C-C chemokine receptor type 8 (CCR8) antagonist antibodies

Abstract

The present application provides isolated antibodies (Ab), e.g., monoclonal antibodies (mAb) that specifically bind to C-C motif chemokine receptor 8 (CCR8), and bispecific antibodies that target CCR8 and CTLA-4, as well as methods for treating cancer in a subject comprising administering to the subject anti-CCR8Ab as monotherapy or in combination with an anti-cancer agent, such as an immune checkpoint inhibitor.

Description

[0001] Related patent applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 412,465, filed Oct. 2, 2022, which is hereby incorporated by reference in its entirety.

[0003] Sequence listing

[0004] The content of the electronic sequence listing (SeqListing-REMD CCR8.xml; size: 91 kilobytes; date of creation: Sep. 30, 2023) is hereby incorporated by reference in its entirety. Technical field

[0005] Two major populations of CD4+ regulatory T cells (Treg cells), defined by whether they express the forkhead box protein 3 transcription factor (Foxp3), are thought to play a key role in maintaining self-tolerance (Barsheshea et al., PNAS, 114(23):6086-6091, 2017). Both the Foxp3+ and Foxp3- subtypes are involved in the regulation of inflammatory autoimmunity and the maintenance of self-tolerance through various mechanisms, including regulating the biological functions of effector TH1 and TH17 CD4+ T cells (Id). The mechanisms by which Tregs promote self-tolerance can be co-opted in the tumor microenvironment to suppress anti-tumor immune responses. Indeed, systemic depletion of Tregs in mice is sufficient to achieve immune-mediated tumor regression (Teng et al., Cancer Research 70(20):7800-9, 2010). Thus, Tregs are thought to play a role in mediating peripheral tolerance to self-antigens, preventing autoimmune diseases, and suppressing anti-tumor immune responses.

[0006] Tregs are found at high frequencies in the tumor tissues of multiple types of solid tumors such as breast cancer, ovarian cancer, renal cell carcinoma (RCC), cervical cancer, prostate cancer, muscle-invasive bladder cancer (MIBC), non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), pancreatic adenocarcinoma (PDAC), brain tumors, head and neck squamous cell carcinoma (HNSCC), and melanoma. Their high frequency within CD4+ T cells in tumor-infiltrating lymphocytes (TILs) or the high ratio of Foxp3+ Tregs to CD8+ cells is associated with poor prognosis in most solid tumors (reviewed in Tanaka, A. and Sakaguchi, S., Cell Res., 27:109-118, 2017).

[0007] Chemokines (chemoattractant cytokines) comprise a family of polypeptides that are structurally and functionally related and are 8-10 kilodaltons in size. Chemokines are involved in a variety of biological functions, including regulation of immune cell proliferation, migration, activation, differentiation, and homing. The biological activity of chemokines is mediated by the family of 7-transmembrane G protein-coupled receptors (GPCRs). CCR4, CCR8, CCR10, and CXCR3 are chemokine receptors responsible for the migration of Treg cells in response to CC and CXC chemokines to the tumor microenvironment (TME): CCR4 is bound by CCL17 and CCL22; CCR8 is bound by CCL1; CCR10 is bound by CCL28; and CXCR3 is activated by CXCL9 / 10 / 11.

[0008] CCR8 (previously also known as CY6, CKR-L1, or TER1) is a chemokine receptor that has recently been identified as a potential specific marker for tumor-infiltrating Tregs, as CCR8 expression is selectively upregulated in these Tregs in a variety of cancers, including breast cancer, colorectal cancer, and lung cancer (Wang L et al., Nature Immunol 20:1220-30, 2019). These CCR8 + Tregs represent a subset of highly activated and inhibitory Tregs, and CCR8 in these tumor types + The high abundance of Tregs is associated with poor prognosis (Id).

[0009] Cancer immunotherapy is based on the concept that the immune system can recognize tumors and eliminate malignant cells. Immunotherapies using agonist, antagonist, or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) have become an area of extensive research and clinical evaluation. Immune checkpoint proteins include CTLA-4, PD-1, PD-L1, LAG-3, TIGIT, and TIM-3, as well as several other proteins (Sharpe et al., Nat Immunol, 8:239-45, 2007). Under normal physiological conditions, immune checkpoints are essential for maintaining self-tolerance (i.e., preventing autoimmunity) and protecting tissues from damage during the immune system's response to pathogen infections. It is now also clear that tumors co-opt certain immune checkpoint pathways as a major mechanism of immune resistance (especially immune tolerance against tumor antigen-specific T cells) (Pardoll DM., Nat Rev Cancer, 12:252-64, 2012). In preclinical models, inhibiting the interaction of PD-1 with its major ligand PD-L1 mediates effective anti-tumor activity (U.S. Patents Nos. 8,008,449 and 7,943,743), and the use of mAb inhibitors of the PD-1 / PD-L1 interaction for the treatment of cancer has become the standard of care for many types of cancer (see, e.g., Topalian et al., Curr Opin Immunol., 24:207-212, 2012; Brahmer et al., N Engl J Med., 366(26):2455-65, 2012; Garon et al., N Engl J Med, 372:2018-2028, 2015; Philips et al., Int. Immunol., 27(1):39-46, 2015; Migden et al., N Engl J Med, 379:341-351, 2018). PD-1 expression has been found on tumor-infiltrating T cells, and PD-L1 expression has been found on tumor cells and myeloid cells within tumors in many murine and human cancers, including human lung cancer, ovarian cancer, and colon cancer, as well as multiple myelomas, and anti-PD-1 antibodies and anti-PD-L1 antibodies developed by, for example, Bristol-Myers Squibb (nivolumab), Merck (pembrolizumab), Regeneron (cemiplimab), Roche (atezolizumab), AstraZeneca (durvalumab) have been approved by the FDA for the treatment of multiple cancer indications. The tolerability of PD-1 pathway blockers and their unique mechanism of action make them an ideal backbone for combination regimen development.Recent clinical data on the combination of CTLA-4 and PD-1 blockade in melanoma patients have shown an increased rate of objective tumor response compared to blocking either checkpoint alone, supporting the view that combination checkpoint blockade may lead to increased clinical benefit (Wolchok et al., N Engl J Med, 366:2443-54, 2012). The combination of Yervoy and Opdivo has been approved for the treatment of certain patients with melanoma, mesothelioma, non-small cell lung cancer, hepatocellular carcinoma, colorectal cancer, and renal cell carcinoma. Despite the observed positive clinical response rates, more patients with advanced solid tumors are resistant or become resistant to immunotherapy (Rizvi et al., Cancer immunology., Science, 348(6230):124-128, 2015).

[0010] Disclosure of the Invention

[0011] According to the present invention, there are provided isolated antibodies and antigen-binding fragments thereof that specifically bind to C-C chemokine receptor type 8 (CCR8), and methods for treating cancer in a subject, the method comprising administering an anti-CCR8 Ab to the subject as a single therapy or in combination with an anti-cancer agent such as an immune checkpoint inhibitor. The inventors propose that the anti-CCR8 mAbs provided herein can be used as safe and effective tumor-infiltrating Treg antagonists and depleting agents that also retain T effector cells (Teff) for optimal anti-tumor responses. CCR8 mAbs provide a safer alternative to other Treg depletion strategies because only tumor-infiltrating Tregs express the high levels of CCR8 required for depletion via ADCC, thus preserving Tregs that maintain immune homeostasis in peripheral tissues. Treg depletion via anti-CTLA-4, anti-CD25, and other Treg surface markers can lead to depletion of both tumor Tregs and peripheral Tregs, which can result in severe autoimmune side effects and potential depletion of beneficial anti-tumor conventional T cells that express a common target antigen.

[0012] In various embodiments, the antibody or antigen-binding fragment is selected from human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, single-chain antibodies, diabodies, triabodies, tetra-bodies, Fab fragments, Fab' fragments, Fab2 fragments, F(ab')2 fragments, domain antibodies, afucosylated antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, IgG1 antibodies having at least one mutation that enhances ADCC / FcR affinity, or IgG4 antibodies having at least one mutation in the hinge region that reduces the propensity to form inter-heavy chain disulfide bonds. In various embodiments, the antibody is a chimeric antibody. In various embodiments, the antibody is a humanized antibody. In various embodiments, the antibody is a fully human antibody. In various embodiments, provided are isolated antibodies and antigen-binding fragments thereof that have high affinity for human CCR8 of SEQ ID NO:1.

[0013] In various embodiments, the antibody or antigen-binding fragment binds to the CCR8 protein with a dissociation constant (K -6 d) of at least about 1×10 -7 −9 -8 M, at least about 1×10 -9 −10 -10 M, at least about 1×10 -11 −11 -12 M, at least about 1×10 D −12

[0014] In various embodiments, the isolated humanized or human monoclonal antibody or antigen-binding fragment thereof of the invention binds to human CCR8 and comprises: (a) a heavy chain CDR1 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO:3, 7, and 9; (b) a heavy chain CDR2 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO:4, 8, 10, and 35-38; (c) a heavy chain CDR3 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO:5, 6, 11, and 39-45; (d) a light chain CDR1 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO:12, 15, and 46-47; (e) a light chain CDR2 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO:13, 16, and 48-49; and (f) a light chain CDR3 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO:14, 17, and 50-52.

[0015] In various embodiments, the isolated humanized or human monoclonal antibody or antigen-binding fragment thereof of the invention binds to human CCR8 and comprises: (1) a heavy chain CDR1 sequence of SEQ ID NO:3; a heavy chain CDR2 sequence of SEQ ID NO:4; a heavy chain CDR3 sequence of SEQ ID NO:5; a light chain CDR1 sequence of SEQ ID NO:12; a light chain CDR2 sequence of SEQ ID NO:13; and a light chain CDR3 sequence of SEQ ID NO:14; or (2) a heavy chain CDR1 sequence of SEQ ID NO:3; a heavy chain CDR2 sequence of SEQ ID NO:4; a heavy chain CDR3 sequence of SEQ ID NO:6; a light chain CDR1 sequence of SEQ ID NO:12; a light chain CDR2 sequence of SEQ ID NO:13; and a light chain CDR3 sequence of SEQ ID NO:14; or (3) a heavy chain CDR1 sequence of SEQ ID NO:7; a heavy chain CDR2 sequence of SEQ ID NO:8; a heavy chain CDR3 sequence of SEQ ID NO:6; a light chain CDR1 sequence of SEQ ID NO:12; a light chain CDR2 sequence of SEQ ID NO:13; and a light chain CDR3 sequence of SEQ ID NO:14; or (4) a heavy chain CDR1 sequence of SEQ ID NO:9; a heavy chain CDR2 sequence of SEQ ID NO:10; a heavy chain CDR3 sequence of SEQ ID NO:11; a light chain CDR1 sequence of SEQ ID NO:15; a light chain CDR2 sequence of SEQ ID NO:16; and a light chain CDR3 sequence of SEQ ID NO:17; or (5) a heavy chain CDR1 sequence of SEQ ID NO:7; a heavy chain CDR2 sequence of SEQ ID NO:35; a heavy chain CDR3 sequence of SEQ ID NO:39; a light chain CDR1 sequence of SEQ ID NO:12; a light chain CDR2 sequence of SEQ ID NO:13; and a light chain CDR3 sequence of SEQ ID NO:14; or (6) a heavy chain CDR1 sequence of SEQ ID NO:3; a heavy chain CDR2 sequence of SEQ ID NO:35; a heavy chain CDR3 sequence of SEQ ID NO:40; a light chain CDR1 sequence of SEQ ID NO:12; a light chain CDR2 sequence of SEQ ID NO:13; and a light chain CDR3 sequence of SEQ ID NO:14; or (7) a heavy chain CDR1 sequence of SEQ ID NO:3; a heavy chain CDR2 sequence of SEQ ID NO:35; a heavy chain CDR3 sequence of SEQ ID NO:40; a light chain CDR1 sequence of SEQ ID NO:12; a light chain CDR2 sequence of SEQ ID NO:13; and a light chain CDR3 sequence of SEQ ID NO:14;or (8) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:4; the heavy chain CDR3 sequence of SEQ ID NO:41; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (9) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:35; the heavy chain CDR3 sequence of SEQ ID NO:42; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (10) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:35; the heavy chain CDR3 sequence of SEQ ID NO:43; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (11) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:35; the heavy chain CDR3 sequence of SEQ ID NO:43; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (12) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:4; the heavy chain CDR3 sequence of SEQ ID NO:44; the light chain CDR1 sequence of SEQ ID NO:46; the light chain CDR2 sequence of SEQ ID NO:48; and the light chain CDR3 sequence of SEQ ID NO:50; or (13) the heavy chain CDR1 sequence of SEQ ID NO:3; the heavy chain CDR2 sequence of SEQ ID NO:4; the heavy chain CDR3 sequence of SEQ ID NO:44; the light chain CDR1 sequence of SEQ ID NO:46; the light chain CDR2 sequence of SEQ ID NO:48; and the light chain CDR3 sequence of SEQ ID NO:50; or (14) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:4; the heavy chain CDR3 sequence of SEQ ID NO:45; the light chain CDR1 sequence of SEQ ID NO:47; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (15) the heavy chain CDR1 sequence of SEQ ID NO:3; the heavy chain CDR2 sequence of SEQ ID NO:38;The heavy chain CDR3 sequence of SEQ ID NO: 44; the light chain CDR1 sequence of SEQ ID NO: 46; the light chain CDR2 sequence of SEQ ID NO: 48; and the light chain CDR3 sequence of SEQ ID NO: 51; or (16) the heavy chain CDR1 sequence of SEQ ID NO: 7; the heavy chain CDR2 sequence of SEQ ID NO: 35; the heavy chain CDR3 sequence of SEQ ID NO: 42; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 13; and the light chain CDR3 sequence of SEQ ID NO: 14; or (17) the heavy chain CDR1 sequence of SEQ ID NO: 7; the heavy chain CDR2 sequence of SEQ ID NO: 36; the heavy chain CDR3 sequence of SEQ ID NO: 45; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 49; and the light chain CDR3 sequence of SEQ ID NO: 14; or (18) the heavy chain CDR1 sequence of SEQ ID NO: 7; the heavy chain CDR2 sequence of SEQ ID NO: 37; the heavy chain CDR3 sequence of SEQ ID NO: 42; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 13; and the light chain CDR3 sequence of SEQ ID NO: 14; or (19) the heavy chain CDR1 sequence of SEQ ID NO: 3; the heavy chain CDR2 sequence of SEQ ID NO: 38; the heavy chain CDR3 sequence of SEQ ID NO: 44; the light chain CDR1 sequence of SEQ ID NO: 46; the light chain CDR2 sequence of SEQ ID NO: 48; and the light chain CDR3 sequence of SEQ ID NO: 52.;

[0016] In various embodiments, the isolated antibody or antigen-binding fragment thereof of the invention binds to human CCR8 and comprises: (a) a heavy chain CDR1 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 3, 7, and 9; (b) a heavy chain CDR2 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 4, 8, 10, and 35 - 38; (c) a heavy chain CDR3 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 5, 6, 11, and 39 - 45; (d) a light chain CDR1 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 12, 15, and 46 - 47; (e) a light chain CDR2 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 13, 16, and 48 - 49; and (f) a light chain CDR3 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 14, 17, and 50 - 52; and (g) a group of four variable region framework regions from human immunoglobulin (IgG). In various embodiments, the antibody may optionally comprise a hinge region. In various embodiments, the framework regions are selected from human germline exons X H , J H , V κ , and J κ sequences. In various embodiments, the antibody is a fully humanized antibody. In various embodiments, the antibody is a fully human antibody.

[0017] In various embodiments, the isolated antibody or antigen-binding fragment thereof of the present invention binds to human CCR8 and comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 22 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 25; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 68; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 68; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 68; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 69; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 69; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 70; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 70; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 60 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 71; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 61 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 71; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 62 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 72; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 63 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 73;or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:64 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:74; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:65 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:75; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:66 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:76; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:67 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:77.

[0018] In various embodiments, the isolated antibody or antigen-binding fragment thereof that binds to human CCR8 of the invention is an isolated chimeric antibody or antigen-binding fragment thereof and comprises: (1) a heavy chain sequence of SEQ ID NO:26 and a light chain sequence of SEQ ID NO:27; or (2) a heavy chain sequence of SEQ ID NO:28 and a light chain sequence of SEQ ID NO:29; or (3) a heavy chain sequence of SEQ ID NO:30 and a light chain sequence of SEQ ID NO:31; or (4) a heavy chain sequence of SEQ ID NO:78 and a light chain sequence of SEQ ID NO:79; or (5) a heavy chain sequence of SEQ ID NO:80 and a light chain sequence of SEQ ID NO:81; or (6) a heavy chain sequence of SEQ ID NO:82 and a light chain sequence of SEQ ID NO:83; or (7) a heavy chain sequence of SEQ ID NO:84 and a light chain sequence of SEQ ID NO:85.

[0019] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the invention binds to human CCR8 and comprises: (a) a heavy chain sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO:86, 88, and 90; and (b) a light chain sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO:87, 89, and 91-92.

[0020] In another aspect, the present invention relates to a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof of the present invention admixed with a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition comprises an isolated human antibody admixed with a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition is formulated for administration via a route selected from the group consisting of subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intraarticular injection, or by infusion.

[0021] In another aspect, the present invention relates to a method of treating a subject suffering from a CCR8-related disorder, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention. In various embodiments, the subject is a human subject. In various embodiments, the CCR8-related disorder is cancer. In various embodiments, the subject was previously responsive to treatment with an anti-cancer therapy but has suffered a relapse after cessation of the therapy (hereinafter referred to as "recurrent cancer"). In various embodiments, the subject has a resistant or refractory cancer. In various embodiments, the cancerous cells are immunogenic tumors (e.g., those tumors for which vaccination with the tumor itself can result in immunity against tumor attack).

[0022] In various embodiments, a method of treating a subject suffering from cancer comprises administering to the subject a therapeutically effective amount of any of the Treg-depleting anti-CCR8 Abs (e.g., mAb, immunoconjugate, or bispecific molecule) disclosed herein, or a pharmaceutical composition comprising any of said Abs (e.g., anti-CCR8 mAb, immunoconjugate, or bispecific molecule), such that the subject is treated.

[0023] In another aspect, the present invention relates to combination therapies designed for treating cancer in a subject. In various embodiments, methods for inhibiting the growth of tumor cells in a subject include administering to the subject a therapeutically effective amount of: (a) any one of the Treg-depleting anti-CCR8 Abs, immunoconjugates, or bispecific molecules disclosed herein, or a pharmaceutical composition comprising any one of the anti-CCR8 Abs, immunoconjugates, or bispecific molecules; and (b) an additional therapy for treating cancer. In various embodiments, the additional therapeutic therapy is a therapeutic agent that is a compound that reduces the suppression of the immune system or increases the stimulation of the immune system such that the growth of tumor cells in the subject is inhibited. In various embodiments, the additional therapy is selected from the group consisting of immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation, wherein the combination therapy provides increased cell killing of tumor cells, i.e., there is a synergistic effect between the isolated antibody or its antigen-binding fragment and the additional therapy when co-administered.

[0024] In another aspect, the present invention relates to a method for enhancing the immune response of a subject to cancerous cells, the method comprising administering to the subject a therapeutically effective amount (as a single therapy or in a combination therapy regimen) of the isolated antibody or its antigen-binding fragment of the present invention. In various embodiments, the present invention provides a method for treating cancerous cells in a subject, the method comprising administering to the subject a therapeutically effective amount (as a single therapy or in a combination therapy regimen) of the antibody or its antigen-binding fragment of the present invention. In various embodiments, the cancerous cells are selected from the group consisting of ovarian cancer, lung cancer, breast cancer, gastric cancer, prostate cancer, colorectal cancer, renal cell cancer, liver cancer, pancreatic cancer, glioblastoma, melanoma, and sarcoma.

[0025] In another aspect, there is provided an isolated immunoconjugate or fusion protein comprising an antibody or antigen-binding fragment conjugated, linked (or otherwise stably associated) to an effector molecule. In various embodiments, the effector molecule is an immunotoxin, cytokine, chemokine, therapeutic agent, or chemotherapeutic agent.

[0026] In another aspect, the invention features bispecific molecules comprising the anti-CCR8 antibodies or antigen-binding fragments thereof of the invention. In various embodiments, the antibodies or antigen-binding fragments of the invention can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand of a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. In various embodiments, the antibodies of the invention can actually be derivatized or linked to more than one other functional molecule to generate a multispecific molecule that binds to more than two different binding sites and / or target molecules. In various embodiments, the bispecific molecule is an anti-CCR8 antibody or antigen-binding fragment thereof of the invention linked to a functional molecule that binds CTLA-4. In various embodiments, the bispecific molecule is a bispecific antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO:93 and a light chain having the amino acid sequence of SEQ ID NO:91. In various embodiments, the bispecific molecule is a bispecific antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO:93 and a light chain having the amino acid sequence of SEQ ID NO:92.

[0027] In another aspect, the antibodies or antigen-binding fragments disclosed herein can be covalently linked (or otherwise stably associated) with additional functional moieties such as markers or moieties that confer desired pharmacokinetic properties. In various embodiments, the markers are selected from the group consisting of fluorescent markers, radioactive markers, and markers having unique nuclear magnetic resonance characteristics.

[0028] In another aspect, the invention provides methods for detecting the presence of human CCR8 peptide in a sample in vitro or in vivo, e.g., for diagnosing human CCR8-related disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a line graph depicting the dose response of an anti-CCR8 antibody in blocking CCR8-mediated calcium flux induced by hCCL1. Reference Ab#1 is a humanized anti-hCCR8 antibody that has been described in the literature.

[0031] Figure 2 is a line graph depicting the dose response of an anti-CCR8 antibody in blocking CCR8-mediated calcium flux induced by hCCL1. Reference Ab#1 and #2 are humanized anti-hCCR8 antibodies that have been described in the literature.

[0032] Figures 3A-3D are a set of graphs showing that the bispecific antibodies FP578-01 and FP578-02 can bind CTLA-4 while they simultaneously bind CCR8.

[0033] Modes of Carrying Out the Invention

[0034] The present invention relates to antigen-binding proteins that specifically bind to human CCR8, such as antibodies or antigen-binding fragments thereof. In one aspect, isolated antibodies and antigen-binding fragments thereof are provided that specifically bind to CCR8, have high affinity for CCR8, exert an inhibitory function on CCR8, have lower immunogenicity in a given species (e.g., human) compared to their unmodified parental antibodies, and can be used to treat human disorders mediated by CCR8. Nucleic acid molecules and derivatives and fragments thereof are also provided that comprise a sequence of a polynucleotide encoding an entire polypeptide or a portion of a polypeptide that binds to CCR8, such as a nucleic acid encoding an entire anti-CCR8 antibody or a portion of an anti-CCR8 antibody, an antibody fragment, or an antibody derivative. Vectors and plasmids comprising such nucleic acids are also provided, as well as cells or cell lines comprising such nucleic acids and / or vectors and plasmids. Methods for preparing, identifying, or isolating an antigen-binding protein that binds to human CCR8 (such as an anti-CCR8 antibody), methods for determining whether an antigen-binding protein binds to CCR8, methods for preparing a composition (such as a pharmaceutical composition) comprising an antigen-binding protein that binds to human CCR8, and methods for administering an antibody or antigen-binding fragment thereof that binds to CCR8 to a subject, e.g., methods for treating a condition mediated by CCR8, are also provided.

[0035] Definition

[0036] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclature and techniques described herein in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are those that are commonly used and well known in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), which is incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature and experimental procedures and techniques described herein in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry are those that are commonly used and well known in the art. Standard techniques are used for chemical synthesis, chemical analysis, the preparation, formulation, and delivery of drugs, and the treatment of subjects.

[0037] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. In various embodiments, a "peptide," "polypeptide," and "protein" is a chain of amino acids in which the α-carbons of the amino acids are joined by peptide bonds. Thus, the terminal amino acid at one end (amino terminus) of the chain has a free amino group, while the terminal amino acid at the other end (carboxyl terminus) of the chain has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free α-amino group on the amino acid at the amino terminus of a peptide, or to the α-amino group of an amino acid at any other position in the peptide (which is an imino group when participating in a peptide bond). Similarly, the term "carboxyl terminus" refers to the free carboxyl group on the carboxyl terminus of a peptide, or to the carboxyl group of an amino acid at any other position in the peptide. Peptides also include substantially any polyamino acid, including but not limited to peptide mimetics such as amino acids joined by ether bonds rather than amide bonds.

[0038] The polypeptides of the present disclosure include polypeptides that have been modified in any way and for any reason, such as: (1) to reduce susceptibility to proteolysis, (2) to reduce susceptibility to oxidation, (3) to alter binding affinity for forming protein complexes, (4) to alter binding affinity, and (5) to confer or modify other physicochemical or functional properties.

[0039] As used herein, an amino acid "substitution" refers to the replacement of one amino acid at a specific position in a parental polypeptide sequence in a polypeptide with a different amino acid. Amino acid substitutions can be generated using genetic methods or chemical methods well known in the art. For example, single amino acid substitutions or more than one amino acid substitution (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in a portion of the polypeptide outside of one or more domains that form intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid that is functionally similar. The following six groups each contain amino acids that are conservative substitutions for one another:

[0040] 1) Alanine (A), Serine (S), and Threonine (T)

[0041] 2) Aspartic acid (D) and Glutamic acid (E)

[0042] 3) Asparagine (N) and Glutamine (Q)

[0043] 4) Arginine (R) and Lysine (K)

[0044] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

[0045] 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0046] A "non-conservative amino acid substitution" refers to the replacement of a member of one of these categories with a member from another category. In making such a change, the hydropathic index of the amino acid can be considered according to various embodiments. Based on the hydrophobicity and charge characteristics of the amino acids, each amino acid has been assigned a hydropathic index. They are: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / Cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9); and Arginine (-4.5).

[0047] Those skilled in the art understand the importance of the hydrophilic amino acid index in conferring the biological functions of protein interactions (see, for example, Kyte et al., 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids can be replaced by other amino acids having similar hydrophilic indices or scores and still retain similar biological activities. In making changes based on the hydrophilic index, in various embodiments, substitutions of amino acids having a hydrophilic index within ±2 are included. In various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0048] Those skilled in the art also understand that similar amino acid substitutions can be effectively made based on hydrophilicity, particularly in cases where the resulting biologically functional protein or peptide is intended for use in immunological embodiments as disclosed herein. In various embodiments, the maximum local average hydrophilicity of a protein (determined by the hydrophilicity of its neighboring amino acids) is related to its immunogenicity and antigenicity, i.e., to the biological properties of the protein.

[0049] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 0.1); glutamic acid (+3.0 ± 0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based on similar hydrophilicity values, in various embodiments, substitutions of amino acids having a hydrophilicity value within ±2 are included, in various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0050] Exemplary amino acid substitutions are listed in Table 1.

[0051] Table 1

[0052]

[0053]

[0054] A person skilled in the art will be able to use well-known techniques to determine suitable polypeptide variants as listed herein. In various embodiments, a person skilled in the art can identify suitable regions of a molecule that can be altered without disrupting activity by targeting regions that are considered unimportant for activity. In other embodiments, a person skilled in the art can identify residues and portions of a molecule that are conserved among similar polypeptides. In additional embodiments, even regions that may be important for biological activity or for structure can undergo conservative amino acid substitutions without disrupting biological activity or adversely affecting polypeptide structure.

[0055] In addition, a person skilled in the art can review structure - function studies that identify residues important for activity or structure in similar polypeptides. Given such comparisons, a person skilled in the art can predict the importance of amino acid residues in a polypeptide corresponding to amino acid residues important for activity or structure in similar polypeptides. A person skilled in the art can choose to make chemically similar amino acid substitutions for such predicted important amino acid residues.

[0056] A person skilled in the art can also analyze the three - dimensional structure and amino acid sequence related to that structure in similar polypeptides. Given such information, a person skilled in the art can predict the arrangement of the amino acid residues of a polypeptide in terms of its three - dimensional structure. In various embodiments, a person skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of a polypeptide, because such residues may be involved in important interactions with other molecules. In addition, a person skilled in the art can generate test variants that contain single amino acid substitutions at each desired amino acid residue. The variants can then be screened using activity assays known to a person skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is found that altering a particular amino acid residue results in disrupted, undesirably reduced, or inappropriate activity, variants having such an alteration can be avoided. In other words, based on the information collected from such routine experiments, a person skilled in the art can easily determine the amino acids at which further substitutions, either alone or in combination with other mutations, should be avoided.

[0057] As used herein, the terms "polypeptide fragment" and "truncated polypeptide" refer to polypeptides having an amino-terminal deletion and / or a carboxyl-terminal deletion as compared to the corresponding full-length protein. In various embodiments, the length of the fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In various embodiments, the length of the fragment can also be, for example, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids. The fragment can also contain one or more additional amino acids at either or both of its termini, for example, sequences of amino acids from a different naturally-occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0058] As used herein, the terms "polypeptide variant", "hybrid polypeptide", and "polypeptide mutant" refer to polypeptides comprising an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the amino acid sequence relative to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. The hybrids of the present disclosure include fusion proteins.

[0059] As described herein, a single mutation will be identified by a specific amino acid substitution at a specific amino acid position within the sequence of wild-type CCR8. For example, for human CCR8 provided as SEQ ID NO:1, a mutation comprising a serine substituting for the full-length wild-type threonine at amino acid 10 is identified as T10S.

[0060] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, such as conjugated to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0061] The terms “% sequence identity” and “% identity” are used interchangeably herein and refer to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity determined by a defined algorithm and means that a given sequence is at least 80% identical to another sequence of another length. In various embodiments, the % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or greater sequence identity to a given sequence. In various embodiments, the % identity is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 95% to about 99%.

[0062] The terms “% sequence homology” and “% homology” are used interchangeably herein and refer to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same as 80% sequence homology determined by a defined algorithm and thus a homolog of a given sequence has a sequence homology greater than 80% relative to the length of the given sequence. In various embodiments, the % homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or greater sequence homology to a given sequence. In various embodiments, the % homology is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 95% to about 99%.

[0063] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, a suite of BLAST programs publicly available on the Internet at the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990 (specifically refer to the publicly available default settings, i.e., parameters w = 4, t = 17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. When evaluating a given amino acid sequence relative to amino acid sequences in GenBank protein sequences and other publicly available databases, the BLASTP program is typically used for sequence searching. The BLASTX program is preferably used to search for nucleic acid sequences that have been translated in all reading frames against amino acid sequences in GenBank protein sequences and other publicly available databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0 and using the BLOSUM-62 matrix.

[0064] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or amino acid sequences would occur by chance. For example, if in a comparison of a test nucleic acid with a reference nucleic acid, the minimum total probability is, for example, less than about 0.1, less than about 0.01, or less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0065] As used herein, the term "modification" refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modification of the polypeptide (e.g., glycosylation).

[0066] The term "therapeutic protein" refers to a protein, polypeptide, antibody, peptide, or fragment or variant thereof having one or more therapeutic and / or biological activities. Therapeutic proteins encompassed by the present invention include, but are not limited to, proteins, polypeptides, peptides, antibodies, and biologics (the terms peptide, protein, and polypeptide may be used interchangeably herein). Specifically contemplated is that the term "therapeutic protein" encompasses the fusion molecules of the present invention.

[0067] As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more genes that originally encoded different proteins, wherein the components of the fusion protein are directly linked to each other by a peptide bond or are linked to each other by a peptide linker. As used herein, the term "fusion" refers to components that are directly linked by a peptide bond or components that are linked via one or more peptide linkers.

[0068] "Linker" refers to a molecule that covalently or by ionic, van der Waals, or hydrogen bonds links two other molecules, such as a nucleic acid molecule that hybridizes to a complementary sequence at the 5' end and hybridizes to another complementary sequence at the 3' end, thus linking two non-complementary sequences. "Cleavable linker" refers to a linker that can be degraded or otherwise cleaved to separate two components linked by the cleavable linker. Cleavable linkers are typically cleaved by enzymes, usually peptidases, proteases, nucleases, lipases, etc. Cleavable linkers can also be cleaved by changes in environmental factors, such as, for example, temperature, pH, salt concentration, etc.

[0069] As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, usually about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n , (SG4) n or G4(SG4) n peptide linkers. "n" is typically a number between 1 and 10, usually between 2 and 4.

[0070] The term "tumor-associated antigen" (TAA) refers to, for example, a cell surface antigen that is selectively expressed or overexpressed in cancer cells relative to most normal cells. As used herein, the terms "TAA variant" and "TAA mutant" refer to a TAA comprising an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the amino acid sequence relative to another TAA sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length.

[0071] As used herein, the term "antibody" refers to a protein that comprises one or more polypeptides and has specificity for a tumor antigen or for a molecule that is overexpressed in a pathological condition, the polypeptides being encoded substantially or partially by immunoglobulin genes or fragments of immunoglobulin genes. Well-known immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as subtypes of these genes and a large number of immunoglobulin variable region genes. Light chains (LCs) are classified as κ or λ. Heavy chains (HCs) are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer comprises the same two pairs of polypeptide chains, each pair having one "light chain" (≈25 kD) and one "heavy chain" (≈50 kD - 70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition.

[0072] In a full-length antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3 (and in some instances, CH4). Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, C L . The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs) that are interspersed therebetween. Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The ranges of the framework regions and CDRs have been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved in species such as humans. The framework region of an antibody, i.e., the combined framework regions of the light and heavy chain components, serves to position and align the CDRs in three-dimensional space. An immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0073] The CDRs are mainly responsible for binding to the epitopes of antigens. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also commonly identified by the chain in which the specific CDR is located. Thus, VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although the CDRs vary between antibodies, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. These positions in the CDRs are called specificity-determining residues (SDRs).

[0074] The Kabat definition is the standard for numbering residues in antibodies and is commonly used to identify CDR regions. The Kabat database is now maintained online, and CDR sequences can be determined. For example, see the IMGT / V-QUEST program version: 3.2.18, March 29, 2011, available on the Internet, and Brochet, X et al., Nucl. Acids Res. 36, W503-508, 2008). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the positions of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17, 1986; Chothia et al., Nature, 342:877-83, 1989. The AbM definition uses a set of integrated computer programs produced by Oxford Molecular Group for modeling antibody structures. See, for example, Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272, 1989; "AbM" TM"A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of a knowledge base and ab initio methods to simulate the tertiary structure of antibodies from primary sequences, such as those described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, 1999. The definition of contacts is based on the analysis of available complex crystal structures. See, for example, MacCallum et al., J. Mol. Biol., 5:732-45, 1996.

[0075] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be generated by digesting a whole antibody with papain. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin typically contains two constant domains, the CH2 domain and the CH3 domain, and optionally contains a CH4 domain. The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance rate of the antibody-antigen-antibody complex (e.g., the neonatal Fc receptor (FcRn) binds to the Fc region of IgG at the acidic pH in the endosome and protects IgG from degradation, thus contributing to the long serum half-life of IgG). Substituting amino acid residues in the Fc portion to alter antibody effector function is known in the art (see, for example, Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821).

[0076] Antibodies exist as intact immunoglobulins or as many well-characterized fragments. Such fragments include Fab fragments, Fab’ fragments, Fab2, F(ab’)2 fragments, single-chain Fv proteins (“scFv”), and disulfide-stabilized Fv proteins (“dsFv”) that bind to a target antigen. The scFv protein is a fusion protein in which the variable region of the light chain of an immunoglobulin and the variable region of the heavy chain of an immunoglobulin are joined by a linker, while in the dsFv, the chains have been mutated to introduce disulfide bonds that stabilize the association of the chains. Although various antibody fragments have been defined in terms of digestion of intact antibodies, one of ordinary skill in the art will understand that such fragments can be synthesized de novo chemically or by using recombinant DNA methods. Thus, as used herein, the term antibody includes, for example, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab’)2 fragments, antibody fragments that exhibit the desired biological activity, disulfide-linked Fv (sdFv), intracellular antibodies (intrabody), and epitope-binding fragments or antigen-binding fragments of any of the foregoing.

[0077] Papain digestion of an antibody produces two identical antigen-binding fragments called “Fab” fragments, each having a single antigen-binding site. A “Fab fragment” contains one light chain as well as the CH1 and variable regions of one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab’ fragment” contains one light chain and a portion of one heavy chain that includes the VH domain and the CH1 domain and also includes the region between the CH1 and CH2 domains such that an interchain disulfide bond can form between the heavy chains of two Fab’ fragments to form an F(ab’)2 molecule.

[0078] Pepsin treatment of an antibody produces an F(ab’)2 fragment that has two antigen-binding sites and is still capable of cross-linking antigens. An “F(ab’)2 fragment” contains two light chains and two heavy chains, the two heavy chains containing a portion of the constant region between the CH1 and CH2 domains such that an interchain disulfide bond forms between the two heavy chains. Thus, the F(ab’)2 fragment contains two Fab’ fragments joined together by a disulfide bond between the two heavy chains.

[0079] The “Fv region” contains the variable regions from both the heavy and light chains but lacks the constant regions.

[0080] "Single-chain antibody" refers to an Fv molecule in which the heavy-chain variable region and the light-chain variable region have been joined by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649, U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference.

[0081] As used herein, the terms "antigen-binding fragment" and "antigen-binding protein" mean any protein that binds to a specific target antigen. "Antigen-binding fragment" includes, but is not limited to, an antibody and its binding portions, such as immunologically functional fragments. Exemplary antigen-binding fragments of an antibody are one or more heavy-chain CDRs and / or one or more light-chain CDRs, or the heavy-chain variable region and / or the light-chain variable region.

[0082] As used herein, the term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen-binding protein is an antigen-binding protein that comprises a portion of an antibody that lacks at least some of the amino acids present in the full-length chain but that is still capable of specifically binding to an antigen (regardless of how that portion was obtained or synthesized). Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding proteins (including full antibodies) for binding to a given epitope. In some embodiments, the fragment is a neutralizing fragment. In one aspect, such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments, will comprise a single heavy chain and / or light chain or a portion thereof. These biologically active fragments can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical cleavage of an antigen-binding protein (including a full antibody). Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, diabodies, Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camel, or rabbit. Also contemplated are functional portions of the antigen-binding proteins disclosed herein, such as one or more CDRs, that can be covalently bound to a second protein or small molecule to produce a therapeutic agent with bifunctional therapeutic properties or with an extended serum half-life for a specific target in the body.

[0083] A diabody is a bivalent antibody that contains two polypeptide chains, each of which contains a VH and a VL region linked by a linker that is too short to permit pairing between the two regions on the same chain, thereby allowing each region to pair with a complementary region on the other polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-48, 1993; and Poljak et al., Structure, 2:1121-23, 1994). If the two polypeptide chains of the diabody are identical, the diabody produced by their pairing will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare diabodies with two different antigen-binding sites. Similarly, a triabody and a tetrabody are antibodies that contain three and four polypeptide chains, respectively, and form three and four antigen-binding sites, respectively, which can be the same or different.

[0084] Bispecific antibodies or fragments can have several configurations. For example, a bispecific antibody can resemble a single antibody (or antibody fragment) but have two different antigen-binding sites (variable regions). In various embodiments, bispecific antibodies can be produced by chemical techniques (Kranz et al., Proc. Natl. Acad. Sci. USA, 78:5807, 1981); by the "polydoma" technique (see, e.g., U.S. Patent No. 4,474,893); or by recombinant DNA techniques. In various embodiments, the bispecific antibodies of the present disclosure can have binding specificities for at least two different epitopes, at least one of which is a tumor-associated antigen. In various embodiments, the antibodies and fragments can also be heteroantibodies. A heteroantibody is two or more antibodies or antigen-binding fragments (e.g., Fab) linked together, each with a different specificity.

[0085] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring that the antibody be produced by any particular method.

[0086] As used herein, the term "chimeric antibody" refers to an antibody that has framework residues from one species (such as human) and CDRs (which typically confer antigen binding) from another species, such as a murine antibody that specifically binds to a targeted antigen.

[0087] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation), such as in the CDRs and particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0088] As used herein, the term "humanized antibody" refers to an antibody that comprises a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds the same antigen as the donor antibody that provides the CDRs. The acceptor framework of the humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. A humanized antibody or other monoclonal antibody may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. In various embodiments, the framework region is selected from human germline exons X H , J H , Vκ and Jκ sequences. For example, the acceptor sequence for humanization of the FR for the V H domain may be selected from germline V H exon V H 1-18 (Matsuda et al., Nature Genetics 3:88-94, 1993) or V H 1-2 (Shin et al., EMBO J. 10:3641-3645, 1991) and for the hinge region (J H ), from exon J H -6 (Mattila et al., Eur. J. Immunol. 25:2578-2582, 1995). In other instances, germline Vκ exon B3 (Cox et al., Eur. J. Immunol. 24:827-836, 1994) and Jκ exon Jκ-1 (Hieter et al., J. Biol. Chem. 257:1516-1522, 1982) may be selected as acceptor sequences for humanization of the V L domain.

[0089] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant, combinatorial human antibody library; antibodies isolated from an animal that is transgenic for human immunoglobulin genes (e.g., a mouse); or antibodies prepared, expressed, produced, or isolated by any other means that include splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions that are derived from human germline immunoglobulin sequences. However, in various embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using transgenic animals with human Ig sequences, to in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur in the in vivo human antibody germline repertoire. All such recombinant means are well known to those of ordinary skill in the art.

[0090] The term "anti-CCR8 antagonist antibody" (which may be interchangeably referred to as "anti-CCR8 antibody") refers to an antibody that is capable of binding to CCR8 and inhibiting CCR8 biological activity and / or one or more downstream pathways mediated by CCR8 signaling. Anti-CCR8 antagonist antibodies include antibodies that block, antagonize, inhibit, or reduce (including significantly reducing) CCR8 biological activity, which includes downstream pathways mediated by CCR8 signaling, such as receptor binding and / or eliciting a cellular response to CCR8. For purposes of the present invention, it will be understood that the term "anti-CCR8 antagonist antibody" includes all previously defined terms, headings, and functional states and characteristics, whereby CCR8 itself, CCR8 biological activity (including but not limited to its ability to mediate any aspect of headache), or the consequences of biological activity are substantially eliminated, reduced, or neutralized to any meaningful extent. In some embodiments, the anti-CCR8 antagonist antibody binds to CCR8 and prevents CCR8 from binding to the CCR8 receptor. In other embodiments, the anti-CCR8 antibody binds to CCR8 and prevents activation of the CCR8 receptor. Examples of anti-CCR8 antagonist antibodies are provided herein.

[0091] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T cell receptor or otherwise interacting with a molecule. Epitope determinants are usually composed of chemically active surface groupings of molecules, such as amino acids or carbohydrates or sugar side chains, and generally have specific three-dimensional structural features as well as specific charge features. Epitopes can be "linear" or "conformational". In a linear epitope, all points of interaction between a protein and an interacting molecule, such as an antibody, are linearly present along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction span amino acid residues that are separated from each other on the protein. Once a desired epitope on an antigen is identified, it is possible to generate an antibody against that epitope, for example, using the techniques described in the present disclosure. Optionally, during the discovery process, the generation and characterization of antibodies can elucidate information about the desired epitope. Then, based on this information, it is possible to competitively screen for antibodies that bind to the same epitope. A way to achieve this is to perform cross-competition studies to find antibodies that competitively bind to each other, such as antibodies that compete to bind an antigen.

[0092] If an antigen-binding protein (including an antibody) binds to an antigen with a high binding affinity as determined by a dissociation constant (K D , or the corresponding Kb, as defined below) value of at least 1×10 -6 M, or at least 1×10 -7 M, or at least 1×10 -8 M, or at least 1×10 -9 M, or at least 1×10 -10 M, or at least 1×10 -11 M, then the antigen-binding protein (including an antibody) "specifically binds" to the antigen. An antigen-binding protein that specifically binds to a human antigen of interest may also be able to bind to the same antigen of interest from other species with the same or different affinities. As used herein, the term "K D " refers to the equilibrium dissociation constant of a particular antigen:antibody interaction.

[0093] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in the protein concentration in a biosensor matrix, for example, using BIACORE TMSystem (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further description, see Jonsson U. et al., Ann. Biol. Clin., 51:19-26, 1993; Jonsson U. et al., Biotechniques, 11:620-627, 1991; Jonsson B. et al., J. Mol. Recognit., 8:125-131, 1995; and Johnsson B. et al., Anal. Biochem, 198:268-277, 1991.

[0094] As used herein, the term "tumor microenvironment" refers to the cellular environment in which a tumor exists, including the surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix (ECM). The components in the tumor microenvironment can regulate the growth of tumor cells, such as their ability to progress and metastasize. The tumor microenvironment can also be affected by the extracellular signals released by the tumor, promoting tumor angiogenesis and inducing peripheral immune tolerance.

[0095] As used herein, the term "immunogenicity" refers to the ability of an antibody or antigen-binding fragment to elicit an immune response (humoral or cellular response) when administered to a recipient, and includes, for example, a human anti-mouse antibody (HAMA) response. When T cells from a subject mount an immune response to the administered antibody, a HAMA response is initiated. Then, the T cells recruit B cells to generate specific "anti-antibody" antibodies.

[0096] As used herein, the term "immune cell" means any cell of the hematopoietic lineage that is involved in regulating an immune response against an antigen (e.g., self-antigen). In various embodiments, the immune cell is, for example, a T cell, B cell, dendritic cell, monocyte, natural killer cell, macrophage, Langerhans cell, or Kupffer cell.

[0097] "Pharmaceutical composition" means a composition suitable for pharmaceutical use in animals. The pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" means the amount of an agent effective to produce the desired pharmacological result. "Pharmaceutically acceptable carrier" means any standard pharmaceutical carrier, vehicle, buffer and excipient, such as phosphate buffered saline solution, aqueous solution of 5% dextrose, and emulsions, such as oil / water emulsion or water / oil emulsion, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Edition 2005, Mack Publishing Co, Easton. "Pharmaceutically acceptable salt" means a salt of a compound that can be formulated for pharmaceutical use, including for example salts of metals (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or salts of organic amines.

[0098] As used herein, "treatment" (and its grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated, and the clinical intervention can be used prophylactically or during a clinical pathologic process. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. As used herein, "alleviating" a disease, disorder or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder or condition. Additionally, reference to "treatment" herein includes reference to curative, palliative and prophylactic treatment.

[0099] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a compound or composition sufficient to treat a particular disorder, condition or disease, such as to improve, alleviate, reduce and / or delay one or more of its symptoms. With respect to NHL and other cancers or other unwanted cell proliferation, an effective amount includes an amount sufficient for: (i) reducing the number of cancer cells; (ii) reducing the tumor size; (iii) inhibiting, to some extent, delaying, slowing and preferably stopping the infiltration of cancer cells into peripheral organs; (iv) inhibiting (i.e., slowing to some extent and preferably stopping) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the occurrence and / or recurrence of a tumor; and / or (vii) alleviating, to some extent, one or more symptoms associated with cancer. An effective amount can be administered in one or more administrations.

[0100] "Adjuvant setting" refers to a clinical setting in which an individual has a history of a proliferative disease, particularly a history of cancer, and typically (but not necessarily) responds to treatment (including but not limited to surgery (such as surgical resection), radiotherapy, and chemotherapy). However, due to their history of a proliferative disease (such as cancer), these individuals are considered at risk of developing the disease. Treatment or administration in the "adjuvant setting" refers to subsequent treatment modalities. The degree of risk (i.e., whether an individual in the adjuvant setting is considered "high risk" or "low risk") depends on several factors, most commonly the extent of the disease at the time of the first treatment.

[0101] The terms "administer" or "cause to be administered" mean the action of a medical professional (such as a physician) or a person controlling the medical care of a patient to control and / or permit the administration of the agent / compound under discussion to the patient. Causing administration can include diagnosing and / or determining an appropriate treatment regimen, and / or prescribing a specific agent / compound for the patient. Such prescribing can include, for example, drafting a prescription form, annotating medical records, etc. "Causing administration" is also contemplated when administration is described herein.

[0102] As used herein, the terms "co-administration", "co-administered", and "in combination with" in reference to the fusion molecules of the invention with one or more other therapeutic agents are intended to mean and do in fact mean the following and include the following: when such components are formulated together into a single dosage form that releases said components to the individual substantially simultaneously, such a combination of the fusion molecules of the invention with one or more therapeutic agents is administered simultaneously to an individual in need of treatment; when such components are formulated separately into separate dosage forms that are taken by the individual substantially simultaneously, whereupon said components are released to the individual substantially simultaneously, such a combination of the fusion molecules of the invention with one or more therapeutic agents is administered to an individual in need of treatment substantially simultaneously; when such components are formulated separately into separate dosage forms that are taken by the individual at successive times with a significant time interval between each administration, whereupon said components are released to the individual at substantially different times, such a combination of the fusion molecules of the invention with one or more therapeutic agents is administered sequentially to an individual in need of treatment; and, when such components are formulated together into a single dosage form that releases said components in a controlled manner, whereupon they are released to the individual simultaneously, successively, and / or overlappingly at the same and / or different times, where each portion can be administered by the same or different routes, such a combination of the fusion molecules of the invention with one or more therapeutic agents is administered sequentially to an individual in need of treatment.

[0103] The terms "patient", "individual", and "subject" are used interchangeably and refer to a mammal, preferably a human or non-human primate, but also a domestic mammal (e.g., canine or feline), a laboratory mammal (e.g., mouse, rat, rabbit, hamster, guinea pig), and an agricultural mammal (e.g., equine, bovine, swine, ovine). In various embodiments, the patient can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other medical staff in a hospital, a psychiatric care facility such as an outpatient clinic, or other clinical settings. In various embodiments, the patient can be an immunocompromised patient or a patient with a weakened immune system, including but not limited to patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressive drugs; and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have a high mutation rate (Lawrence et al., Nature, 499(7457):214–218, 2013).

[0104] The term "immunotherapy" refers to cancer treatment, including but not limited to treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic antibodies, antagonistic antibodies, or blocking antibodies against co-stimulatory molecules or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, SIRP, CD40, CD47, Siglec8, Siglec 9, Siglec 15, TIGIT, and VISTA; treatment with bispecific T cell engager antibodies such as blinatumomab; treatment involving administration of biological response modifiers such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatment with therapeutic vaccines such as sipuleucel-T; treatment with Bacillus Calmette-Guérin (BCG); treatment with dendritic cell vaccines or tumor antigen peptide vaccines; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor-infiltrating lymphocytes (TIL); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic anti-tumor T cells); treatment with TALL-104 cells; and treatment with immune stimulants such as Toll-like receptor (TLR) agonists CpG and imiquimod.

[0105] "Resistant or refractory cancer" refers to tumor cells or cancers that do not respond to a previous anti-cancer therapy, where the previous anti-cancer therapy includes, for example, chemotherapy, surgery, radiotherapy, stem cell transplantation, and immunotherapy. Tumor cells can be resistant or refractory at the start of treatment or can become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond to treatment at the start of treatment or initially respond to treatment for a short period but lose response. Refractory tumor cells also include tumors that respond to anti-cancer therapy treatment but lose response to subsequent rounds of therapy. For the purposes of the present invention, refractory tumor cells also include tumors that appear to be inhibited by anti-cancer therapy treatment but recur up to 5 years (and sometimes up to 10 years or longer) after treatment is stopped. Anti-cancer therapy can use a single chemotherapeutic agent, radiotherapy alone, targeted therapy alone, immunotherapy alone, surgery alone, or combinations thereof. For ease of description and not limitation, it should be understood that refractory tumor cells and resistant tumors are interchangeable.

[0106] As used herein, "specifically binds" means that the binding to an antigen is selective and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind a specific antigen can be measured by an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art such as surface plasmon resonance (SPR) techniques.

[0107] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). The affinity of molecule X for its partner Y can generally be presented by the dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). A particular method for measuring affinity is surface plasmon resonance (SPR).

[0108] As used herein, the term "reduced binding" refers to a decrease in the affinity of the respective interaction, as measured, for example, by SPR. In contrast, "increased binding" refers to an increase in the binding affinity of the respective interaction.

[0109] As used herein, the term "polymer" generally includes, but is not limited to, homopolymers; copolymers such as, for example, block, graft, random, and alternating copolymers; and terpolymers; as well as mixtures and modifications thereof. Additionally, unless otherwise explicitly defined, the term "polymer" shall include all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and atactic symmetries.

[0110] "Polynucleotide" means a polymer comprising nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include analogs that contain non-naturally occurring bases, nucleotides that engage with other nucleotides through linkages other than the naturally occurring phosphodiester bond, or nucleotides that contain bases attached through linkages other than the phosphodiester bond. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, dithiophosphates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using automated DNA synthesizers. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, typically no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T".

[0111] In this article, polynucleotide sequences are described using conventional symbols: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is called the 5'-direction. The 5' to 3' direction in which nucleotides are added to a nascent RNA transcript is called the transcription direction. The DNA strand having the same sequence as the mRNA is called the "coding strand"; the sequence at the 5' that is on the DNA strand having the same sequence as the mRNA transcribed from that DNA and is located 5' of the 5'-end of the RNA transcript is called the "upstream sequence"; the sequence at the 3' that is on the DNA strand having the same sequence as the RNA and is at the 3'-end of the coding RNA transcript is called the "downstream sequence".

[0112] "Vector" refers to a polynucleotide that can be used to introduce another nucleic acid linked thereto into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), into which additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell and are thus replicated together with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0113] "Regulatory sequence" refers to a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid operably linked thereto. A regulatory sequence can, for example, exert its effect directly on the nucleic acid being regulated or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Other examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (e.g., level, timing, or location of expression).

[0114] "Host cell" is a cell that can be used to express the polynucleotides of the present disclosure. The host cell can be a prokaryote, such as Escherichia coli (E. coli), or the host cell can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma. Generally, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with the nucleic acid to be expressed. The host cell can also be a cell that contains the nucleic acid but does not express the nucleic acid at the desired level, unless regulatory sequences are introduced into the host cell such that the regulatory sequences become operably linked to the nucleic acid. It will be understood that the term host cell refers not only to a particular subject cell but also to the progeny or potential progeny of such a cell. Since certain modifications can occur in subsequent generations due to, for example, mutation or environmental influences, such progeny may not actually be identical to the parental cell but are still included within the scope of the term as used herein.

[0115] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that by virtue of its origin or source from which it is derived (1) is not associated with the components that are naturally associated with it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cell system different from the cell of its natural origin will be "isolated" from its naturally associated components. It is also possible to render the molecule substantially free of the naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of the molecule can be determined in many ways well known in the art. For example, the purity of a polypeptide sample can be determined using techniques well known in the art using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0116] A protein or polypeptide is "substantially pure", "substantially homogeneous" or "substantially purified" when at least about 60% to 75% of the sample exhibits a single species of polypeptide. The polypeptide or protein can be monomeric or multimeric. A substantially pure polypeptide or protein will generally comprise about 50%, 60%, 70%, 80% or 90% w / w of the protein sample, more usually about 95%, and preferably will be more than 99% pure. Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of the protein sample followed by visualization of individual polypeptide bands after staining the gel with a stain well known in the art. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0117] As used herein, the term "heterologous" refers to a composition or state that is not native or does not exist in nature, which can be achieved, for example, by replacing an existing native composition or state with a component or state from another source. Similarly, protein expression in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0118] Unless the context clearly indicates otherwise, the singular forms "a", "or" and "the" as used herein and in the appended claims include plural referents. It is to be understood that aspects and embodiments of the present disclosure described herein include "consisting of these aspects and embodiments" and / or "consisting essentially of these aspects and embodiments".

[0119] As used herein, reference to a "about" value or parameter includes (and describes) variations that are directed to the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0120] C-C chemokine receptor type 8 (CCR8)

[0121] CCR8 is a G protein-coupled 7-transmembrane CC chemokine receptor protein, expressed in the thymus, spleen, etc. The gene encoding this protein is located on human chromosome 3p21. Human CCR8 consists of 355 amino acids (J. Immunol., 1996, Vol. 157, No. 7, pp. 2759-63). CCL1 is known to be an endogenous ligand of CCR8 (J. Biol. Chem., 1997, Vol. 272, No. 28, pp. 17251-4). The human CCR8 cDNA consists of the nucleotide sequence represented by GenBank Acc No. NM_005201.3, and the mouse CCR8 cDNA consists of the nucleotide sequence represented by GenBank Acc No. NM_007720.2.

[0122] As used herein, the term "CCR8" includes human CCR8 (hCCR8), variants, isotypes, and species homologs of hCCR8, and analogs having at least one common epitope with hCCR8. In various embodiments, hCCR8 as used herein may comprise the amino acid sequence set forth in SEQ ID NO:1, which is: MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL (SEQ ID NO:1)

[0123] In various embodiments, CCR8 comprises an amino acid sequence having, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% observed homology with the human CCR8 sequence of SEQ ID NO:1. In some embodiments, CCR8 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 1x, at least 1.5x, at least 2x, at least 2.5x, or at least 3x the activity of the human CCR8 of SEQ ID NO:1. As used herein, variants of CCR8 can be described by reference to the addition, deletion, or substitution of an amino acid residue present at a given position in the 360 - amino - acid sequence of SEQ ID NO:1. Thus, for example, the term "T10S" means that the "T" (threonine, in standard single - letter code) residue at position 10 in SEQ ID NO:1 has been replaced by an "S" (serine, in standard single - letter code) residue.

[0124] Antibody

[0125] Methods for generating novel antibodies that bind to human CCR8 are known to those of skill in the art. For example, methods for generating monoclonal antibodies that specifically bind to CCR8 can include administering to a mouse an amount of an immunogenic composition that includes CCR8 that is effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify hybridomas that produce monoclonal antibodies that specifically bind to CCR8. After obtaining, the hybridomas can be propagated in cell culture, optionally under culture conditions in which cells derived from the hybridomas produce monoclonal antibodies that specifically bind to CCR8. The monoclonal antibodies can be purified from the cell culture. Then, a variety of different techniques for testing antigen:antibody interactions to identify particularly desired antibodies are available.

[0126] Other suitable methods for generating or isolating antibodies with the requisite specificity can be used, including, for example, methods for selecting recombinant antibodies from libraries, or methods that rely on immunizing transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-2555, 1993; Jakobovits et al., Nature, 362:255-258, 1993; Lonberg et al., U.S. Patent No. 5,545,806; Surani et al., U.S. Patent No. 5,545,807.

[0127] Antibodies can be engineered in a variety of ways. They can be prepared as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs TM ), Fab and F(ab’)2 fragments, and the like. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications list many types of antibodies and methods for engineering such antibodies. See, for example, U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.

[0128] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, the gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with a restriction enzyme to remove the region encoding murine Fc and replaced with the equivalent portion of the gene encoding the human Fc constant region (see Robinson et al., International Patent Publication PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al., Science, 240:1041-1043, 1988; Liu et al., PNAS USA, 84:3439-3443, 1987; Liu et al., J. Immunol. 139:3521-3526, 1987; Sun et al., PNAS USA, 84:214-218, 1987; Nishimura et al., Canc. Res. 47:999-1005, 1987; Wood et al., Nature, 314:446-449, 1985; and Shaw et al., J. Natl Cancer Inst., 80:1553-1559, 1988).

[0129] Methods for humanizing antibodies have been described in the art. In practice, humanized antibodies are usually human antibodies in which some of the hypervariable region residues and possibly some of the framework region residues have been replaced with residues from the analogous sites in a rodent antibody. Thus, such "humanized" antibodies are chimeric antibodies in which substantially less than one complete human variable region has been replaced with the corresponding sequences from a non-human species. To some extent, this can be achieved in connection with humanization techniques and display techniques using appropriate libraries. It will be understood that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art (see, e.g., Winter et al., ImmunolToday, 14:43-46, 1993; and Wright et al., Crit. Reviews in Immunol., 12:125-168, 1992). Antibodies of interest can be engineered by recombinant DNA techniques to replace the CH1, CH2, CH3, hinge domain, and / or framework domain with the corresponding human sequences (see WO 92 / 02190 and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,792; 5,714,350; and 5,777,085). In addition, the construction of chimeric immunoglobulin genes using Ig cDNA is known in the art (Liu et al., P.N.A.S. 84:3439, 1987; J. Immunol. 139:3521, 1987). mRNA is isolated from antibody-producing hybridomas or other cells and used to generate cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (U.S. Patent Nos. 4,683,195 and 4,683,202). Alternatively, libraries are prepared and screened to isolate the sequences of interest. The DNA sequences encoding the variable regions of the antibodies are then fused to human constant region sequences. The sequences of the human constant regions of the genes can be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. Publication No. 91-3242. The human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions such as complement fixation or antibody-dependent cell cytotoxicity activity. In various embodiments, the isotype is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant region κ or λ can be used. The chimeric, humanized antibodies are then expressed by conventional methods.

[0130] U.S. Patent No. 5,693,761 to Queen et al. discloses improvements to the Winter et al. approach to antibody humanization and is based on the premise that avidity loss is attributed to problems with the structural motifs of the humanized framework, which due to steric or other chemical incompatibilities, interfere with the CDRs folding into the binding-competent conformations found in murine antibodies. To address this problem, Queen taught the use of linear peptide sequences of human framework sequences closely homologous to the framework sequences of the murine antibody to be humanized. Thus, Queen's method focused on comparing the framework sequences between species. Generally, all available human variable region sequences were compared to a particular murine sequence, and the percent identity between corresponding framework residues was calculated. The human variable region with the highest percentage was selected to provide the framework sequence for the humanization project. Queen also taught that it was important to retain certain amino acid residues from the murine framework that were crucial for supporting the CDRs in a binding-competent conformation. The potential importance was evaluated from molecular models. Candidate residues for retention were typically those that were adjacent to the CDR in the linear sequence or physically within those of any CDR residue.

[0131] In other methods, when a low-avidity humanized construct is obtained, the importance of specific framework amino acid residues is determined experimentally by reverting individual residues to the murine sequence and measuring antigen binding, as described by Riechmann et al., 1988. Another exemplary method for identifying important amino acids in the framework sequence is disclosed by Carter et al., U.S. Patent No. 5,821,337 and Adair et al., U.S. Patent No. 5,859,205. These references disclose specific Kabat residue positions in the framework that may need to be replaced with the corresponding murine amino acids in a humanized antibody to maintain avidity.

[0132] Another method of antibody humanization, called "framework shuffling", relies on generating a combinatorial library that has non-human CDR variable regions in a pool that are in-frame fused to a single human germline framework (Dall’Acqua et al., Methods, 36:43, 2005). The library is then screened to identify clones encoding humanized antibodies that retain good binding.

[0133] The selection of the human variable regions (both light and heavy chains) to be used in the preparation of the desired humanized antibody is very important for reducing antigenicity. According to a method known as "best fit", the variable region sequences of rodent antibodies are screened against the entire library of known human variable domain sequences. The human sequence that is most closely related to the rodent sequence is then accepted as the human framework region (framework region) for the humanized antibody (Sims et al., J. Immunol., 151:2296, 1993; Chothia et al., J. Mol. Biol., 196:901, 1987). Another method uses a particular framework region that is a consensus sequence of all human antibodies derived from a particular subgroup of light chain variable regions or heavy chain variable regions. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285, 1992; Presta et al., J. Immunol., 151:2623, 1993).

[0134] The selection of non-human residues to be substituted into the human variable regions can be influenced by a variety of factors. These factors include, for example, the rarity of the amino acid at a particular position, the likelihood of interaction with the CDR or antigen, and the likelihood of participation in the interface between the light chain variable domain interface and the heavy chain variable domain interface. (See, for example, U.S. Patent Nos. 5,693,761, 6,632,927, and 6,639,055). One way to analyze these factors is by using three-dimensional models of the non-human and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of the selected candidate immunoglobulin sequences are available. Examination of these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, e.g., analysis of the residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, non-human residues can be selected and substituted for human variable region residues in order to achieve the desired antibody characteristics, such as increased affinity for one or more target antigens.

[0135] Methods for preparing fully human antibodies have been described in the art. By way of example, methods for generating CCR8 antibodies or antigen-binding fragments thereof include the steps of synthesizing a library of human antibodies on phage, screening the library with CCR8 or an antibody-binding portion thereof, isolating phage that bind CCR8, and obtaining the antibody from the phage. By way of another example, methods for preparing a library of antibodies for use in phage display technology include the steps of immunizing a non-human animal comprising a human immunoglobulin locus with CCR8 or an antigenic portion thereof to generate an immune response, extracting antibody-producing cells from the immunized animal, isolating RNA encoding the heavy and light chains of the antibody of the invention from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector such that the antibody is expressed on the phage. The recombinant anti-CCR8 antibodies of the invention can be obtained in this manner.

[0136] The recombinant human anti-CCR8 antibodies of the invention can also be isolated by screening a recombinant combinatorial antibody library. Preferably, the library is a scFv phage display library generated using human VL and VH cDNAs prepared from mRNA isolated from B cells. Methods for preparing and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene SurfZAP TMPhage display kits, catalog number 240612). There are also other methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., U.S. Patent No. 5,223,409; PCT Publications WO 92 / 18619, WO 91 / 17271, WO 92 / 20791, WO 92 / 15679, WO 93 / 01288, WO 92 / 01047, WO 92 / 09690; Fuchs et al., Bio / Technology, 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas 3:81-85, 1992; Huse et al., Science, 246:1275-1281, 1989; McCafferty et al., Nature 348:552-554, 1990; Griffiths et al., EMBO J. 12:725-734, 1993; Hawkins et al., J. Mol. Biol. 226:889-896, 1992; Clackson et al., Nature 352:624-628, 1991; Gram et al., Proc. Natl. Acad. Sci. USA 89:3576-3580, 1992; Garrad et al., Bio / Technology 9:1373-1377, 1991; Hoogenboom et al., Nuc. Acid Res. 19:4133-4137, 1991; and Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991, each incorporated herein by reference for the purpose of teaching the preparation and screening of phage display libraries).

[0137] Human antibodies are also produced by immunizing non-human transgenic animals with human IgE antigen, which non-human transgenic animals contain some or all of the human immunoglobulin heavy and light chain loci in their genomes, such as XenoMouse TM animals (Abgenix, Inc. / Amgen, Inc. - Fremont, Calif.). XenoMouse TMA mouse is an engineered mouse strain that contains large fragments of the human immunoglobulin heavy and light chain loci and is defective in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21, 1994; and U.S. Patent Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364, 6,162,963, and 6,150,584. See also WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, WO 96 / 33735, WO 98 / 16654, WO 98 / 24893, WO 98 / 50433, WO 99 / 45031, WO 99 / 53049, WO 00 / 09560, and WO 00 / 037504. XenoMouse TM The mouse generates an adult-like human repertoire of fully human antibodies and produces antigen-specific human antibodies. In some embodiments, the XenoMouse TM mouse contains approximately 80% of the human antibody V gene repertoire. In other embodiments, the XenoMouse TM mouse also contains approximately all of the human λ light chain locus. See Mendez et al., Nature Genetics, 15:146-156, 1997; Green and Jakobovits, J. Exp. Med. 188:483-495 (1998) and WO 98 / 24893 (each incorporated by reference in its entirety for the purpose of teaching the preparation of fully human antibodies). In another aspect, the present invention provides a method for preparing anti-CCR8 antibodies from a non-human, non-mouse animal by immunizing a non-human transgenic animal containing a human immunoglobulin locus with a CCR8 antigen. One can produce such animals using the methods described in the documents mentioned above.

[0138] Characterization of antibody-antigen binding

[0139] The binding of the antibodies of the present invention to human CCR8 can be tested by, for example, standard ELISA. For example, microtiter plates are coated with purified CCR8 in PBS or cells overexpressing human CCR8, and then blocked with 5% bovine serum albumin in PBS. A dilution of the antibody (e.g., a plasma dilution from a mouse immunized with CCR8) is added to each well and incubated at 37°C or 4°C for 1-2 hours. The plates are washed with PBS / Tween, and then incubated with a second reagent conjugated to alkaline phosphatase (e.g., for human antibodies, a goat anti-human IgG Fc specific polyclonal reagent) at 37°C for 1 hour. After washing, the plates are developed with pNPP substrate (1 mg / ml) and analyzed at an OD of 405 nm - 650 nm. Preferably, the mice that produce the highest titers will be used for fusion. ELISA can also be used to screen hybridomas that show positive reactivity with the CCR8 immunogen. Hybridomas that bind to CCR8 with high affinity are subcloned and further characterized. One clone that retains the reactivity (by ELISA) of the parental cells can be selected from each hybridoma for preparation of a cell bank of 5-10 vials stored at -140°C and for antibody purification.

[0140] To determine whether the selected anti-CCR8 monoclonal antibodies bind to distinct epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Competitive studies using unlabeled and biotinylated monoclonal antibodies can be performed using CCR8-coated ELISA plates as described above. Binding of the biotinylated mAb can be detected with a streptavidin-alkaline phosphatase probe. To determine the isotype of the purified antibody, isotype ELISA can be performed using reagents specific for the antibody of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated with 1 μg / ml of anti-human immunoglobulin at 4°C overnight. After blocking with 1% BSA, the plates are reacted with 1 μg / ml or less of the test monoclonal antibody or purified isotype control at ambient temperature for 1 to 2 hours. The wells can then be reacted with a probe conjugated to alkaline phosphatase specific for human IgG1 or human IgM. The plates are developed and analyzed as described above.

[0141] The anti-CCR8 human IgG can be further tested for reactivity with the CCR8 antigen by Western blot. Briefly, CCR8 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked with 10% fetal bovine serum, and probed with the monoclonal antibody to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

[0142] Identification of anti-CCR8 antibodies

[0143] The present invention provides monoclonal antibodies and antigen-binding fragments thereof that specifically bind to the CCR8 antigen.

[0144] The present invention also includes antibodies that bind to the same epitope as the anti-CCR8 antibody of the present invention. To determine whether an antibody can competitively bind to the same epitope as the epitope bound by the anti-CCR8 antibody of the present invention, a cross-blocking assay, such as a competitive ELISA, can be performed. In an exemplary competitive ELISA, CCR8 coated on the wells of a microtiter plate is pre-incubated with or without a candidate competitive antibody, and then the biotinylated anti-CCR8 antibody of the present invention is added. The amount of the labeled anti-CCR8 antibody that binds to the CCR8 antigen in the wells is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody can be labeled with a radioactive label or a fluorescent label or some other detectable and measurable label. The amount of the labeled anti-CCR8 antibody that binds to the antigen will have an indirect correlation with the ability of the candidate competitive antibody (test antibody) to competitively bind to the same epitope, i.e., the greater the affinity of the test antibody for the same epitope, the less the labeled antibody will bind to the antigen-coated wells. If the candidate antibody can block the binding of the CCR8 antibody by at least 20%, preferably at least 20%-50%, and even more preferably at least 50% compared to a control performed in parallel in the absence of the candidate competitive antibody, the candidate competitive antibody is considered to be an antibody that binds to substantially the same epitope as the anti-CCR8 antibody of the present invention or competes for binding to the same epitope. It will be understood that variations of this assay can be performed to achieve the same quantitative values.

[0145] The amino acid sequences of the heavy chain variable region CDRs and light chain variable region CDRs of the various murine mAbs (mAbs A1-A19) produced as described herein are shown in Table 2 below.

[0146] Table 2 Heavy chain CDR

[0147]

[0148]

[0149] Light chain CDR

[0150]

[0151]

[0152] In various embodiments of the present invention, the antibody or antigen-binding fragment is a murine antibody comprising a combination of heavy chain variable region sequences and light chain variable region sequences listed in Table 3:

[0153] Table 3 Mouse Ab clones

[0154]

[0155]

[0156] In various embodiments, the antibodies of the present invention include antibodies that bind to the same epitope as murine antibodies MAb1-MAb19.

[0157] In various embodiments of the present invention, the antibody or antigen-binding fragment is a murine-human chimeric antibody (derived from murine antibody A1 (“41E1C2A5”) and human IgG1) that comprises the heavy chain sequence of SEQ ID NO:26:

[0158]

[0159] and the light chain sequence of SEQ ID NO:27:

[0160]

[0161] In various embodiments of the present invention, the antibody or antigen-binding fragment is a murine-human chimeric antibody (derived from murine antibody A3 (“80E4D1F11”) and human IgG1) that comprises the heavy chain sequence of SEQ ID NO:28:

[0162]

[0163] and the light chain sequence of SEQ ID NO:29:

[0164]

[0165] In various embodiments of the present invention, the antibody or antigen-binding fragment is a murine-human chimeric antibody (derived from murine antibody A4 (“419C7B3B2”) and human IgG1) that comprises the heavy chain sequence of SEQ ID NO:30:

[0166]

[0167] and the light chain sequence of SEQ ID NO:31:

[0168]

[0169] In various embodiments of the present invention, the antibody or antigen-binding fragment is a murine-human chimeric antibody (derived from murine antibody A18 (“504E12D8D12”) and human IgG1), which comprises the heavy chain sequence of SEQ ID NO:78:

[0170]

[0171] and the light chain sequence of SEQ ID NO:79:

[0172]

[0173] In various embodiments of the present invention, the antibody or antigen-binding fragment is a murine-human chimeric antibody (derived from murine antibody A10 (“516D7D12”) and human IgG1), which comprises the heavy chain sequence of SEQ ID NO:80:

[0174]

[0175] and the light chain sequence of SEQ ID NO:81:

[0176]

[0177] In various embodiments of the present invention, the antibody or antigen-binding fragment is a murine-human chimeric antibody (derived from murine antibody A11 (“525F2F3F11”) and human IgG1), which comprises the heavy chain sequence of SEQ ID NO:82:

[0178]

[0179] and the light chain sequence of SEQ ID NO:83:

[0180]

[0181] In various embodiments of the present invention, the antibody or antigen-binding fragment is a murine-human chimeric antibody (derived from murine antibody A13 (“531B9B1C9”) and human IgG1), which comprises the heavy chain sequence of SEQ ID NO:84:

[0182]

[0183] and the light chain sequence of SEQ ID NO:85:

[0184]

[0185] The antibody or antigen-binding fragment thereof of the present invention may comprise any constant region known in the art. The light chain constant region may be, for example, a κ or λ type light chain constant region, such as a human κ or λ type light chain constant region. The heavy chain constant region may be, for example, an α, δ, ε, γ or μ type heavy chain constant region, such as an IgA, IgD, IgE, IgG and IgM type heavy chain constant region. In various embodiments, the light chain constant region or heavy chain constant region is a fragment, derivative, variant or mutein of a naturally occurring constant region.

[0186] Techniques for deriving different subclasses or isotypes of antibodies from an antibody of interest, i.e., subclass switching, are known. Thus, an IgG antibody can be derived from, for example, an IgM antibody and vice versa. Such techniques allow the preparation of new antibodies that have the antigen-binding properties of a given antibody (parent antibody) but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques can be used. Cloned DNA encoding a specific antibody polypeptide, for example, DNA encoding the constant domain of an antibody of a desired isotype, can be used in such procedures. See also Lanitto et al., Methods Mol. Biol. 178:303-16, 2002.

[0187] In various embodiments, the antibody of the present invention further comprises a light chain κ or λ constant domain or a fragment thereof, and further comprises a heavy chain constant domain or a fragment thereof. The sequences of the light chain constant region and heavy chain constant region used in the exemplified antibodies and the polynucleotides encoding them are provided below.

[0188] Light chain (κ) constant region

[0189]

[0190] Light chain (λ) constant region

[0191]

[0192] Heavy chain constant region

[0193]

[0194] In various embodiments of the present invention, the antibody or antigen-binding fragment is a humanized antibody (“41E1C2A5-HC3-LC4”), which comprises the heavy chain sequence of SEQ ID NO:86:

[0195]

[0196] and the light chain sequence of SEQ ID NO:87:

[0197]

[0198] In various embodiments of the present invention, the antibody or antigen-binding fragment is a humanized antibody (“41E1C2A5-HC4-LC2”), which comprises the heavy chain sequence of SEQ ID NO:88:

[0199]

[0200] and the light chain sequence of SEQ ID NO:89:

[0201]

[0202] In various embodiments of the present invention, the antibody or antigen-binding fragment is a humanized antibody (“504E12D8D12-HC1-LC1”), which comprises the heavy chain sequence of SEQ ID NO:90:

[0203]

[0204] and the light chain sequence of SEQ ID NO:91:

[0205]

[0206] In various embodiments of the present invention, the antibody or antigen-binding fragment is a humanized antibody (“504E12D8D12-HC1-LC1(G34A)”), which comprises the heavy chain sequence of SEQ ID NO:90 and the light chain sequence of SEQ ID NO:92:

[0207]

[0208] The antibodies of the present invention can also be described or designated according to their cross-reactivity. Antibodies that bind to CCR8 are also included in the present invention, and the CCR8 has at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity to human CCR8 (as calculated using methods known in the art and described herein).

[0209] The present invention also includes antibodies that bind to the same epitope as the anti-CCR8 antibody of the present invention. To determine whether an antibody can competitively bind to the same epitope as the epitope bound by the anti-CCR8 antibody of the present invention, a cross-blocking assay, such as a competitive ELISA, can be performed. In an exemplary competitive ELISA, CCR8 coated on the wells of a microtiter plate is pre-incubated with or without a candidate competitive antibody, and then the biotinylated anti-CCR8 antibody of the present invention is added. The amount of the labeled anti-CCR8 antibody that binds to the CCR8 antigen in the wells is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody can be labeled with a radioactive label, a fluorescent label, or some other detectable and measurable label. The amount of the labeled anti-CCR8 antibody that binds to the antigen will have an indirect correlation with the ability of the candidate competitive antibody (test antibody) to competitively bind to the same epitope, i.e., the greater the affinity of the test antibody for the same epitope, the less the labeled antibody will bind to the antigen-coated wells. If the candidate antibody can block the binding of the CCR8 antibody by at least 20%, at least 30%, at least 40%, or at least 50% compared to a control performed in parallel in the absence of the candidate competitive antibody, the candidate competitive antibody is considered to be an antibody that binds to substantially the same epitope as the anti-CCR8 antibody of the present invention or competes for binding to the same epitope. It will be understood that variations of this assay can be performed to achieve the same quantitative values.

[0210] In certain alternative embodiments, the antibodies of the present invention can be modified by modifying one or both variable regions (i.e., V H and / or V Lone or more residues in [[ID=]], or by modifying residues in one or more constant regions, such as to alter one or more effector functions of the antibody, are engineered. In various embodiments, the variable regions of the antibody will be modified by CDR grafting using framework sequences that can be obtained from public DNA databases containing germline antibody gene sequences or published references (e.g., Tomlinson, I.M. et al., J. Mol. Biol. 227:776-798, 1992; and Cox, J.P.L. et al., Eur. J. Immunol. 24:827-836, 1994; the contents of each are expressly incorporated herein by reference). In various embodiments, the antibody can be modified using site-directed mutagenesis or PCR-mediated mutagenesis to introduce one or more mutations in VH and / or VL that improve binding affinity and / or reduce immunogenicity. In various embodiments, the antibody can be modified in the Fc region for the purpose of altering the serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity of the antibody. In various embodiments, the antibody can be modified for the purpose of modifying the glycosylation of the antibody. Methods for making each of the modifications described herein, as well as other methods, are well known to those of skill in the art.

[0211] Pharmaceutical composition

[0212] In another aspect, the invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described above. The pharmaceutical compositions, methods, and uses of the invention thus also include embodiments of combination (co-administration) with other active agents, as detailed below.

[0213] Typically, the antibodies or antigen-binding fragments thereof of the present invention are suitable for administration as a formulation in combination with one or more pharmaceutically acceptable excipients. As used herein, the term 'excipient' is used to describe any ingredient other than one or more compounds of the present invention. The choice of one or more excipients will depend to a large extent on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include an isotonic agent, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride, in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting agents or emulsifying agents, preservatives, or buffers, which extend the shelf life of the antibody or enhance the potency of the antibody. The pharmaceutical compositions and methods of the present invention will be apparent to those skilled in the art. For example, such compositions and methods of preparation can be found in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). It is preferred to manufacture the pharmaceutical compositions under GMP conditions.

[0214] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in a single unit dose or in more than one single unit dose. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a pre-determined amount of the active ingredient. The amount of the active ingredient is usually equal to the dose of the active ingredient to be administered to a subject or a convenient fraction of such a dose, such as, for example, half or one-third of such a dose.

[0215] Any method recognized in the art for the administration of peptides, proteins, or antibodies can be suitably used for the antibodies and moieties of the present invention.

[0216] The pharmaceutical compositions of the present invention are generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically breaching the tissue of a subject and administering the pharmaceutical composition through the breach in the tissue, thus generally resulting in direct administration into the bloodstream, into muscle, or into visceral organs. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection of the composition, by application through a surgical incision, by application through a non-surgical wound permeable to tissue, etc. In particular, parenteral administration is expected to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular / intracardiac, intraurethral, intracranial, intraarticular injection or infusion; and renal dialysis infusion techniques. Various embodiments include intravenous and subcutaneous routes.

[0217] Formulations of pharmaceutical compositions suitable for parenteral administration often generally comprise the active ingredient in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in an ampoule or in a multi-dose container containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, etc. Such formulations may also contain one or more additional ingredients including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) and then parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates, and buffers (preferably to a pH of from 3 to 9), but for some applications they may be more suitably formulated as sterile non-aqueous solutions or in a dry form for use with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions such as aqueous propylene glycol or dextrose solutions. If desired, such dosage forms may be suitably buffered. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form, or in liposomal preparations. Formulations for parenteral administration may be formulated for immediate release and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0218] For example, in one aspect, a sterile injectable solution can be prepared by incorporating the required amount of the anti-CCR8 antibody into a suitable solvent having one or a combination of the ingredients listed above, followed by filter sterilization if desired. Generally, a dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional required ingredients from its previously sterile filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prolonged absorption of an injectable composition can be brought about by including agents that delay absorption, such as monostearate and gelatin, in the composition.

[0219] The antibodies of the invention can also be administered intranasally or by inhalation, generally in the form of a dry powder (alone, as a mixture, or as mixed component particles, e.g., mixed with a suitable pharmaceutically acceptable excipient), administered from a dry powder inhaler, as an aerosol spray from a pressure vessel, pump, spray, nebulizer (preferably a nebulizer using electrohydrodynamics to produce a fine mist), or atomizer, with or without a suitable propellant, or as a nasal drop.

[0220] The pressure vessel, pump, spray, nebulizer, or atomizer generally contains a solution or suspension of the antibody of the invention, the solution or suspension containing, for example, suitable agents for dispersing, dissolving, or prolonging the release of the active agent, and one or more propellants as solvents.

[0221] Before use in dry powder or suspension formulations, the pharmaceutical product is typically micronized to a size suitable for delivery by inhalation (generally less than 5 microns). This can be carried out by any suitable comminution method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[0222] Capsules, blisters, and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound of the invention, a suitable powder matrix, and a performance modifier.

[0223] Suitable flavorings such as menthol and levomenthol, or sweeteners such as saccharin or sodium saccharin, can be added to those formulations of the invention intended for inhaled / intranasal administration.

[0224] Formulations for inhalation / nasal administration can be formulated for immediate release and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0225] In the case of dry powder inhalers and aerosols, the dose unit is determined by means of a valve that delivers a metered amount. The units according to the invention are generally arranged to administer a metered dose of the antibody of the invention or to "puff" the antibody of the invention. The total daily dose will usually be administered as a single dose, or more usually, as divided doses throughout the day.

[0226] The antibodies and antibody portions of the invention can also be formulated for administration by the oral route. Oral administration can involve swallowing so that the compound enters the gastrointestinal tract, and / or buccal, sublingual, or sublingual administration, by which the compound enters the bloodstream directly from the mouth.

[0227] Formulations suitable for oral administration include solid, semi-solid, and liquid systems such as tablets; soft or hard capsules containing multi-particles or nanoparticles, liquids, or powders; lozenges (including those filled with liquid); chews; gels; rapidly dispersing dosage forms; films; ovules; sprays, and buccal / mucoadhesive patches.

[0228] Pharmaceutical compositions intended for oral use can be prepared by any method known in the art for manufacturing pharmaceutical compositions, and such compositions can contain one or more agents selected from the group consisting of sweetening agents to provide a pharmaceutically elegant and palatable formulation. For example, to prepare an orally deliverable tablet, the antibody or its antigen-binding fragment is mixed with at least one pharmaceutical excipient, and the solid formulation is compressed according to known methods to form a tablet for delivery to the gastrointestinal tract. Tablet compositions are usually formulated with additives such as sugar or cellulose carriers, binders such as starch paste or methylcellulose, fillers, disintegrants, or other additives commonly used in the manufacture of medical formulations. To prepare an orally deliverable capsule, DHEA is mixed with at least one pharmaceutical excipient, and the solid formulation is placed in a capsule container suitable for delivery to the gastrointestinal tract. Compositions containing an antibody or its antigen-binding fragment can be prepared as generally described in Remington’s Pharmaceutical Sciences, 18th Edition, 1990 (Mack Publishing Co., Easton, Pa. 18042), Chapter 89, which is incorporated herein by reference.

[0229] In various embodiments, the pharmaceutical composition is formulated as an orally deliverable tablet comprising an antibody or antigen-binding fragment thereof mixed with a non-toxic pharmaceutically acceptable excipient suitable for manufacturing a tablet. These excipients can be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as, for example, corn starch, gelatin or gum arabic, and lubricants such as magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide a sustained action over a longer period of time. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate alone or in combination with waxes can be used.

[0230] In various embodiments, the pharmaceutical composition is formulated as a hard gelatin capsule in which the antibody or antigen-binding fragment thereof is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as a soft gelatin capsule in which the antibody or antigen-binding fragment thereof is mixed with an aqueous medium or an oil medium such as arachis oil, peanut oil, liquid paraffin or olive oil.

[0231] Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations can be used as fillers in soft or hard capsules (made from, for example, gelatin or hydroxypropylmethylcellulose) and generally contain a carrier such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or a suitable oil, and one or more emulsifying and / or suspending agents. Liquid formulations can also be prepared by reconstituting, for example, a solid from a sachet.

[0232] Therapeutic use

[0233] In another aspect, the invention relates to a method of treating a subject suffering from a CCR8-related disorder, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention. In various embodiments, the subject is a human subject. In various embodiments, the CCR8-related disorder is cancer. In various embodiments, the cancerous cells are selected from the group consisting of ovarian cancer, lung cancer, breast cancer, gastric cancer, prostate cancer, colorectal cancer, renal cell carcinoma, liver cancer, pancreatic cancer, glioblastoma, melanoma and sarcoma. In various embodiments, the subject has previously responded to treatment with an anti-cancer therapy but has suffered a relapse after cessation of the therapy (hereinafter referred to as "recurrent cancer"). In various embodiments, the subject has a resistant or refractory cancer. In various embodiments, the cancerous cells are immunogenic tumors (e.g., those tumors for which vaccination with the tumor itself can result in immunity against tumor attack).

[0234] In various embodiments, a method for treating a subject suffering from cancer comprises administering to the subject a therapeutically effective amount of any one of the Treg-depleting anti-CCR8 Abs (e.g., mAb, immunoconjugate, or bispecific molecule) disclosed herein, or a pharmaceutical composition comprising any one of said Abs (e.g., anti-CCR8 mAb, immunoconjugate, or bispecific molecule), such that the subject is treated.

[0235] In another aspect, the invention relates to a combination therapy designed for treating cancer in a subject. In various embodiments, a method for inhibiting the growth of tumor cells in a subject comprises administering to the subject a therapeutically effective amount of: (a) any one of the Treg-depleting anti-CCR8 Abs, immunoconjugates, or bispecific molecules disclosed herein, or a pharmaceutical composition comprising any one of said anti-CCR8 Abs, immunoconjugates, or bispecific molecules; and (b) an additional therapy for treating cancer. In various embodiments, the additional therapeutic therapy is a therapeutic agent that is a compound that reduces the inhibition of the immune system or increases the stimulation of the immune system, such that the growth of tumor cells in the subject is inhibited. In various embodiments, the additional therapy is selected from the group consisting of immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation, wherein the combination therapy provides increased cell killing of tumor cells, i.e., there is a synergistic effect between the isolated antibody or its antigen-binding fragment and the additional therapy when co-administered.

[0236] In another aspect, the invention relates to a method for enhancing the immune response of a subject to cancerous cells, the method comprising administering to the subject a therapeutically effective amount (as a single therapy or in a combination therapy regimen) of the isolated antibody or its antigen-binding fragment of the invention. In various embodiments, the invention provides a method for treating cancerous cells in a subject, the method comprising administering to the subject a therapeutically effective amount (as a single therapy or in a combination therapy regimen) of the antibody or its antigen-binding fragment of the invention. In various embodiments, the cancerous cells are selected from the group consisting of ovarian cancer, lung cancer, breast cancer, gastric cancer, prostate cancer, colorectal cancer, renal cell carcinoma, liver cancer, pancreatic cancer, glioblastoma, melanoma, and sarcoma.

[0237] In various embodiments, the cancers to be treated include, but are not limited to, solid tumors, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal area cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, and combinations of the foregoing cancers. In various embodiments, the cancerous cells are immunogenic tumors (e.g., those tumors for which vaccination with the tumor itself can result in immunity against tumor attack). In various embodiments, the cancers are selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), colorectal cancer (CRC), kidney cancer, bladder cancer, non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon cancer, ovarian cancer, and lung cancer.

[0238] In various embodiments, the solid tumor is a cancer selected from HNSCC, cervical cancer, CRC, NSCLC-SCC, NSCLC-ADC, pancreatic cancer, stomach cancer, bladder cancer, and breast cancer.

[0239] In various embodiments, the cancer is a hematological malignancy, which includes liquid tumors derived from either of two major blood cell lineages, the myeloid cell lineage (which gives rise to granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphocytic cell lineage (which gives rise to B cells, T cells, NK cells, and plasma cells), including all types of leukemia, lymphoma, and myeloma. Hematological malignancies that can be treated using the methods of the present invention include, for example, cancers selected from the following: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), advanced, metastatic, refractory, and / or relapsed hematological malignancies, and any combination of the foregoing hematological malignancies.

[0240] In various embodiments, the antibodies and antigen-binding fragments thereof of the invention can be used to directly kill or ablate cancerous cells in vivo. Direct killing includes administering an antibody (optionally fused with a cytotoxic agent) to a subject in need of such treatment. In various embodiments, the cancer includes cancer cells that express CCR8 at a higher level than non-cancerous cells of comparable tissue. Since the antibody recognizes CCR8 on the cancer cells, any such cells bound by the antibody are destroyed. When the antibody is used alone to kill or ablate cancer cells, such killing or ablation can be affected by the initial endogenous host immune functions such as CDC and / or ADCC. Assays for determining whether an antibody kills cells in this manner are within the capabilities of those skilled in the art.

[0241] In various embodiments, the antibodies and antigen-binding fragments thereof of the invention can be used to promote growth inhibition and / or proliferation inhibition of cancerous tumor cells. These methods can inhibit or prevent the growth of cancer cells in the subject, such as by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. Thus, in the case where the cancer is a solid tumor, such modulation can reduce the size of the solid tumor by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0242] Inhibition of cancer cell proliferation can be measured by cell-based assays, such as bromodeoxyuridine (BRDU) incorporation (Hoshino et al., Int. J. Cancer 38, 369, 1986; Campana et al., J. Immunol. Meth. 107:79, 1988); 3H]-thymidine incorporation (Chen, J., Oncogene 13:1395-403, 1996; Jeoung, J., J. Biol. Chem. 270:18367-73, 1995); the dye Alamar Blue (obtainable from Biosource International) (Voytik-Harbin et al., In Vitro Cell Dev Biol Anim 34:239-46, 1998). Anchorage-independent growth of cancer cells was evaluated by colony formation assay in soft agar (such as by counting the number of cancer cell colonies formed on top of the soft agar) (see Examples and Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989).

[0243] Inhibition of cancer cell growth in a subject can be evaluated by monitoring cancer growth in a subject (such as an animal model or a human subject). An exemplary monitoring method is the tumorigenicity assay. In one example, the xenograft comprises human cells from a pre-existing tumor or from a tumor cell line. Tumor xenograft assays are known in the art and are described herein (see, e.g., Ogawa et al., Oncogene 19:6043-6052, 2000). In another embodiment, tumorigenicity is monitored using the hollow fiber assay, which is described in U.S. Patent No. 5,698,413, which is incorporated herein by reference in its entirety.

[0244] The percentage of inhibition is calculated by comparing cancer cell proliferation, anchorage-independent growth, or cancer cell growth under modulator treatment with that under negative control conditions (usually without modulator treatment). For example, where the number of cancer cells or cancer cell colonies (colony formation assay), or PRDU or 3 H]-thymidine incorporation is A (under modulator treatment) and C (under negative control conditions), the percentage of inhibition will be (C - A) / C × 100%.

[0245] Examples of tumor cell lines derived from human tumors and useful in in vitro and in vivo studies include, but are not limited to, leukemia cell lines (e.g., CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR, P388, and P388 / ADR); non-small cell lung cancer cell lines (e.g., A549 / ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522, and LXFL 529); small cell lung cancer cell lines (e.g., DMS114 and SHP-77); colon cancer cell lines (e.g., COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620, DLD-1, and KM20L2); central nervous system (CNS) cancer cell lines (e.g., SF-268, SF-295, SF-539, SNB-19, SNB-75, U251, SNB-78, and XF 498); melanoma cell lines (e.g., LOX I MVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, RPMI-7951, and M19-MEL); ovarian cancer cell lines (e.g., IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3); renal cancer cell lines (e.g., 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31, RXF-631, and SN12K1); prostate cancer cell lines (e.g., PC-3 and DU-145); breast cancer cell lines (e.g., MCF7, NCI / ADR-RES, MDA-MB-231 / ATCC, HS 578T, MDA-MB-435, BT-549, T-47D, and MDA-MB-468); and thyroid cancer cell lines (e.g., SK-N-SH).

[0246] A "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a therapeutic agent that will relieve one or more symptoms of a disorder being treated to some extent.

[0247] A therapeutically effective dose can be initially evaluated by determining the IC 50 from cell culture assays. Then, doses can be formulated to achieve in an animal model the IC as determined in cell culture 50The circulating plasma concentration range. Such information can be used to more precisely determine useful doses in humans. The levels in plasma can be measured, for example, by HPLC. The exact composition, route of administration, and dose can be selected by an individual physician in view of the condition of the subject.

[0248] The dosage regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally decreased or increased as indicated by the exigencies of the therapeutic situation. To facilitate administration and dosage uniformity, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as a single dose for the mammalian subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure will be determined primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0249] Accordingly, those skilled in the art will understand that, based on the disclosure provided herein, the dosage and dosing regimen are adjusted according to methods well known in the art of therapy. That is, the maximum tolerable dose can be readily determined, and the effective amount that provides a detectable therapeutic benefit to the subject can also be determined, as can the time requirements for administering each dose to provide a detectable therapeutic benefit to the subject. Accordingly, although certain doses and dosing regimens are exemplified herein, these examples in no way limit the doses and dosing regimens that can be provided to a subject in practicing the present disclosure.

[0250] It should be noted that the dosage value can vary with the type and severity of the condition to be alleviated and can include a single dose or more than one dose. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges listed herein are only exemplary and are not intended to limit the scope of the claimed composition or its practice. In addition, the dosage regimen of the compositions of the present disclosure can be based on multiple factors, including the type of disease, the age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibody used of the subject. Accordingly, the dosage regimen can vary widely but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects such as toxic effects and / or experimental values. Thus, the present disclosure includes intra-subject dose-escalation as determined by a person skilled in the art. Determining the appropriate dosage and regimen is well known in the relevant art and will be understood to be within the grasp of a person skilled in the art once the teachings disclosed herein are provided.

[0251] For administration to human subjects, of course depending on the mode of administration, the total monthly dosage of the antibody or its antigen-binding fragment of the present disclosure can be in the range of 0.5 - 1200 mg / subject, 0.5 - 1100 mg / subject, 0.5 - 1000 mg / subject, 0.5 - 900 mg / subject, 0.5 - 800 mg / subject, 0.5 - 700 mg / subject, 0.5 - 600 mg / subject, 0.5 - 500 mg / subject, 0.5 - 400 mg / subject, 0.5 - 300 mg / subject, 0.5 - 200 mg / subject, 0.5 - 100 mg / subject, 0.5 - 50 mg / subject, 1 - 1200 mg / subject, 1 - 1100 mg / subject, 1 - 1000 mg / subject, 1 - 900 mg / subject, 1 - 800 mg / subject, 1 - 700 mg / subject, 1 - 600 mg / subject, 1 - 500 mg / subject, 1 - 400 mg / subject, 1 - 300 mg / subject, 1 - 200 mg / subject, 1 - 100 mg / subject, or 1 - 50 mg / subject. For example, the intravenous monthly dosage may require about 1 - 1000 mg / subject. In various embodiments, the antibody or its antigen-binding fragment of the present disclosure can be administered at about 1 - 200 mg / subject, 1 - 150 mg / subject, or 1 - 100 mg / subject. The total monthly dosage can be administered as a single dose or divided doses and can, at the discretion of the physician, fall outside the typical ranges given herein.

[0252] In various embodiments, the non-limiting daily dose range of a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure can be from 0.001 mg / kg body weight to 100 mg / kg body weight, from 0.001 mg / kg body weight to 90 mg / kg body weight, from 0.001 mg / kg body weight to 80 mg / kg body weight, from 0.001 mg / kg body weight to 70 mg / kg body weight, from 0.001 mg / kg body weight to 60 mg / kg body weight, from 0.001 mg / kg body weight to 50 mg / kg body weight, from 0.001 mg / kg body weight to 40 mg / kg body weight, from 0.001 mg / kg body weight to 30 mg / kg body weight, from 0.001 mg / kg body weight to 20 mg / kg body weight, from 0.001 mg / kg body weight to 10 mg / kg body weight, from 0.001 mg / kg body weight to 5 mg / kg body weight, from 0.001 mg / kg body weight to 4 mg / kg body weight, from 0.001 mg / kg body weight to 3 mg / kg body weight, from 0.001 mg / kg body weight to 2 mg / kg body weight, from 0.001 mg / kg body weight to 1 mg / kg body weight, from 0.010 mg / kg body weight to 50 mg / kg body weight, from 0.010 mg / kg body weight to 40 mg / kg body weight, from 0.010 mg / kg body weight to 30 mg / kg body weight, from 0.010 mg / kg body weight to 20 mg / kg body weight, from 0.010 mg / kg body weight to 10 mg / kg body weight, from 0.010 mg / kg body weight to 5 mg / kg body weight, from 0.010 mg / kg body weight to 4 mg / kg body weight, from 0.010 mg / kg body weight to 3 mg / kg body weight, from 0.010 mg / kg body weight to 2 mg / kg body weight, from 0.010 mg / kg body weight to 1 mg / kg body weight, from 0.1 mg / kg body weight to 50 mg / kg body weight, from 0.1 mg / kg body weight to 40 mg / kg body weight, from 0.1 mg / kg body weight to 30 mg / kg body weight, from 0.1 mg / kg body weight to 20 mg / kg body weight, from 0.1 mg / kg body weight to 10 mg / kg body weight, from 0.1 mg / kg body weight to 5 mg / kg body weight, from 0.1 mg / kg body weight to 4 mg / kg body weight, from 0.1 mg / kg body weight to 3 mg / kg body weight, from 0.1 mg / kg body weight to 2 mg / kg body weight, 0.1 mg / kg body weight to 1 mg / kg body weight, 1 mg / kg body weight to 50 mg / kg body weight, 1 mg / kg body weight to 40 mg / kg body weight, 1 mg / kg body weight to 30 mg / kg body weight, 1 mg / kg body weight to 20 mg / kg body weight, 1 mg / kg body weight to 10 mg / kg body weight, 1 mg / kg body weight to 5 mg / kg body weight, 1 mg / kg body weight to 4 mg / kg body weight, 1 mg / kg body weight to 3 mg / kg body weight, 1 mg / kg body weight to 2 mg / kg body weight, or 1 mg / kg body weight to 1 mg / kg body weight.

[0253] For repeated administration for several days or longer, depending on the situation, treatment is continued until the desired symptom suppression occurs or until a sufficient treatment level is reached, such as pain relief. Exemplary dosing regimens include administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of the anti-CCR8 antibody, or a maintenance dose of about 1 mg / kg administered every other week. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern the practitioner wishes to achieve. For example, in some embodiments, once-weekly to four-times-weekly dosing is contemplated. The progress of the therapy is readily monitored by conventional techniques and assays. The dosing regimen (including one or more CCR8 antagonists used) can vary over time. In various embodiments, the appropriate dose of the anti-CCR8 antagonist antibody will depend on the anti-CCR8 antagonist antibody (or composition thereof) used, the type and severity of the headache (e.g., migraine) to be treated, whether the agent is being administered for prophylactic or therapeutic purposes, previous therapies, the patient's clinical history and response to the agent, and the judgment of the attending physician. Generally, the clinician will administer the anti-CCR8 agonist antibody until a dose is reached that achieves the desired result. The dose and / or frequency can vary over the course of treatment.

[0254] It should be noted that the dose values can vary with the type and severity of the condition to be alleviated. It should also be understood that for any particular subject, the specific dosing regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dose ranges listed herein are merely exemplary and are not intended to limit the scope of the claimed composition or practice.

[0255] In various embodiments, the total dose administered will achieve plasma antibody concentrations in the range of, for example, about 1 μg / ml to 1000 μg / ml, about 1 μg / ml to 750 μg / ml, about 1 μg / ml to 500 μg / ml, about 1 μg / ml to 250 μg / ml, about 10 μg / ml to 1000 μg / ml, about 10 μg / ml to 750 μg / ml, about 10 μg / ml to 500 μg / ml, about 10 μg / ml to 250 μg / ml, about 20 μg / ml to 1000 μg / ml, about 20 μg / ml to 750 μg / ml, about 20 μg / ml to 500 μg / ml, about 20 μg / ml to 250 μg / ml, about 30 μg / ml to 1000 μg / ml, about 30 μg / ml to 750 μg / ml, about 30 μg / ml to 500 μg / ml, about 30 μg / ml to 250 μg / ml.

[0256] The toxicity and therapeutic index of the pharmaceutical compositions of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as those used to determine the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) of standard pharmaceutical procedures. The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the therapeutic index can be expressed as the ratio LD 50 / ED 50 . Compositions that exhibit a large therapeutic index are generally preferred.

[0257] In various embodiments, the pharmaceutical composition is administered in a single dose or in more than one dose depending on the dose and frequency that the subject can tolerate and requires. In any case, the composition should provide a sufficient amount of at least one antibody or antigen-binding fragment thereof disclosed herein to effectively treat the subject. The dose can be administered once, but can be applied periodically until a therapeutic result is achieved or until side effects warrant discontinuation of the therapy.

[0258] The frequency of administration of the antibody or antigen-binding fragment pharmaceutical composition depends on the nature of the therapy and the particular disease being treated. The subject can be treated at regular intervals, such as weekly or monthly, until the desired therapeutic result is achieved. Exemplary frequencies of administration include, but are not limited to: once weekly without interruption; once weekly, every other week; once every 2 weeks; once every 3 weeks; once weekly without interruption for 2 weeks, then once monthly; once weekly without interruption for 3 weeks, then once monthly; once monthly; once every other month; once every 3 months; once every 4 months; once every 5 months; or once every 6 months, or once a year.

[0259] Combination therapy

[0260] As used herein, the terms "co-administration," "co-administered," and "in combination with" with respect to an antibody or antigen-binding fragment thereof of the present disclosure and one or more other therapeutic agents are intended to mean and do in fact mean the following and include the following: when such a combination of an antibody or antigen-binding fragment thereof of the present disclosure and one or more therapeutic agents is formulated together as a single dosage form that releases the components to a subject in need of treatment substantially simultaneously, administering such components to the subject simultaneously; when such a combination of an antibody or antigen-binding fragment thereof of the present disclosure and one or more therapeutic agents is formulated separately from one another as separate dosage forms that are taken by a subject in need of treatment substantially simultaneously, whereupon the components are released to the subject substantially simultaneously, administering such components to the subject substantially simultaneously; when such a combination of an antibody or antigen-binding fragment thereof of the present disclosure and one or more therapeutic agents is formulated separately from one another as separate dosage forms that are taken by a subject in need of treatment at successive times with a significant time interval between each administration, whereupon the components are released to the subject at substantially different times, administering such components to the subject sequentially; and, when such a combination of an antibody or antigen-binding fragment thereof of the present disclosure and one or more therapeutic agents is formulated together as a single dosage form that releases the components in a controlled manner, whereupon they are released to a subject in need of treatment simultaneously, continuously, and / or overlappingly at the same and / or different times, and where each portion may be administered by the same or different routes, administering such components to the subject sequentially.

[0261] In another aspect, the invention relates to a combination therapy designed to treat cancer or an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof of the invention and b) one or more additional therapies selected from the group consisting of: immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, vaccination regimens, and stem cell transplantation, wherein the combination therapy provides increased cell killing of tumor cells, i.e., there is a synergistic effect between the isolated antibody or antigen-binding fragment and the additional therapy when co-administered.

[0262] In various embodiments, the immunotherapy is selected from the group consisting of: treatment with agonistic antibodies, antagonistic antibodies, or blocking antibodies against co-stimulatory molecules or co-inhibitory molecules (immune checkpoints) such as PD-1, PD-L1, OX-40, CD137, GITR, LAG3, TIM-3, CD40, TIGIT, CD47, SIRPα, and VISTA; treatment with bispecific T cell engaging antibodies Treatments such as blinatumomab; treatments involving administration of a biological response modifier such as IL-2, IL-7, IL-12, IL-15, IL-21, GM-CSF, STING agonists, and IFN-α, IFN-β, and IFN-γ; treatments using a therapeutic vaccine such as sipuleucel-T; treatments using a dendritic cell vaccine or a tumor antigen peptide vaccine; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using tumor-infiltrating lymphocytes (TIL); treatments using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic anti-tumor T cells); treatments using TALL-104 cells; and treatments using immune stimulants such as Toll-like receptor (TLR) agonists CpG and imiquimod.

[0263] In various embodiments, additional therapies include an antibody that specifically binds an immune checkpoint protein antigen (from a list including but not limited to: CD276, CD272, CD152, CD223, CD279, CD274, TIM-3, and B7-H4) or any immune checkpoint protein antigen antibody taught in the art. In various embodiments, PD-1 inhibitors for use in combination therapy methods are selected from the group consisting of but not limited to: pembrolizumab (Merck), nivolumab (Bristol-Myers Squibb), cemiplimab (Regeneron), dostarlimab (GlaxoSmithKline), and retifanlimab (Incyte). In various embodiments, the PD-1 inhibitor is pembrolizumab. In various embodiments, the PD-1 inhibitor is nivolumab. In various embodiments, the PD-1 inhibitor is cemiplimab. In various embodiments, the PD-1 inhibitor is dostarlimab. In various embodiments, the PD-1 inhibitor is retifanlimab. In various embodiments, a PD-1 inhibitor is administered at between about 0.1 mg / kg and about 10 mg / kg. In various embodiments, a PD-1 inhibitor is administered at between about 1 mg / kg and about 15 mg / kg.

[0264] A large number of conventional compounds have been shown to have anti-neoplastic activity. These compounds have been used as agents in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant T cells in leukemia or myeloid malignancies. Although chemotherapy is effective in treating many types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments can act synergistically and allow for a reduction in the dose of each treatment, thereby reducing the harmful side effects produced by each compound at higher doses. In other cases, malignancies that are refractory to treatment can respond to combination therapy of two or more different treatments.

[0265] When the antibodies or antigen-binding fragments disclosed herein are administered concomitantly or sequentially in combination with another conventional anti-neoplastic agent, such antibodies or antigen-binding fragments can enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular tolerance to such anti-neoplastic agent. This allows for a reduction in the dose of the anti-neoplastic agent, thereby reducing undesirable side effects, or restoring the effectiveness of the anti-neoplastic agent in tolerant T cells.

[0266] Drug compounds that can be used in combination antitumor therapies include, for illustration purposes only: aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole,Octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0267] These chemotherapeutic anti-tumor compounds can be classified into groups such as the following according to their mechanism of action: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folic acid antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); anti-proliferative / anti-mitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule-disrupting agents such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA-damaging agents (actinomycin, amsacrine, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, paclitaxel, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracycline, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; anti-proliferative / anti-mitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazines - dacarbazine (DTIC); anti-proliferative / anti-mitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole);Anticoagulants (e.g., heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agent; breveldin; immunosuppressive agents (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (retinoic acid); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, teniposide, epirubicin, etoposide, idarubicin, and mitoxantrone, topotecan, irinotecan); corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0268] In various embodiments, chemotherapy comprises chemotherapeutic agents selected from the group consisting of: daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, mechlorethamine, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyurea, taxanes (such as paclitaxel and docetaxel) and / or anthracycline antibiotics, and combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP, or FOLFOX.

[0269] In various embodiments, the small molecule kinase inhibitor targeted therapy comprises a small molecule kinase inhibitor selected from the group consisting of Bruton's tyrosine kinase (BTK) inhibitors, phosphatidylinositol-3-kinase (PI3K) inhibitors, SYK inhibitors (e.g., entospletinib), AKT inhibitors, mTOR inhibitors, Src inhibitors, JAK / STAT inhibitors, Ras / Raf / MEK / ERK inhibitors, and Aurora inhibitors (see D’Cruz et al., Expert Opin Pharmacother, 14(6):707-21, 2013).

[0270] In various embodiments, the combination therapy comprises co-administering an antibody or an antigen-binding fragment thereof and one or more additional therapies. In various embodiments, the antibody or antigen-binding fragment thereof composition and one or more additional therapies are administered sequentially, i.e., the antibody or antigen-binding fragment thereof composition is administered before or after the administration of one or more additional therapies.

[0271] In various embodiments, the administration of the antibody or antigen-binding fragment thereof composition and one or more additional therapies is simultaneous, i.e., the administration time periods of the antibody or antigen-binding fragment thereof composition and one or more additional therapies overlap with each other.

[0272] In various embodiments, the administration of the antibody or antigen-binding fragment thereof composition and one or more additional therapies is not simultaneous. For example, in various embodiments, the administration of the antibody or antigen-binding fragment thereof composition is terminated before the administration of one or more additional therapies. In various embodiments, the administration of one or more additional therapies is terminated before the administration of the antibody or antigen-binding fragment thereof composition.

[0273] When the antibodies or antigen-binding fragments thereof disclosed herein are co-administered concomitantly or sequentially in combination with one or more additional therapies, such antibodies or antigen-binding fragments thereof may enhance the therapeutic effect of one or more additional therapies or overcome cellular tolerance to one or more additional therapies. This allows for a reduction in the dose of one or more additional therapies or a shortening of the duration of one or more additional therapies, thereby reducing undesirable side effects, or restoring the efficacy of one or more additional therapies.

[0274] Diagnostic use

[0275] In another aspect, the present invention provides methods for detecting the presence of human CCR8 peptide in a sample, in vitro or in vivo, e.g., for diagnosing human CCR8-related disorders. In some methods, this is achieved by contacting the sample to be tested, as well as a control sample, with a human sequence antibody or a human monoclonal antibody or an antigen-binding portion thereof (or a bispecific or multispecific molecule) of the present invention under conditions that permit the formation of a complex between the antibody and human CCR8. Complex formation is then detected (e.g., using ELISA) in both samples, and any statistically significant difference in complex formation between the samples indicates the presence of human CCR8 antigen in the test sample.

[0276] In various embodiments, methods are provided for detecting a CCR8-related disorder in a subject or for determining a diagnosis of a CCR8-related disorder. The method comprises contacting a biological sample from the subject with an isolated antibody or an antigen-binding fragment thereof of the present invention, and detecting the binding of the isolated human monoclonal antibody or an antigen-binding fragment thereof to the sample. An increase in the binding of the isolated human monoclonal antibody or an antigen-binding fragment thereof to the sample, compared to the binding of the isolated human monoclonal antibody or an antigen-binding fragment thereof to a control sample, detects a CCR8-related disorder in the subject or determines a diagnosis of a CCR8-related disorder in the subject. The control can be a sample from a subject known not to have a CCR8-related disorder, or a standard value. The sample can be any sample, including but not limited to tissue from a biopsy, autopsy, and pathological specimen. The biological sample also includes tissue sections, e.g., frozen sections collected for histological purposes. The biological sample also includes body fluids such as blood, serum, plasma, sputum, and spinal fluid.

[0277] In one embodiment, a kit is provided for detecting CCR8 in a biological sample such as a blood sample. A kit for detecting a polypeptide will generally comprise a human antibody that specifically binds to CCR8, such as any of the antibodies disclosed herein. In some embodiments, antibody fragments such as Fv fragments are included in the kit. For in vivo use, the antibody can be an scFv fragment. In additional embodiments, the antibody is labeled (e.g., labeled with a fluorescent, radioactive, or enzyme label).

[0278] In one embodiment, the kit includes instructional materials that disclose means for using an antibody that specifically binds to CCR8. The instructional materials can be written in electronic form (such as a computer floppy disk or compact disc) or can be visual (such as a video file). The kit can also include additional components that facilitate the particular application for which the kit is designed. Thus, for example, the kit can additionally contain means for detecting a label (such as an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, a suitable secondary label such as a secondary antibody, etc.). The kit can additionally contain buffers and other reagents commonly used in practicing the particular method. Such kits and suitable components are well known to those skilled in the art.

[0279] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay can vary with the particular format used, methods for detecting CCR8 in a biological sample generally include the step of contacting the biological sample with an antibody that specifically reacts with CCR8 under immunological reaction conditions. The antibody is allowed to specifically bind under immunological reaction conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected either directly or indirectly.

[0280] In various embodiments, the antibody or antigen-binding fragment can be labeled or unlabeled for diagnostic purposes. Generally, diagnostic assays require detection of the formation of a complex resulting from the binding of the antibody to CCR8. The antibody can be labeled directly. A number of labels can be used, including but not limited to radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, hapten). Many suitable immunoassays are known to those skilled in the art (see, for example, U.S. Patent Nos. 3,817,827; 3,850,752; 3,901,654; and 4,098,876). When unlabeled, the antibody can be used in assays such as agglutination assays. The unlabeled antibody can also be used in combination with additional (one or more) suitable reagents that can be used to detect the antibody, such as a labeled antibody (e.g., a secondary antibody) that reacts with a first antibody (e.g., an anti-idiotype antibody or other antibody specific for the unlabeled immunoglobulin) or other suitable reagents (e.g., labeled protein A).

[0281] The antibodies or antigen-binding fragments provided herein can also be used in methods for detecting the susceptibility of a mammal to certain diseases. By way of illustration, the method can be used to detect the susceptibility of a mammal to a disease that progresses based on the amount of CCR8 present on cells and / or the number of CCR8-positive cells in the mammal. In one embodiment, the present application provides a method for detecting the susceptibility of a mammal to a tumor. In this embodiment, a sample to be tested is contacted with an antibody that binds to CCR8 or a portion thereof under conditions suitable for binding of the antibody thereto, wherein the sample comprises cells that express CCR8 in a normal individual. The binding of the antibody and / or the amount of binding is detected, which indicates the susceptibility of the individual to a tumor, wherein a higher level of the receptor is associated with an increased susceptibility of the individual to a tumor.

[0282] In various embodiments, the antibody or antigen-binding fragment is attached to a label that can be detected (e.g., the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor). The active moiety can be a radioactive agent such as: radioactive heavy metals such as iron chelates, radioactive chelates of gadolinium or manganese, positron emitters of oxygen, nitrogen, iron, carbon, or gallium, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 132 I or 99 Tc. The binding agent attached to such moieties can be used as an imaging agent and administered in an amount effective for diagnostic use in mammals such as humans, and then the localization and accumulation of the imaging agent are detected. The localization and accumulation of the imaging agent can be detected by radioisotope scintigraphy, magnetic resonance imaging, computed tomography, or positron emission tomography.

[0283] Immunoscintigraphy using an antibody or antigen-binding fragment against CCR8 can be used to detect and / or diagnose cancer and the vascular system. For example, against 99 technetium, 111 indium, or 125Monoclonal antibodies of iodine-labeled CCR8 markers can be effectively used for such imaging. As will be apparent to those skilled in the art, the amount of radioisotope to be administered depends on the radioisotope. A person of ordinary skill in the art can easily formulate the amount of the imaging agent to be administered based on the specific activity and energy of a given radionuclide used as the active moiety. Generally, 0.1-100 millicuries, or 1-10 millicuries, or 2-5 millicuries are administered per dose of the imaging agent. Accordingly, the disclosed compositions are useful as imaging agents that comprise a targeting moiety conjugated to a radioactive moiety that comprises 0.1-100 millicuries, in some embodiments 1-10 millicuries, in some embodiments 2-5 millicuries, in some embodiments 1-5 millicuries.

[0284] Antibody-drug conjugates (ADCs) and immunoconjugates

[0285] Antibody-drug conjugates (ADCs) combine the binding specificity of an antibody with the potency of a drug such as a cytotoxic agent, an anti-cancer drug, and an immunosuppressive drug. The use of ADCs allows for the targeted delivery of the drug, which may result in unacceptable levels of toxicity to normal cells if administered as an unconjugated drug. The mechanism of action of an ADC is that the antibody recognizes and binds to a specific antigen, triggering a series of reactions and then entering the cytoplasm through endocytosis, where the highly cytotoxic drug dissociates from the antibody after being degraded by lysosomal enzymes to kill cancer cells. Compared with traditional chemotherapy that indiscriminately damages both cancer cells and normal tissues, targeted drug delivery can enable the drug to act directly on cancer cells and reduce damage to normal cells.

[0286] This application also provides an ADC that comprises the novel antibody and antigen-binding fragment of the present invention linked to a second molecule selected from the group consisting of a cytotoxic agent, an anti-cancer drug, or an immunosuppressive drug.

[0287] Cytotoxic compounds intended for use in antibody-drug conjugates inhibit various essential cellular targets such as microtubules (maytansine alkaloids, auristatins, taxanes: U.S. Patent Nos. 5,208,020; 5,416,064; 6,333,410; 6,441,163; 6,340,701; 6,372,738; 6,436,931; 6,596,757; 7,276,497; 7,301,019; 7,303,749; 7,368,565; 7,473,796; 7,585,857; 7,598,290; 7,495,114; 7,601,354, U.S. Patent Application Nos. 20100092495, 20100129314, 20090274713, 20090076263, 20080171865) and DNA (calicheamicin, doxorubicin, CC-1065 analogs: U.S. Patent Nos. 5,475,092; 5,585,499; 5,846,545; 6,534,660; 6,756,397; 6,630,579; 7,388,026; 7,655,660; 7,655,661).

[0288] This application also provides immunoconjugates or fusion proteins comprising an antibody of the invention or an antigen-binding fragment thereof directly or indirectly conjugated (or linked) to an effector molecule. In this regard, the terms "conjugated" or "linked" are meant to render two polypeptide molecules into one contiguous polypeptide molecule. The linkage can be effected by chemical or recombinant means. In one embodiment, the linkage is chemical, wherein the reaction between the antibody moiety and the effector molecule results in the formation of a covalent bond between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. In various embodiments, the antibody or antigen-binding fragment is linked to the effector molecule. In other embodiments, the antibody or antigen-binding fragment linked to the effector molecule is further linked to a lipid, protein, or peptide to increase its half-life in vivo. Thus, in various embodiments, the antibodies of the present disclosure can be used to deliver a variety of effector molecules.

[0289] The effector molecule can be a detectable label, an immunotoxin, a cytokine, a chemokine, a therapeutic agent, or a chemotherapeutic agent.

[0290] Specific, non-limiting examples of immunotoxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, cholix toxin, or modified toxins thereof, or other agents that directly or indirectly inhibit cell growth or kill cells.

[0291] "Cytokines" are a class of proteins or peptides released by one cell population that act as intercellular mediators on another cell. Cytokines can act as immunomodulators. Examples of cytokines include lymphokines, monokines, growth factors, and traditional polypeptide hormones. Thus, embodiments can utilize interferons (e.g., IFN-α, IFN-β, and IFN-γ); members of the tumor necrosis factor superfamily (TNFSF); human growth hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle-stimulating hormone (FSH); thyroid-stimulating hormone (TSH); luteinizing hormone (LH); hepatocyte growth factor; prostaglandins, fibroblast growth factors; prolactin; placental lactogen, OB protein; TNF-α; TNF-β; integrins; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-derived growth factor; TGF-α; TGF-β; insulin-like growth factors-I and -II; erythropoietin (EPO); colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL-1 to IL-36), kit-ligand or FLT-3, angiostatin, thrombospondin, or endostatin; immune checkpoint proteins, including CTLA-4, PD-1, PD-L1, LAG-3, TIGIT, and TIM-3, and several other substances (Sharpe et al., Nat Immunol, 8:239-45, 2007). These cytokines include proteins from natural sources or from recombinant cell cultures and bioactive equivalents of native sequence cytokines.

[0292] In various embodiments, the effector molecule is selected from the list provided in Table 4. Each relevant reference is incorporated herein by reference for the purpose of identifying the tumor markers mentioned.

[0293] Table 4 Exemplary tumor markers

[0294]

[0295]

[0296]

[0297] In various embodiments, the effector molecule is selected from the list provided in Table 5. These targets can also be used for cancer targeting applications.

[0298] Table 5 Targets of autoimmune and inflammatory disorders or cancer

[0299]

[0300]

[0301] Chemokines can also be conjugated to the antibodies disclosed herein. Chemokines are a superfamily of small (about 4K D to about 14K D ) inducible and secreted pro-inflammatory cytokines that act mainly as chemoattractants and activators of specific leukocyte cell subtypes. The production of chemokines is induced by inflammatory cytokines, growth factors, and pathogenic stimuli. Chemokine proteins are divided into subfamilies (α, β, and δ) based on conserved amino acid sequence motifs and into four highly conserved groups - CXC, CC, C, and CX3C - based on the position of the first two cysteines proximal to the amino terminus. To date, more than 50 chemokines have been discovered and at least 18 human seven-transmembrane domain (7TM) chemokine receptors. Chemokines used include, but are not limited to, RANTES, MCAF, MCP-1, and fractalkine.

[0302] The therapeutic agent can be a chemotherapeutic agent. A person skilled in the art can readily determine the chemotherapeutic agent to be used (for example, see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison’s Principles of Internal Medicine, 14th Edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc; Baltzer L., Berkery R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd Edition, St. Louis, Mosby Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds.): The Cancer Chemotherapy Handbook, 4th Edition, St. Louis, Mosby Year Book, 1993). Useful chemotherapeutic agents for preparing immunoconjugates include auristatin, dolastatin, MMAE, MMAF, AFP, DM1, AEB, doxorubicin, daunorubicin, methotrexate, melphalan, chlorambucil, vinca alkaloids, 5-fluorouridine, mitomycin-C, paclitaxel, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, etoposide, BCNU, irinotecan, camptothecin, bleomycin, idarubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel, and their salts, solvents, and derivatives. In various embodiments, the chemotherapeutic agent is auristatin E (also known as dolastatin-10 in the art) or a derivative thereof and its pharmaceutically acceptable salts or solvates. Typical auristatin derivatives include DM1, AEB, AEVB, AFP, MMAF, and MMAE. The synthesis and structure of auristatin E and its derivatives and the linker are described, for example, in U.S. Patent Application Publication No. 20030083263; U.S. Patent Application Publication No. 20050238629; and U.S. Patent No. 6,884,869 (each of which is incorporated herein by reference in its entirety). In various embodiments, the therapeutic agent is an auristatin or an auristatin derivative.In various embodiments, the auristatin derivative is dovaline-valine-dolaisoleunine-dolaproine-phenylalanine (MMAF) or monomethyl auristatin E (MMAE). In various embodiments, the therapeutic agent is a maytansinoid or a maytansinol analog. In various embodiments, the maytansinoid is DM1.

[0303] Any number of means known to those skilled in the art can be used to link the effector molecule to the antibody or antigen-binding fragment of the invention. Both covalent and non-covalent attachment means can be used. The procedure for attaching the effector molecule to the antibody varies according to the chemical structure of the effector molecule. Polypeptides generally contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to result in the binding of the effector molecule. Optionally, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization can include attaching any one of a number of linker molecules, such as those available from Pierce Chemical Company, Rockford, III. The linker can be any molecule used to link the antibody to the effector molecule. The linker is capable of forming covalent bonds with both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers or peptide linkers. When the antibody and the effector molecule are polypeptides, the linker can be attached to the component amino acids through the side groups of the component amino acids (such as through a disulfide bond with cysteine), or to the α-carbon amino and carboxyl groups of the terminal amino acids.

[0304] In some cases, it is desirable to release the effector molecule from the antibody when the immunoconjugate has reached its target site. Thus, in these cases, the immunoconjugate will contain a cleavable linkage near the target site. Enzymatic activity or the conditions to which the immunoconjugate is subjected within the target cell or near the target site can cause cleavage of the linker to release the effector molecule from the antibody.

[0305] Procedures for conjugating antibodies with effector molecules have been previously described and are within the capabilities of those skilled in the art. For example, procedures for preparing enzymatically active polypeptides of immunotoxins are described in WO84 / 03508 and WO85 / 03508, which are hereby incorporated by reference for the purpose of their specific teachings. Other techniques are described in Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); Shih et al., U.S. Patent No. 5,057,313; Shih Cancer Res. 51:4192, International Publication WO 02 / 088172; U.S. Patent No. 6,884,869; International Patent Publication WO 2005 / 081711; U.S. Published Application 2003-0130189A and U.S. Patent Application No. 20080305044, each of which is incorporated by reference herein for the purpose of teaching such techniques.

[0306] The immunoconjugates of the present invention retain the immunoreactivity of the antibody or antigen-binding fragment, e.g., the antibody or antigen-binding fragment has approximately the same or only slightly reduced ability to bind antigen after conjugation as before conjugation.

[0307] Bispecific molecule

[0308] Bispecific antibodies are antibodies that include two different antigen-binding sites of a monoclonal antibody, and the two different antigen-binding sites of the monoclonal antibody can bind two different antigens or two different sites of one antigen. In addition to simultaneously blocking two different signaling pathways and thereby enhancing tumor cell killing, bispecific antibodies can potentially increase binding specificity by interacting with two different cell surface antigens rather than one. Nowadays, it is considered an effective molecule for the next generation of cancer therapy. It can minimize the regulatory and commercial issues arising from the administration of multiple therapeutic molecules. It also has the potential for new activities not present in mixtures of parental antibodies. Several bispecific antibodies are on the market, and many are in clinical development.

[0309] In another aspect, the invention features bispecific molecules comprising the anti-CCR8 antibody or antigen-binding fragment thereof of the invention. The antibody or antigen-binding fragment thereof of the invention can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand of a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the invention can in fact be derivatized or linked to more than one other functional molecule to generate a multispecific molecule that binds to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To generate the bispecific molecules of the invention, the antibody of the invention can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent association, or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule is produced. In various embodiments, the invention includes bispecific molecules capable of binding to both effector cells expressing FcγR or FcαR (e.g., monocytes, macrophages, or polymorphonuclear cells (PMN)) and target cells expressing PD. In such embodiments, the bispecific molecule targets cells expressing CCR8 to the effector cells and triggers Fc receptor-mediated effector cell activity, e.g., phagocytosis of cells expressing CCR8, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anions. Methods for preparing the bispecific molecules of the invention are well known in the art.

[0310] In various embodiments, the second functional molecule is an antibody, antibody fragment, or protein or peptide that exhibits antigen binding to an immune checkpoint protein present on the surface of an immune cell. In various embodiments, the immune checkpoint protein antigen is selected from the group consisting of, but not limited to: CD276, CD272, CD152, CD223, CD279, CD274, CD40, SIRPα, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec-7, Siglec-8, Siglec-9, Siglec-15, TIGIT, and VISTA. In various embodiments, D1 can comprise an antibody against an immune checkpoint protein antigen present on the surface of a tumor cell, the immune checkpoint protein antigen being selected from the group consisting of, but not limited to: PD-L1, B7-H3, and B7-H4.

[0311] In various embodiments of the invention, the antibody or antigen-binding fragment is a CCR8 / CTLA4 bispecific antibody comprising the heavy chain sequence of SEQ ID NO:93:

[0312]

[0313] and a light chain sequence selected from the group consisting of SEQ ID NO:91 and SEQ ID NO:92.

[0314] Polynucleotide and antibody expression

[0315] The present application also provides a polynucleotide comprising a nucleotide sequence encoding an anti-CCR8 antibody or an antigen-binding fragment thereof. Due to the degeneracy of the genetic code, many nucleic acid sequences encode each antibody amino acid sequence. The present application also provides a polynucleotide that hybridizes, for example, under stringent hybridization conditions as defined herein or less stringent hybridization conditions, to a polynucleotide encoding an antibody that binds to human CCR8.

[0316] Stringent hybridization conditions include, but are not limited to, hybridizing DNA bound to a filter in 6×SSC at about 45°C, followed by washing one or more times in 0.2×SSC / 0.1% SDS at about 50°C - 65°C, highly stringent conditions such as hybridizing DNA bound to a filter in 6×SSC at about 45°C, followed by washing one or more times in 0.1×SSC / 0.2% SDS at about 60°C, or any other stringent hybridization conditions known to those skilled in the art (see, e.g., Ausubel, F.M. et al., eds. 1989 Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY, pp. 6.3.1 - 6.3.6 and 2.10.3).

[0317] The polynucleotide can be obtained and the nucleotide sequence of the polynucleotide determined by any method known in the art. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), briefly, which includes synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR. In one embodiment, the codons used include those that are typical for humans or mice (see, e.g., Nakamura, Y., Nucleic Acids Res. 28:292 (2000)).

[0318] The polynucleotides encoding the antibodies can also be generated from nucleic acids from a suitable source. If a clone containing the nucleic acid encoding a specific antibody is not available, but the sequence of the antibody molecule is known, then the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody, or nucleic acids isolated from any tissue or cell expressing the antibody, preferably polyA+RNA, such as a hybridoma cell selected to express the antibody), which is obtained by PCR amplification using synthetic primers that hybridize to the 3'-end and 5'-end of the sequence, or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, for example, a cDNA clone from a cDNA library encoding the antibody. Then, the amplified nucleic acids generated by PCR can be cloned into a replicable cloning vector by any method well known in the art.

[0319] The invention also relates to host cells expressing the CCR8 and / or anti-CCR8 antibodies of the invention. Many types of host expression systems known in the art can be used to express the antibodies of the invention, including prokaryotic (bacterial) expression systems and eukaryotic expression systems (such as yeast, baculovirus, plants, mammalian and other animal cells, transgenic animals and hybridoma cells), as well as phage display expression systems.

[0320] The antibodies of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells. To recombinantly express an antibody, a host cell is transformed, transduced, infected, etc. with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and / or heavy chains of the antibody such that the light and / or heavy chains are expressed in the host cell. The heavy and light chains can be expressed independently from different promoters operably linked in one vector, or alternatively, the heavy and light chains can be expressed independently from different promoters operably linked in two vectors, one vector expressing the heavy chain and one vector expressing the light chain. Optionally, the heavy and light chains can be expressed in different host cells.

[0321] In addition, the recombinant expression vector can encode a signal peptide that facilitates the secretion of the antibody light and / or heavy chain from the host cell. The antibody light and / or heavy chain genes can be cloned into the vector such that the signal peptide is operably linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Preferably, the recombinant antibody is secreted into the culture medium of the host cells, from which the antibody can be recovered or purified.

[0322] The isolated DNA encoding HCVR can be converted into a full-length heavy chain gene by operably linking the DNA encoding HCVR to another DNA molecule encoding a heavy chain constant region. The sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments containing these regions can be obtained by, for example, standard PCR amplification. The heavy chain constant region can be of any type (e.g., IgG, IgA, IgE, IgM, or IgD), class (e.g., IgG1, IgG2, IgG3, and IgG4), or subclass constant region and any allelic variants thereof, as described in Kabat (see above).

[0323] The isolated DNA encoding the LCVR region can be converted into a full-length light chain gene (and into a Fab light chain gene) by operably linking the DNA encoding LCVR to another DNA molecule encoding a light chain constant region. The sequences of human and other mammalian light chain constant region genes are known in the art. DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a κ or λ constant region.

[0324] In addition to one or more antibody heavy and / or light chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of one or more antibody chain genes in a host cell. The term "regulatory sequence" is intended to include, as needed, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of one or more antibody chain genes. The design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and / or polyomavirus.

[0325] In addition, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate the replication of the vector in a host cell (e.g., origin of replication) and one or more selectable marker genes. The selectable marker genes facilitate the selection of host cells that have been introduced with the vector. For example, generally, the selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate on the host cells that have been introduced with the vector. Preferred selectable marker genes include the dihydrofolate reductase (dhfr) gene (for use in dhfr-negative host cells selected / amplified with methotrexate), the neo gene (for G418 selection), and GS in glutamine synthetase (GS)-negative cell lines (such as NSO) used for selection / amplification.

[0326] To express the light chain and / or heavy chain, one or more expression vectors encoding the heavy chain and / or light chain are introduced into a host cell by standard techniques such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, transduction, infection, etc. Although it is theoretically possible to express the antibodies of the present invention in prokaryotic or eukaryotic host cells, eukaryotic cells are preferred, and mammalian host cells are most preferred because such cells are more likely to assemble and secrete properly folded and immunologically active antibodies. Preferred mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO cells) [including dhfr-negative CHO cells as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-20, 1980, used with a DHFR selectable marker as described in Kaufman and Sharp, J. Mol. Biol. 159:601-21, 1982], NSO myeloma cells, COS cells, and SP2 / 0 cells. When the recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, the antibody is secreted into the culture medium in which the host cell is grown under conditions known in the art. The antibody can be recovered from the host cell and / or the culture medium using standard purification methods.

[0327] The present invention provides a host cell comprising a nucleic acid molecule according to the present invention. Preferably, the host cell of the present invention comprises one or more vectors or constructs comprising the nucleic acid molecule of the present invention. For example, the host cell of the present invention is a cell into which the vector of the present invention has been introduced, the vector comprising a polynucleotide encoding the LCVR of the antibody of the present invention and / or a polynucleotide encoding the HCVR of the present invention. The present invention also provides a host cell into which two vectors of the present invention have been introduced; one vector comprises a polynucleotide encoding the LCVR of the antibody of the present invention, and one vector comprises a polynucleotide encoding the HCVR present in the antibody of the present invention, and each polynucleotide is operably linked to an enhancer / promoter regulatory element (e.g., derived from SV40, CMV, adenovirus, etc., such as the CMV enhancer / AdMLP promoter regulatory element or the SV40 enhancer / AdMLP promoter regulatory element) to drive high-level transcription of the gene.

[0328] Following expression, the intact antibodies, individual light and heavy chains, or other immunoglobulin forms of the invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation, ion exchange, affinity (e.g., protein A), reverse phase, hydrophobic interaction column chromatography, hydroxyapatite chromatography, gel electrophoresis, etc. Standard procedures for the purification of therapeutic antibodies, such as those described by Feng Li, Joe X. Zhou, Xiaoming Yang, Tim Tressel, and Brian Lee in the article entitled “Current Therapeutic Antibody Production and Process Optimization” (BioProcessing Journal, September / October 2005) (incorporated by reference in its entirety for the purpose of teaching the purification of therapeutic antibodies). Additionally, standard techniques for removing viruses from recombinant expressed antibody preparations are also known in the art (see, e.g., Gerd Kern and Mani Krishnan, “Viral Removal by Filtration: Points to Consider” (Biopharm International, October 2006)). The effectiveness of filtration for removing viruses from a therapeutic antibody formulation is known to depend at least in part on the concentration of protein and / or antibody in the solution to be filtered. The purification process for the antibodies of the invention can include a step of filtration to remove viruses from the main stream of one or more chromatographic operations. Preferably, the chromatographic main stream containing the antibodies of the invention is diluted or concentrated to give a total protein concentration and / or total antibody concentration of about 1 g / L to about 3 g / L prior to filtration through a pharmaceutical grade nanofilter to remove viruses. Even more preferably, the nanofilter is a DV20 nanofilter (e.g., Pall Corporation; East Hills, N.Y.). Substantially pure immunoglobulins that are preferably at least about 90%, about 92%, about 94%, or about 96% homogeneous, and most preferably about 98% to about 99% or higher homogeneous are used for pharmaceutical purposes. Once partially purified or purified to homogeneity as desired, then the sterile antibodies can be therapeutically used as directed herein.

[0329] In view of the foregoing discussion, the invention also relates to antibodies obtainable by a method comprising the steps of culturing a host cell such that a nucleic acid is expressed and optionally, recovering the antibody from the host cell culture medium, the host cell including but not limited to mammalian, plant, bacterial, transgenic animal, or transgenic plant cells that have been transformed with a polynucleotide or vector comprising a nucleic acid molecule encoding an antibody of the invention.

[0330] In certain aspects, the present application provides hybridoma cell lines, and monoclonal antibodies produced by these hybridoma cell lines. The disclosed cell lines have other uses in addition to being used to produce monoclonal antibodies. For example, the cell lines can be fused with other cells (such as appropriately drug-labeled human myeloma, mouse myeloma, human-mouse hybrid myeloma, or human lymphoblastoid cells) to produce additional hybridomas, and thus provide the transfer of genes encoding monoclonal antibodies. In addition, the cell lines can be used as a source of nucleic acids encoding anti-CCR8 immunoglobulin chains, which can be isolated and expressed (e.g., after being transferred to other cells using any suitable technique) (see, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Winter, U.S. Patent No. 5,225,539). For example, clones containing rearranged anti-CCR8 light or heavy chains can be isolated (e.g., by PCR), or cDNA libraries can be prepared from mRNA isolated from the cell lines, and cDNA clones encoding anti-CCR8 immunoglobulin chains can be isolated. Thus, nucleic acids encoding the heavy and / or light chains of an antibody or portions thereof can be obtained and used according to recombinant DNA techniques for producing specific immunoglobulins, immunoglobulin chains, or variants thereof (e.g., humanized immunoglobulins) in a variety of host T cells or in vitro translation systems. For example, nucleic acids encoding variants such as humanized immunoglobulins or immunoglobulin chains, including cDNA or derivatives thereof, can be placed into a suitable prokaryotic or eukaryotic vector (e.g., an expression vector), and introduced into a suitable host T cell by appropriate methods (e.g., transformation, transfection, electroporation, infection) such that the nucleic acid is operably linked to one or more expression control elements (e.g., in the vector or integrated into the host T cell genome). For production, the host T cells can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide. If desired, the encoded protein can be recovered and / or isolated (e.g., from the host T cell or the medium). It will be understood that the production methods include expression in host T cells of transgenic animals (see, e.g., WO 92 / 03918 of GenPharm International published on March 19, 1992) (incorporated by reference in its entirety).

[0331] Host cells can also be used to produce portions or fragments of whole antibodies by conventional techniques, such as Fab fragments or scFv molecules. For example, it may be desirable to transfect host cells with DNA encoding the light or heavy chain of the antibody of the invention. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that are not necessary for binding to human CCR8. The antibodies of the invention also include molecules expressed from such truncated DNA molecules.

[0332] Methods for expressing single-chain antibodies and / or refolding them into the appropriate active form, including single-chain antibodies, from bacteria such as Escherichia coli have been described and are well known and applicable to the antibodies disclosed herein (see, for example, Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989, all incorporated herein by reference).

[0333] Typically, functional heterologous proteins from Escherichia coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants and subsequent refolding. During the solubilization step, as is well known in the art, a reducing agent must be present to cleave disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithiothreitol). Reoxidation of disulfide bonds can occur in the presence of low molecular weight thiol reagents in both reduced and oxidized forms, as described in Saxena et al., Biochemistry 9:5015-5021, 1970, incorporated herein by reference, and particularly as described by Buchner et al. (supra).

[0334] Renaturation is typically achieved by diluting the denatured and reduced protein (e.g., 100-fold) into a refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[0335] As a modification to the purification protocol for diabodies, the heavy and light chain regions are solubilized and reduced separately and then combined in a refolding solution. Exemplary yields are obtained when the two proteins are mixed in a molar ratio such that one protein is not more than 5-fold in molar excess relative to the other. After redox shuffling is complete, excess oxidized glutathione or other oxidized low molecular weight compounds can be added to the refolding solution.

[0336] In addition to recombinant methods, the antibodies, labeled antibodies, and antigen-binding fragments thereof disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid-phase synthesis of polypeptides less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid-phase synthesis are described by: Barany and Merrifield, The Peptides: Analysis, Synthesis, Biology. Volume 2: Special Methods in Peptide Synthesis, Part A. Pages 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963 and Stewart et al., Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chem. Co., Rockford, Ill., 1984. Larger length proteins can be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods for forming peptide bonds by activation of the carboxyl terminus (such as by using the coupling agent N,N'-dicyclohexylcarbodiimide) are well known in the art.

[0337] The following examples are provided to more fully illustrate the invention, but should not be construed as limiting the scope of the invention.

[0338] Example 1

[0339] Generation of a Mouse Monoclonal Antibody Specifically Targeting Human CCR8

[0340] A human CCR8 expression plasmid was constructed, and CHO-K1 cells overexpressing hCCR8 were generated to be used as an immunogen for generating anti-hCCR8 monoclonal antibodies. BALB / c, C57BL / 6, A / J, and SJL mice were immunized every two weeks (a total of 6 or more immunizations) with plasmid DNA containing hCCR8, cells expressing hCCR8, or membrane proteins. The immunogen preparation was injected subcutaneously or intraperitoneally. Serum was collected from the immunized mice and tested by flow cytometry on both CHO-K1 cells expressing hCCR8 and parental CHO-K1 cells. Mice with a significant level (titer) of anti-hCCR8 antibody were selected for hybridoma fusion. Briefly, spleen cells were harvested 3-4 days after the final immunization for fusion with the myeloma cell line SP2 / 0 from the ATCC (American Type Culture Collection). Electro-fusion was used to obtain hybridoma cells.

[0341] Antigen binding of hybridoma supernatants was screened by flow cytometry and / or cell-based ELISA. In the cell-based ELISA, both CHO-K1 cells and cells expressing hCCR8 were incubated with the supernatant. The cells were washed with PBS and then incubated with a secondary antibody, goat anti-mouse IgG-HRP. After washing the cells, TMB was added. Absorbance readings were taken at 450 nm using a plate reader. Primary hybridoma clones with the highest OD450 ratio in cells expressing hCCR8 to CHO-K1 cells were selected by limiting dilution for subcloning.

[0342] Subcloned supernatants were tested by cell-based ELISA to confirm the presence of antibodies that specifically bind hCCR8. Only subclones with an OD450 ratio higher than 1.9 were selected for further analysis.

[0343] Human CCL1 (hCCL1) is the dominant ligand of CCR8. Subcloned supernatants were tested in a cell-based CCR8 CHO-K1 β-arrestin bioassay (Eurofins) to determine the antagonist activity of the anti-CCR8 antibodies, i.e., blocking the CCR8 downstream signaling induced by hCCL1. Briefly, approximately 10,000 cells were seeded into wells of a 96-well plate and incubated at 37 °C, 5% CO2 for 24 - 48 hours. The supernatant was added and incubated for 30 minutes to allow the antibody to bind to hCCR8 on the cells. After adding 2 nM of hCCL1, the plate was incubated at 37 °C, 5% CO2 for 90 minutes to stimulate the production of β-arrestin. Finally, the detection solution was added and the plate was incubated at room temperature in the dark for 1 hour. The plate was read on a Varioskan LUX multimode microplate reader (ThermoFisher Scientific). Subclones showing at least 50% inhibition of β-arrestin production in the assay were selected for sequencing (see Table 3).

[0344] Total RNA was isolated from hybridoma cells according to the manufacturer's instructions (Vazyme). Then, total RNA was reverse transcribed into cDNA using isotype-specific antisense primers or universal primers according to the technical manual of SMARTScribe reverse transcriptase (TaKaRa). Antibody fragments of the heavy and light chains were amplified according to the standard operating procedure for rapid amplification of cDNA ends (RACE) from ProBio. The amplified antibody fragments were cloned into standard cloning vectors separately. Colony PCR was performed to screen for clones with inserts of the correct size and sequenced. The mouse mAb clones listed in Table 3 contain the heavy chain variable region (VH) and / or light chain variable region (VL) and / or CDR sequences listed in SEQ ID NO: 3 - 25 and 35 - 77.

[0345] Example 2

[0346] Anti-hCCR8 antibodies block the binding of hCCL1 to hCCR8

[0347] Mouse antibodies 41E1C2A5, 46A5C4B1, and 80E4D1F11, as well as chimeric antibodies (human IgG1) 504E12D8D12, 516D7D12, 525F2F3F11, and 531B9B1C9, were evaluated in the CCR8 CHO-K1 β-arrestin assay to determine antagonist activity for blocking hCCL1-induced CCR8 signaling activation. Data were plotted using GraphPad Prism version 9 (San Diego, CA), and IC50 values were determined by non-linear regression curve fitting and summarized in Table 6.

[0348] Table 6 Antagonist activity of anti-hCCR8 antibodies measured by β-arrestin assay

[0349]

[0350] Example 3

[0351] Anti-hCCR8 antibodies do not bind to human CCR4

[0352] Mouse antibodies 41E1C2A5, 46A5C4B1, and 80E4D1F11, as well as chimeric antibodies 504E12D8D12, 516D7D12, 525F2F3F11, and 531B9B1C9, specifically bound to CHO-K1 cells expressing hCCR8 and blocked the binding of hCCL1 to hCCR8, indicating that they are CCR8-specific antibodies. Antibody binding to human CCR4 (hCCR4) was further screened by flow cytometry. Antibodies were incubated with CHO-K1 cells and CHO-K1 cells expressing hCCR4 at 4 °C for 60 minutes. Cells were washed thoroughly with PBS buffer and then incubated with FITC-conjugated goat anti-mouse IgG Fc antibody or anti-human IgG Fc antibody. The mean fluorescence intensity (MFI) of antibody binding on the cells was analyzed by flow cytometry (Table 7). The results showed that anti-hCCR8 antibodies did not bind hCCR4.

[0353] Table 7 MFI of antibody binding measured by flow cytometry

[0354]

[0355]

[0356] Example 4

[0357] ADCC activity of anti-hCCR8 chimeric antibodies

[0358] The ADCC activity of anti-hCCR8 chimeric antibodies was measured by a reporter gene bioassay (BPS Bioscience). Briefly, CHO-K1 cells expressing hCCR8 were seeded at a density of 12,000 cells / well in 100 μl of assay medium in a 96-well assay plate and incubated overnight at 37 °C and 5% CO2. After discarding the medium, 60 μl of serially diluted anti-CCR8 antibody was added and incubated for 1 hour. Then 40 μl of ADCC / NFAT-reporter gene-Jurkat cells (∼75,000 cells) were added. After incubation for 5 - 6 hours, 100 μl of luciferase substrate was added to each well, and the plate was gently shaken at room temperature for 15 minutes to 1 hour. Luminescence was measured on a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific). Each treatment was run in triplicate. Data analysis was performed using GraphPad Prism (version 9) to determine the EC50.

[0359] The results showed that the murine-human chimeric (human IgG1) antibodies 41E1C2A5, 504E12D8D12, and 531B9B1C9 had potent ADCC activity (Table 8).

[0360] Table 8

[0361] EC50 of the ADCC activity of chimeric anti-hCCR8 antibodies

[0362]

[0363] Example 5

[0364] Humanization of murine anti-hCCR8 antibodies

[0365] Humanization of murine anti-hCCR8 mAb 41E1C2A5

[0366] Murine anti-hCCR8 mAb 41E1C2A5 was humanized by CDR grafting and back mutation. The structure of the parental antibody was modeled by a computer-aided homology modeling program (MOE). Based on sequence similarity, the CDRs were grafted into the framework of the most closely related human germline. Human germline IGHV3-73*01 was selected for the heavy chain, and IGKV2-28*01 was selected for the light chain. Several residues in the heavy and light chain frameworks were mutated back to the corresponding residues in the murine antibody to preserve the antibody structure.

[0367] The heavy and light chains were designed and paired with each other to generate antibodies by transient expression for affinity ranking by flow cytometry. Briefly, 50 μl of CHO-K1 cells expressing hCCR8 (1×105 (Cells / well) were loaded onto 96-well plates. A 3-fold dilution series (11 points) of each antibody was prepared with a final starting concentration of 45 μg / ml. The antibodies were incubated with the cells at 4 °C for 1 hour. After thorough washing, the cells were incubated with Alexa Fluor 647-conjugated goat anti-human IgG (H+L) antibody. The geometric mean was measured by flow cytometry. A sigmoidal curve was generated using nonlinear regression curve fitting (4PL) in GraphPad Prism to yield the EC50. The humanized antibodies 41E1C2A5-HC3+LC4 (SEQ ID NO:86 and SEQ ID NO:87) and 41E1C2A5-HC4+LC2 (SEQ ID NO:88 and SEQ ID NO:89) had binding affinities comparable to those of the chimeric 41E1C2A5 mAb.

[0368] Humanization of murine anti-hCCR8 mAb 504E12D8D12

[0369] Murine anti-hCCR8 mAb 504E12D8D12 was humanized by transplanting the CDRs into the framework of the closest human germline IGHV3-73*01 (heavy chain) and IGKV2-18*01 (light chain). Back mutations were made in the framework sequences. In the light chain CDR1 of 504E12D8D12, there was a potential deamidation motif NG. Mutations N33Q and G34A were made to eliminate the potential deamidation propensity. Heavy and light chains were designed and paired to generate antibodies for affinity ranking. The humanized antibodies 504E12D8D12-HC1+LC1 (SEQ ID NO:90 and SEQ ID NO:91) and 504E12D8D12-HC1+LC1 (G34A) (SEQ ID NO:90 and SEQ ID NO:92) had binding affinities comparable to those of the chimeric 504E12D8D12 mAb.

[0370] Example 6

[0371] Humanized antibodies block calcium flux mediated by hCCR8

[0372] The humanized antibodies were tested by FLIPR calcium flux assay to evaluate their antagonist activity in blocking hCCL1-induced hCCR8-mediated calcium flux. Briefly, CHO-K1 cells (ProBio) expressing hCCR8 were seeded onto 384-well assay plates and incubated at 37 °C, 5% CO2 for 16 - 20 hours. After incubation, the plates were placed at room temperature. The dye loading solution (FLIPR Calcium 4 Assay Kit, Molecular Devices) was prepared with GPCR buffer and 20 μl was transferred to each well. Serial dilutions of the antibodies were prepared and 10 μl was added. After incubation at 37 °C, 5% CO2 for 1 hour, hCCL1 was added to each well such that the final concentration was equal to the EC80. The fluorescence signal was monitored using the FLIPR Tetra system. Anti-hCCR8 antibodies described in the literature (Reference Ab#1 and Reference Ab#2) were also tested in the assay. Data were recorded and analyzed using the ScreenWorks (version 3.1) program. A sigmoidal curve was generated using non-linear regression curve fitting (4PL) in GraphPad Prism to calculate the IC50.

[0373] The dose-response curves of the humanized antibodies 41E1C2A5-HC3+LC4 and 41E1C2A5-HC4+LC2 are shown in Figure 1 and the IC50 values are summarized in Table 9.

[0374] Table 9 Antagonist activity of humanized mAbs derived from 41E1C2A5 measured by calcium flux assay

[0375]

[0376] The dose-response curves of the humanized antibodies 504E12D8D12-HC1+LC1 and 504E12D8D12-HC1+LC1(G34A) are shown in Figure 2 and the IC50 values are summarized in Table 10. Compared with the reference antibodies, 504E12D8D12-HC1+LC1 and 504E12D8D12-HC1+LC1(G34A) have stronger or similar antagonist activity.

[0377] Table 10

[0378] Antagonist activity of humanized mAbs derived from 504E12D8D12 measured by calcium flux assay

[0379]

[0380] Example 7

[0381] The humanized antibody binds to hCCR8 with high affinity

[0382] The humanized antibodies 504E12D8D12-HC1+LC1 and 504E12D8D12-HC1+LC1(G34A) were tested by kinetic exclusion assay (KinExA, Sapidyne Instruments) to determine the binding affinity to hCCR8-overexpressing cells. Briefly, polystyrene particles (#442178, Sapidyne Instruments) were coated with goat anti-human IgG Fc-specific Fab fragments (#109-007-008, Jackson ImmunoResearch Lab) according to the manufacturer's protocol. The antibody was incubated overnight at room temperature with serial dilutions of HEK293 cells expressing hCCR8 by gentle rotation to reach equilibrium. The final antibody concentrations were adjusted to 0.05 nM and 1 nM, respectively. The supernatant was collected and run on a KinExA3200 (Sapidyne Instruments), and the antibody was captured by the antibody-coated polystyrene particles. The captured antibody was detected with an Alexa Fluor 647-conjugated goat anti-human IgG (H+L) antibody (#109-605-003, Jackson ImmunoResearch Lab). Data were recorded and analyzed using KinExA Pro software (version 4.3.20).

[0383] The humanized antibodies 504E12D8D12-HC1+LC1 and 504E12D8D12-HC1+LC1(G34A) were found to have high binding affinity for hCCR8 (Table 11).

[0384] Table 11 Dissociation constant of humanized antibody determined by KinExA

[0385]

[0386]

[0387] Example 8

[0388] Bispecific antibody targeting CCR8 and CTLA-4

[0389] Construct a bispecific antibody targeting CCR8 and CTLA-4

[0390] The heavy chain designated as FP578-HC (SEQ ID NO:93) was prepared by fusing a humanized anti-hCTLA-4 single domain antibody (sdAb) (amino acid residues 470-599 of SEQ ID NO:93) to the C-terminus of heavy chain 504E12D8D12-HCl (amino acid residues 1-454 of SEQ ID NO:93) via a linker (amino acid residues 455-469 of SEQ ID NO:93). FP578-HC was then paired with 504E12D8D12-LC1(G34A) (SEQ ID NO:92) to generate the bispecific antibody FP578-01. FP578-HC was also paired with 504E12D8D12-LC1 (SEQ ID NO:91) to generate the bispecific antibody FP578-02, respectively.

[0391] The bispecific antibody can bind CTLA-4 while binding CCR8

[0392] The bispecific antibodies FP578-01 and FP578-02, as well as the anti-hCCR8 antibodies 504E12D8D12-HC1+LC1(G34A) and 504E12D8D12-HC1+LC1, were incubated with HEK293 cells expressing hCCR8 at 4 °C for 1 hour. The cells were washed and then incubated with biotinylated recombinant human CTLA-4-Fc chimera (#786704, BioLegend) at 4 °C for 1 hour. After thoroughly washing the cells, CTLA-4 was detected by flow cytometry using phycoerythrin (PE)-conjugated streptavidin (#405203, BioLegend).

[0393] The results showed that both bispecific antibodies FP578-01 and FP578-02 could bind CTLA-4 while binding hCCR8 on HEK293 cells expressing hCCR8. Specific binding of the anti-hCCR8 antibody to CTLA-4 was not observed (Figure 3A - Figure 3D).

[0394] The bispecific antibody has a high binding affinity for hCCR8

[0395] The affinities of the bispecific antibodies FP578-01 and FP578-02 were determined by kinetic exclusion assay as described above. The data showed the K of the bispecific antibodies and the anti-CCR8 antibodies D were comparable (Table 12).

[0396] Table 12 Affinity of bispecific antibody determined by KinExA

[0397]

[0398] Bispecific antibodies block the binding of hCCL1 to hCCR8

[0399] As described above, the bispecific antibodies FP578-01 and FP578-02 were tested in a cell-based CCR8 CHO-K1 β-arrestin assay to determine antagonist activity. The data showed that FP578-01 and FP578-02 were as potent as the anti-hCCR8 antibody in blocking hCCR8 downstream signaling induced by hCCL1 (Table 13).

[0400] Table 13 Antagonist activity of bispecific antibody measured by β-arrestin assay

[0401]

[0402] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the invention. It will be apparent to those of skill in the art that all such variations and equivalents, whether now existing or later developed, are considered to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification indicate the level of skill of those of ordinary skill in the art to which the invention pertains. For all purposes, all patents, patent applications, and publications are hereby incorporated by reference in their entirety, and to the extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The invention illustratively described herein can be suitably practiced in the absence of any one or more of the elements not specifically disclosed herein. Accordingly, it is to be understood that, although the invention has been specifically disclosed by preferred embodiments and optional features, those of skill in the art may seek alterations and variations of the concepts disclosed herein, and such alterations and variations are considered to be within the scope of the invention as defined by the appended claims.

[0403] Sequence Listing

[0404] The nucleic acid and amino acid sequences listed in the appended sequence listing are shown using the standard letter abbreviations for nucleotide bases and the single-letter codes for amino acids as defined in 37 C.F.R. 1.822.

[0405] SEQ ID NO:1 is the amino acid sequence containing human CCR8.

[0406] SEQ ID NO:2 is the amino acid sequence containing human CCL1.

[0407] SEQ ID NO: 3, 7, and 9 are the amino acid sequences of the heavy chain CDR1 in monoclonal antibodies that specifically bind to CCR8.

[0408] SEQ ID NO: 4, 8, and 10 are the amino acid sequences of the heavy chain CDR2 in monoclonal antibodies that specifically bind to CCR8.

[0409] SEQ ID NO: 5, 6, and 11 are the amino acid sequences of the heavy chain CDR3 in monoclonal antibodies that specifically bind to CCR8.

[0410] SEQ ID NO: 12 and 15 are the amino acid sequences of the light chain CDR1 in monoclonal antibodies that specifically bind to CCR8.

[0411] SEQ ID NO: 13 and 16 are the amino acid sequences of the light chain CDR2 in monoclonal antibodies that specifically bind to CCR8.

[0412] SEQ ID NO: 14 and 17 are the amino acid sequences of the light chain CDR3 in monoclonal antibodies that specifically bind to CCR8.

[0413] SEQ ID NO: 18, 20, 22, and 24 are the amino acid sequences of the heavy chain variable regions in murine monoclonal antibodies that specifically bind to CCR8.

[0414] SEQ ID NO: 19, 21, 23, and 25 are the amino acid sequences of the light chain variable regions in murine monoclonal antibodies that specifically bind to CCR8.

[0415] SEQ ID NO: 26, 28, and 30 are the amino acid sequences of the heavy chains in murine-human chimeric antibodies that specifically bind to CCR8.

[0416] SEQ ID NO: 27, 29, and 31 are the amino acid sequences of the light chains in murine-human chimeric antibodies that specifically bind to CCR8.

[0417] SEQ ID NO: 32 is the amino acid sequence of the light chain constant region.

[0418] SEQ ID NO: 33 is the amino acid sequence of the light chain constant region.

[0419] SEQ ID NO: 34 is the amino acid sequence of the heavy chain constant region.

[0420] SEQ ID NO: 35 - 38 are the amino acid sequences of the heavy chain CDR2 in monoclonal antibodies that specifically bind to CCR8.

[0421] SEQ ID NO: 39 - 45 is the amino acid sequence of the heavy chain CDR3 in a monoclonal antibody that specifically binds to CCR8.

[0422] SEQ ID NO: 46 - 47 is the amino acid sequence of the light chain CDR1 in a monoclonal antibody that specifically binds to CCR8.

[0423] SEQ ID NO: 48 - 49 is the amino acid sequence of the light chain CDR2 in a monoclonal antibody that specifically binds to CCR8.

[0424] SEQ ID NO: 50 - 52 is the amino acid sequence of the light chain CDR3 in a monoclonal antibody that specifically binds to CCR8.

[0425] SEQ ID NO: 53 - 67 is the amino acid sequence of the heavy chain variable region in a murine monoclonal antibody that specifically binds to CCR8.

[0426] SEQ ID NO: 68 - 77 is the amino acid sequence of the light chain variable region in a murine monoclonal antibody that specifically binds to CCR8.

[0427] SEQ ID NO: 78, 80, 82 and 84 are the amino acid sequences of the heavy chains of a murine - human chimeric antibody that specifically binds to CCR8.

[0428] SEQ ID NO: 79, 81, 83 and 85 are the amino acid sequences of the light chains of a murine - human chimeric antibody that specifically binds to CCR8.

[0429] SEQ ID NO: 86, 88 and 90 are the amino acid sequences of the heavy chains of a humanized antibody that specifically binds to CCR8.

[0430] SEQ ID NO: 87, 89, 91 and 92 are the amino acid sequences of the light chains of a humanized antibody that specifically binds to CCR8.

[0431] SEQ ID NO: 93 is the amino acid sequence of the heavy chain of a CCR8 / CTLA4 bispecific antibody.

[0432] Sequence Listing

[0433] SEQ ID NO: 1 - CCR8 Amino Acid Sequence

[0434] MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVV

[0435] CKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMF

[0436] FITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGVLQC

[0437] YSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVI

[0438] ASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEKFK

[0439] KHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL

[0440] SEQ ID NO:2 - Amino acid sequence of CCL1

[0441] KSMQVPFSRCCFSFAEQEIPLRAILCYRNTSSICSNEGLIFKLKRGKEACALDTVGWVQRH

[0442] RKMLRHCPSKRK

[0443] SEQ ID NO:3 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR1

[0444] AYAMN

[0445] SEQ ID NO:4 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2

[0446] RIRSKSNNYATYYADSVKD

[0447] SEQ ID NO:5 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3

[0448] GGTYGSSSYFDY

[0449] SEQ ID NO:6 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3

[0450] GGTYGSTSYFDY

[0451] SEQ ID NO:7 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR1

[0452] TYAMN

[0453] SEQ ID NO:8 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2

[0454] RIRSKSNNYATYYADSVKA

[0455] SEQ ID NO:9 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR1

[0456] DYNMD

[0457] SEQ ID NO:10 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2

[0458] AINPNNGGTGYTQKFKG

[0459] SEQ ID NO:11 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3

[0460] RGVYMFAY

[0461] SEQ ID NO:12 - Amino acid sequence of mouse monoclonal antibody light chain CDR1

[0462] RSSKSLLHSNGNTYLY

[0463] SEQ ID NO:13 - Amino acid sequence of mouse monoclonal antibody light chain CDR2

[0464] RMSNLAS

[0465] SEQ ID NO:14 - Amino acid sequence of mouse monoclonal antibody light chain CDR3

[0466] MQHLEYPFT

[0467] SEQ ID NO:15 - Amino acid sequence of mouse monoclonal antibody light chain CDR1

[0468] KSSQSLLHSDGKTYLN

[0469] SEQ ID NO:16 - Amino acid sequence of mouse monoclonal antibody light chain CDR2

[0470] LVSKLDS

[0471] SEQ ID NO:17 - Amino acid sequence of mouse monoclonal antibody light chain CDR3

[0472] WQGTHFPYT

[0473] SEQ ID NO:18 - Amino Acid Sequence of the Heavy Chain Variable Region of a Murine Monoclonal Antibody

[0474] EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNYA

[0475] TYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYFCVRGGTYGSSSYFDYWGQGT

[0476] TLTVSS

[0477] SEQ ID NO:19 - Amino Acid Sequence of the Light Chain Variable Region of a Murine Monoclonal Antibody

[0478] DIVMTQAAPSVPVTPGESVSIPCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0479] GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLQIR

[0480] SEQ ID NO:20 - Amino Acid Sequence of the Heavy Chain Variable Region of a Murine Monoclonal Antibody

[0481] EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNYA

[0482] TYYADSVKDRFIISRDDSESMLYLQMNNLKTEDTAMYFCVRGGTYGSTSYFDYWGQGTT

[0483] LTVSS

[0484] SEQ ID NO:21 - Amino Acid Sequence of the Light Chain Variable Region of a Murine Monoclonal Antibody

[0485] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0486] GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK

[0487] SEQ ID NO:22 - Amino Acid Sequence of the Heavy Chain Variable Region of a Murine Monoclonal Antibody

[0488] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYA

[0489] TYYADSVKARFTISRDDSESMLYLQMNNLKTEDTAMYFCVRGGTYGSTSYFDYWGQGT

[0490] TLTVSS

[0491] SEQ ID NO:23 - Amino acid sequence of the light chain variable region of murine monoclonal antibody

[0492] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0493] GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK

[0494] SEQ ID NO:24 - Amino acid sequence of the heavy chain variable region of murine monoclonal antibody

[0495] EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGAINPNNGGTG

[0496] YTQKFKGKATLTVDKSSSTAFMELRSLTSEDSAVYYCARRGVYMFAYWGQGTLVTVSA

[0497] SEQ ID NO:25 - Amino acid sequence of the light chain variable region of murine monoclonal antibody

[0498] DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLIYLVSKLDS

[0499] GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK

[0500] SEQ ID NO:26 - Amino acid sequence of the heavy chain of murine - human chimeric antibody

[0501] EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNYA

[0502] TYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYFCVRGGTYGSSSYFDYWGQGT

[0503] TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

[0504] VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

[0505] LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

[0506] EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

[0507] PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0508] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0509] SEQ ID NO:27 - Amino acid sequence of the light chain of a murine - human chimeric antibody

[0510] DIVMTQAAPSVPVTPGESVSIPCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0511] GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLQIRRTVAAPSVFI

[0512] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0513] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0514] SEQ ID NO:28 - Amino acid sequence of the heavy chain of a murine - human chimeric antibody

[0515] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYA

[0516] TYYADSVKARFTISRDDSESMLYLQMNNLKTEDTAMYFCVRGGTYGSTSYFDYWGQGT

[0517] TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

[0518] VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

[0519] LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

[0520] EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

[0521] PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0522] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0523] SEQ ID NO:29 - Amino acid sequence of the light chain of a murine - human chimeric antibody

[0524] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0525] GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFI

[0526] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0527] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0528] SEQ ID NO:30 - Mouse - Human Chimeric Antibody Heavy Chain Amino Acid Sequence

[0529] EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGAINPNNGGTG

[0530] YTQKFKGKATLTVDKSSSTAFMELRSLTSEDSAVYYCARRGVYMFAYWGQGTLVTVSAA

[0531] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL

[0532] YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

[0533] FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST

[0534] YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT

[0535] KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

[0536] GNVFSCSVMHEALHNHYTQKSLSLSPGK

[0537] SEQ ID NO:31 - Mouse - Human Chimeric Antibody Light Chain Amino Acid Sequence

[0538] DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLIYLVSKLDS

[0539] GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKRTVAAPSVFI

[0540] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0541] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0542] SEQ ID NO:32 - Amino acid sequence of the light chain constant region

[0543] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

[0544] SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0545] SEQ ID NO:33 - Amino acid sequence of the light chain constant region

[0546] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ

[0547] SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0548] SEQ ID NO:34 - Amino acid sequence of the heavy chain constant region

[0549] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

[0550] LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

[0551] VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

[0552] TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

[0553] TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ

[0554] QGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0555] SEQ ID NO:35 - Mouse monoclonal antibody heavy chain CDR2 amino acid sequence

[0556] RIRTKSNNYATYYADSVKD

[0557] SEQ ID NO:36 - Mouse monoclonal antibody heavy chain CDR2 amino acid sequence

[0558] RIRTKSNNYATFYADSVKD

[0559] SEQ ID NO:37 - Mouse monoclonal antibody heavy chain CDR2 amino acid sequence

[0560] RIRTKSNNYATYYAASVKD

[0561] SEQ ID NO:38 - Mouse monoclonal antibody heavy chain CDR2 amino acid sequence

[0562] RIRSKSNNFATYYADSVKD

[0563] SEQ ID NO:39 - Mouse monoclonal antibody heavy chain CDR3 amino acid sequence

[0564] GGSGIKYVRYFDV

[0565] SEQ ID NO:40 - Mouse monoclonal antibody heavy chain CDR3 amino acid sequence

[0566] GGSGIRYVKYFDV

[0567] SEQ ID NO:41 - Mouse monoclonal antibody heavy chain CDR3 amino acid sequence

[0568] GGSGISYVRYFDV

[0569] SEQ ID NO:42 - Mouse monoclonal antibody heavy chain CDR3 amino acid sequence

[0570] GGSGLNYVRYFDV

[0571] SEQ ID NO:43 - Mouse monoclonal antibody heavy chain CDR3 amino acid sequence

[0572] GGSGLRYVRYFDV

[0573] SEQ ID NO: 44 - Amino acid sequence of the CDR3 of the heavy chain of a murine monoclonal antibody

[0574] QTYGSRDYAMDY

[0575] SEQ ID NO: 45 - Amino acid sequence of the CDR3 of the heavy chain of a murine monoclonal antibody

[0576] GGSGIRYVRYFDV

[0577] SEQ ID NO: 46 - Amino acid sequence of the CDR1 of the light chain of a murine monoclonal antibody

[0578] RSSQSLVHSNGNTYLH

[0579] SEQ ID NO: 47 - Amino acid sequence of the CDR1 of the light chain of a murine monoclonal antibody

[0580] RSSKSLQHSNGNIYLY

[0581] SEQ ID NO: 48 - Amino acid sequence of the CDR2 of the light chain of a murine monoclonal antibody

[0582] KVSNRFS

[0583] SEQ ID NO: 49 - Amino acid sequence of the CDR2 of the light chain of a murine monoclonal antibody

[0584] RMSDLAS

[0585] SEQ ID NO: 50 - Amino acid sequence of the CDR3 of the light chain of a murine monoclonal antibody

[0586] CQSTHVPPYT

[0587] SEQ ID NO: 51 - Amino acid sequence of the CDR3 of the heavy chain of a murine monoclonal antibody

[0588] SQSTHVPPYT

[0589] SEQ ID NO: 52 - Amino acid sequence of the CDR3 of the light chain of a murine monoclonal antibody SQNTHVPPYT

[0590] SEQ ID NO: 53 - Amino acid sequence of the variable region of the heavy chain of a murine monoclonal antibody

[0591] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0592] TYYADSVKDRFTISRDDSENILYLQMNNLKTEDTAMYYCVRGGSGIKYVRYFDVWGTGT

[0593] TVTVSS

[0594] SEQ ID NO:54 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0595] EVQLVESGGGLVQPRGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0596] TYYADSVKDRFTISRDDSESMLYLQMINLKTEDTAMYYCVRGGSGIRYVKYFDVWGTGT

[0597] TVTVSS

[0598] SEQ ID NO:55 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0599] EVQLVESGGGLVQPGGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0600] TYYADSVKDRFTISRDDSESMLYLQMINLKTEDTAMYYCVRGGSGIRYVKYFDVWGTGT

[0601] TVTVSS

[0602] SEQ ID NO:56 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0603] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYA

[0604] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRGGSGISYVRYFDVWGTGT

[0605] TVTVSS

[0606] SEQ ID NO:57 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0607] EVQLVESGGGLVQPKGSLKLSCAASGFSFKTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0608] TYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDVWGTG

[0609] TTVTVSS

[0610] SEQ ID NO:58 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0611] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0612] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDVWGTG

[0613] TTVTVSS

[0614] SEQ ID NO:59 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0615] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0616] TYYADSVKDRFTISRDDSENMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDVWGTG

[0617] TTVTVSS

[0618] SEQ ID NO:60 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0619] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLDWVARIRSKSNNYA

[0620] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYFCVRQTYGSRDYAMDYWGQGT

[0621] SVTVSS

[0622] SEQ ID NO:61 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0623] EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLDWVARIRSKSNNYA

[0624] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYFCVRQTYGSRDYAMDYWGQGT

[0625] SVTVSS

[0626] SEQ ID NO:62 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0627] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYA

[0628] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRGGSGIRYVRYFDVWGTG

[0629] TTVTVSS

[0630] SEQ ID NO:63 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0631] EVQLVESGGGLVQPRGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNFA

[0632] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRQTYGSRDYAMDYWGQG

[0633] TSVTVSS

[0634] SEQ ID NO:64 - Amino acid sequence of the heavy chain variable region of a murine monoclonal antibody

[0635] EVQLVESGGGLVQPKGSLKLSCAASGFSFKTYAMNWVRQAPGEGLEWVARIRTKSNNYA

[0636] TYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDVWGPG

[0637] TTVTVSS

[0638] SEQ ID NO: 65 - Amino Acid Sequence of the Heavy Chain Variable Region of a Murine Monoclonal Antibody

[0639] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0640] TFYADSVKDRFTISRHDSESMLYLQMNNLKTEDTAMYYCVRGGSGIRYVRYFDVWGTGT

[0641] TVTVSS

[0642] SEQ ID NO: 66 - Amino Acid Sequence of the Heavy Chain Variable Region of a Murine Monoclonal Antibody

[0643] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0644] TYYAASVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDVWGTG

[0645] TTVTVSS

[0646] SEQ ID NO: 67 - Amino Acid Sequence of the Heavy Chain Variable Region of a Murine Monoclonal Antibody

[0647] EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNFA

[0648] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRQTYGSRDYAMDYWGQG

[0649] TSVTVSS

[0650] SEQ ID NO: 68 - Amino Acid Sequence of the Light Chain Variable Region of a Murine Monoclonal Antibody

[0651] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0652] GVPERFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK

[0653] SEQ ID NO:69 - Amino Acid Sequence of the Light Chain Variable Region of a Murine Monoclonal Antibody

[0654] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0655] GVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK

[0656] SEQ ID NO:70 - Amino Acid Sequence of the Light Chain Variable Region of a Murine Monoclonal Antibody

[0657] DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0658] GVPERFSGSGSGSAFTLRVSRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK

[0659] SEQ ID NO:71 - Amino Acid Sequence of the Light Chain Variable Region of a Murine Monoclonal Antibody

[0660] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRF

[0661] SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCCQSTHVPPYTFGGGTKLEIK

[0662] SEQ ID NO:72 - Amino Acid Sequence of the Light Chain Variable Region of a Murine Monoclonal Antibody

[0663] DIVMTQAAPSVPVTPGESVSISCRSSKSLQHSNGNIYLYWFLQRPGQSPQLLIYRMSNLAS

[0664] GVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK

[0665] SEQ ID NO:73 - Amino Acid Sequence of the Light Chain Variable Region of a Murine Monoclonal Antibody

[0666] DVVMTQTPLSLPVSLGDRASISCRSSQSLVHSNGNTYLHWYLQKPGQSPRLLIYKVSNRF

[0667] SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPYTFGGGTKLEIK

[0668] SEQ ID NO:74 - Amino acid sequence of the light chain variable region of a murine monoclonal antibody

[0669] DIVMTQAAPSVLVTPGESVSFSCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIFRMSNLAS

[0670] GVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK

[0671] SEQ ID NO:75 - Amino acid sequence of the light chain variable region of a murine monoclonal antibody

[0672] DIVMTQAAPSVTVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSDLAS

[0673] GVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK

[0674] SEQ ID NO:76 - Amino acid sequence of the light chain variable region of a murine monoclonal antibody

[0675] DIVMTQAAPSVFVIPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0676] GVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK

[0677] SEQ ID NO:77 - Amino acid sequence of the light chain variable region of a murine monoclonal antibody

[0678] DVVMTQTPLSLPVSLGDRASISCRSSQSLVHSNGNTYLHWYLQKPGQSPRLLIYKVSNRF

[0679] SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPYTFGGGTKLEIK

[0680] SEQ ID NO: 78 - Amino Acid Sequence of the Heavy Chain of a Mouse - Human Chimeric Antibody

[0681] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0682] TYYAASVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDVWGTG

[0683] TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

[0684] AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0685] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0686] REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0687] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0688] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0689] SEQ ID NO: 79 - Amino Acid Sequence of the Light Chain of a Mouse - Human Chimeric Antibody

[0690] DIVMTQAAPSVFVIPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0691] GVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRTVAAPSVFIF

[0692] PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0693] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0694] SEQ ID NO:80 - Amino acid sequence of the heavy chain of a murine - human chimeric antibody

[0695] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0696] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDVWGTG

[0697] TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

[0698] AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0699] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0700] REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0701] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0702] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0703] SEQ ID NO:81 - Amino acid sequence of the light chain of a murine - human chimeric antibody

[0704] DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0705] GVPERFSGSGSGSAFTLRVSRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRTVAAPSVFI

[0706] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0707] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0708] SEQ ID NO:82 - Amino acid sequence of the heavy chain of the murine - human chimeric antibody

[0709] EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYA

[0710] TYYADSVKDRFTISRDDSENMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDVWGTG

[0711] TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

[0712] AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0713] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0714] REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0715] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0716] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0717] SEQ ID NO:83 - Amino acid sequence of the light chain of the murine - human chimeric antibody

[0718] DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS

[0719] GVPERFSGSGSGSAFTLRVSRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRTVAAPSVFI

[0720] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0721] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0722] SEQ ID NO:84 - Amino acid sequence of the heavy chain of a murine - human chimeric antibody

[0723] EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLDWVARIRSKSNNYA

[0724] TYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYFCVRQTYGSRDYAMDYWGQGT

[0725] SVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

[0726] VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

[0727] LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

[0728] EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

[0729] PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0730] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0731] SEQ ID NO:85 - Amino acid sequence of the light chain of a murine - human chimeric antibody

[0732] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRF

[0733] SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCCQSTHVPPYTFGGGTKLEIKRTVAAPSV

[0734] FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0735] SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0736] SEQ ID NO:86 - Amino acid sequence of the heavy chain of a humanized antibody

[0737] EVQLVESGGGLVQPGGSLKLSCAASGFSFNAYAMNWVRQASGKGLEWVARIRSKSNNYA

[0738] TYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYFCVRGGTYGSSSYFDYWGQGTT

[0739] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0740] LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

[0741] LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0742] EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

[0743] SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

[0744] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0745] SEQ ID NO:87 - Amino acid sequence of the light chain of a humanized antibody

[0746] DIVMTQSPLSLPVTPGEPASIPCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASG

[0747] VPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFIFP

[0748] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0749] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0750] SEQ ID NO:88 - Amino acid sequence of the heavy chain of a humanized antibody

[0751] EVQLVESGGGLVQPGGSLKLSCAASGFSFNAYAMNWVRQASGKGLEWVARIRSKSNNYA

[0752] TYYADSVKDRFTISRDDSENTAYLQMNSLKTEDTAVYFCVRGGTYGSSSYFDYWGQGTT

[0753] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0754] LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

[0755] LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0756] EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

[0757] SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

[0758] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0759] SEQ ID NO:89 - Amino acid sequence of the light chain of the humanized antibody

[0760] DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASG

[0761] VPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFIFP

[0762] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0763] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0764] SEQ ID NO:90 - Amino acid sequence of the heavy chain of the humanized antibody

[0765] EVQLVESGGGLVQPGGSLKLSCAASGFSFNTYAMNWVRQASGKGLEWVGRIRTKSNNYA

[0766] TYYAASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSGLNYVRYFDVWGQG

[0767] TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

[0768] AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0769] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0770] REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0771] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0772] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0773] SEQ ID NO:91 - Amino acid sequence of the light chain of a humanized antibody

[0774] DIVMTQTPPSLPVNPGEPASISCRSSKSLLHSNGNTYLYWYLQKPGQSPQLLIYRMSNLAS

[0775] GVPDRFSGSGSGSDFTLKISWVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFI

[0776] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0777] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0778] SEQ ID NO:92 - Amino acid sequence of the light chain of a humanized antibody

[0779] DIVMTQTPPSLPVNPGEPASISCRSSKSLLHSNANTYLYWYLQKPGQSPQLLIYRMSNLAS

[0780] GVPDRFSGSGSGSDFTLKISWVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFI

[0781] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0782] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0783] SEQ ID NO:93 - Amino acid sequence of the heavy chain of the CCR8 / CTLA4 bispecific antibody

[0784] EVQLVESGGGLVQPGGSLKLSCAASGFSFNTYAMNWVRQASGKGLEWVGRIRTKSNNYA

[0785] TYYAASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSGLNYVRYFDVWGQG

[0786] TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

[0787] AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0788] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0789] REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0790] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0791] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEVQLVES

[0792] GGGLVQPGGSLRLSCAASGYTYSRHCLGWFRQAPGKGREAVSTIDSDGSTSYADSVKGR

[0793] FTISRDNAKNTLYLQMNSLRPEDTAVYYCAIGPNPRYCSGAPNTRGAEHYFGYWGQGTL

[0794] VTVSS。

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to human CCR8 and comprises: (a) a heavy-chain CDR1 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 3, 7, and 9; (b) a heavy-chain CDR2 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 4, 8, 10, and 35 - 38; (c) a heavy-chain CDR3 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 5, 6, 11, and 39 - 45; (d) a light-chain CDR1 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 12, 15, and 46 - 47; (e) a light-chain CDR2 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 13, 16, and 48 - 49; and (f) a light-chain CDR3 sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 14, 17, and 50 - 52.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) the heavy chain CDR1 sequence of SEQ ID NO: 3; the heavy chain CDR2 sequence of SEQ ID NO: 4; the heavy chain CDR3 sequence of SEQ ID NO: 5; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 13; and the light chain CDR3 sequence of SEQ ID NO: 14; or (2) the heavy chain CDR1 sequence of SEQ ID NO: 3; the heavy chain CDR2 sequence of SEQ ID NO: 4; the heavy chain CDR3 sequence of SEQ ID NO: 6; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 13; and the light chain CDR3 sequence of SEQ ID NO: 14; or (3) the heavy chain CDR1 sequence of SEQ ID NO: 7; the heavy chain CDR2 sequence of SEQ ID NO: 8; the heavy chain CDR3 sequence of SEQ ID NO: 6; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 13; and the light chain CDR3 sequence of SEQ ID NO: 14; or (4) the heavy chain CDR1 sequence of SEQ ID NO: 9; the heavy chain CDR2 sequence of SEQ ID NO: 10; the heavy chain CDR3 sequence of SEQ ID NO: 11; the light chain CDR1 sequence of SEQ ID NO: 15; the light chain CDR2 sequence of SEQ ID NO: 16; and the light chain CDR3 sequence of SEQ ID NO: 17; or (5) the heavy chain CDR1 sequence of SEQ ID NO: 7; the heavy chain CDR2 sequence of SEQ ID NO: 35; the heavy chain CDR3 sequence of SEQ ID NO: 39; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 13; and the light chain CDR3 sequence of SEQ ID NO: 14; or (6) the heavy chain CDR1 sequence of SEQ ID NO: 3; the heavy chain CDR2 sequence of SEQ ID NO: 35; the heavy chain CDR3 sequence of SEQ ID NO: 40; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 13; and the light chain CDR3 sequence of SEQ ID NO: 14; or (7) the heavy chain CDR1 sequence of SEQ ID NO: 3; the heavy chain CDR2 sequence of SEQ ID NO: 35; the heavy chain CDR3 sequence of SEQ ID NO: 40; the light chain CDR1 sequence of SEQ ID NO: 12; the light chain CDR2 sequence of SEQ ID NO: 13; and the light chain CDR3 sequence of SEQ ID NO: 14; or (8) the heavy chain CDR1 sequence of SEQ ID NO: 7;The heavy chain CDR2 sequence of SEQ ID NO:4; the heavy chain CDR3 sequence of SEQ ID NO:41; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (9) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:35; the heavy chain CDR3 sequence of SEQ ID NO:42; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (10) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:35; the heavy chain CDR3 sequence of SEQ ID NO:43; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (11) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:35; the heavy chain CDR3 sequence of SEQ ID NO:43; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (12) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:4; the heavy chain CDR3 sequence of SEQ ID NO:44; the light chain CDR1 sequence of SEQ ID NO:46; the light chain CDR2 sequence of SEQ ID NO:48; and the light chain CDR3 sequence of SEQ ID NO:50; or (13) the heavy chain CDR1 sequence of SEQ ID NO:3; the heavy chain CDR2 sequence of SEQ ID NO:4; the heavy chain CDR3 sequence of SEQ ID NO:44; the light chain CDR1 sequence of SEQ ID NO:46; the light chain CDR2 sequence of SEQ ID NO:48; and the light chain CDR3 sequence of SEQ ID NO:50; or (14) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:4; the heavy chain CDR3 sequence of SEQ ID NO:45; the light chain CDR1 sequence of SEQ ID NO:47; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (15) the heavy chain CDR1 sequence of SEQ ID NO:3; the heavy chain CDR2 sequence of SEQ ID NO:38; the heavy chain CDR3 sequence of SEQ ID NO:44;The light chain CDR1 sequence of SEQ ID NO:46; the light chain CDR2 sequence of SEQ ID NO:48; and the light chain CDR3 sequence of SEQ ID NO:51; or (16) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:35; the heavy chain CDR3 sequence of SEQ ID NO:42; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (17) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:36; the heavy chain CDR3 sequence of SEQ ID NO:45; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:49; and the light chain CDR3 sequence of SEQ ID NO:14; or (18) the heavy chain CDR1 sequence of SEQ ID NO:7; the heavy chain CDR2 sequence of SEQ ID NO:37; the heavy chain CDR3 sequence of SEQ ID NO:42; the light chain CDR1 sequence of SEQ ID NO:12; the light chain CDR2 sequence of SEQ ID NO:13; and the light chain CDR3 sequence of SEQ ID NO:14; or (19) the heavy chain CDR1 sequence of SEQ ID NO:3; the heavy chain CDR2 sequence of SEQ ID NO:38; the heavy chain CDR3 sequence of SEQ ID NO:44; the light chain CDR1 sequence of SEQ ID NO:46; the light chain CDR2 sequence of SEQ ID NO:48; and the light chain CDR3 sequence of SEQ ID NO:52.; 3. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 - 2, wherein the antibody or antigen-binding fragment thereof is selected from human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, diabodies, triabodies, tetra-bodies, Fab fragments, Fab' fragments, Fab2 fragments, F(ab)'2 fragments, domain antibodies, non-fucosylated antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, IgG1 antibodies having at least one mutation that enhances ADCC / FcR affinity, or IgG4 antibodies having at least one mutation in the hinge region that reduces the tendency to form intra-H chain disulfide bonds.

4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the isolated antibody or antigen-binding fragment thereof has a density of at least about 1×10 -6 M, at least about 1×10 -7 M, at least about 1×10 -8 M, at least about 1×10 -9 M, at least about 1×10 -10 M, at least about 1×10 -11 M, or at least about 1×10 -12 The dissociation constant (K D ) binds to the CCR8 protein.

5. An isolated antibody or antigen-binding fragment thereof, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to human CCR8 comprising: a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 22 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 25; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 68; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 68; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 68; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 69; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 69; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 70; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 70; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 60 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 71; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 61 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 71; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 62 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 72; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 63 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 73;or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 64 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 74; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 75; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 66 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 76; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 67 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:

77.

6. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 - 4, further comprising a set of four variable region framework regions from human immunoglobulin (IgG).

7. An isolated chimeric antibody or antigen-binding fragment thereof, wherein the isolated chimeric antibody or antigen-binding fragment thereof specifically binds to human CCR8 and comprises: (1) a heavy-chain sequence of SEQ ID NO: 26 and a light-chain sequence of SEQ ID NO: 27; or (2) a heavy-chain sequence of SEQ ID NO: 28 and a light-chain sequence of SEQ ID NO: 29; or (3) a heavy-chain sequence of SEQ ID NO: 30 and a light-chain sequence of SEQ ID NO: 31; or (4) a heavy-chain sequence of SEQ ID NO: 78 and a light-chain sequence of SEQ ID NO: 79; or (5) a heavy-chain sequence of SEQ ID NO: 80 and a light-chain sequence of SEQ ID NO: 81; or (6) a heavy-chain sequence of SEQ ID NO: 82 and a light-chain sequence of SEQ ID NO: 83; or (7) a heavy-chain sequence of SEQ ID NO: 84 and a light-chain sequence of SEQ ID NO:

85.

8. An isolated humanized antibody or antigen-binding fragment thereof, wherein the isolated humanized antibody or antigen-binding fragment thereof specifically binds to human CCR8 and comprises a heavy chain sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 86, 88, and 90; and a light chain sequence selected from the group consisting of the amino acid sequences defined by SEQ ID NO: 87, 89, and 91-92.

9. The isolated humanized antibody or antigen-binding fragment thereof according to claim 8, comprising the heavy chain sequence of SEQ ID NO: 86 and the light chain sequence of SEQ ID NO:

87.

10. The isolated humanized antibody or antigen-binding fragment thereof according to claim 8, comprising the heavy chain sequence of SEQ ID NO: 88 and the light chain sequence of SEQ ID NO:

89.

11. The isolated humanized antibody or antigen-binding fragment thereof according to claim 8, comprising the heavy chain sequence of SEQ ID NO: 90 and the light chain sequence of SEQ ID NO:

91.

12. The isolated humanized antibody or antigen-binding fragment thereof according to claim 8, comprising the heavy chain sequence of SEQ ID NO: 90 and the light chain sequence of SEQ ID NO:

92.

13. A pharmaceutical composition, the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-12 mixed with a pharmaceutically acceptable carrier.

14. An isolated immunoconjugate, the isolated immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-12 conjugated to an effector molecule.

15. The isolated immunoconjugate according to claim 14, wherein the effector molecule is selected from the group consisting of immunotoxins, cytokines, chemokines, therapeutic agents, and chemotherapeutic agents.

16. A pharmaceutical composition, the pharmaceutical composition comprising the isolated immunoconjugate according to any one of claims 14 to 15 mixed with a pharmaceutically acceptable carrier.

17. An antibody-drug conjugate (ADC), the antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-12 conjugated to a second molecule selected from the group consisting of cytotoxic agents, anti-cancer drugs, and immunosuppressive drugs.

18. A pharmaceutical composition, the pharmaceutical composition comprising the ADC according to claim 17 mixed with a pharmaceutically acceptable carrier.

19. A bispecific antibody, the bispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-12 conjugated to a second functional molecule to produce a bispecific antibody that binds to at least two different binding sites or target molecules.

20. The bispecific antibody according to claim 19, comprising the heavy chain of SEQ ID NO: 93 and the light chain of SEQ ID NO:

92.

21. The bispecific antibody according to claim 19, comprising the heavy chain of SEQ ID NO: 93 and the light chain of SEQ ID NO:

91.

22. A pharmaceutical composition, the pharmaceutical composition comprising a bispecific antibody according to any one of claims 19 - 21, admixed with a pharmaceutically acceptable carrier.

23. A method of treating a subject suffering from a CCR8 - related disorder, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 13, 16, 18, and 22.

24. A method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 13, 16, 18, and 22.

25. The method according to claim 24, wherein the cancer is selected from the group consisting of ovarian cancer, lung cancer, breast cancer, gastric cancer, prostate cancer, colorectal cancer, renal cell carcinoma, liver cancer, pancreatic cancer, glioblastoma, melanoma, and sarcoma.

26. The method according to any one of claims 24 - 25, wherein the subject is selected from subjects with recurrent cancer and subjects with resistant or refractory cancer.

27. A method for treating a subject suffering from cancer, the method comprising: a) administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 13, 16, 18, and 22; and b) one or more additional therapies selected from the group consisting of immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, vaccination regimens, and stem cell transplantation, wherein the combination therapy provides increased cell killing of tumor cells.

28. The method according to claim 27, wherein the cancer is selected from the group consisting of ovarian cancer, lung cancer, breast cancer, gastric cancer, prostate cancer, colorectal cancer, renal cell carcinoma, liver cancer, pancreatic cancer, glioblastoma, melanoma, and sarcoma.

29. The method according to any one of claims 27 - 28, wherein the subject is selected from subjects with recurrent cancer and subjects with resistant or refractory cancer.

Images