) and a heavy chain CDR.

[0078] In another embodiment, the disclosure provides an antigen binding unit comprising a light chain CDR and a heavy chain CDR, wherein said antigen binding unit specifically binds to CCR8 and / or blocks binding of CCL1 to CCR8.

[0079] In yet another embodiment, the disclosure provides an antigen binding unit comprising a light chain CDR and a heavy chain CDR, and a fragment crystallizable (Fc) region operably linked to an antigen binding unit, wherein the antigen binding unit specifically binds to CCR8 and / or blocks binding of CCL1 to CCR8.

[0080] In yet another embodiment, the disclosure provides an antigen binding unit comprising a light chain CDR and a heavy chain CDR, and a fragment crystallizable (Fc) region, wherein the Fc region comprises a mutation at at least one amino acid position and is operably linked to an antigen binding unit, wherein the antigen binding unit specifically binds to CCR8 and / or blocks binding of CCL1 to CCR8, and wherein the mutation in the Fc region enhances antibody-dependent cellular cytotoxicity (ADCC).

[0081] In some embodiments, the fragment crystallizable (Fc) region comprises at least a portion of a human immunoglobulin constant region (Fc), with or without a mutation. In some embodiments, the mutation comprises an S239D mutation, an I332E mutation, an L235V mutation, an F243L mutation, an R292P mutation, a Y300L mutation, a P396L mutation, or a combination thereof.

[0082] In some embodiments, the fragment crystallizable (Fc) region comprises at least a portion of human IgG1 (Fc) with no mutations (SEQ ID NO: 119) or with mutations including S239D / I332E (SEQ ID NO: 120) or L235V / F243L / R292P / Y300L / P396L (SEQ ID NO: 121), wherein the mutations in the Fc region enhance antibody-dependent cellular cytotoxicity (ADCC).

[0083] In some embodiments, the fragment crystallizable (Fc) region comprises an amino acid sequence selected from SEQ ID NOs: 119-121: SEQ ID NO:119 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:120 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:121 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0084] In any of the embodiments disclosed herein, the antigen binding unit comprises a light chain CDR (designated as vL or LC in Tables 1-3). The light chain CDR may be a complementarity determining region of the light chain of the antigen binding unit. The light chain CDR may comprise a contiguous sequence of amino acid residues, or a contiguous sequence of two or more amino acid residues, optionally flanked by non-complementarity determining regions, such as framework regions. In some examples, the light chain CDR comprises two or more light chain CDRs, referred to as light chain CDR-1, CDR-2, etc. In a preferred example, the light chain CDR comprises three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively. In some examples, a group of CDRs present on a common light chain may be collectively referred to as light chain CDRs.

[0085] In other embodiments, the antigen-binding unit comprises a heavy chain CDR (referred to as vH or HC in Tables 1-3). The heavy chain CDR may be a complementarity determining region of the heavy chain of the antigen-binding unit. The heavy chain CDR may comprise a contiguous sequence of amino acid residues, or a contiguous sequence of two or more adjacent amino acid residues, separated by non-complementarity determining regions, such as framework regions, as needed. In some examples, the heavy chain CDR comprises two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, etc. In a preferred example, the heavy chain CDR comprises three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively. In some examples, a group of CDRs present on a common heavy chain may be collectively referred to as heavy chain CDRs.

[0086] In other embodiments, the subject antigen-binding unit specifically binds to CCR8. As used herein, CCR8 can also refer to orthologs, homologs, codon-optimized, truncated, fragmented, mutated, or other known derivatives of the CCR8 sequence. For example, CCR8 can be human CCR8, mouse CCR8, or cynomolgus CCR8.

[0087] The binding specificity can be determined by the complementarity determining regions or CDRs (such as light chain CDRs or heavy chain CDRs). In many cases, the binding specificity is determined by the light chain CDRs and the heavy chain CDRs. A particular combination of heavy and light chain CDRs provides a particular binding pocket that confers higher affinity and / or specificity for CCR8 compared to other reference antigens.

[0088] The binding of an antigen-binding unit to CCR8 can be characterized or expressed by any method known in the art. For example, the binding can be characterized by binding affinity, which can be the strength of the interaction between the antigen-binding unit and the antigen. The binding affinity can be determined by any method known in the art, such as an in vitro binding assay. For example, the binding affinity of the antigen-binding units disclosed herein can be determined when assayed in an in vitro binding assay using cells expressing CCR8. The binding affinity of a subject antigen-binding unit can be expressed in terms of Kd, which is the equilibrium dissociation constant between an antibody and its respective antigen. In some cases, the antigen-binding units disclosed herein specifically bind to CCR8 with a Kd in the range of about 10 μM to about 1 fM. For example, an antigen binding unit can specifically bind to CCR8 with a Kd of less than about 10 μM, 1 μM, 0.1 μM, 10 nM, 1 nM, 0.1 nM, 10 pM, 1 pM, 0.1 pM, 10 fM, 1 fM, 0.1 fM, or 0.1 fM.

[0089] In some embodiments, the antigen binding unit reduces or blocks binding of CCR8 to CCL1, thereby inhibiting recruitment of regulatory T cells and / or recruitment of signaling factors, such as β-arrestin.

[0090] In some embodiments, an antigen binding unit comprises a light chain CDR and a heavy chain CDR. A subject antigen binding unit can comprise any of the sequences listed in Table 1. Additionally or alternatively, a subject antigen binding unit can comprise a sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (including any range between these endpoints, e.g., 60% to 85%) to any of the sequences listed in Tables 1-3.

[0091] In some embodiments, an antigen binding unit comprises a light chain CDR and a heavy chain CDR, wherein the light chain CDR and the heavy chain CDR comprise LC-CDRs and HC-CDRs selected from any combination of the light chain CDR (vL or LC) or heavy chain CDR (vH or HC) sequences listed in Tables 1 and 2, respectively.

[0092] [Table 1-1]

[0093] [Table 1-2]

[0094] [Table 1-3]

[0095] [Table 1-4]

[0096] [Table 1-5]

[0097] [Table 1-6]

[0098] [Table 2-1]

[0099] [Table 2-2]

[0100] [Table 2-3]

[0101]

Table 2-4

[0102]

Table 2-5

[0103]

Table 2-6

[0104]

Table 2-7

[0105]

Table 2-8

[0106]

Table 2-9

[0107]

Table 3-1

[0108]

Table 3-2

[0109]

Table 3-3

[0110]

Table 3-4

[0111] [Table 3-5]

[0112] [Table 3-6]

[0113] [Table 3-7]

[0114] [Table 3-8]

[0115] In some embodiments, the subject antigen-binding units are monoclonal antigen-binding units, polyclonal antigen-binding units, humanized antigen-binding units, chimeric antigen-binding units, monovalent antigen-binding units, multivalent antigen-binding units, bispecific antigen-binding units, or any combination thereof. Antigen-binding units can take a variety of formats, including, but not limited to, scFv, Fab', single-chain Fab (scFab'), Fd or F(ab')2, sFC, Fv, ccFv. Such antibody-binding units can be generated from whole immunoglobulins by lysine, pepsin, papain, or other protease cleavage.

[0116] Additionally, antigen-binding units can be engineered using recombinant immunoglobulin technology. For example, "Fv" immunoglobulins can be generated by linking a variable light chain region to a variable heavy chain region via a peptide linker. For example, the peptide linker can be a polyglycine or another sequence that does not form an α-helix or β-sheet motif. As described in U.S. Pat. No. 6,147,203, Vv immunoglobulins can be generated by linking a variable light chain region to a variable heavy chain region via a peptide linker. For example, the peptide linker can be a polyglycine or another sequence that does not form an α-helix or β-sheet motif. H Area and V LFv can also be made with stabilizing disulfide bonds between regions, and the patent is fully incorporated herein by reference. Any of these antigen-binding units can be used. In some embodiments, the antigen-binding unit can be a complete immunoglobulin with two light chains paired with two heavy chains.

[0117] The antigen-binding unit may be a heteromultimer comprising a light chain polypeptide and a heavy chain polypeptide. Examples of antigen-binding units include, but are not limited to, (i) a ccFv fragment stabilized by a heterodimerization sequence disclosed in U.S. Pat. No. 6,833,441 (incorporated herein in its entirety), (ii) other monovalent and multivalent molecules comprising at least one ccFv fragment described herein, (iii) a Fab fragment consisting of VL, VH, CL, and CH1 domains, (iv) an Fd fragment consisting of VH and CH1 domains, (v) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bonds at the hinge region, and (vii) a diabody.

[0118] Polyclonal antibodies can be generated by standard protocols by injecting the antigen composition into a production animal. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. When using whole proteins or larger portions of proteins, antibodies can be generated by immunizing production animals with the protein and a suitable adjuvant (e.g., Freund's adjuvant, Freund's complete adjuvant, oil-in-water emulsion, etc.). When using smaller peptides, it is advantageous to conjugate the peptide to a larger molecule to create an immunostimulatory conjugate. Common conjugated proteins that are commercially available for such purposes include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). Peptides derived from the complete sequence can be used to generate antibodies against specific epitopes. Alternatively, to generate antibodies against relatively short peptide portions of protein targets, conjugating the polypeptide to a carrier protein (e.g., ovalbumin, BSA, KLH) can elicit a superior immune response.

[0119] Polyclonal or monoclonal antigen-binding units or antibodies can be produced from animals genetically engineered to produce human immunoglobulins. Transgenic animals can be produced by first producing a "knock-out" animal that does not produce the animal's natural antibodies, and then stably transforming the animal with human antibody loci (e.g., by using human artificial chromosomes). In such cases, only human antibodies are produced by the animal. Techniques for producing such animals and obtaining antibodies therefrom are described in U.S. Patent Nos. 6,162,963 and 6,150,584, which are fully incorporated herein by reference. Such antibodies can be referred to as human xenoantibodies.

[0120] Alternatively, the antigen binding units can be generated from a phage library containing human variable regions, see U.S. Patent No. 6,174,708, which is incorporated herein by reference in its entirety.

[0121] In some aspects of the embodiments disclosed herein, the antigen-binding unit is produced by a hybridoma. For example, the antigen-binding unit disclosed herein can be produced by a hybridoma selected from the group consisting of hybridomas expressing one of the antigen-binding units listed in Table 1.

[0122] In the case of monoclonal antigen-binding units or monoclonal antibodies, hybridomas can be formed by isolating stimulated immune cells, such as cells from the spleen of an inoculated animal. These cells can then be fused with immortalized cells, such as myeloma cells or transformed cells, that can replicate indefinitely in cell culture, thereby generating immortal immunoglobulin-secreting cell lines. The immortalized cell lines utilized can be selected to be deficient in enzymes required for utilization of specific nutrients. Many such cell lines (such as myelomas) are known to those skilled in the art and include, for example, thymidine kinase (TK) or hypoxanthine-guanine phosphoriboxyltransferase (HGPRT). These deficiencies allow the selection of fused cells for their ability to grow, for example, on hypoxanthine aminopterin thymidine medium (HAT).

[0123] Additionally, antigen-binding units may be produced by genetic engineering. Humanized, chimeric, or xenogeneic human antigen-binding units are contemplated, which would elicit less of an immune response when administered to humans.

[0124] The antigen-binding units disclosed herein may be less prone to elicit undesirable immune responses in humans, such as anaphylactic shock, and less prone to priming immune responses that would prevent repeated administration of an antibody therapeutic or imaging agent (e.g., human anti-mouse antibody "HAMA" responses). Such antigen-binding units include, but are not limited to, humanized, chimeric, or xenogeneic human antigen-binding units.

[0125] Chimeric antigen binding units or chimeric antibodies can be produced, for example, by recombinant means combining mouse variable light and heavy chain regions (VL and VH) obtained from a mouse (or other animal derived) hybridoma clone with human constant light and heavy chain regions to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art and can be accomplished by standard means (e.g., as described in U.S. Pat. No. 5,624,659, incorporated herein by reference in its entirety).

[0126] The term "humanized" as applied to non-human (e.g., rodent or primate) antibodies refers to hybrid immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies are most often human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, or primate having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance and to minimize immunogenicity when introduced into the human body. In some examples, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, such as human IgG1.

[0127] Humanized antibodies can be designed to contain human-like immunoglobulin domains and incorporate only the complementarity determining regions of an animal-derived antibody. This can be achieved by carefully examining the sequences of the hypervariable loops of the variable regions of the monoclonal antigen-binding units or monoclonal antibodies and adapting them to the structure of human antigen-binding units or human antibody chains. See, for example, U.S. Patent No. 6,187,287, which is incorporated herein by reference in its entirety.

[0128] Methods for humanizing non-human antibodies are well known in the art. A "humanized" antibody is an antibody in which at least part of the sequence has been altered from its original form to resemble a human immunoglobulin. In some versions, the heavy (H) and light (L) chain constant (C) regions are replaced with human sequences. This may be a fusion polypeptide comprising a variable (V) region and a heterologous immunoglobulin C region. In some versions, the complementarity determining regions (CDRs) comprise non-human antibody sequences, and the V framework regions are also converted to human sequences. See, for example, EP0329400. In some versions, the V regions are humanized by designing consensus sequences of human and mouse V regions and converting residues outside the CDRs that differ between the consensus sequences.

[0129] In principle, framework sequences from humanized antibodies can be used as templates for CDR grafting, but it has been demonstrated that direct replacement of CDRs into such frameworks can significantly reduce binding affinity to the antigen. Glaser et al.(1992) J.Immunol.149:2606;Tempest et al.(1992) Biotechnology 9:266;and Shalaby et al.(1992) J.Exp.Med.17:217. The more homologous a human antibody (HuAb) is to the original murine antibody (muAb), the less likely it is that the human framework will introduce affinity-reducing distortions into the murine CDRs. Based on sequence homology searches against antibody sequence databases, HuAb IC4 shows good framework homology with muM4TS.22. However, other highly homologous HuAbs are also suitable, especially kappa light chains from human subgroup I or heavy chains from human subgroup III. Kabat et al.(1987). Various computer programs such as ENCAD (Levitt et al. (1983) J. Mol. Biol. 168:595) can be used to predict the ideal sequence of the V region. HuAbs with various variable (V) regions have been considered. It is within the skill of one of ordinary skill in the art to determine suitable V region sequences and optimize these sequences. Methods for obtaining antibodies with reduced immunogenicity are also described in U.S. Pat. No. 5,270,202 and European Patent No. 699,755.

[0130] Humanized antibodies can be prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are familiar to those skilled in the art. Computer programs are available to illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows the analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., residues that influence the ability of the candidate immunoglobulin to bind to an antigen. In this way, FR residues can be selected and combined from the consensus and import sequences to achieve desired antibody properties, such as improved affinity for the target antigen.

[0131] The process of humanization of a subject's antigen-binding unit can be as follows: The most suitable germline acceptor heavy and light chain variable regions are selected based on the homology, canonical structure, and physical properties of the human antibody germline to be grafted. Computer modeling of the mVH / VL to the grafted hVH / VL is performed to generate a prototype humanized antibody sequence. If modeling indicates that framework backmutations are required, a second variant is generated containing the indicated FW changes. DNA fragments encoding the selected germline framework and mouse CDRs are synthesized. The synthesized DNA fragments are subcloned into an IgG expression vector and the sequence is confirmed by DNA sequencing. The humanized antibody is expressed in cells such as 293F and the protein is tested in MDM phagocytosis assays, antigen binding assays, etc. The humanized antigen-binding units are compared in antigen-binding affinity to the parent antigen-binding units, for example by FACS on cells expressing the target antigen. If the affinity is more than 2-fold lower than the parent antigen-binding unit, a second round of humanized variants can be generated and tested as described above.

[0132] As mentioned above, antigen-binding units are either "monovalent" or "multivalent". The former have one binding site per antigen-binding unit, whereas the latter contain multiple binding sites that can bind to multiple antigens of the same or different types. Depending on the number of binding sites, an antigen-binding unit can be bivalent (two antigen-binding sites), trivalent (three antigen-binding sites), tetravalent (four antigen-binding sites), etc.

[0133] Multivalent antigen-binding units can be further classified based on binding specificity. A "monospecific" antigen-binding unit is a molecule capable of binding to one or more antigens of the same type. A "multispecific" antigen-binding unit is a molecule with binding specificity for at least two different antigens. Such molecules usually only bind to two different antigens (i.e., bispecific antigen-binding units), but as used herein, antibodies with additional specificities, such as trispecific antibodies, are also encompassed by this term. The present disclosure further provides multispecific antigen-binding units. A multispecific antigen-binding unit is a multivalent molecule capable of binding to at least two different antigens. Preferred multispecific antigen-binding units are bispecific and trispecific molecules that exhibit binding specificity for two and three different antigens, respectively.

[0134] In some embodiments, the antigen binding unit is a bispecific antigen binding unit, and the antigen binding unit specifically binds to CCR8 and a second antigen. In some examples, the second antigen binds to CCR8 of a different species. For example, the bispecific antigen binding unit can bind to human CCR8 and cynomolgus monkey CCR8. Alternatively, the bispecific antigen binding unit can include a binding property that binds, for example, to human CCR8, and a blocking property that prevents, for example, CCL1 from binding to human CCR8. Other suitable second antigens include, but are not limited to, tumor cell antigens, immune cell antigens, cytotoxic trigger molecules, toxins, fibrinolytic agents, cell surface receptors, infectious disease targets, vaccine adjuvants, diagnostic agents, detection molecules, and reporter molecules.

[0135] Polynucleotides and Vectors In some embodiments, an isolated nucleic acid is provided that encodes any of the antigen binding units disclosed herein. In another embodiment, a vector is provided that comprises a nucleic acid sequence that encodes any of the antigen binding units disclosed herein. In some embodiments, an isolated nucleic acid is provided that encodes the light chain CDRs and the heavy chain CDRs of the antigen binding units disclosed herein.

[0136] The antigen-binding unit of interest can be prepared by recombinant DNA technology, synthetic chemical technology, or a combination thereof. For example, sequences encoding the desired components of the antigen-binding unit, including light chain CDR and heavy chain CDR, are usually cloned and assembled into expression vectors using standard molecular techniques known in the art. These sequences can be assembled from other vectors encoding the protein sequence of interest, from fragments generated by PCR using the respective template nucleic acid, or by synthetic oligonucleotides encoding the sequence of interest. The expression system can be created by transfecting an expression vector containing the antigen-binding unit of interest into a suitable cell.

[0137] Nucleotide sequences corresponding to various regions of the light or heavy chains of existing antibodies can be readily obtained and sequenced using conventional techniques, including but not limited to hybridization, PCR, and DNA sequencing. Hybridoma cells producing monoclonal antibodies serve as a preferred source of antibody nucleotide sequences. A vast number of hybridoma cells producing an array of monoclonal antibodies can be obtained from public or private repositories. The largest depository is the American Type Culture Collection (atcc.org), which offers a variety of well-characterized hybridoma cell lines. Alternatively, antibody nucleotides can be obtained from immunized or non-immunized rodents or humans forming organs such as spleen or peripheral blood lymphocytes. Specific techniques applicable to the extraction and synthesis of antibody nucleotides are described in Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837, Larrick et al. (1989) Biochem. Biophys. Res. Commun. 160:1250-1255, Sastry et al. (1989) Proc. Natl. Acad. Sci., USA 86:5728-5732, and U.S. Patent No. 5,969,108.

[0138] Polynucleotides encoding antigen-binding units can also be modified, for example, by replacing homologous non-human sequences with coding sequences for human heavy and light chain constant regions, thus preparing chimeric antibodies that retain the binding specificity of the original antigen-binding units.

[0139] It is also understood that the polynucleotides include those that encode functional equivalents of the exemplified polypeptides and fragments thereof. Functionally equivalent polypeptides include those that enhance, reduce, or do not significantly affect the properties of the polypeptide encoded thereby. Functional equivalents may be polypeptides with conservative amino acid substitutions, analogs, including fusions, and variants.

[0140] Due to the degeneracy of the genetic code, there may be considerable variation in the nucleotides of the antigen-binding unit coding sequence, as well as in the sequences suitable for constructing polynucleotides and vectors. Sequence variants may have modified DNA or amino acid sequences, one or more substitutions, deletions, or additions, the net effect of which is to retain the desired antigen-binding activity. For example, various substitutions may be made in the coding region that do not change the encoded amino acid or result in conservative changes. These substitutions are included in the disclosure of this specification. Conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative substitutions effectively change one or more amino acid residues contained in the generated polypeptide, but are not expected to interfere with the antigen-binding activity of the generated antigen-binding unit. Nucleotide substitutions that do not change the encoded amino acid residues are useful for optimizing gene expression in various systems. Appropriate substitutions are known to those of skill in the art and are made, for example, to reflect the preferred codon usage in the expression system.

[0141] Optionally, the recombinant polynucleotide can contain heterologous sequences that facilitate detection of expression and purification of the gene product. Examples of such sequences are known in the art and include sequences encoding reporter proteins such as β-galactosidase, β-lactamase, chloramphenicol acetyltransferase (CAT), luciferase, green fluorescent protein (GFP), and derivatives thereof. Other heterologous sequences that facilitate purification can encode epitopes such as Myc, HA (from influenza virus hemagglutinin), His-6, FLAG, or the Fc portion of immunoglobulin, glutathione S-transferase (GST), and maltose binding protein (MBP).

[0142] The polynucleotides disclosed herein can be linked to various chemically functional moieties as described above. Commonly used moieties include labels capable of generating detectable signals, signal peptides, agents that enhance immunological reactivity, agents that facilitate binding to solid supports, vaccine carriers, biological response modifiers, paramagnetic labels and drugs. These moieties can be covalently linked to the polynucleotides by recombinant or other means known in the art.

[0143] The polynucleotide can include additional sequences, such as additional coding sequences within the same transcription unit, regulatory elements such as promoters, ribosome binding sites, polyadenylation sites, additional transcription units under the control of the same or different promoters, sequences allowing for cloning, expression, and transformation of host cells, and any such constructs desirable to provide the embodiment.

[0144] Polynucleotides can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequence data provided herein to obtain desired polynucleotides by using a DNA synthesizer or ordering from a commercial service.

[0145] The polynucleotide containing the desired sequence can be inserted into a suitable vector, which can then be introduced into a suitable host cell for replication and amplification. Thus, a variety of vectors containing one or more polynucleotides are provided. Also provided is a selectable library of expression vectors that includes at least one vector encoding the antigen-binding unit disclosed herein.

[0146] The vector generally contains the transcriptional or translational control sequences necessary for expressing the antigen-binding unit. Suitable transcriptional or translational control sequences include, but are not limited to, an origin of replication, a promoter, an enhancer, a repressor binding region, a transcription initiation site, a ribosome binding site, a translation initiation site, a transcription and translation termination site, etc.

[0147] The choice of promoter depends largely on the host cell into which the vector is introduced. It is also possible to utilize the promoter normally associated with the desired light or heavy chain gene, as long as such control sequences are compatible with the host cell system. Cell-specific or tissue-specific promoters can also be used. Those skilled in the art have described and used a wide variety of tissue-specific promoters. Examples of promoters that function in selective animal cells include hepatocyte-specific promoters and cardiac muscle-specific promoters. Depending on the choice of recipient cell type, those skilled in the art will know other suitable cell-specific or tissue-specific promoters that can be applied to the construction of the expression vector.

[0148] Using known molecular cloning or genetic engineering techniques, suitable transcription control sequences, enhancers, terminators, or any other genetic elements known in the art can be incorporated in operative relationship with the complete, optionally expressed, selectable fusion gene. In addition to the above elements, vectors can contain selectable markers (e.g., genes encoding proteins necessary for the survival or growth of a host cell transformed with the vector), although such marker genes can be carried on another polynucleotide sequence co-introduced into the host cell.

[0149] The polynucleotides and vectors have several specific uses. They are useful, for example, in expression systems for the production of antigen-binding units. Such polynucleotides are useful as primers for the amplification of desired polynucleotides. Furthermore, polynucleotides are useful in pharmaceutical compositions, including vaccines, diagnostics, and drugs.

[0150] Host cells can be used, inter alia, as repositories of subject polynucleotides, vectors, or as vehicles for producing and screening desired antigen-binding units based on antigen-binding specificity.

[0151] Thus, there is provided a method for identifying an antigen-binding unit that immunoreacts with a desired antigen, which method can include the steps of: (a) providing a library of genetically diverse antigen-binding units comprising at least one subject antigen-binding unit; (b) contacting the library of antigen-binding units with a desired antigen; and (c) detecting specific binding between the antigen-binding unit and said antigen, thereby identifying an antigen-binding unit that immunoreacts with the desired antigen.

[0152] The ability of an antigen-binding unit to specifically bind to a desired antigen can be tested by a variety of procedures well established in the art. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Gherardi et al. (1990) J. Immunol. Meth. 126:61-68. Usually, an antigen-binding unit exhibiting a desired binding specificity can be directly detected by immunoassay, for example, by reacting a labeled antigen-binding unit with an antigen immobilized on a solid support or substrate. In general, the substrate to which the antigen is attached is made of a material that exhibits low levels of non-specific binding during immunoassay. Exemplary solid supports are made of one or more of the following types of materials: plastic polymers, glass, cellulose, nitrocellulose, semiconductor materials, and metals. In some examples, the substrate is a Petri dish, a chromatography bead, a magnetic bead, or the like.

[0153] In such solid-phase assays, unreacted antigen-binding units are removed by washing. However, in liquid-phase assays, unreacted antigen-binding units are removed by filtration or other separation techniques such as chromatography. After binding of the antigen to the labeled antigen-binding units, the amount of bound label is measured. A variation of this technique is the competitive assay, in which the antigen is bound to the original binding molecule until saturation. When a population of antigen-binding units from a subject is introduced into the complex, only those that exhibit a higher binding affinity are able to compete and therefore remain bound to the antigen.

[0154] Alternatively, specific binding to a particular antigen can be assessed by cell sorting, which involves presenting the desired antigen to the cells to be sorted, then labeling the target cells with an antigen-binding unit conjugated to a detectable drug, followed by separation of the labeled cells from the unlabeled cells in a cell sorter. An elegant method of cell sorting is fluorescence-activated cell sorting (FACS): cells moving in a single file in a thin stream are passed through a laser beam, and the fluorescence of each cell that binds to a fluorescently labeled antigen-binding unit is measured.

[0155] Subsequent analysis of the eluted antigen-binding units may include protein sequencing to delineate the amino acid sequences of the light and heavy chains. Based on the deduced amino acid sequence, the cDNA encoding the antibody polypeptide can be obtained by recombinational cloning methods including PCR, library screening, homology searches in existing nucleic acid databases, or any combination thereof. Commonly used databases include, but are not limited to, GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, HTGS, etc.

[0156] If the library of antigen-binding units is displayed on phage or bacterial particles, the selection is preferably performed using affinity chromatography. This method typically involves binding and then capturing the library of phage antigen-binding units to antigen-coated plates, column matrices, cells, or biotinylated antigens in solution. The phages or bacteria bound to the solid phase are washed and then eluted with soluble haptens, acid, or alkali. Alternatively, the antigen-binding units can be dissociated from the affinity matrix by increasing the concentration of antigen. For certain antigen-binding units with very high affinity or avidity for the antigen, efficient elution may require high pH or mildly reducing solutions, as described in WO92 / 01047.

[0157] The efficiency of selection may depend on a combination of several factors, including the kinetics of dissociation during washing and whether multiple antigen-binding units on a single phage or bacterium can simultaneously bind to antigen on the solid support. For example, antibodies with fast dissociation rates (weak binding affinity) can be retained by shortening washing times, using multivalent display, and increasing the coating density of the antigen on the solid support. Conversely, selection of antigen-binding units with slow dissociation rates (good binding affinity) can be facilitated by increasing washing times, using monovalent phage, and decreasing the coating density of the antigen.

[0158] If desired, the library of antigen-binding units can be preselected against an unrelated antigen to counterselect unwanted antigen-binding units. The library may also be preselected against a related antigen, for example to isolate anti-idiotypic antigen-binding units.

[0159] host cell In some embodiments, the present disclosure provides a host cell that expresses any one of the antigen binding units disclosed herein. The subject host cell typically comprises a nucleic acid encoding any one of the antigen binding units disclosed herein.

[0160] In one embodiment, a host cell is provided that is transfected with the above-mentioned polynucleotide, vector, or library of vectors.Vector can be introduced into suitable prokaryotic or eukaryotic cells by any of several suitable means, including electroporation, microprojectile bombardment; lipofection, infection (if the vector is bound to an infectious agent), transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances.The choice of means for introducing vector often depends on the characteristics of the host cell.

[0161] For most animal cells, any of the above methods are suitable for vector delivery.Preferred animal cells are vertebrate cells, preferably mammalian cells, that can express exogenously introduced gene products in large amounts, for example at milligram levels.Non-limiting examples of preferred cells include NIH3T3 cells, COS cells, HeLa cells, and CHO cells.

[0162] Once introduced into a suitable host cell, expression of the antigen-binding unit can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of the light chain CDR or heavy chain CDR, or the antigen-binding unit, can be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based techniques (see, e.g., U.S. Pat. Nos. 5,405,783, 5,412,087, and 5,445,934) using probes complementary to any region of the antigen-binding unit polynucleotide.

[0163] Vector expression can also be determined by examining the expressed antigen-binding units. For protein analysis, various techniques are available in the art. These include, but are not limited to, radioimmunoassay, ELISA (enzyme-linked immunoradiometric assay), "sandwich" immunoassay, immunoradiometric assay, in situ immunoassay (e.g., using colloidal gold, enzyme, or radioisotope labels), Western blot analysis, immunoprecipitation, immunofluorescence, and SDS-PAGE.

[0164] Preparation of antigen-binding units In some embodiments, there is provided a method of producing any of the antigen binding units disclosed herein, comprising culturing a host cell expressing the antigen binding unit under conditions suitable for expression of the antigen binding unit, and isolating the antigen binding unit expressed by the host cell.

[0165] The expressed antigen-binding unit can be isolated using various protein purification techniques known in the art. Generally, the antigen-binding unit is isolated from the medium as a secreted polypeptide, but if it is directly produced without a signal peptide, it can be recovered from host cell lysates or bacterial periplasm. If the antigen-binding unit is membrane-bound, it can be solubilized by an appropriate detergent solution commonly used by those skilled in the art. The recovered antigen-binding unit may be further purified by salt precipitation (e.g., with ammonium sulfate), ion exchange chromatography (e.g., a cation or anion exchange column run at neutral pH and eluted with a step gradient of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography with tag affinity columns or affinity resins such as protein A, protein G, hydroxyapatite, anti-immunoglobulin, etc.

[0166] Additionally, derivatized immunoglobulins to which detectable moieties such as chemical linkers, fluorescent dyes, enzymes, substrates, chemiluminescent moieties, specific binding moieties such as streptavidin, avidin, or biotin, or drug conjugates may be added, may be utilized in the methods and compositions.

[0167] Further disclosed herein are antigen-binding units conjugated to a chemically functional moiety. Typically, this moiety is a label capable of generating a detectable signal. These conjugated antigen-binding units are useful in detection systems, such as, for example, tumor load quantification, metastatic imaging, and tumor imaging. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, substrate cofactors, and inhibitors. For examples of patents that teach the use of such labels, see U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. These moieties can be covalently bound to the antigen-binding unit, recombinantly bound, or conjugated to the antigen-binding unit via a second reagent, such as a second antibody, protein A, or a biotin-avidin complex.

[0168] Other functional moieties include signal peptides, agents that enhance immune reactivity, agents that facilitate binding to solid supports, vaccine carriers, biological response modifiers, paramagnetic labels and drugs. Signal peptides are short amino acid sequences that direct the passage of newly synthesized proteins to the cell membrane (usually the endoplasmic reticulum of eukaryotic cells, the inner membrane or both the inner and outer membranes of bacteria). Signal peptides can be present at the N-terminal portion of a polypeptide or at the C-terminal portion of a polypeptide and can be enzymatically removed during the biosynthesis of the polypeptide and its secretion from the cell. Such peptides can be incorporated into antigen-binding units to allow the secretion of the synthesized molecule.

[0169] Agents that enhance immune reactivity include, but are not limited to, bacterial superantigens. Agents that facilitate binding to solid supports include, but are not limited to, biotin and avidin. Immunogen carriers include, but are not limited to, physiologically acceptable buffers. Biological response modifiers include cytokines, particularly tumor necrosis factor (TNF), interleukin-2, interleukin-4, granulocyte macrophage colony stimulating factor, and gamma interferon.

[0170] Suitable drug moieties include antitumor agents, non-limiting examples of which include radioisotopes, vinca alkaloids such as vinblastine, vincristine, vindesine sulfate, adriamycin, bleomycin sulfate, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, dactinomycin D, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, lomustine, mechloroethamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbazepam hydrochloride, streptozotocin, taxol, thioguanine, uracil mustard, and the like.

[0171] Immunotoxins containing antigen-binding units can be produced by recombinant means. The production of various immunotoxins is well known in the art, and methods are described, for example, in "Monoclonal Antibody-toxin Conjugates: Aiming the Magic Bullet," Thorpe et al. (1982) Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190; Vitatta (1987) Science 238: 1098-1104, and Winter and Milstein (1991) Nature 349: 293-299. Suitable toxins include, but are not limited to, ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins such as restrictocin, and phospholipase enzymes. See generally, "Chimeric Toxins," Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985).

[0172] Chemically functional moieties can be made recombinantly, for example, by creating a fusion gene that codes for the antigen-binding unit and the functional moiety. Alternatively, the antigen-binding unit can be chemically linked to the moiety by any of a variety of established chemical procedures. For example, if the moiety is a protein, the linkage can be achieved by heterobifunctional crosslinkers, such as SPDP, carbodiimide glutaraldehyde, and the like. The moiety can be covalently linked or conjugated via a second reagent, such as a second antibody, protein A, or biotin-avidin complex. Paramagnetic moieties and their conjugation to antibodies are well known in the art. See, for example, Miltenyi et al. (1990) Cytometry 11:231-238.

[0173] Methods of Use and Treatment CCR8-specific antigen binding units and pharmaceutical compositions containing same can be used in a variety of applications, including, but not limited to, therapeutic and diagnostic applications.

[0174] In one embodiment, a pharmaceutical composition is provided comprising a pharma- ceutically acceptable excipient and any of the antigen-binding units disclosed herein.

[0175] In another embodiment, a method of eradicating immune cells is provided, comprising contacting a population of immune cells with an effective amount of an antigen binding unit described herein.

[0176] In yet another embodiment, a method of eradicating immune cells is provided, comprising contacting a population of immune cells with an effective amount of a pharmaceutical composition described herein.

[0177] In embodiments, the immune cells are present in a subject, such as, for example, a human patient in need of treatment for a disease caused by aberrant immune cells and / or in need of removal of immune cells.

[0178] In an embodiment, the immune cells are regulatory T cells (Tregs). Tregs may exist in a subject as resident tissue Tregs, for example, in tumor tissue as a subpopulation of resident tumor infiltrating lymphocytes (TILs).

[0179] In embodiments, migration of Tregs, elimination of Tregs by antibody-dependent cellular cytotoxicity (ADCC), or both.

[0180] In embodiments, a method of treating cancer in a subject comprises administering an effective amount of an antigen binding unit described herein to a subject in need thereof, and optionally repeating the administration step for a period of time, e.g., once daily, once weekly, once monthly, periodically for 1, 2, 3, 4, 5, 6 months, or until the subject is free of cancer.

[0181] In embodiments, a method of treating cancer in a subject comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and any of the antigen binding units described herein, in embodiments, the administering step is repeated according to a regimen effective to treat cancer, as evidenced by the subject being free of cancer.

[0182] In an embodiment, the subject is a human patient in need of anti-cancer therapy. The cancer can be a blood cancer or a solid tumor. Blood cancer includes, but is not limited to, leukemia, lymphoma (such as non-Hodgkin's lymphoma), and myeloma. Solid tumors include, but are not limited to, colorectal cancer (CRC), spleen cancer, breast cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, melanoma, bile duct cancer, gallbladder cancer, thyroid cancer, sarcoma, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, thymic cancer, liver cancer, testicular cancer, urothelial cancer, esophageal tumor, and gastric tumor. In most cases, the effective amount is determined empirically by testing methods well known in the art. In one aspect, the antigen-binding unit is administered at a dosage of about 0.1 mg to about 10 mg per kg of body weight.

[0183] Treatment of cancer can be evidenced by inhibition of cancer cell proliferation, including, but not limited to, inhibition of cancer cell proliferation, and reduction in the incidence of non-cancerous cells becoming cancerous cells. Achievement of inhibition of cancer cell proliferation can be determined by [ 3 The efficacy of the agent can be readily determined using known assays, including, but not limited to, incorporation of [H]-thymidine, counting cell numbers over a period of time, detection and / or measurement of markers associated with AML, etc. Whether an agent or a particular amount of an agent is effective in treating cancer can be assessed using a variety of known cancer diagnostic assays, including, but not limited to, biopsy, contrast x-ray, CAT scan, detection of tumor markers associated with cancer in the individual's blood, etc. The agent can be administered systemically or locally, and is typically administered systemically.

[0184] In embodiments, the treatment of cancer can be evidenced by a reduction in tumor volume. Tumor volume can be determined using any method known in the art. For example, tumor volume can be determined by measuring the tumor using a caliper. In such cases, two dimensions of the tumor can be measured, and the tumor volume can be calculated using the formula V=0.5axb 2 The diameter of the sphere can be determined using the formula: a = b , where a and b are the first and second diameters. In some cases, the first diameter is the major diameter or the larger of the two diameters. In some cases, the second diameter is the minor diameter or the smaller of the two diameters.

[0185] In embodiments, the treatment of cancer can be evidenced by a reduction in tumor volume. In some cases, the tumor volume is reduced by a percentage ranging from 1% to 100%. In some instances, the tumor volume is reduced by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some instances, the tumor volume is reduced by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0186] In embodiments, the comparison of the effect of the subject antigen-binding unit and the reference antigen-binding unit can be determined by calculating the anti-tumor effect. In such cases, the tumor volume can be measured as described above. Alternatively, different parameters of tumor size or another suitable characteristic of the tumor can be determined or measured. When dealing with a quantifiable characteristic such as tumor volume, the anti-tumor effect can be determined using the formula: T / C, where T is the measurement selected for the treatment group (e.g., tumor volume) and C is the measurement selected for the control group (e.g., tumor volume). The anti-tumor effect can be determined over any desired period of time and can be determined using an average value from any desired number of samples. The anti-tumor effect can be expressed as a number or a percentage. In some examples, the anti-tumor effect may be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some examples, the anti-tumor effect may be at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some examples, the anti-tumor effect is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0187] The compositions disclosed herein, e.g., antigen-binding units and pharmaceutical compositions, can be administered using any medically appropriate procedure, such as intravascular (intravenous, intraarterial, intracapillary) administration, injection into lymph nodes, etc. Intravascular injection can be by intravenous or intraarterial injection. The effective amount of the composition administered to a particular patient will depend on a variety of factors, some of which vary from patient to patient and can be empirically determined. The dosage of the composition will depend on the determined treatment regimen, the route of administration, the nature of the therapeutic agent, the sensitivity of the tumor to the therapeutic agent, etc. The available LD for the antigen-binding units disclosed herein can be used to administer the compositions to a patient.50 Using animal data and other information, the clinician can determine the maximum safe dosage for an individual depending on the route of administration. For example, an intravenous dose may be higher than a topical dose, given the greater volume of bodily fluid into which the therapeutic composition is administered. Similarly, compositions that are rapidly cleared from the body may be administered at higher doses or in repeated doses to maintain therapeutic concentrations. Using routine skill, a competent clinician can optimize the dosage of a particular composition.

[0188] Methods of combination therapy are contemplated, using agents known to modulate other pathways, or other components of the same pathway, or sets of overlapping target enzymes, in combination with the subject's antigen-binding units or pharmaceutical compositions comprising the subject's antigen-binding units. In one embodiment, such therapies include, but are not limited to, combinations of one or more antigen-binding units of the present disclosure with chemotherapeutic agents, therapeutic antibodies, and radiation therapy to provide synergistic or additive therapeutic effects.

[0189] Currently, many chemotherapeutic agents are known in the art and can be used in combination with a subject antigen binding unit, hi some embodiments, the chemotherapeutic agent is selected from the group consisting of antimitotic agents, alkylating agents, metabolic inhibitors, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antihormones, angiogenesis inhibitors, and antiandrogens.

[0190] Non-limiting examples include chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as Gleevec® (imatinib mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib), and adriamycin, as well as many other chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylolmelamine; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine, methylmel ... Nitrogen mustards such as chlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomysins, actinomycin, autramycin, azaserine, bleomycin, cactinomycin C, calicheamicin, carabicin, carminomycin, carzinophilin, and casodex. TM), chromomycin, dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, and other antibiotics; methotrexate and 5-fluorouracil (5-FU ); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calstarone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides glycoside); aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetateacetate);etoglucid;gallium nitrate;hydroxyurea;lentinan;lonidamine;mitoguazone;mitoxantrone;mopidamol;nitracrine;pentostatin;phenamet;pirarubicin;podophyllinic acid;2-ethylhydrazide;procarbazine;PSK.RTM;razoxane;sizofuran;spirogermanium;tenuazonic acid acid); triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, NJ), docetaxel (TAXOTERET™, Rhone-Poulenc Rorer, Antony, France); retinoic acid; esperamicin; capecitabine; as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. Suitable chemotherapeutic cell modulating agents also include anti-hormonal agents that modulate or inhibit the action of hormones on tumors, such as tamoxifen (Nolvadex™), raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY.antiestrogens such as 117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); These include ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11); the topoisomerase inhibitor RFS2000; and difluoromethylornithine (DMFO). Optionally, the antigen binding unit or pharmaceutical composition of the disclosure is selected from the group consisting of Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Tasotere®, ABVD, AVICINE, abagovomab, acridine carboxamide, adecatumab, 17-N-allylamino-17-demethoxygeldanamycin, radium 223 chloride (alfaradin), alvocidib, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, amonafide, anthracenedione, anti-CD22 immunotoxin, antineoplastic drugs, antineoplastic herbs, apaziquone, atiprimod, azathioprine, belotecan, bendamustine, BIBW2992, biricodar, brostallicin, bryostatin, buthionine sulfoximine, CBV (chemotherapy), calyculin, cell cycle non-specific antitumor agent, dichloroacetic acid, discodermolide, elsamitrucin, enocitabine, epothilone, eribulin, everolimus, exatecan, exisulind, ferruginol, forodesine, fosfestrol, ICE chemotherapy regimen, IT-101, imexon, Imiquimod, indolocarbazole, irofulven, laniquidar, larotaxel, lenalidomide, lucanthone, lurtotecan, mafosfamide, mitozolomide, nafoxidine, nedaplatin, olaparib, ortataxel, PAC-1, papaya, pixantrone, proteasome inhibitors, rebeccamycin, resiquimod, rubitecan, SN-38, salinosporamide A, sapacitabine, Stanford V It may be used in combination with commonly prescribed anticancer drugs such as prasugrel, swainsonine, talaporfin, tariquidar, tegafur-uracil, temodar, tesetaxel, triplatin tetranitrate, tris(2-chloroethyl)amine, troxacitabine, uramustine, vadimezan, vinflunine, ZD6126 or zosuquidar.

[0191] The present disclosure further relates to methods of using the subject antigen binding units or pharmaceutical compositions provided herein in combination with radiation therapy to inhibit abnormal cell growth or treat hyperproliferative diseases in a mammal. Techniques for administering radiation therapy are known in the art and these techniques can be used in the combination therapy described herein.

[0192] Radiation therapy can be administered by one of several methods or a combination of methods, including, but not limited to, external beam radiation therapy, internal radiation therapy, implant radiation, stereotactic radiosurgery, total body radiation therapy, radiotherapy, and permanent or temporary interstitial brachytherapy. As used herein, the term "brachytherapy" refers to radiation therapy administered by spatially confined radioactive material inserted into the body at or near the site of a tumor or other proliferative tissue disease. This term includes, but is not limited to, exposure to radioisotopes (e.g., At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and radioisotopes of Lu). Radiation sources suitable for use as cell conditioning agents of the present disclosure include both solid and liquid sources. As non-limiting examples, the radioactive source can be a radionuclide such as I-125, I-131, Yb-169, Ir-192 as a solid source, I-125 as a solid source, or other radionuclides that emit photons, beta particles, gamma rays, or other therapeutic radiation. The radioactive material can be a fluid made from any solution of the radionuclide, such as a solution of I-125 or I-131, or the radioactive fluid can be produced using a slurry of a suitable fluid containing small particles of a solid radionuclide, such as Au-198, Y-90, etc. Additionally, the radionuclide can be embodied in a gel or radioactive microspheres.

[0193] The antigen binding units or pharmaceutical compositions of the disclosure may be used in combination with one or more agents selected from anti-angiogenic agents, signal transduction inhibitors, anti-proliferative agents, glycolysis inhibitors, or autophagy inhibitors.

[0194] Antiangiogenic agents, such as MMP-2 (matrix metalloprotease 2) inhibitors, MMP-9 (matrix metalloprotease 9) inhibitors, and COX-11 (cyclooxygenase 11) inhibitors, can be used in combination with the antigen binding units of the present disclosure and the pharmaceutical compositions described herein. Antiangiogenic agents include, for example, rapamycin, temsirolimus (CCI-779), everolimus (RAD001), sorafenib, sunitinib, and bevacizumab. Examples of useful COX-II inhibitors include Celebrex (Celebrex™, alecoxib), valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are disclosed in WO 96 / 33172 (published October 24, 1996), WO 96 / 27583 (published March 7, 1996), European Patent Application No. 97304971.1 (filed July 8, 1997), European Patent Application No. 99308617.2 (filed October 29, 1999), WO 98 / 07697 (published February 26, 1998), WO 98 / 03516 (published March 7, 1996), and the like. WO98 / 34918 (published August 13, 1998), WO98 / 34915 (published August 13, 1998), WO98 / 33768 (published August 6, 1998), WO98 / 30566 (published July 16, 1998), European Patent Publication No. 606,046 (published July 13, 1994), European Patent Publication No. 931,788 (published July 28, 1999), WO90 / 0571 9 (published 31 May 1990), WO99 / 52910 (published 21 October 1999), WO99 / 52889 (published 21 October 1999), WO99 / 29667 (published 17 June 1999), PCT International Application No. PCT / IB98 / 01113 (filed 21 July 1998), European Patent Application No. 99302232.1 (filed 25 March 1999), UK Patent Application No. 9912961 No. 5,861,510 (published January 19, 1999), and European Patent Publication No. 780,386 (published June 25, 1997), all of which are incorporated herein by reference in their entireties.Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-1. More preferred are those that selectively inhibit MMP-2 and / or AMP-9 compared to other matrix metalloproteases (e.g., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-II, MMP-12, and MMP-13). Specific examples of MMP inhibitors useful in the present disclosure include AG-3340, RO 32-3555, and RS 13-0830.

[0195] Autophagy inhibitors include, but are not limited to, chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil™), bafilomycin A1, 5-amino-4-imidazolecarboxamide riboside (AICAR), okadaic acid, autophagy-inhibiting algal toxins that inhibit type 2A or type 1 protein phosphatases, analogs of cAMP, and agents that increase cAMP levels, such as adenosine, LY204002, N6-mercaptopurine riboside, vinblastine, etc. Additionally, antisense or siRNA that inhibit the expression of proteins, including but not limited to ATG5 (involved in autophagy), can also be used.

[0196] In some embodiments, the antigen-binding units and pharmaceutical compositions described herein are formulated or administered in combination with a liquid or solid tissue barrier, also known as a lubricant. Examples of tissue barriers include, but are not limited to, polysaccharides, polyglycans, Seprafilm, Interceed, hyaluronic acid, etc.

[0197] In some embodiments, the pharmaceutical agent administered in combination with a subject antigen binding unit may be any suitable pharmaceutical agent usefully delivered by inhalation, for example, analgesics such as codeine, dihydromorphine, ergotamine, fentanyl, or morphine; antianginal preparations such as diltiazem; antiallergic agents such as cromoglycate, ketotifen, or nedocromil; anti-infectives such as cephalosporins, penicillins, streptomycin, sulfonamides, tetracyclines, or pentamidine; antihistamines such as methapyrilene; anti-inflammatory agents such as beclomethasone, flunisolide, budesonide, tipredane, triamcinolone acetonide, or fluticasone; antitussives such as noscapine; bronchodilators such as ephedrine, adrenaline, fenoterol, formoterol, isoforms, or combinations thereof. prenaline, metaproterenol, phenylephrine, phenylpropanolamine, pirbuterol, reproterol, rimiterol, salbutamol, salmeterol, terbutaline, isoetharine, tulobuterol, orciprenaline or (-)-4-amino-3,5-dichloro-α-[[[6-[2-(2-pyridinyl)ethoxy]hexyl]-amino]methyl]benzenemethanol; diuretics, such as amiloride; anticholinergics, such as ipratropium, atropine or oxitropium; hormones, such as cortisone, hydrocortisone or prednisolone; xanthines, such as aminophylline, choline theophyllinate, lysine theophyllinate or theophylline; and therapeutic proteins and peptides, such as insulin or glucagon. It will be apparent to those skilled in the art that, where appropriate, the agents are used in the form of a salt (e.g. as an alkali metal or amine salt or as an acid addition salt) or an ester (e.g. a lower alkyl ester) or a solvate (e.g. a hydrate) to optimize the activity and / or stability of the agent.

[0198] Other exemplary therapeutic agents useful in combination therapy include, but are not limited to, the agents listed above, radiation therapy, hormone antagonists, hormones and their releasing factors, thyroid and antithyroid agents, estrogens and progestins, androgens, adrenocorticotropic hormones; corticosteroids and their synthetic analogs; adrenal cortical hormones, insulin, oral hypoglycemic agents, inhibitors of the synthesis and action of pancreatic endocrine pharmacology, agents affecting mineralization and bone metabolism: calcium, phosphate, parathyroid hormone, vitamin D, calcitonin, water-soluble vitamins, vitamin B complex, ascorbic acid, fat-soluble vitamins, vitamins such as vitamins A, K, E, growth factors, cytokines, chemokines, muscarinic receptor agonists and antagonists; anticholinesterase agents; agents acting on the neuromuscular junction and / or autonomic ganglia; catecholamines, sympathomimetics, and adrenergic receptor agonists or antagonists; and 5-hydroxytryptamine (5-HT, serotonin) receptor agonists and antagonists.

[0199] Therapeutic agents may further include histamine and histamine antagonists, bradykinin and bradykinin antagonists, 5-hydroxytryptamine (serotonin), lipid substances produced by the biotransformation of selective hydrolysis products of membrane phospholipids, eicosanoids, prostaglandins, thromboxanes, leukotrienes, aspirin, nonsteroidal anti-inflammatory drugs, analgesics and antipyretics, agents that inhibit the synthesis of prostaglandins and thromboxanes, selective inhibitors of inducible cyclooxygenase, selective inhibitors of inducible cyclooxygenase-2, autacoids, paracrine hormones, somatostatin, gastrin, cytokines that mediate interactions involved in humoral and cellular immune responses, lipid-derived autacoids, eicosanoids, beta adrenergic agonists, ipratropium, glucocorticoids, methylxanthines, sodium channel blockers, opioid receptor agonists, calcium channel blockers, membrane stabilizers, and leukotriene inhibitors.

[0200] Additional therapeutic agents contemplated herein include diuretics, vasopressin, agents that affect renal water retention, rennin, angiotensin, agents useful in the treatment of myocardial ischemia, antihypertensive agents, angiotensin-converting enzyme inhibitors, beta-adrenergic receptor antagonists, agents to treat hypercholesterolemia, and agents to treat dyslipidemia.

[0201] Other therapeutic agents contemplated include agents used to control stomach acid, agents for peptic ulcers, agents for gastroesophageal reflux disease, prokinetic agents, antiemetics, agents for irritable bowel syndrome, agents for diarrhea, agents for constipation, agents for inflammatory bowel disease, agents for biliary tract disease, agents for pancreatic disease, agents for the treatment of protozoal infections, agents for the treatment of malaria, amebiasis, giardiasis, trichomoniasis, trypanosomiasis, and / or leishmaniasis, and / or agents for the chemotherapy of helminthiasis. Other treatments include antibiotics, sulfa drugs, trimethoprim-sulfamethoxazole quinolones, drugs for urinary tract infections, penicillins, cephalosporins, and other drugs, beta-lactam antibiotics, drugs containing aminoglycosides, protein synthesis inhibitors, drugs used in chemotherapy for tuberculosis, Mycobacterium avium complex, and leprosy, antifungals, and antivirals including nonretrovirals and antiretrovirals.

[0202] Examples of therapeutic antibodies that can be combined with the antigen binding units of the present disclosure include, but are not limited to, anti-receptor tyrosine kinase antibodies (cetuximab, panitumumab, trastuzumab), anti-CD20 antibodies (rituximab, tositumomab), immune checkpoint inhibitors (anti-PD-1, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, such as, for example, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and ipilimumab), and other antibodies such as alemtuzumab, bevacizumab, gemtuzumab, etc. In some embodiments, combination treatment of the CCR8-specific antigen binding units disclosed herein with anti-PD-1 induces significant and / or synergistic tumor growth inhibition and survival benefit, as exemplified in Examples 27 and 28.

[0203] Additionally, therapeutic agents used in immunomodulation, such as immunomodulators, immunosuppressants, tolerogens, and immunostimulants, are contemplated by the methods herein, as are therapeutic agents acting on blood and blood-forming organs, hematopoietic agents, growth factors, minerals, and vitamins, anticoagulants, thrombolytic agents, and antiplatelet agents.

[0204] To treat kidney cancer, the antigen binding unit of the present disclosure can be combined with sorafenib and / or avastin. To treat endometrial disease, the antigen binding unit of the present disclosure can be combined with doxorubicin, taxotere (taxol), and / or cisplatin (carboplatin). To treat ovarian cancer, the antigen binding unit of the present disclosure can be combined with cisplatin (carboplatin), taxotere, doxorubicin, topotecan, and / or tamoxifen. To treat breast cancer, the antigen binding unit of the present disclosure can be combined with taxotere (taxol), gemcitabine (capecitabine), tamoxifen, letrozole, tarceva, lapatinib, PD0325901, avastin, herceptin, OSI-906, and / or OSI-930. For the treatment of lung cancer, the antigen binding units of the invention may be combined with Taxotere (Taxol), Gemcitabine, Cisplatin, Pemetrexed, Tarceva, PD0325901, and / or Avastin.

[0205] Additional therapeutic agents that can be combined with the antigen binding units of the disclosure are described in Goodman and Gilman's "The Pharmacological Basis of Therapeutics" Tenth Edition edited by Hardman, Limbird and Gilman, or the Physician's Desk Reference, both of which are incorporated herein by reference in their entireties.

[0206] The antigen binding units described herein can be used in combination with the agents described herein or other suitable agents depending on the condition being treated. Thus, in some embodiments, one or more antigen binding units of the present disclosure are combined with other agents as described above. When used in combination therapy, the antigen binding units described herein are administered simultaneously or separately with the second agent. This combined administration includes simultaneous administration of the two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate sole administration. That is, the antigen binding units described herein and any of the agents described above can be formulated together in the same dosage form and administered simultaneously. Alternatively, the antigen binding units of the present disclosure and any of the agents described above can be administered simultaneously, where both agents are present in separate formulations. In another alternative, the antigen binding units can be administered sequentially with any one or more of the agents described above, or vice versa. In some embodiments of the separate administration protocol, the antigen binding units of the present disclosure and any of the agents described above are administered minutes, hours, or days apart.

[0207] Further description of the development and use of antigen binding units, polynucleotides, vectors, and host cells is provided in the Examples section below, which are provided as a guide to the skilled artisan and are not intended to be limiting in any way.

[0208] Working Example The following examples are offered by way of illustration and not by way of limitation of various embodiments.

[0209] Example 1: Transient transfection of human CCR8 or cynomolgus CCR8 Stable cell lines HEK293 and CHO-K1 overexpressing human CCR8, cynomolgus monkey CCR8, or mouse CCR8 were generated at Genomeditech Inc. and used for immunization and antibody screening purposes. The lymphoma cell line HuT78 (TIB-161) expressing CCR8 was obtained from ATCC.

[0210] 24 h before transfection, cells were cultured at approximately 5–8 × 10 6 Cells were cultured at a density of 2 × 10 cells / mL with viability of at least 95%. On the day of transfection, cells were cultured at a density of 2 × 10 cells / mL in ExpiCHO-S expression medium (50 mL) in shake flasks. 6 Cells were seeded at a density of 1000 cells / mL. 40 μL of plasmid DNA and 160 μL of EXPIFECTAMINE CHO reagent were incubated at room temperature and the solution was slowly transferred to a shake flask while gently swirling the flask. Cells were incubated on an orbital shaker at 37°C in a CO2 incubator.

[0211] The 433H monoclonal antibody (BD Biosciences, #644092), which specifically binds to human CCR8, and 10A11-1, described in WO2020138489, were used as reference antibodies.

[0212] Example 2: Alignment of human, cynomolgus and mouse CCR8 sequences Human CCR8, cynomolgus monkey, and mouse CCR8 sequences were obtained from Uniprot and aligned (Figure 1). Pairwise alignment of human and cynomolgus monkey CCR8 yielded 94.37% sequence identity, but significant differences were observed in the N-terminal extracellular domain (ECD) and C-terminal regions between mouse and human CCR8 (percent identity = 70.99%) and between mouse and cynomolgus monkey CCR8 (percent identity = 71.55%). The sequence homology between cynomolgus monkey CCR8 ECD and human CCR8 ECD is 68%, whereas the sequence homology between mouse CCR8 ECD and human CCR8 ECD is 52%. Nevertheless, the acidic and tyrosine sulfated ECDs exhibit negatively charged clusters and have been successfully used to generate cross-reactive antibodies.

[0213] Example 3: Immunization and Hybridomas Immunization of Balb / c and SJL mice to generate antibodies was performed using the gene gun method. Gene gun cartridges for gene immunization were prepared by mixing human CCR8 plasmid with other immune adjuvant plasmids including mouse GM-CSF and mouse GM-FLT3L (Tables 4-6). To induce antibodies that cross-react with both human and cynomolgus CCR8, mice were immunized five times with human CCR8 plasmid and then with cynomolgus CCR8 plasmid. Serum titers of immunized mice were detected by FACS using human CCR8 overexpressing cell lines 7 days after each immunization. Mice with the highest titers were selected for the final boost with HEK293-cynomolgus CCR8 cell lines 3 days before hybridoma fusion.

[0214] [Table 4]

[0215] [Table 5]

[0216] [Table 6]

[0217] Hybridoma fusion and subcloning Mouse spleen cells isolated from high titer Balb / c mice and myeloma fusion partners were fused by electric field-induced electrofusion using a Cyto Pulse large chamber cell fusion electroporator (BTX, ECM2001). Single cell suspensions of splenic lymphocytes from immunized mice were fused with half the number of sp2 / 0-Ag14 (ATCC CRL1581) non-secreting mouse myeloma cells. The resulting cells were plated at 2.0x10 cells / well in flat-bottom 96-well cell culture plates in 200ul selective DMEM medium supplemented with high glucose (GIBICO, Cat. No.: 11995-065) and 20% FBS (GIBICO, Cat. No.: 10091-148), plus 50X HAT (GIBICO, Cat. No.: 21060-017). 4 After culturing for 7 days in a CO2 incubator, the medium containing HAT in the 96-well cell culture plate was replaced with medium containing HT supplement (100X), liquid [GIBICO, Cat. No.: 11067030] and 10% FBS.

[0218] After 10 days, primary screening by FACS was performed. For this screening, 50 μl of the supernatant from the fusion plate was transferred to a 96-well round-bottom plate (Corning Incorporated, 3799) and mixed with the cynomolgus CCR8-overexpressing cell line. After 1 h of incubation at 4°C, the plate was centrifuged and the cells were washed twice with FACS buffer (PBS containing 1.5% FBS). Secondary antibody (goat anti-mouse IgG-labeled AF647) was then added and incubated with the cells for 30 min at 4°C. After two additional washes, the cells were brought into cell suspension and read on a BD FACS Celesta reader.

[0219] Hybridoma cells from positive wells that showed strong binding signals to CCR8-overexpressing cells in FACS screening were transferred to 24-well plates. After culturing for 3-5 days, cell supernatants from individual wells were characterized by FACS and other functional assays.

[0220] Parental hybridoma cell lines identified as positive antibodies were subcloned by limiting dilution. After 7 days of culture, positive monoclonal cell lines were selected by FACS. Monoclonal antibodies were generated from promising clones and further characterized. After ranking and validation by various assays, several hybridoma cells were selected for sequencing and further analysis.

[0221] Example 4 Screening of hybridoma clones by FACS binding and AF647-CCL1 binding blocking assays Binding of hybridoma supernatants to CHO-K1-human CCR8 and ExpiCHO-S cynomolgus CCR8 cells. A total of 660 96-well plates were seeded from hybridomas derived from immunized animals and screened by FACS binding. A total of 338 clones were identified as positive based on geometric mean fluorescence intensity (MFI) fold (MFI fold = CCR8 positive cell MFI / CCR8 negative cell MFI) >5.

[0222] To obtain antibodies that cross-react with human and cynomolgus CCR8, hybridomas from mice immunized with human and cynomolgus CCR8 DNA were screened by FACS. Briefly, 50 μL of ExpiCHO-S cells (cell density: 2×10) transfected with human CCR8 or cynomolgus CCR8 were cultured in 1000 mL of PBS. 6 Cells / mL, viability >90%, were incubated with an equal volume of hybridoma supernatant in a 96-well plate (Corning) for 1 h at 4°C. After washing with FACS buffer (DPBS with 2% FBS), the cell / antibody mixture was stained with secondary antibody (Alexa Flour® 647-conjugated rabbit anti-mouse IgG, Jackson ImmunoResearch). Finally, the mixture was washed and resuspended in FACS buffer and subjected to FACS analysis on a BD FACS Celesta. Raw data were analyzed with FlowJo software. Thirteen hybridomas derived from immunized mice showed strong binding to CHO-K1-human CCR8 and CHO-K1-cynomolgus CCR8 cells (Table 7).

[0223] Blocking activity of hybridoma supernatants against CHO-K1-human CCR8 cells . CHO-K1-human CCR8 cells were incubated with hybridoma supernatants and 4 nM AF647-labeled human CCL1 for 60 min at 4°C. Cells were washed three times with FACS buffer (PBS buffer containing 2% FBS) and AF647 signals were detected on a BD FACS Celesta instrument. Data were analyzed using Flowjo V10 software. Percentage of binding inhibition was plotted against antibody concentration. Thirteen hybridomas derived from immunized mice showed significant blocking activity against CHO-K1-human CCR8 cells (Table 7).

[0224] [Table 7]

[0225] Example 5: Binding and Isolation of Purified Mouse Antibodies blocking Activity The binding affinity of the purified antibodies was measured on human CCR8 and cynomolgus CCR8 overexpressing cells. Briefly, a mixture of CFSE (Life technologies, Catalog No.: C34554) labeled CHO-K1-human CCR8 cells and CHO-K1 cells, a mixture of CFSE labeled HEK293-human CCR8 cells and HEK293 cells, or a mixture of CFSE labeled HEK293-cynomolgus CCR8 cells and HEK293 cells was used in the assay. The mixture ratio was 1:1. A total of 5x10 cells were added to each well. 4The mixed cells were seeded in a 96-well plate and washed once with FACS buffer (DPBS containing 1% FBS). The cells were incubated with serially diluted purified hybridoma antibodies for 1 h at 4°C. Antibodies were prepared in 3-fold serial dilutions ranging from 200 nM to 0.003 nM in FACS buffer. After primary antibody incubation, the cells were washed three times with FACS buffer. The cells were then stained with secondary antibodies (Alexa Flu647-conjugated rabbit anti-mouse IgG, Jackson ImmunoResearch, #315606046) diluted 1:600 ​​in FACS buffer and incubated for 0.5 h at 4°C. After washing three times, the Alexa Fluor 647 signal of the stained cells was detected with a BD FACS Celesta and the MFI was determined. FlowJo software was used for analysis. Data were plotted as logarithm of antibody concentration versus mean fluorescence signal. EC 50 Values ​​were calculated in GraphPad Prism 8 (GraphPad Software) using log(agonist) vs. response variable slope (4-parameter) curve fitting. Twelve purified mouse antibodies, including 149F2C10, 153D4G2, 160D1E3, 164G10D6, 204A2G12, 206F1B2, 258H7F3, 262C2G7, 234G9C12, 191E12H8, 273H2G6, and 348E2D11, showed dose-dependent binding to CHO-K1-human CCR8 cells and HEK293-human CCR8 cells. These antibodies also cross-reacted with cynomolgus CCR8 and showed dose-dependent binding to HEK293-cynomolgus CCR8 cells. All antibodies did not bind to CHO-K1 and HEK293 cells. EC of selected antibodies against human CCR8 50 The EC values ​​of selected antibodies against cynomolgus monkey CCR8 are less than 5 nM, except for 262C2G7 and 234G9C12. 50 is less than 1 nM except for 348E2D11 (Table 8).

[0226] [Table 8]

[0227] A blocking curve was generated to rank the CCL1 blocking activity of the hybridoma antibodies. Briefly, a total of 5x10 antibodies were added to each well of a 96-well plate. 4 CHO-K1-human CCR8 cells or HEK293-human CCR8 cells were seeded. The cells were incubated with serially diluted purified hybridoma antibodies and 4 nM AF647-labeled human CCL1 (Almac, #CAF-07) for 1 h at 4 °C. Antibodies were prepared in 3-fold serial dilutions ranging from 200 nM to 0.01 nM in FACS buffer. After incubation, the cells were washed three times with FACS buffer. The Alexa Fluor 647 signal of the stained cells was detected with a BD FACS Celesta and the MFI was determined. Analysis was performed using FlowJo software. Data were plotted as logarithm of antibody concentration against mean fluorescence signal. IC 50 Values ​​were determined using log(agonist) versus response variable slope (four parameters) curve fitting in GraphPad Prism 8 (GraphPad Software). As shown in Table 9, twelve purified mouse antibodies, including 149F2C10, 153D4G2, 160D1E3, 164G10D6, 204A2G12, 206F1B2, 258H7F3, 262C2G7, 234G9C12, 191E12H8, 273H2G6, and 348E2D11, were able to block CCL1 binding.

[0228] [Table 9]

[0229] Example 6: Purified antibodies inhibit CCL1-induced β-arrestin recruitment In response to stimuli, binding of a ligand such as CCL1 to CCR8-GPCR can activate G protein-independent signaling such as β-arrestin recruitment. This leads to internalization of the chemokine receptor. The β-arrestin assay kit was purchased from Discover X. Briefly, CCR8 is fused in frame with the small enzyme donor fragment ProLink (PK) and co-expressed in cells stably expressing a fusion protein of β-arrestin and a larger N-terminal deletion mutant of β-galactosidase (termed enzyme acceptor or EA). Activation of CCR8 stimulates the binding of β-arrestin to PK-tagged CCR8, forcing the complementation of the two enzyme fragments to form the active β-galactosidase enzyme. This interaction increases enzyme activity, which can be measured using chemiluminescent Path Hunter detection reagents. Briefly, cells are incubated with ECs containing CCL1 (CN-07, Almac) and then incubated with ECs containing 100 mM KCl. 80 (4 nM) to activate β-arrestin recruitment, and antibodies of the present application or reference antibody 433H were added to assess their ability to block activated β-arrestin recruitment. β-arrestin recruitment was blocked by reference antibody 433H, while purified antibodies 149F2C10, 153D4G2, 160D1E3, 164G10C6, 204A2G12, and 206F1B2 showed partial inhibition of β-arrestin recruitment (Table 10).

[0230] [Table 10]

[0231] Example 7: Binding and blocking activity of antibodies with deleted PTM sites The antibody sequences generated by hybridoma technology were analyzed for post-translational modifications (PTMs), which can cause problems during the development of therapeutic proteins, such as increased heterogeneity, reduced biological activity, decreased stability, immunogenicity, fragmentation, and aggregation. The potential impact of a PTM depends on its location and, in some cases, exposure to solvent. The CDRs of all sequences were analyzed for asparagine deamination, aspartic acid isomerization, free cysteine ​​thiol groups, N-glycosylation, oxidation, and fragmentation due to potential hydrolysis sites.

[0232] In the case of 149F2C10, Asn 28 -Gly 29 A (NG) deamidation site is present, which may cause stability issues. To reduce the risk of deamidation, a new variant was designed that removes the NG site. The binding activity of the NG site-removed mutant was tested in cells along with the parental clone as a control.

[0233] The binding affinity of NG site-deleted antibodies was measured in human CCR8 and cynomolgus CCR8 overexpressing cells. Briefly, CFSE-labeled HEK293-human CCR8 cells or CFSE-labeled HEK293-cynomolgus CCR8 cells were mixed with HEK293 cells at a mixing ratio of 1:1. A total of 5x10 4The mixed cells were seeded in a 96-well plate and washed once with FACS buffer (DPBS with 1% FBS). The cells were incubated with serially diluted purified hybridoma antibodies for 1 h at 4 °C. Antibodies were prepared in 3-fold serial dilutions ranging from 100 nM to 0.0017 nM or 25.3165 nM to 0.0004 nM in FACS buffer. 433H was set as a positive control. After primary antibody incubation, the cells were washed three times with FACS buffer. Then, the cells were stained with secondary antibody (Alexa Flu647-conjugated rabbit anti-mouse IgG, Jackson ImmunoResearch, #315606046) at 1:600 ​​dilution in FACS buffer and incubated for 0.5 h at 4 °C. The Alexa Fluor 647 signal of the stained cells was detected with a BD FACS Celesta and the MFI was measured. The data were analyzed using FlowJo software. The data were plotted as the logarithm of the antibody concentration versus the mean fluorescence signal. EC 50 Values ​​were calculated using log(agonist) versus response variable slope (4-parameter) curve fitting in GraphPad Prism 8 (GraphPad Software).

[0234] A blocking curve was generated to rank the blocking activity of the PTM site-removing antibodies. Briefly, a total of 5x10 4 CHO-K1-human CCR8 cells were seeded in a 96-well plate. The cells were incubated with serially diluted purified hybridoma antibodies and 4 nM AF647-labeled human CCL1 (Almac, #CAF-07) for 1 h at 4 °C. Antibodies were prepared in 3-fold serial dilutions ranging from 100 nM to 0.0051 nM in FACS buffer. After incubation, the cells were then washed three times with FACS buffer. The Alexa Fluor 647 signal of the stained cells was detected with a BD FACS Celesta and the MFI was measured. Analysis was performed using FlowJo software. Data were plotted as logarithm of antibody concentration versus mean fluorescence signal. IC 50Values ​​were calculated using log(agonist) versus response variable slope (4-parameter) curve fitting in GraphPad Prism 8 (GraphPad Software).

[0235] As shown in FIG. 2 and Table 11, 149F2C10_G29A showed the highest binding activity to HEK293-human CCR8 and HEK293-cynomolgus monkey CCR8 among the PTM site-removed antibodies, and had the highest EC 50 The IC50 and IC60 values ​​were 0.129 nM and 0.173 nM, respectively. None of the antibodies showed nonspecific binding to HEK293 cells. As shown in FIG. 3 and Table 11, 149F2C10_G29A showed the highest blocking activity against CHO-K1-human CCR8 among the PTM site-deleted antibodies, with IC50 values ​​of 0.129 nM and 0.173 nM, respectively. 50 was 0.725 nM.

[0236] [Table 11]

[0237] Example 8: Humanization of anti-CCR8 antibodies Humanization of the selected candidates 149F2C10_G29A (abbreviated as "149") and 348E2D11 (abbreviated as "348") was performed by grafting the CDR residues of the mouse antibodies into human germline frameworks. First, the sequences of the VH and VL regions of the selected candidates were compared with the human germline sequences, and the most compatible germline acceptors were selected based on homology, canonical structure, and physical properties. Then, homology modeling was used to generate structural models of the candidates. The CDR regions of both the heavy and light chains of the candidate antibodies were fixed, and the mouse frameworks were replaced with the selected human germline frameworks. Residues that differed between the mouse and human frameworks, potentially affecting the CDR conformation or the VH / VL interface, were back-mutated. DNA fragments encoding the designed humanized variants were synthesized and subcloned into an IgG expression vector. The DNA sequences were confirmed by sequencing. Various combinations of humanized heavy and light chains were co-transfected and expressed in CHO-K1 cells. Finally, nine humanized variants were derived from 149F2C10_G29A: Hu149-1, Hu149-2, Hu149-3, Hu149-4, Hu149-5, Hu149-6, Hu149-7, Hu149-8, and Hu149-9. Nine humanized variants were derived from 348E2D11: Hu348-1, Hu348-2, Hu348-3, Hu348-4, Hu348-5, Hu348-6, Hu348-7, Hu348-8, and Hu348-9. The humanized antibodies were compared to the parental antibodies for antigen-binding affinity by FACS using cells expressing the target antigen.

[0238] Example 9: Binding and blocking activity of humanized 149 A total of 5 x 10 per well 4Cells were seeded in 96-well plates and washed once with FACS buffer (DPBD with 1.5% FBS). Antibodies were prepared in 3-fold serial dilutions ranging from 100 nM to 0.0017 nM in FACS buffer. Cells were incubated with 50 μL of diluted antibody for 1 h at 4 °C. Control groups included cells incubated with human IgG1, 433H, or mouse IgG2a. After primary antibody incubation, cells were washed three times with FACS buffer. Cells were then stained with Alexa Flu647-conjugated anti-human IgG secondary antibody (Jackson ImmunoResearch, #109606170) or Alexa Flu647-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch, #115605072), diluted 1:600 ​​in FACS buffer and incubated for 0.5 h at 4 °C. After three washes, binding was measured by flow cytometry.

[0239] The binding affinity of the humanized 149 antibodies was tested using HEK293-human CCR8 cells and HEK293-cynomolgus CCR8 cells. The blocking activity of the humanized 149 antibodies was tested using CHO-K1-human CCR8 cells. As shown in Tables 12-13, Hu149-4 and Hu149-9 showed the highest binding activity among the humanized antibodies against HEK293-human CCR8 cells and HEK293-cynomolgus CCR8 cells in two separate experimental settings. As shown in Tables 12-13, Hu149-4 and Hu149-9 showed the highest blocking activity among the humanized antibodies against CHO-K1-human CCR8 cells, with IC 50 were 0.137 nM and 0.119 nM, respectively.

[0240] [Table 12]

[0241] [Table 13]

[0242] Example 10: Binding and blocking activity of humanized 348 The binding affinity of the humanized 348 antibody was tested using HEK293-human CCR8 and ExpiCHO-S-cynomolgus CCR8 cells. The blocking activity of the humanized 348 antibody was tested using CHO-K1-human CCR8 cells. As shown in Table 14, Hu348-1, Hu348-2, Hu348-3, Hu348-4, Hu348-8, Hu348-9, parental antibodies CM348 and 433H showed comparable binding affinity to HEK293-human CCR8 and EC 50 were 0.122 nM, 0.119 nM, 0.108 nM, 0.109 nM, 0.144 nM, 0.182 nM, 0.197 nM and 0.091 nM, respectively. Interestingly, Hu348-4 had an EC 50 The optimal binding to ExpiCHO-S cynomolgus CCR8 cells was significantly improved compared to the IC value of 0.087 nM (Table 14). Hu348-4 and the parent antibody CM348 showed similar CCL1 binding blocking activity, with IC 50 were 0.173 nM and 0.151 nM, respectively (Table 14).

[0243] [Table 14]

[0244] Example 11: Selected humanized antibodies do not bind to human CCR1 or CCR4 CCR1 is a receptor for CC-type chemokines. It binds to MIP-1-α, MIP-1-δ, RANTES, and MCP-3, and less efficiently to MIP-1-β or MCP-1, and then transmits signals by increasing intracellular calcium ion levels. CCR1 is involved in stem cell proliferation.

[0245] CCR4 is a high affinity receptor for the CC-type chemokines CCL17 / TARC, CCL22 / MDC, and CKLF isoform 1 / CKLF1. The activity of this receptor is mediated by G(i) proteins that activate the phosphatidylinositol calcium second messenger system. It can function as a chemoattractant homing receptor on circulating memory lymphocytes and as a coreceptor for some primary HIV-2 isolates. In the central nervous system (CNS), CCR4 can mediate survival of hippocampal neurons.

[0246] The sequences of human CCR8, CCR1, and CCR4 were obtained from Uniprot. The sequences were aligned for sequence identity calculation. Pairwise alignment of CCR1 and CCR8 yielded a sequence identity of 39.33%, while the percentage is similar between CCR4 and CCR8 (percentage of identity = 43.13%). The sequence homology between CCR1 ECD and CCR8 ECD was 22.9%, while the sequence homology between CCR4 ECD and CCR8 ECD was 31.4%.

[0247] ExpiCHO-S cells were transiently transfected with human CCR1 or human CCR4. A total of 5 × 10 4Cells were seeded in 96-well plates and washed once with FACS buffer (DPBS with 1.5% FBS). Antibodies were prepared in 3-fold serial dilutions from 200 nM to 0.00338 nM in FACS buffer. Cells were incubated with 50 μL of diluted antibody for 1 h at 4 °C. Control groups included cells incubated with human IgG1, 433H, or mouse IgG2a. After primary antibody incubation, cells were washed three times with FACS buffer. Cells were then stained with Alexa Flu647-labeled anti-human IgG secondary antibody (Jackson ImmunoResearch, #109606170) or Alexa Flu647-labeled anti-mouse IgG secondary antibody (Jackson ImmunoResearch, #115605072) diluted 1:600 ​​for 0.5 h at 4 °C in FACS buffer. Flow cytometry was performed to measure binding. As shown in FIG. 4 and Table 15, the anti-CCR1 antibody (5F10B29) showed dose-dependent binding to ExpiCHO-CCR1 cells and EC 50 The EC value was 24.7 nM, whereas all selected humanized antibodies showed no or minimal binding to Expi CHO-CCR1 cells. The anti-CCR4 antibody (1G1) showed dose-dependent binding to Expi CHO-CCR4 cells, with an EC 50 was 11.7 nM, whereas all selected humanized antibodies showed no or minimal binding to Expi CHO-CCR4 cells.

[0248] [Table 15]

[0249] Example 12: Antibody-dependent cellular cytotoxicity (ADCC) function of selected humanized antibodies Human PBMC-based ADCC activity of humanized antibodies was tested in CHO-K1 human CCR8 cells. Cryopreserved PBMCs (AllCells) from healthy subjects were thawed 1 day before the assay and cultured overnight in RPMI160 medium containing 10% FBS and 200 IU IL-2 (R&D, Cat. No.: 202-IL) in a CO2 incubator. Target cells were labeled with CFSE (Life technologies, Cat. No.: C34554) at a final concentration of 2.5 μM for 15 min. After staining, the cell concentration was increased to 6 × 10 4 Adjust to 1 x 10 cells / mL 6 The cells were mixed with twice the amount of PBMCs adjusted to 1000 cells / mL (effector cell / target cell ratio was 40:1). Then, 150 μL of the mixed target and effector cell suspension and 50 μL of the diluted antibody were mixed into each well. Duplicate wells were prepared for each antibody concentration. Target cells alone were used as a control group. After incubation at 37°C and 5% CO2 for 5 hours, 1 μg / mL PI (Invitrogen, Cat. No.: 51-66211E) was added and analyzed by flow cytometry (BD FACS Celesta). Specific cytotoxicity was calculated by the following formula: specific cytotoxicity = PI positive cells with antibody (%) - PI positive cells without antibody (%). Hu149-4 HuIgG1, Hu149-9 HuIgG1, and parental antibody CM149 HuIgG1 showed similar ADCC activity against CHO-K1 cells overexpressing human CCR8. Humanized Hu348-4 HuIgG1 and parental antibody CM348 HuIgG1 showed similar ADCC activity against CHO-K1 cells overexpressing human CCR8 (Figure 5, Table 16).

[0250] [Table 16]

[0251] Example 13: Selected humanized antibodies inhibit CCL1-induced β-arrestin recruitment In the first experiment, CHO-K1 cells co-expressing human CCR8 tagged with ProLink (PK) and Enzyme Acceptor (EA) were incubated with antibodies or CCL1 (CN-07, Almac) for 90 min, followed by addition of detection reagent (93-0001, DiscoverX). CCL1 induced β-arrestin recruitment in a dose-dependent manner, and EC 50 was 0.288 nM. Reference antibodies 433H, CM149, or Hu149-4 did not induce β-arrestin recruitment (FIG. 6).

[0252] CHO-K1 CCR8 β-arrestin cells were cultured with CCL1 (CN-07, Almac) for EC 80 β-arrestin recruitment was activated by stimulation with β-arrestin agonist (4 nM) and selected humanized or reference antibodies were added to assess their ability to inhibit activated β-arrestin recruitment. Because the subtype of the reference antibody 433H was mouse IgG2a, the subtype of all tested antibodies was changed to mouse IgG2b. As shown in Table 17, Hu149-9 and Hu10A11-1 showed similar inhibitory activity against β-arrestin recruitment, with IC 50 The IC values ​​of 433H and Hu149-9 were 8.64 nM and 17.5 nM, respectively. 433H showed a better inhibitory activity against β-arrestin recruitment than Hu149-9, and the IC 50 were 0.23 nM and 8.64 nM, respectively.

[0253] [Table 17]

[0254] Example 14: Humanized antibodies inhibit CCL1-induced cell migration The activity of humanized anti-CCR8 antibodies to inhibit CCL1-induced cell migration was evaluated. CHO-K1-human CCR8 cells with or without anti-CCR8 antibodies were added to the upper chamber of a Transwell 96-well permeable support. The chemoattractant CCL1 was added to the lower chamber. The cells passed through the membrane from the top and attached to the underside of the membrane. The cells were detached from the membrane using trypsin and quantified by Cell Titer Glo. As shown in Figure 7, Hu149-4-mIgG2a, Hu149-9-mIgG2a, and the reference antibody 433H were tested in the migration assay. Hu149-4-mIgG2a, Hu149-9-mIgG2a, and 433H inhibited CCL1-induced cell migration. IC 50 were 2.95 nM, 1.92 nM, and 1.25 nM, respectively (Figure 7, Table 18). Inhibition rate (%) = (1-(chemotaxis index (sample)-1 / chemotaxis (without antibody)-1)) x 100 Chemotaxis index = CTG value (sample) / CTG value (without CCL1)

[0255] [Table 18]

[0256] Example 15: Affinity Maturation of Exemplary Anti-CCR8 Antibody HU149-9 To improve the binding affinity of the humanized variant 149-9 to human CCR8, we utilized phage display technology for affinity maturation. The parent antibody sequence was used as a template and assembled into a VH-VL format with a (G4S)3 linker and cloned into the pComb3XSS phagemid vector.

[0257] The phagemid vector containing the scFv was transformed into TG1, and the antigen binding of the phage-displayed 149-9 scFv was verified by FACS. Two CDR mutagenesis libraries targeting CDRH3 and CDRL3 were constructed separately by soft mutagenesis-guided primer design and overlap PCR. The CDR positions were defined by the Kabat numbering system. The amino acid residues in the CDR3 loop were randomized with the NNK code. The mutation rate at each position was maintained at approximately 50% to maximally retain the original binding epitope. During affinity-driven cell-based panning, stringent and mild washing conditions were incorporated to reduce nonspecific binding and wild-type occupancy.

[0258] Example 16: Screening of affinity matured mutants by FACS binding 50-100 random clones for each library were sequenced and sequence alignment with the wild-type sequence was used to assess the quality of the mutant libraries, including mutation positions and mutation rates. Up to 5500 single clones were selected for preparation of periplasmic extracts (PPE) and FACS binding screening (Figure 8A-B). Clones with high signal / background ratios were subjected to sequencing, and 14 scFvs were purified and subsequently confirmed by FACS binding followed by human IgG1 conversion containing combinations of beneficial mutations from the heavy and light chains. After transient expression in HEK293 cells, IgG in the culture supernatant was subjected to one-step protein A purification. The purified IgG was further characterized by purity by SDS-PAGE, aggregation by SEC-HPLC, and enrichment by UV280. Antigen binding was verified by FACS (Tables 19-20). Considering that ADCC effect may contribute significantly to Treg depletion, Fc mutations were introduced, including S239D / I332E (US2005054832A1) and L235V / F243L / R292P / Y300L / P396L (AU2004204494B2).

[0259] [Table 19]

[0260] [Table 20]

[0261] Example 17: Binding and blocking activity of selected affinity matured antibodies The binding affinity of affinity matured antibody Hu149 was compared to that of the parent antibody by FACS analysis (Sartourius, iQue3) using CHO-K1-human CCR8 and HEK293-cynomolgus CCR8 cells. The blocking activity of affinity matured antibody Hu149 was also evaluated in CHO-K1-human CCR8 cell line. As shown in Table 21, all tested affinity matured antibodies except Hu149-21 showed comparable binding affinity and blocking activity to CHO-K1-human CCR8 and HEK293-cynomolgus CCR8 cells.

[0262] [Table 21]

[0263] The binding affinity of selected affinity-matured anti-CCR8 antibodies was measured using HuT78 cells expressing endogenous human CCR8. Briefly, a total of 5 × 10 4 Cells were seeded in 96-well plates and washed once with FACS buffer (DPBS with 1.5% FBS). Antibodies were prepared in 4-fold serial dilutions ranging from 200 nM to 0.0122 nM in FACS buffer. Cells were incubated with 50 μL of diluted antibody for 1 h at 4 °C. After primary antibody incubation, cells were washed three times with FACS buffer. Cells were then stained with Alexa Flu647-labeled anti-human IgG secondary antibody (Jackson ImmunoResearch, #109606170) diluted 1:600 ​​for 0.5 h at 4 °C in FACS buffer. After three washes, flow cytometry (Sartourius, iQue3) was performed to measure the binding affinity of the antibodies.

[0264] As shown in Table 22, Hu149-11, Hu149-12, and Hu149-14 have improved binding to HuT78 cells compared to the parent antibody Hu149-9, and the EC 50 were 0.278 nM, 0.170 nM, and 0.184 nM, respectively. As shown in Table 22, the maximum MFI values ​​of Hu149-16, Hu149-18, Hu149-19, Hu149-20, Hu149-21, Hu149-22, Hu149-23, and Hu149-24 were significantly higher than that of Hu149-9. Whether this is due to nonspecific binding needs further investigation.

[0265] [Table 22]

[0266] Example 18: Binding of selected affinity matured antibodies to human PBMCS Because CCR8 is highly expressed on tumor-infiltrating regulatory T cells but not on healthy or effector T cells, PBMCs from healthy individuals can be used to detect nonspecific binding of CCR8 antibodies. Briefly, PBMCs were thawed and blocked with Fc blocker for 10 min at 4°C, 200 nM antibodies and PBMCs were incubated for 1 h at 4°C, and stained with FITC-anti-CD3, BV421-anti-CD4, and AF647-anti-human Fc for 30 min at 4°C. Stained cells were analyzed by flow cytometry. Tables 23-24 show that compared to the hIgG1 isotype control, Hu149-11 showed no or minimal binding to CD4+ and CD4- T cells, whereas Hu149-18, Hu149-19, Hu149-20, Hu149-21, Hu149-22, Hu149-23, and Hu149-24 showed significant binding to CD4+ and CD4- T cells. Based on the FACS staining and gating strategy, CD4- T cells are considered here as CD8+ T cells.

[0267] [Table 23]

[0268] [Table 24]

[0269] Example 19: Affinity matured antibodies inhibit CCL1-induced β-arrestin recruitment CHO-K1 CCR8 β-arrestin cells were cultured with CCL1 (CN-07, Almac) for EC 80 β-arrestin recruitment was activated by stimulation with β-arrestin agonist (4 nM) and selected humanized or reference antibodies were added to assess their ability to block activated β-arrestin recruitment. As shown in Figure 9 and Table 25, Hu149-11, Hu149-12, Hu149-18 and 433H showed similar inhibitory activity against β-arrestin recruitment, with IC 50 were 0.800 nM, 0.350 nM, 0.610 nM and 0.890 nM, respectively.

[0270] [Table 25]

[0271] Example 20: Affinity matured antibodies inhibit CCL1-induced cell migration The activity of mature anti-CCR8 antibodies to inhibit CCL1-induced cell migration was evaluated. CHO-K1-human CCR8 cells with or without anti-CCR8 antibodies were added to the upper chamber of a Transwell 96-well permeable support. The chemoattractant CCL1 was added to the lower chamber. Cells passed through the top of the membrane and attached to the underside of the membrane. Cells were detached from the membrane using trypsin and quantified by Cell Titer Glo. Inhibition rate (%) = (1-(chemotaxis index (sample)-1 / chemotaxis (without antibody)-1)) x 100 Chemotaxis index = CTG value (sample) / CTG value (without CCL1)

[0272] As shown in Figure 10, Hu149-11G1, Hu149-12G1, and Hu348-4m2G1 were tested in migration assays. 433H was used as a positive control. Hu149-11G1, Hu149-12G1, and Hu348-4m2G1 inhibited cell migration induced by CCL1. IC 50 were 0.849 nM, 0.953 nM, and 1.42 nM, respectively (Table 26).

[0273] [Table 26]

[0274] Example 21: Binding and blocking activity of FC variants of humanized anti-CCR8 antibodies The binding and blocking activity of humanized anti-CCR8 antibodies with different Fc variants was evaluated by cross-reactivity in CHO-K1-human CCR8 cell line and HEK293-cynomolgus CCR8 cell line mentioned above. As shown in Table 27, Hu149-11G1m and Hu149-11G1x showed similar binding and blocking affinity to the parent antibody Hu149-11G1. In addition, Hu348-4-m2G1m and Hu348-4-m2G1x also showed similar binding and blocking activity compared to the parent antibody Hu348-4-m2G1. Hu149-11G1m, Hu149-11G1x, Hu348-4-m2G1m, and Hu348-4-m2G1x also showed better binding activity to CHO-cynomolgus CCR8 cells compared to the parent antibody Hu10A11-1. As shown in FIG. 11 and Table 28, Hu149-11G1m and Hu149-11G1x also showed better binding activity and higher binding signal to HuT78 cells compared to Hu10A11-1. Hu348-4-m2G1m and Hu348-4-m2G1x also showed better binding activity to HuT78 cells compared to Hu10A11-1.

[0275] [Table 27]

[0276] [Table 28]

[0277] Example 22: ADCC activity of Fc variants of humanized anti-CCR8 antibodies Human FcγRIIIa is dimorphic at residue 158. One allele (V158) encodes a high Fc affinity receptor variant with a valine at amino acid residue 158, whereas the other allele (F158) encodes a low Fc affinity receptor variant with a phenylalanine at amino acid residue 158. Jurkat T cells expressing the firefly luciferase gene under the control of the NFAT response element and the low affinity FcγRIIIa variant (F158, Cat. No. 60540) and Jurkat T cells expressing the firefly luciferase gene under the control of the NFAT response element and the high affinity FcγRIIIa variant (V158, Cat. No. 60541) were purchased from BPS biosciences.

[0278] One day prior to the assay, CD16a / NFAT-Jurkat cells (BPS 60640 or 60541) were seeded in assay medium and grown for one day. On the day of the assay, 1x10 4 Target cells at 6x10 cells / well were seeded in a white opaque 96-well plate. Anti-CCR8 human antibody IgG1 or control antibody was added, mixed and incubated for 1 hour at 37°C with 5% CO2. CD16a / NFAT reporter Jurkat cells were harvested by centrifugation and resuspended in assay medium. 4Cells / well were added to the target wells and incubated with either anti-CCR8 or a non-specific negative control antibody. 100 μL of assay medium was added to control wells without cells (for measurement of background luminescence). Plates were incubated for 5 h at 37 °C in a CO2 incubator. After the 5 h incubation, 50 μl of One-Step Luciferase Reagent was added per well and gently shaken at room temperature for 30 min. Luminescence was measured using a luminometer. Data analysis: The average background luminescence (control wells without cells) was subtracted from the luminescence measurements of all wells. Nonlinear regression analysis was performed in GraphPad Prism 8 (GraphPad Software) to determine EC 50 values ​​were calculated.

[0279] As shown in Figures 12-13 and Table 29, Hu149-11G1 (wild-type variant) and Hu10A11-1 (wild-type variant) showed similar ADCC activity on CHO-human CCR8 cells, which express CCR8 at high levels, as V or F variant CD16a / NFAT-Jurkat cells. However, when HuT78 cells, which endogenously express CCR8 and have a lower expression level of CCR8 than CHO-CCR8, were used as target cells, Hu149-11G1 (wild-type variant) was 2.5-5.5 times more potent than Hu10A11-1 (wild-type variant). EC of Hu149-11G1 (wild-type variant) and Hu10A11-1 (wild-type variant) with V variant CD16a / NFAT-Jurkat cells in Hut78 50 The EC values ​​of Hu149-11G1 (wild-type variant) and Hu10A11-1 (wild-type variant) with F variant CD16a / NFAT- Jurkat cells in Hut78 were 0.586 nM and 1.47 nM, respectively. 50 were 0.402 nM and 2.20 nM, respectively (FIGS. 14-15, Table 29). As shown in FIGS. 12-15 and Table 29, the ADCC activity of Hu149-11G1m (an ADCC-enhanced variant having L235V / F243L / R292P / Y300L / P396L Fc mutations) was far superior to that of Hu149-11G1.

[0280] [Table 29]

[0281] Human PBMC-based ADCC activity of humanized antibodies was tested in CHO-K1 human CCR8 cells. Cryopreserved PBMCs (AllCells) from healthy subjects were thawed 1 day before the assay and cultured overnight in a CO2 incubator in RPMI1640 medium containing 10% FBS and 200 IU IL-2 (R&D, Cat. No.: 202-IL). Target cells were labeled with CFSE (Life technologies, Cat. No.: C34554) at a final concentration of 2.5 μM for 15 min. After staining, the cell concentration was increased to 6 × 10 4 Adjust to 1 x 10 cells / mL 6 The cells were mixed with 2x the volume of PBMCs adjusted to 1000 cells / mL (effector cell / target cell ratio 40:1). Then, 150 μL of the mixed suspension of target and effector cells and 50 μL of the diluted antibody were mixed in each well. Duplicate wells were prepared for each antibody concentration. Target cells alone were used as a control group. After 5 hours of incubation at 37°C and 5% CO2, 1 μg / mL PI (Invitrogen, Cat. No.: 51-66211E) was added and analyzed by flow cytometry (BD FACS Celesta). Specific cytotoxicity was calculated by the following formula: specific cytotoxicity = PI positive cells with antibody (%) - PI positive cells without antibody (%).

[0282] As shown in FIG. 16 and Table 30, Hu-149-11G1 (wild-type variant) has higher ADCC activity and EC 50 The ADCC activity of Hu149-11G1m (an ADCC-enhanced variant with L235V / F243L / R292P / Y300L / P396L Fc mutations) was significantly improved against CHO-K1-human CCR8 cells compared with Hu149-11G1, and the EC 50were 0.0000850 nM and 0.0139 nM, respectively.

[0283] [Table 30]

[0284] Example 23: FC variants of humanized anti-CCR8 antibodies inhibit CCL1-induced β-arrestin recruitment CHO-K1 CCR8 β-arrestin cells were cultured with CCL1 (CN-07, Almac) for EC 80 β-arrestin recruitment was activated by stimulation with β-arrestin agonist (4 nM) and selected humanized and reference antibodies were added to assess their ability to inhibit activated β-arrestin recruitment. As shown in Figure 17 and Table 31, Hu149-11G1m was more potent than Hu10A11-1 in inhibiting β-arrestin recruitment, with an IC 50 The IC values ​​were 2.09 nM and 37.1 nM, respectively, and the maximum inhibition rates were 72.7% and 43.6%, respectively. Hu149-11G1m and Hu149-12G1m had similar β-arrestin recruitment inhibitory activities, and the IC 50 were 2.09 nM and 1.16 nM, respectively.

[0285] [Table 31]

[0286] Example 24: Binding of FC variants of humanized anti-CCR8 antibodies to human peripheral blood mononuclear cells (PBMCS) Non-specific binding of the Fc variants of humanized anti-CCR8 antibodies was detected using PBMCs from healthy individuals. Briefly, PBMCs were thawed and blocked with Fc blocker for 10 min at 4°C, PBMCs were incubated with 200 nM antibody for 1 h at 4°C, and stained with FITC-anti-CD3, BV421-anti-CD4, and AF647-anti-human Fc for 30 min at 4°C. The stained cells were analyzed by flow cytometry. As shown in Table 32, the Fc variants of Hu149-11 and Hu149-12 showed no or minimal binding to CD4+ and CD4- T cells. Hu348-4-m2G1m showed significant binding to CD4+ and CD4- T cells.

[0287] [Table 32]

[0288] Example 25: Expression of CCR8 in various immune cell populations Frozen human dissociated tumor cells (hDTCs) from patients with kidney, bladder, breast, colorectal, and melanoma were purchased from Discovery Life Sciences. Cells were thawed according to the vendor's instructions and filtered through a 70 μm cell strainer. Cells were then counted and stained with Zombie Violet™ fixable viability dye (Biolegend, Catalog No.: 423114, 1:1000 in PBS) for 15 min at room temperature. Cells were washed with FACS staining buffer (Biolegend, Catalog No.: 420201) and 1-2 million hDTCs / well in 100 μl were seeded into each well of a 96-well plate. hDTCs were first pre-stained with Human TruStain FcX (BioLegend, Catalog No.: 422302). Cells were then surface stained and intracellular stained using True-Nuclear Transcription Factor Staining Buffer Set (Biolegend, Cat#: 424401) and following the manufacturer's protocol. FMO controls were included with each hDTC sample to ensure accurate gating. Samples were acquired using a BD LSRFortessa X-20 and analyzed using FlowJo (BD Biosciences). A complete list of flow cytometry antibodies is available in Table 33.

[0289] [Table 33]

[0290] Flow cytometry was used to evaluate the expression pattern of CCR8 protein in different immune cell populations. Experiments were performed in both hDTCs and donor-matched PBMCs. CCR8 was not detected on the surface of major immune cell populations in PBMCs, including FoxP3+Treg cells, CD8+T cells, CD11b+myeloid cells, B cells, and NK cells (Figure 18). In contrast, high expression of CCR8 was detected in the FoxP3+Treg population of donor-matched hDTCs (Figure 19). CCR8 was slightly induced in CD8T cells in hDTCs and was not detected in other populations, such as CD11b+myeloid cells, B cells, and NK cells. CCR8 was not detected by FoxP3 conventional CD4+T cells in either peripheral blood or hDTCs. These data indicate that CCR8 is only upregulated in Tregs in the tumor microenvironment, which may be possibly due to TCR-mediated activation of Tregs. When CCR8 expression was examined in hDTCs of different cancer types, two subsets of CCR8+ Tregs were observed, namely CCR8 lo (subset 1) and CCR8 hi (subset 2) populations. Even within the same cancer type, Tregs from different donors showed heterogeneity; some donors had only subset 1 (panels E, G, H, L, N in Figure 20) or both subsets 1 and 2 (panels A, B, C, D, F, I, J, K, M in Figure 20). It has been reported that CCR8 expression is positively correlated with several activation markers and immune checkpoint molecules, such as Lag3, Klrg1, Tnfrsf4, Tnfrsf9, Il2ra, Helios, costimulatory molecule Cd81, secretory molecule Areg, and Il10(1), indicating that CCR8 hi Tregs (subset 2) are probably a highly suppressive phenotype.

[0291] Example 26: CCR8 antibody treatment significantly reduces tumor volume Six- to eight-week-old female transgenic mice engineered to express the human CCR8 gene instead of the mouse CCR8 gene were purchased from Biocytogen (Wakefield, MA) and allowed to acclimate for one week before the start of the study. The mouse colorectal cancer cell line MC38 was inoculated into the right flank of the mice at 0.5x10 6 Cells were implanted subcutaneously at 100 μl per mouse. Prior to inoculation, cells were subcultured no more than three times in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Upon reaching 80–85% confluence, cells were harvested and plated at 5x10 in a 1:1 mixture of serum-free RPMI 1640 and Matrigel. 6 Resuspended at 100 cells / ml.

[0292] After cell implantation, mice were monitored twice weekly for tumor growth. For tumor measurements, the length and width of each tumor were measured with a caliper, and the volume was calculated according to the following formula: tumor volume (mm 3 ) = (width (mm) x length (mm) 2 ) / 2. On the day treatment began, all tumors were measured, outliers were removed, and mice were randomly assigned to treatment groups. For anti-CCR8 treatment, a human IgG1 antibody was developed and engineered with an engineered human IgG1 Fc (to enhance ADCC activity). As a control, mice received a nonspecific human IgG1 antibody with the same Fc mutations as the experimental antibody. Treatment was performed at 10-20°C for 12 days, with a mean tumor volume of approximately 120 mm in each group. 3 The treatments were administered intraperitoneally (ip) 7 days after tumor inoculation on study day 0. Treatments were then administered on study days 3, 7, and 11.

[0293] Tumors were grown to a volume of 10% of the animal's body weight, or approximately 2000 mm 3Measurements continued at least twice weekly until tumor volumes exceeded 100%. Changes in tumor size are shown by graphing the individual tumor volumes for each treatment group on day 15 after animals were first treated with Hu149-11G1m (Figure 21). Treatment with Hu149-11G1m significantly reduced tumor growth compared to control IgG1 when the therapeutic agent was administered at 3 or 10 mg / kg (p<0.05). No significant mean tumor growth inhibition was observed with Hu149-11G1m when administered at 1, 0.3, or 0.1 mg / kg. P values ​​were calculated using unpaired two-tailed t-test analysis of tumor volumes calculated at the end of the study (day 15). Referring to Figure 21, statistical significance was determined by unpaired two-tailed t-test comparing Hu149-11G1m to the human IgG1 control, *p<0.05, **p<0.01, and ns p>0.05.

[0294] Example 27: Antitumor effect of CCR8 antibody in combination with anti-PD-1 antibody on MC38 tumor model in HCCR8 knock-in transgenic mice In this study, we investigated the in vivo antitumor efficacy of CCR8 antibodies alone and in combination with PD-1 antibodies. More specifically, we tested the efficacy of Hu149-11G1m in human CCR8 transgenic mice carrying MC38 tumors. MC38 is a murine colon cancer cell line. Mice were inoculated subcutaneously with MC38, and tumor-bearing mice were administered Hu149-11G1m twice weekly, either alone at a dose of 3 mg / kg or in combination with anti-PD-1 antibody (Biocell, Catalog No.: CP151) at a dose of 5 mg / kg. Tumor growth was monitored throughout the study, and survival was recorded at the end of the study. Mice with complete tumor regression were re-exposed to tumor to test the durability of the antitumor efficacy. As detailed below, superior survival was achieved and significant depletion of Tregs was observed in the combination treatment group.

[0295] MC38 mouse colon carcinoma cells were cultured in DMEM medium supplemented with 10% FBS and 1x penicillin / streptomycin. After several in vitro passages, tumor cells were harvested using TrypLE Express reagent, counted, and diluted to 10E06 cells / mL in PBS. An equal volume of Matrigel was added to the cell solution to a final concentration of 5E06 cells / mL.

[0296] In this study, transgenic mice with human genes were used. Based on the C57Bl / 6 mouse strain, these animals had the human CCR8 gene inserted into the mouse CCR8 locus. Transgenic hCCR8 mice were inoculated by injecting 100 μL of cell suspension into the right hind flank. After 7 days of observation, the tumor volumes of the mice were measured and 10 animals were assigned to four treatment groups. The average tumor volume in each group was approximately 100 mm. 3 Tumor-bearing mice were injected twice weekly with Hu149-11G1m 3mpk alone or in combination with anti-PD-1. Human IgG1 with matching Fc served as a negative control (hIgG1 control). Tumor volume (TV) was determined for tumors with a volume of 1500 mm 3 Tv was assessed twice weekly using the formula TV=0.5xLxWxW until it approached 0.05, after which mice were humanely euthanized.

[0297] On day 105, 17 mice with eradicated tumors were rechallenged with 5E05 MC38 tumor cells, 10 from the combination treatment group and 7 from the anti-PD-1 treatment group. Tumor growth was monitored until day 131. All mice were tumor free.

[0298] The results showed that MC38 tumors harvested from inoculated C57Bl / 6 transgenic mice indeed expressed human CCR8 on Tregs. As shown in Figure 22, treatment with 3 mg / kg Hu149-11G1m showed a moderate antitumor response similar to the effect of anti-PD-1 antibody. Combination treatment with 3 mg / kg Hu149-11G1m and 5 mg / kg anti-PD-1 further reduced the mean tumor volume, and such antitumor effects persisted after cessation of drug treatment. Mice with complete tumor regression showed no tumor growth 15 weeks after tumor rechallenge (Figure 22). As shown in Figure 23, the combination treatment group showed a higher survival rate. Importantly, the results also showed that there was no substantial mean weight loss in any of the groups treated with Hu149-11G1m alone or in combination with anti-PD-1 antibody. Collectively, these data demonstrate that CCR8 antibodies such as Hu149-11G1m can mediate antitumor activity in vivo and further confer sensitivity to anti-PD-1 antibodies. The antitumor effects are durable.

[0299] Example 28: Antitumor activity of CCR8 antibodies in combination with anti-PD-1 antibodies in CCR8 knock-in transgenic mice inoculated with E0771 tumors In this study, immunocompetent mice engineered with the human CCR8 gene instead of the mouse gene were inoculated with murine E0771 breast cancer cells. Tumor-bearing mice were treated with Hu149-11G1m alone or in combination with an anti-PD-1 antibody (Biocell, catalog no. CP151) and tumor growth was assessed.

[0300] In the current study, human gene knock-in transgenic mice were used. Based on the C57Bl / 6 mouse strain, these animals had the human CCR8 gene inserted at the mouse CCR8 locus. The E0771 cell line is a spontaneously arising medullary mammary carcinoma from C57BL / 6 mice. hCCR8 transgenic C57BL / 6 mice were inoculated with the E0771 mouse breast cancer cell line, and tumor-bearing mice were administered Hu149-11G1m twice weekly at a dose of 3 mg / kg as a single agent or in combination with an anti-PD-1 antibody at a dose of 5 mg / kg. Tumor growth was monitored throughout the study period, and survival rates were recorded at the end of the study. Evaluation of tumor-infiltrating lymphocytes (TIL profiling) was also performed to understand how Hu149-11G1m affects immune cells in the tumor microenvironment (TME). CD4, CD8, and Treg were evaluated after isolating fresh tumors.

[0301] E0771 mouse breast cancer cells were cultured in RPMI medium supplemented with 10% FBS and 1x penicillin / streptomycin. After several in vitro passages, tumor cells were harvested using TrypLE Express reagent, counted, and diluted to 20E06 cells / mL in PBS. An equal volume of Matrigel was added to the cell solution to a final concentration of 10E06 cells / mL.

[0302] Transgenic hCCR8 mice were inoculated by injecting 50 μL of the cell suspension into the right mammary fat pad. After 7 days of observation, the tumor volumes of the mice were measured and 56 animals were assigned to four treatment groups (14 mice each). The average tumor volume in each group was approximately 80 mm. 3 Tumor-bearing mice were injected twice weekly with the indicated dose levels of Hu149-11G1m, anti-PD-1, or 10 mg / kg human IgG1. Tumor volumes were calculated using the formula TV=0.5xLxWxW for tumor volumes of 1500 mm 3 Mice were evaluated twice weekly until they reached a maturity of 10 days, after which they were humanely euthanized.

[0303] Seven days after tumor implantation, a subset of mice (n=4 / treatment group) was selected to harvest tumor tissues and perform TIL profiling. Tumor tissues were homogenized and digested with a tumor dissociation kit (Miltenyi 130-096-730). Three million cells from each sample were stained and flow cytometry analysis was performed.

[0304] The remaining mice (n = 10 / treatment group) continued to be monitored twice weekly for health checks and tumor volume measurements. 3 All mice without IFN-γ-γ were maintained for survival evaluation. As detailed below, better survival was achieved and significant Treg depletion was observed in the combination treatment group.

[0305] Results: Combination treatment with 3 mg / kg Hu149-11G1m and 5 mg / kg anti-PD-1 further reduced the mean tumor volume, and such anti-tumor effects persisted even after drug treatment was discontinued (see Figure 24). Table 34 shows the tumor volumes of individual animals at day 22 after tumor inoculation. Hu149-11G1m monotherapy and combination treatment with anti-PD-1 antibody significantly reduced tumor growth compared to the negative control (human IgG1 with matching Fc) (p<0.05 and p<0.001). Furthermore, at the later time point of day 26 after tumor inoculation (Table 35), the combination treatment group showed significantly better anti-tumor effects than the Hu149-11G1m and anti-PD-1 monotherapy group (p<0.01 and p<0.05). As shown in Figure 25, the combination treatment also showed improved survival. Furthermore, there was no substantial mean weight loss in either the groups treated with Hu149-11G1m alone or in combination with anti-PD-1 antibodies. Collectively, these data demonstrate that CCR8 antibodies such as Hu149-11G1m can mediate antitumor activity in vivo and further confer sensitivity to anti-PD-1 antibodies.

[0306] [Table 34]

[0307] [Table 35]

[0308] In the present study, we also found that administration of Hu149-11G1m and anti-PD-1 antibody significantly depleted Tregs in the tumor and increased the CD8+T / Treg ratio (Figure 26) (p<0.05, 4 animals per treatment group were selected to harvest tumor tissues and collect immune cells to stain for flow analysis). There were no significant changes in other immune cell populations.

[0309] Example 29: Binding of FC variants of humanized anti-CCR8 antibodies to TALL-1 cells TALL-1 is a human T-cell leukemia cell line that endogenously expresses CCR8 at levels comparable to those observed in tumor-infiltrating Treg cells and was further used to examine the binding activity of Fc variants of humanized anti-CCR8 antibodies. Briefly, a total of 5 × 10 4 Cells were seeded in a 96-well plate and washed once with FACS buffer (DPBS with 1.5% FBS). Antibodies were prepared in 3-fold serial dilutions ranging from 100 nM to 0.00508 nM in FACS buffer. Cells were incubated with 50 μL of diluted antibody for 40 min at 4 °C. After primary antibody incubation, cells were washed twice with FACS buffer. Then, Alexa Flu647-labeled anti-human IgG Fc secondary antibody (Jackson ImmunoResearch, #109606170) was diluted 1:500 in FACS buffer and stained for 40 min at 4 °C. After washing twice with FACS buffer, flow cytometry (BD FACS Celesta) was performed to measure the binding affinity of the antibodies.

[0310] As shown in Table 36, Hu149-11G1m, Hu149-11G1 (wild-type Fc variant), and TPP21360 (wild-type Fc variant) showed similar binding activity to TALL-1 cells and EC 50were 0.328 nM, 0.315 nM, and 0.461 nM, respectively. The maximum MFI values ​​of Hu149-11G1m, Hu149-11G1 (wild-type variant), and TPP21360 (wild-type variant, see WO2021152186) were also comparable.

[0311] [Table 36]

[0312] Example 30: ADCC activity of anti-CCR8 antibodies To compare the ADCC activity of anti-CCR8 antibodies against TALL-1 cells, which express CCR8 at a level comparable to that observed in tumor-infiltrating Treg cells, an ADCC reporter bioassay was performed. In this setting, the anti-CCR8 antibodies, Hu149-11G1, Hu10A11-1, and TPP21360, all have wild-type IgG1 Fc, and an isotype control was used in this study. The method was as described previously (see Example 22). As shown in Table 37, Hu149-11G1 was significantly more potent than Hu10A11-1 and TPP21360 in the assay. The EC of Hu149-11G1 was significantly higher than that of Hu10A11-1 and TPP21360 in the assay. 50 were 0.0658 nM and 0.120 nM, respectively, when 158V CD16a / NFAT-Jurkat cells and 158F CD16a / NFAT-Jurkat cells were used as surrogate effector cells. 50 Values ​​were not applicable in this assay.

[0313] [Table 37]

[0314] To compare the ADCC activity of anti-CCR8 antibodies, a human PBMC-based ADCC assay was further performed. Human PBMCs (AllCells) were cultured overnight in T75 flasks in RPMI 1640 medium containing 10% heat-inactivated FBS and 200 IU IL-2 (R&D, catalog number: 202-IL). 2 × 10 4 TALL-1 cells were co-cultured with pre-activated PBMCs (1:20 ratio) in the presence of various concentrations of antibodies, and the cells were incubated in RPMI 1640 containing 2% heat-inactivated FBS for 4 h. Upon completion of incubation, lysis buffer was added to the assay plate and incubated for 30 min, then LDH working solution (DOJINDO, Cat. No: CK12) was added to the assay plate and incubated for 25 min. After adding stop solution to the assay plate, the absorbance at 490 nm was measured with a microplate reader. Nonlinear regression analysis was performed with Prism 8 (GraphPad Software) to obtain the EC 50 Values ​​were calculated (log(agonist) vs. response-variable slope (four parameters)). Specific cytotoxicity was calculated by the following formula: % specific cytotoxicity=(ER-ER0) / [(TMR-VCC)-(TSR-CMB)]×100. ER indicates experimental release, ER0 indicates experimental release, no antibody, TMR indicates maximum release of target cells, VCC indicates volume-corrected control, TSR indicates spontaneous release of target cells, and CMB indicates medium background control.

[0315] As shown in Table 38, Hu-149-11G1 (wild-type Fc variant) was 45.7-fold more potent than TPP21360 (wild-type Fc variant) in the ADCC assay, with an EC 50 The EC values ​​of Hu149-11G1m and Hu149-11G1m were 0.0121 nM and 0.553 nM, respectively, and the maximum specific lysis rates were 13.8% and 10.2%, respectively. Hu149-11G1m (ADCC-enhanced variant) showed significantly improved ADCC activity against TALL-1 cells compared with Hu149-11G1 (wild-type variant), and the EC values ​​of Hu149-11G1m were 0.0121 nM and 0.553 nM, respectively, and the maximum specific lysis rates were 13.8% and 10.2%, respectively. 50The specific lysis rate was 0.000682 nM and the maximum specific lysis rate was 32.2%.

[0316] [Table 38]

[0317] While preferred embodiments of the present application have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present application. It is understood that various alternatives to the embodiments of the present application described herein may be used in practicing the present application. The appended claims define the scope of the present application, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. An antigen-binding unit comprising a light chain (LC) variable region and a heavy chain (HC) variable region, the heavy chain variable region comprises HC-complementarity determining region (CDR) 1, HC-CDR2, and HC-CDR3; the light chain variable region comprises LC-CDR1, LC-CDR2, and LC-CDR3; the HC-CDR1 comprises a sequence selected from SEQ ID NOs: 13, 71, 83, 115, and 117, the HC-CDR2 comprises a sequence selected from SEQ ID NOs: 14, 72, 77, 84, 89, 91, 92, 97, 114, 116, and 118, and the HC-CDR3 comprises a sequence selected from SEQ ID NOs: 15, 19-22, 73, 78, 81, 85, 93, 98, 112, and 113; Antigen-binding unit.

2. The antigen binding unit of claim 1, wherein the LC-CDR1 comprises a sequence selected from SEQ ID NOs: 16, 74, 79, 86, 90, 94, 99, and 102 to 109, the LC-CDR2 comprises a sequence selected from SEQ ID NOs: 17, 75, 80, 87, 95, 100, 110, and 111, and the LC-CDR3 comprises a sequence selected from SEQ ID NOs: 18, 23 to 25, 76, 82, 88, 96, and 101.

3. The HC-CDR1, HC-CDR2, and HC-CDR3 are, as a group, respectively, selected from the following groups: SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:19; SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20; SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21; SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:22; SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; SEQ ID NO:71, SEQ ID NO:77, and SEQ ID NO:78; SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:81; SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85; SEQ ID NO:13, SEQ ID NO:89, and SEQ ID NO:15; The antigen-binding unit of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 91, and 15; SEQ ID NOs: 71, 92, and 93; SEQ ID NOs: 13, 97, and 98; SEQ ID NOs: 13, 97, and 112; SEQ ID NOs: 13, 97, and 113; SEQ ID NOs: 13, 114, and 15; SEQ ID NOs: 115, 114, and 15; SEQ ID NOs: 83, 116, and 85; SEQ ID NOs: 117, 116, and 85; and SEQ ID NOs: 83, 118, and 85.

4. The LC-CDR1, LC-CDR2, and LC-CDR3 are, as a group, respectively, selected from the following groups: SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:23; SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:24; SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:25; SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76; SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:76; SEQ ID NO:79, SEQ ID NO:75, and SEQ ID NO:82; SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88; SEQ ID NO:90, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96; SEQ ID NO:79, SEQ ID NO:75, and SEQ ID NO: 76; SEQ ID NO:99, SEQ ID NO:100, and SEQ ID NO:101; SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:103, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:104, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:105, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:106, SEQ ID NO:17, and SEQ ID NO:18; SEQ ID NO:107, SEQ ID NO:100, and SEQ ID NO:101; SEQ ID NO:108, SEQ ID NO:100, and SEQ ID NO:101; SEQ ID NO:109, SEQ ID NO:100, and SEQ ID NO:101; and SEQ ID NO:99, SEQ ID NO:111, and SEQ ID NO:

101.

5. The HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 are, as a group, respectively, selected from the following groups: SEQ ID NOs: 13, 14, 15, 16, 17, and 18; SEQ ID NOs: 13, 14, 19, 16, 17, and 18; SEQ ID NOs: 13, 14, 20, 16, 17, and 18; SEQ ID NOs: 13, 14, 21, 16 , 17, and 18; SEQ ID NOs: 13, 14, 22, 16, 17, and 18; SEQ ID NOs: 13, 14, 15, 16, 17, and 23; SEQ ID NOs: 13, 14, 15, 16, 17, and 24; SEQ ID NOs: 13, 14, 15, 16, 17, and 25; SEQ ID NOs: 13, 14, 19, 16, 17, and 23; SEQ ID NOs: 13, 14, 20, 16, 17, and 23; SEQ ID NOs: The antigen-binding unit of claim 3, comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 13, 14, 21, 16, 17, and 23; SEQ ID NOs: 13, 14, 22, 16, 17, and 23; SEQ ID NOs: 13, 89, 15, 90, 17, and 18; SEQ ID NOs: 13, 89, 15, 102, 17, and 18; SEQ ID NOs: 13, 89, 15, 103, 17, and 18; SEQ ID NOs: 13, 89, 15, 104, 17, and 18; SEQ ID NOs: 13, 89, 15, 16, 17, and 18; SEQ ID NOs: 13, 89, 15, 105, 17, and 18; SEQ ID NOs: 13, 89, 15, 106, 17, and 18; SEQ ID NOs: 13, 114, 15, 16, 17, and 18; and SEQ ID NOs: 115, 114, 15, 16, 17, and 18.

6. The antigen binding unit of claim 3, wherein the HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 comprise, as a group, an amino acid sequence selected from the group of SEQ ID NOs: 83, 84, 85, 86, 87, and 88; SEQ ID NOs: 83, 116, 85, 86, 87, and 88; SEQ ID NOs: 117, 116, 85, 86, 87, and 88; and SEQ ID NOs: 83, 118, 85, 86, 87, and 88, respectively.

7. The antigen binding unit of claim 1, wherein the heavy chain variable region and the light chain variable region comprise, as a group, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 2; SEQ ID NOs: 4 and 2; SEQ ID NOs: 5 and 2; SEQ ID NOs: 6 and 2; SEQ ID NOs: 7 and 2; SEQ ID NOs: 1 and 8; SEQ ID NOs: 1 and 9; SEQ ID NOs: 1 and 10; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 8; SEQ ID NOs: 5 and 8; SEQ ID NOs: 6 and 8; SEQ ID NOs: 11 and 8; SEQ ID NOs: 12 and 8; SEQ ID NOs: 34 and 35; SEQ ID NOs: 34 and 43; SEQ ID NOs: 34 and 44; SEQ ID NOs: 34 and 45; SEQ ID NOs: 34 and 46; SEQ ID NOs: 34 and 47; SEQ ID NOs: 34 and 48; SEQ ID NOs: 56 and 2; SEQ ID NOs: 57 and 2; SEQ ID NOs: 58 and 2; SEQ ID NOs: 59 and 2; SEQ ID NOs: 56 and 60; SEQ ID NOs: 57 and 60; SEQ ID NOs: 58 and 60; and SEQ ID NOs: 59 and 60.

8. The antigen binding unit of claim 1, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences selected from the group consisting of SEQ ID NOs: 32 and 33; SEQ ID NOs: 61 and 62; SEQ ID NOs: 61 and 63; SEQ ID NOs: 61 and 64; SEQ ID NOs: 61 and 65; SEQ ID NOs: 66 and 62; SEQ ID NOs: 67 and 62; SEQ ID NOs: 68 and 62; SEQ ID NOs: 69 and 62; and SEQ ID NOs: 70 and 62, respectively.

9. The antigen-binding unit of claim 1, wherein the antigen-binding unit is a monoclonal antibody, a humanized antibody, a chimeric antibody, a scFv, a Fab', a single-chain Fab (scFab'), a Fd, or a F(ab')2.

10. 2. The antigen binding unit of claim 1, comprising an IgG1 framework and optionally comprising one or more mutations in the IgG1 framework Fc region, wherein optionally the one or more mutations comprise a S239D mutation, a I332E mutation, a L235V mutation, a F243L mutation, a R292P mutation, a Y300L mutation, or a P396L mutation.

11. The antigen-binding unit of claim 1, wherein the antigen-binding unit binds to at least one of human C-C motif chemokine receptor 8 (CCR8) or cynomolgus monkey CCR8, or blocks binding of chemokine C-C motif ligand 1 (CCL1) to human CCR8 or cynomolgus monkey CCR8.

12. A pharmaceutical composition comprising the antigen-binding unit of claim 1 and a pharmaceutically acceptable excipient.

13. An isolated nucleic acid encoding the antigen-binding unit of claim 1.

14. A vector or host cell comprising a nucleic acid sequence encoding the antigen-binding unit of claim 1.

15. The antigen-binding unit of claim 1 for use in treating cancer, comprising: administering an effective amount of the antigen-binding unit of claim 1 to a subject in need of treatment; Optionally, the antigen-binding unit is administered at a dosage of about 0.1 mg / kg to about 10 mg / kg.