CC chemokine receptor type 8 (CCR8) antagonist antibody

Abstract

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

Description

[Technical Field]

[0001] Related patent applications This application claims the benefit of U.S. Provisional Application No. 63 / 412,465, filed October 2, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The contents of the electronic sequence listing (SeqListing-REMD CCR8.xml; size: 91 kb; created on: September 30, 2023) are incorporated herein by reference in their entirety.

[0003] Two major populations of CD4+ regulatory T cells (Treg cells), defined by whether they express the forkhead box protein 3 (Foxp3) transcription factor, are thought to play a key role in maintaining self-tolerance (Barsheshea et al., PNAS, 114(23):6086-6091, 2017). Both Foxp3+ and Foxp3- subtypes participate in regulating inflammatory autoimmunity and maintaining self-tolerance through various mechanisms, including regulating the biological functions of effector TH1 and TH17 CD4+ T cells (ibid.). The mechanisms utilized by Tregs to promote self-tolerance may be employed in the tumor microenvironment to suppress anti-tumor immune responses. Indeed, systemic depletion of Tregs in mice is sufficient to enable immune-mediated tumor regression (Teng et al., Cancer Research 70(20):7800-9, 2010). As such, Tregs are thought to play a role in mediating peripheral tolerance to self-antigens, preventing autoimmune disease, and suppressing anti-tumor immune responses.

[0004] Tregs are frequently found in tumor tissues of various 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 tumor, head and neck squamous cell carcinoma (HNSCC), and melanoma. Their high frequency among CD4+ T cells within tumor-infiltrating lymphocytes (TILs) or a high ratio of Foxp3+ Tregs to CD8+ cells is associated with poor prognosis in most solid tumors (for review, see Tanaka, A. and Sakaguchi, S., Cell Res., 27:109-118, 2017).

[0005] Chemokines (chemoattractant cytokines) comprise a family of structurally and functionally related polypeptides of 8–10 kilodaltons. Chemokines are involved in diverse biological functions, including regulating immune cell proliferation, migration, activation, differentiation, and homing. The biological activity of chemokines is mediated by a family of seven-transmembrane G protein-coupled receptors (GPCRs). CCR4, CCR8, CCR10, and CXCR3 are chemokine receptors responsible for Treg cell migration into the tumor microenvironment (TME) in response to CC and CXC chemokines: 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.

[0006] CCR8 (previously known as CY6, CKR-L1, or TER1) is a chemokine receptor recently identified as a potential specific marker for tumor-infiltrating Tregs, and CCR8 expression is selectively upregulated in these Tregs in multiple cancers, including breast, colorectal, and lung (Wang L, et al., Nature Immunol 20:1220-30, 2019). + Tregs represent a highly activated and suppressive subpopulation of Tregs, as well as CCR8 in these tumor types. +High abundance of Tregs is associated with poor prognosis.

[0007] Tumor immunotherapy is based on the concept that the immune system has the ability to recognize tumors and eliminate malignant cells. Immunotherapy using agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints) is an area that has undergone 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 others (Sharpe et al., Nat Immunol, 8:239-45, 2007). Under normal physiological conditions, immune checkpoints are crucial for maintaining self-tolerance (i.e., preventing autoimmunity) and protecting tissues from damage when the immune system responds to pathogenic infections. It is also clear that tumors co-opt certain immune checkpoint pathways as a primary mechanism of immune resistance, particularly against tumor antigen-specific T cells (Pardoll DM., Nat Rev Cancer, 12:252-64, 2012). Inhibition of PD-1 interaction with its primary ligand PD-L1 mediates potent antitumor activity in preclinical models (U.S. Pat. Nos. 8,008,449 and 7,943,743), and the use of mAb inhibitors of the PD-1 / PD-L1 interaction to treat cancer has become the standard of care for many types of cancer (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 intratumoral myeloid cells in many mouse and human cancers, including human lung, ovarian, and colon cancers and various myelomas, and anti-PD-1 and anti-PD-L1 antibodies developed by, for example, Bristol-Myers Squibb (nivolumab), Merck (pembrolizumab), Regeneron (cemiplimab), Roche (atezolizumab), and AstraZeneca (durvalumab) have been approved by the FDA to treat a variety of cancer conditions. The tolerability of PD-1 pathway blockers and their unique mechanisms of action make them ideal scaffolds for the development of combination regimens. Recent clinical data on combined CTLA-4 and PD-1 blockade in melanoma patients demonstrated an increased rate of objective tumor responses compared with blockade of either checkpoint alone, supporting the concept that combined checkpoint blockade may result in 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 positive observed rates of clinical responses, many patients with advanced solid tumors are resistant or become refractory to immunotherapy (Rizvi et al., Cancer immunology., Science, 348(6230):124-128, 2015). Summary of the Invention

[0008] The present invention provides isolated antibodies and antigen-binding fragments thereof that specifically bind to CC chemokine receptor type 8 (CCR8), as well as methods for treating cancer in subjects, comprising administering anti-CCR8 Abs to the subject as monotherapy or in combination with anticancer agents, such as immune checkpoint inhibitors.The inventors propose that the anti-CCR8 mAbs provided herein can be used as safe and effective tumor-infiltrating Treg antagonizing and depleting agents that also preserve effector T cells (Teff) for optimal anti-tumor responses.Because only tumor-infiltrating Tregs express high levels of CCR8, which is required for ADCC-mediated depletion, CCR8 mAbs provide a safer alternative to other Treg depletion strategies, sparing Tregs in peripheral tissues that function to maintain immune homeostasis. Depletion of Tregs via anti-CTLA-4, anti-CD25, and other Treg surface markers can result in the depletion of both tumor and peripheral Tregs, which can lead to severe autoimmune side effects, as well as the potential depletion of beneficial anti-tumor conventional T cells expressing shared target antigens.

[0009] In various embodiments, the antibody or antigen-binding fragment is selected from a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a single-chain antibody, a diabody, a triabody, a tetrabody, a Fab fragment, a Fab' fragment, a Fab2 fragment, a F(ab)'2 fragment, a domain antibody, a non-fucosylated antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgG1 antibody with at least one mutation that enhances ADCC / FcR affinity, or an IgG4 antibody with at least one mutation in the hinge region that reduces the tendency to form intra-H 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, an isolated antibody having high affinity for human CCR8 of SEQ ID NO: 1, and an antigen-binding fragment thereof, are provided.

[0010] In various embodiments, the antibody or antigen-binding fragment is at least about 1 x 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 Dissociation constant of M (K D ) binds to the CCR8 protein.

[0011] In various embodiments, the isolated humanized or human monoclonal antibody, or antigen-binding fragment thereof, of the present invention binds to human CCR8 and comprises: (a) a heavy chain CDR1 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 3, 7, and 9; (b) a heavy chain CDR2 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 4, 8, 10, and 35-38; (c) a heavy chain CDR3 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 5, 6, 11, and 39-45; (d) a light chain CDR1 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 12, 15, and 46-47; (e) a light chain CDR2 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 13, 16, and 48-49; and (f) a light chain CDR3 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 14, 17, and 50-52.

[0012] In various embodiments, the isolated humanized or human monoclonal antibody or antigen-binding fragment thereof of the present invention binds to human CCR8 and is selected from the group consisting of (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 sequence number 35; the heavy chain CDR3 sequence of sequence number 39; the light chain CDR1 sequence of sequence number 12; the light chain CDR2 sequence of sequence number 13; and the light chain CDR3 sequence of sequence number 14; or (6) the heavy chain CDR1 sequence of sequence number 3; the heavy chain CDR2 sequence of sequence number 35; the heavy chain CDR3 sequence of sequence number 40; the light chain CDR1 sequence of sequence number 12; the light chain CDR2 sequence of sequence number 13; and the light chain CDR3 sequence of sequence number 14; or (7) the heavy chain CDR1 sequence of sequence number 3; the heavy chain CDR2 sequence of sequence number 35; the heavy chain CDR3 sequence of sequence number 40; the light chain CDR2 sequence of sequence number 12 CDR1 sequence; light chain CDR2 sequence of SEQ ID NO: 13; and light chain CDR3 sequence of SEQ ID NO: 14; or (8) heavy chain CDR1 sequence of SEQ ID NO: 7; heavy chain CDR2 sequence of SEQ ID NO: 4; heavy chain CDR3 sequence of SEQ ID NO: 41; light chain CDR1 sequence of SEQ ID NO: 12; light chain CDR2 sequence of SEQ ID NO: 13; and light chain CDR3 sequence of SEQ ID NO: 14; or (9) heavy chain CDR1 sequence of SEQ ID NO: 7; heavy chain CDR2 sequence of SEQ ID NO: 35; heavy chain CDR3 sequence of SEQ ID NO: 42; light chain CDR1 sequence of SEQ ID NO: 12; light chain CDR2 sequence of SEQ ID NO: 13; and 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 sequence a heavy chain CDR3 sequence of SEQ ID NO: 44; a light chain CDR1 sequence of SEQ ID NO: 46; a light chain CDR2 sequence of SEQ ID NO: 48; and a light chain CDR3 sequence of SEQ ID NO: 50; or (13) 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: 44; a light chain CDR1 sequence of SEQ ID NO: 46; a light chain CDR2 sequence of SEQ ID NO: 48; and a light chain CDR3 sequence of SEQ ID NO: 50; or (14) a heavy chain CDR1 sequence of SEQ ID NO: 7; a heavy chain CDR2 sequence of SEQ ID NO: 4; a heavy chain CDR3 sequence of SEQ ID NO: 45; a light chain CDR1 sequence of SEQ ID NO: 47; a light chain CDR1 sequence of SEQ ID NO: 13 R2 sequence; 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 CDR1 sequence; heavy chain CDR2 sequence of SEQ ID NO: 36; heavy chain CDR3 sequence of SEQ ID NO: 45; light chain CDR1 sequence of SEQ ID NO: 12; light chain CDR2 sequence of SEQ ID NO: 49; and light chain CDR3 sequence of SEQ ID NO: 14; or (18) heavy chain CDR1 sequence of SEQ ID NO: 7; heavy chain CDR2 sequence of SEQ ID NO: 37; heavy chain CDR3 sequence of SEQ ID NO: 42; light chain CDR1 sequence of SEQ ID NO: 12; light chain CDR2 sequence of SEQ ID NO: 13; and light chain CDR3 sequence of SEQ ID NO: 14; or (19) heavy chain CDR1 sequence of SEQ ID NO: 3; heavy chain CDR2 sequence of SEQ ID NO: 38; 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.

[0013] In various embodiments, the isolated antibody or antigen-binding fragment thereof of the present invention binds to human CCR8 and comprises (a) a heavy chain CDR1 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 3, 7, and 9; (b) a heavy chain CDR2 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 4, 8, 10, and 35-38; (c) a heavy chain CDR3 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 5, 6, 11, and 39-45; (d) a light chain CDR1 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 12, 15, and 46-47; (e) a light chain CDR2 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 13, 16, and 48-49; and (f) a light chain CDR3 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 14, 17, and 50-52; and (g) any of a set of four variable region framework regions from a human immunoglobulin (IgG). In various embodiments, the antibody can optionally include a hinge region. In various embodiments, the framework regions are derived from human germline exon X H , J H , Vκ and Jκ sequences. In various embodiments, the antibody is a fully humanized antibody. In various embodiments, the antibody is a fully human antibody.

[0014] 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: 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;

[0015] In various embodiments, the isolated antibody or antigen-binding fragment thereof of the present invention binds to human CCR8 and is an isolated chimeric antibody or antigen-binding fragment thereof, and comprises (1) the heavy chain sequence of SEQ ID NO:26 and the light chain sequence of SEQ ID NO:27; or (2) the heavy chain sequence of SEQ ID NO:28 and the light chain sequence of SEQ ID NO:29; or (3) the heavy chain sequence of SEQ ID NO:30 and the light chain sequence of SEQ ID NO:31; or (4) the heavy chain sequence of SEQ ID NO:78 and the light chain sequence of SEQ ID NO:79; or (5) the heavy chain sequence of SEQ ID NO:80 and the light chain sequence of SEQ ID NO:81; or (6) the heavy chain sequence of SEQ ID NO:82 and the light chain sequence of SEQ ID NO:83; or (7) the heavy chain sequence of SEQ ID NO:84 and the light chain sequence of SEQ ID NO:85.

[0016] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human CCR8 and comprises (a) a heavy chain sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 86, 88, and 90; and (b) a light chain sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 87, 89, and 91-92.

[0017] In another aspect, the invention relates to a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment of the invention in admixture with a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition comprises an isolated human antibody in admixture 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, intrasynovial injection, or via infusion.

[0018] In another aspect, the present invention relates to a method for treating a subject suffering from a CCR8-associated disorder, 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-associated disorder is cancer. In various embodiments, the subject previously responded to treatment with an anti-cancer therapy but suffered a relapse upon cessation of the therapy (hereinafter "recurrent cancer"). In various embodiments, the subject has a resistant or refractory cancer. In various embodiments, the cancerous cells are an immunogenic tumor (e.g., a tumor in which vaccination with the tumor itself can lead to immunity against tumor challenge).

[0019] In various embodiments, a method of treating a subject afflicted with cancer comprises administering to the subject a therapeutically effective amount of any one of the Treg-depleting anti-CCR8 Abs, e.g., mAbs, immunoconjugates, or bispecific molecules disclosed herein, or a pharmaceutical composition comprising any one of said Abs, e.g., anti-CCR8 mAbs, immunoconjugates, or bispecific molecules, such that the subject is treated.

[0020] In another aspect, the present invention relates to a combination therapy designed to treat cancer in a subject. In various embodiments, a method for inhibiting tumor cell growth 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 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 immune system inhibition or increases immune system stimulation, such that tumor cell growth is inhibited in the subject. 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, and the combination therapy provides increased cell killing of tumor cells, i.e., synergism exists between the isolated antibody or antigen-binding fragment and the additional therapy when administered in combination.

[0021] In another aspect, the invention relates to a method of enhancing an immune response to cancerous cells in a subject, comprising administering to the subject (as a monotherapy or in a combination treatment regimen) a therapeutically effective amount of an isolated antibody or antigen-binding fragment of the invention. In various embodiments, the invention relates to a method of treating cancerous cells in a subject, comprising administering to the subject (as a monotherapy or in a combination treatment regimen) a therapeutically effective amount of an antibody or antigen-binding fragment thereof 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.

[0022] In another aspect, an isolated immunoconjugate or fusion protein is provided 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.

[0023] In another aspect, the present invention features bispecific molecules comprising an anti-CCR8 antibody of the invention, or an antigen-binding fragment thereof. In various embodiments, an antibody of the invention, or an antigen-binding fragment thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., a ligand for another antibody or receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. In various embodiments, an antibody of the invention may 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 of the invention, or an antigen-binding fragment thereof, linked to a functional molecule that binds to 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.

[0024] In another aspect, the antibodies or antigen-binding fragments disclosed herein may be covalently linked (or otherwise stably associated) with an additional functional moiety, such as a label or a moiety that confers desirable pharmacokinetic properties. In various embodiments, the label is selected from the group consisting of a fluorescent label, a radioactive label, and a label with a distinctive nuclear magnetic resonance signature.

[0025] In another embodiment, the present invention provides methods for detecting the presence of human CCR8 peptides in a sample in vitro or in vivo, for example, to diagnose a human CCR8-associated disorder. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a line graph depicting the dose response of anti-CCR8 antibodies in blocking CCR8-mediated calcium flux induced by hCCL1. Reference Ab #1 is a humanized anti-hCCR8 antibody described in the literature. [Figure 2] Figure 2 is a line graph depicting the dose response of anti-CCR8 antibodies in blocking CCR8-mediated calcium flux induced by hCCL1. Reference Abs #1 and #2 are humanized anti-hCCR8 antibodies described in the literature. [Figure 3] FIG. 3 is a set of diagrams showing that bispecific antibodies FP578-01 and FP578-02 can bind to CTLA-4 while simultaneously binding to CCR8. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to antigen-binding proteins, such as antibodies, or antigen-binding fragments thereof, that specifically bind to human CCR8. In one embodiment, isolated antibodies and antigen-binding fragments thereof are provided that specifically bind to CCR8, have high affinity for CCR8, function to inhibit CCR8, are less immunogenic in a given species (e.g., humans) than their unmodified parent antibodies, and can be used to treat human disorders mediated by CCR8. Nucleic acid molecules comprising polynucleotide sequences encoding all or part of polypeptides that bind to CCR8, as well as derivatives and fragments thereof, such as nucleic acids encoding all or part of anti-CCR8 antibodies, antibody fragments, or antibody derivatives, are also provided. Vectors and plasmids containing such nucleic acids, and cells or cell lines containing such nucleic acids and / or vectors and plasmids are also provided. Also provided are methods for producing, identifying, or isolating antigen-binding proteins that bind to human CCR8, e.g., anti-CCR8 antibodies, methods for determining whether an antigen-binding protein binds to CCR8, methods for producing compositions, e.g., pharmaceutical compositions, comprising antigen-binding proteins that bind to human CCR8, and methods for administering antibodies that bind to CCR8, or antigen-binding fragments thereof, to a subject, e.g., methods for treating conditions mediated by CCR8.

[0028] definition Unless otherwise defined herein, scientific and technical terms used in the context of the present invention have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms include plurals, and plural terms include the singular. Generally, the technical terms used in the context of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and these techniques, 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 the various general and more specific references referenced and discussed throughout this specification. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are carried out according to manufacturer's specifications as commonly accomplished in the art or as described herein. The technical terms used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0029] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, a "peptide," "polypeptide," and "protein" is a chain of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (the amino terminus) thus has a free amino group, and the terminal amino acid at the other end of the chain (the carboxy terminus) 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 the α-amino group (imino group when participating in a peptide bond) of an amino acid at any other position within the peptide. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other position within the peptide. Peptides also include essentially any polyamino acid, including, but not limited to, peptidomimetics, e.g., those in which amino acids are joined by ether bonds rather than amide bonds.

[0030] Polypeptides of the present disclosure include polypeptides that have been modified in any way and for any reason, such as, for example, (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 impart or modify other physicochemical or functional properties.

[0031] As used herein, an amino acid "substitution" refers to the replacement of one amino acid in a polypeptide at a specific position in a parent polypeptide sequence with a different amino acid. Amino acid substitutions can be generated using genetic or chemical methods well known in the art. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in a portion of a polypeptide outside the domain that forms intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid with a functionally similar amino acid in a polypeptide. The following six groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), and Threonine (T) 2) Aspartic acid (D) and glutamic acid (E) 3) Asparagine (N) and Glutamine (Q) 4) Arginine (R) and Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V) 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0032] "Non-conservative amino acid substitutions" refer to the replacement of a member of one of these classes with a member from another class. In making such changes, according to various embodiments, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are as follows: 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).

[0033] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on hydropathic index, various embodiments include substitution of amino acids whose hydropathic index is within ±2, in various embodiments within ±1, and in various embodiments within ±0.5.

[0034] It is also understood in the art that substitutions of like amino acids can be usefully made on the basis of hydrophilicity, particularly when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0035] The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 +-.1); glutamic acid (+3.0 +-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 +-.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). When making changes based on similar hydrophilicity values, in various embodiments, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are included, in various embodiments, within ±1, and in various embodiments, within ±0.5 are included.

[0036] Exemplary amino acid substitutions are listed in Table 1. TIFF2025534340000002.tif173170

[0037] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein. In various embodiments, those skilled in the art can identify suitable sections of the molecule that can be changed without destroying activity by targeting regions that are not believed to be important for activity. In other embodiments, those skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides. In further embodiments, even sections that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0038] Additionally, one skilled in the art can review structure-function studies to identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one skilled in the art can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues in the similar polypeptide that are important for activity or structure. One skilled in the art can select substitutions of chemically similar amino acids for such predicted important amino acid residues.

[0039] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence relative to that structure in similar polypeptides. Taking such information into account, those skilled in the art can predict the alignment of amino acid residues of a polypeptide relative to the three-dimensional structure of the polypeptide. In various embodiments, those skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the polypeptide, because such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art may generate test variants containing single amino acid substitutions at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is discovered that a change to a specific amino acid residue results in destroyed, undesirably reduced, or unsuitable activity, the variant with such a change can be avoided. In other words, based on the information gathered from such routine experiments, those skilled in the art can easily determine amino acids for which further substitutions should be avoided, either alone or in combination with other mutations.

[0040] The terms "polypeptide fragment" and "truncated polypeptide," as used herein, refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion compared to the corresponding full-length protein. In various embodiments, 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 length. In various embodiments, fragments can also be, for example, up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 25, up to 10, or up to 5 amino acids in length. A fragment can further comprise one or more additional amino acids at either or both of its termini, for example, a sequence 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).

[0041] The terms "polypeptide variant," "hybrid polypeptide," and "polypeptide mutant," as used herein, refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted compared to another polypeptide sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues 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. Hybrids of the present disclosure include fusion proteins.

[0042] As described herein, a single mutation is identified by a unique 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 containing a serine substituting the full-length wild-type threonine at amino acid 10 is identified as T10S.

[0043] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugated to another chemical moiety, e.g., polyethylene glycol, albumin (e.g., human serum albumin), etc., phosphorylated, and glycosylated.

[0044] The term "sequence identity %" is used interchangeably herein with the term "identity %" and refers 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, meaning that a given sequence is at least 80% identical to another sequence. 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 more sequence identity to a given sequence. In various embodiments, the percent identity is within 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%.

[0045] The term "sequence homology %" is used interchangeably herein with the term "homology %" and refers 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 is the same as 80% sequence homology determined by a defined algorithm, and thus, a homolog of a given sequence has a sequence homology of more than 80% over a certain 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 more sequence homology to the given sequence. In various embodiments, the percent homology is within 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%.

[0046] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, the BLAST series of programs published on the NCBI website on the Internet, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN.Also see Altschul et al., J. Mol. Biol. 215:403-10, 1990 (particularly referring to the published default settings, i.e., parameters w=4, t=17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997.Sequence searches are typically performed using the BLASTP program, where a given amino acid sequence is compared with the amino acid sequences in GenBank Protein Sequences and other public databases.The BLASTX program is preferred for searching nucleic acid sequences translated in all reading frames against the amino acid sequences in GenBank Protein Sequences and other public 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 utilize the BLOSUM-62 matrix.

[0047] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid with the reference nucleic acid is, for example, less than about 0.1, less than about 0.01, or less than about 0.001.

[0048] The term "alteration" (modification), as used herein, refers to any manipulation of the peptide backbone (eg, amino acid sequence) or post-translational modification of a polypeptide (eg, glycosylation).

[0049] 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 are used interchangeably herein). It is specifically contemplated that the term "therapeutic protein" encompasses the fusion molecules of the present invention.

[0050] The term "fusion protein," as used herein, refers to a fusion polypeptide molecule comprising two or more genes that originally encoded separate proteins, in which the components of the fusion protein are joined to each other by peptide bonds, either directly or through a peptide linker. The term "fused," as used herein, refers to components joined by peptide bonds, either directly or through one or more peptide linkers.

[0051] A "linker" refers to a molecule that joins two other molecules covalently or through ionic, van der Waals, or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes at its 5'-end to one complementary sequence and at its 3'-end to another complementary sequence, thereby joining two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded or otherwise cleaved to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, and lipases. Cleavable linkers may also be cleaved by environmental cues, such as changes in temperature, pH, salt concentration, and the like.

[0052] The term "peptide linker," as used herein, refers to a peptide containing two or more amino acids, typically about 1 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 a G4 (SG4)n peptide linker, where "n" is generally a number from 1 to 10, typically 2 to 4.

[0053] The term "tumor-associated antigen" (TAA) refers to a cell surface antigen, for example, that is selectively expressed by cancer cells or overexpressed in cancer cells compared to most normal cells. The terms "TAA variant" and "TAA mutant," as used herein, refer to a TAA comprising an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted compared to another TAA sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues 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.

[0054] The term "antibody" is used herein to refer to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes and having specificity for tumor antigens or molecules overexpressed in pathological conditions. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as subtypes of these genes and the myriad immunoglobulin variable region genes. Light chains (LC) are classified as either kappa or lambda. Heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon, 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 is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.

[0055] 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 cases, 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 LThe VH and VL regions can be further divided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs is defined. The sequences of framework regions of different light or heavy chains are relatively conserved within species, e.g., humans. The framework region of an antibody, which is the combination of the framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. Immunoglobulin molecules 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.

[0056] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while a 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 CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs).

[0057] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. Kabat's database is currently maintained online, and CDR sequences can be determined. (See, for example, the IMGT / V-QUEST program, version 3.2.18, March 29, 2011, available online, and Brochet, X. et al., Nucl. Acids Res. 36, W503-508, 2008.) The Chothia definition is similar to the Kabat definition, but takes into account the location 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 unified set of computer programs produced by the Oxford Molecular Group to model antibody structure. For example, Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272, 1989; “AbM TM See, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from its primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, 1999. The contact definition is based on an analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45, 1996.

[0058] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally contains two constant domains, a CH2 domain and a 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 antibody and antigen-antibody complexes (e.g., neonatal FcR (FcRn) binds to the Fc region of IgG at the acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG). The replacement of amino acid residues in the Fc portion to alter antibody effector functions is known in the art (see, eg, Winter et al., US Pat. Nos. 5,648,260 and 5,624,821).

[0059] Antibodies exist as intact immunoglobulins or as a number of 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 target antigens. scFv proteins are fusion proteins in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are joined by a linker; in dsFv, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. While various antibody fragments are defined in terms of the digestion of intact antibodies, those skilled in the art will understand that such fragments may also be synthesized de novo chemically or by utilizing recombinant DNA methodologies. 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, camelidized antibodies, chimeric antibodies, single-chain Fvs (scFvs), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments exhibiting the desired biological activity, disulfide-linked Fvs (sdFvs), intrabodies, and epitope- or antigen-binding fragments of any of the above.

[0060] Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. A "Fab fragment" contains one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. A "Fab' fragment" contains one light chain and a portion of one heavy chain containing the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that interchain disulfide bonds can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0061] Pepsin treatment of an antibody yields an F(ab')2 fragment that has two antigen-binding sites and still has the ability to cross-link antigen. The "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the two heavy chains. The F(ab')2 fragment is therefore composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0062] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0063] A "single-chain antibody" is an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in WO 88 / 01649, U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0064] The terms "antigen-binding fragment" and "antigen-binding protein" as used herein refer to any protein that binds to a specified target antigen. "Antigen-binding fragment" includes, but is not limited to, antibodies and binding portions thereof, such as immunologically functional fragments. Exemplary antigen-binding fragments of antibodies are heavy and / or light chain CDRs, or heavy and / or light chain variable regions.

[0065] The term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen-binding protein, as used herein, refers to a species of antigen-binding protein that includes a portion of an antibody (regardless of how the portion was obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain but still has the ability to specifically bind to an antigen. Such fragments are biologically active in that they can bind to a target antigen and compete with other antigen-binding proteins, including intact antibodies, for binding to a given epitope. In some embodiments, the fragment is a neutralizing fragment. In one aspect, such a fragment retains at least one CDR present in a full-length light or heavy chain, and in some embodiments, includes a single heavy and / or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of antigen-binding proteins, including intact antibodies. 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, camelid, or rabbit. It is further contemplated that functional portions of the antigen-binding proteins disclosed herein, e.g., one or more CDRs, can be covalently linked to a second protein or small molecule to create therapeutic agents that are directed to unique targets in the body, have bifunctional therapeutic properties, or have extended serum half-lives.

[0066] Diabodies are bivalent antibodies comprising two polypeptide chains, each of which is too short to allow pairing between the two regions on the same chain and therefore comprises a VH and VL region joined by a linker that allows each region to pair with a complementary region on another 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). When the two polypeptide chains of a diabody are identical, the resulting diabody has two identical antigen-binding sites. Polypeptide chains with different sequences can be used to create diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, and form three and four antigen-binding sites, which can be the same or different.

[0067] Bispecific antibodies or fragments can be in several configurations. For example, a bispecific antibody may 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 "polydoma" technology (see, e.g., U.S. Pat. No. 4,474,893); or by recombinant DNA technology. In various embodiments, 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, antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies or antibody-binding fragments (e.g., Fabs) linked together, where each antibody or fragment has a different specificity.

[0068] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to 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 production of the antibody by any particular method.

[0069] The term "chimeric antibody," as used herein, refers to an antibody that has framework residues from one species, e.g., human, and CDRs (which generally confer antigen binding properties) from another species, e.g., a murine antibody, that specifically binds to a targeted antigen.

[0070] The term "human antibody," as used herein, 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 (e.g., mutations introduced in vitro by random or site-specific mutagenesis or in vivo by somatic mutation), e.g., in the CDRs, and particularly in CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., a mouse, have been grafted onto human framework sequences.

[0071] The term "humanized antibody," as used herein, refers to an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. A humanized 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 constructed from human germline exon X. H , J H , V K and J. K For example, V H For humanization of the FR domain, the acceptor sequence is the authentic 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 the hinge region (J H ) for Exon J H -6 (Mattila et al., Eur. J. Immunol. 25:2578-2582, 1995). In another example, a germline V K Exon B3 (Cox et al., Eur. J. Immunol. 24:827-836, 1994) and J K Exxon J K -1 (Hieter et al., J. Biol. Chem. 257:1516-1522, 1982) is V L It can be selected as an acceptor sequence for humanization of the domain.

[0072] The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, created, 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 (e.g., a mouse) transgenic for human immunoglobulin genes; or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In various embodiments, however, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within the human antibody germline repertoire in vivo. All such recombinant means are well known to those of skill in the art.

[0073] The term "anti-CCR8 antagonist antibody" (interchangeably referred to as "anti-CCR8 antibody") refers to an antibody capable of binding to CCR8 and inhibiting CCR8 biological activity and / or downstream pathways mediated by CCR8 signaling. Anti-CCR8 antagonist antibodies include antibodies that block, antagonize, suppress, or reduce (including significantly) CCR8 biological activity, including downstream pathways mediated by CCR8 signaling, such as receptor binding to CCR8 and / or eliciting cellular responses. For purposes of the present invention, the term "anti-CCR8 antagonist antibody" is expressly understood to encompass all previously identified terms, titles, and functional states and characteristics in which CCR8 itself, CCR8 biological activity (including, but not limited to, the ability to mediate any aspect of headache), or the consequences of biological activity are substantially neutralized, reduced, or neutralized to any meaningful extent. In some embodiments, anti-CCR8 antagonist antibodies bind to CCR8 and prevent 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.

[0074] The term "epitope," as used herein, includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitope determinants generally consist of chemically active surface groupings of molecules, such as amino acids or carbohydrate or sugar side chains, and generally have specific charge characteristics in addition to specific three-dimensional structural features. Epitopes may be "linear" or "conformational." In a linear epitope, all of the points of interaction between the protein and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another. Once a desired epitope on an antigen has been determined, it is possible to generate antibodies against that epitope, for example, using the techniques described in this disclosure. Alternatively, during the discovery process, antibody generation and characterization may reveal information about the desired epitope. From this information, antibodies can then be competitively screened for binding to the same epitope. An approach to achieve this is to perform cross-competition studies to find antibodies that bind competitively with each other, e.g., antibodies compete for binding to an antigen.

[0075] Antigen-binding proteins, including antibodies, are 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 The dissociation constant of M (as defined below, K D An antigen-binding protein "specifically binds" to an antigen if it binds to the antigen with high binding affinity as determined by its affinity (Kb) or corresponding Kb value. An antigen-binding protein that specifically binds to a human antigen of interest may also be able to bind the same antigen of interest from other species with the same or different affinity. D" as used herein refers to the equilibrium dissociation constant of a particular antigen:antibody interaction.

[0076] The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows the analysis of real-time biomolecular specific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).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.

[0077] As used herein, the term "tumor microenvironment" refers to the cellular environment in which a tumor resides, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix (ECM). Components in the tumor microenvironment can modulate the proliferation, for example, progression and metastasis ability of tumor cells. The tumor microenvironment can also be influenced by tumors, which release extracellular signals, promote tumor angiogenesis, and induce peripheral immune tolerance.

[0078] The term "immunogenicity," as used herein, refers to the ability of an antibody or antigen-binding fragment to elicit an immune response (humoral or cellular) when administered to a recipient, including, for example, a human anti-mouse antibody (HAMA) response. A HAMA response is initiated when T cells from a subject mount an immune response to an administered antibody. The T cells then recruit B cells to produce specific "anti-antibody" antibodies.

[0079] The term "immune cell," as used herein, refers to any cell of the hematopoietic lineage that is involved in regulating the immune response to an antigen (e.g., an autoantigen). In various embodiments, the immune cell is, for example, a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, a Langerhans cell, or a Kupffer cell.

[0080] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. A pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. A "pharmacologically effective amount" refers to an amount of an agent effective to produce the intended pharmacological result. A "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as phosphate-buffered saline solution, a 5% aqueous solution of dextrose, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co., Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0081] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or palliation of the disease state, and improvement in remission or prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative, palliative, and preventative treatment.

[0082] The term "effective amount" or "therapeutically effective amount" as used herein refers to the amount of a compound or composition that is sufficient to treat a specified disorder, condition or disease, for example, to ameliorate, 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: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down, and preferably stop the cancer cell infiltration into peripheral organs to some extent; (iv) inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumor; and / or (vii) alleviate one or more of the symptoms associated with cancer to some extent.An effective amount can be administered in one or more administrations.

[0083] The "adjuvant setting" refers to a clinical situation in which an individual has a history of a proliferative disease, particularly cancer, and has generally (though not necessarily) been responsive to treatment, including, but not limited to, surgery (e.g., surgical resection), radiation therapy, and chemotherapy. However, because of their history of a proliferative disease (e.g., cancer), these individuals are considered at risk for developing the disease. Treatment or administration in the "adjuvant setting" refers to a subsequent mode of treatment. 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 usually the extent of the disease when first treated.

[0084] The phrases "administering" or "causing to be administered" refer to the act of managing and / or allowing the administration of an agent / compound of interest to a patient by a medical professional (e.g., a physician) or someone managing the medical care of a patient. Causing to be administered can involve determining a diagnosis and / or an appropriate treatment regimen, and / or prescribing a particular agent / compound for the patient. Such prescribing can include, for example, issuing a prescription, annotating a medical record, and the like. When administering is described herein, "causing to be administered" is also contemplated.

[0085] As used herein, the terms "co-administration," "co-administered," and "in combination with," referring to a fusion molecule of the invention and one or more other therapeutic agents, are intended to mean, and refer to and include, the simultaneous administration of a fusion molecule of the invention and a combination of therapeutic agents to an individual in need of treatment, where such components are formulated together in a single dosage form that releases the components to said individual at substantially the same time; and the substantially simultaneous administration of a fusion molecule of the invention and a combination of therapeutic agents to an individual in need of treatment, where such components are formulated separately from one another in separate dosage forms that are taken by said individual at substantially the same time and that release the components to said individual at substantially the same time. the sequential administration of a combination of a fusion molecule and a therapeutic agent of the present invention to an individual in need of treatment, where such components are formulated apart from one another to be in separate dosage forms taken by the individual at successive times with a significant time interval between each administration, such that the components are released to the individual at substantially different times; and the sequential administration of a combination of a fusion molecule and a therapeutic agent of the present invention to an individual in need of treatment, where such components are formulated together to be in a single dosage form that releases the components in a controlled manner, such that they are released to the individual at the same and / or different times in a concurrent, sequential, and / or overlapping manner, where each portion may be administered by the same or different routes.

[0086] The terms "patient," "individual," and "subject" may be used interchangeably and may refer to a mammal, preferably a human or non-human primate, but may also refer to domestic mammals (e.g., canines or felines), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., equines, bovines, porcines, ovines). In various embodiments, a patient may be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) under the care of a physician or other healthcare professional in a hospital, psychiatric care facility, outpatient setting, or other clinical setting. In various embodiments, a patient may be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, patients with primary immunodeficiency, AIDS; cancer and transplant patients receiving 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 that have been reported to have high rates of mutations (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0087] The term "immunotherapy" includes treatments using depleting antibodies against specific tumor antigens; treatments using antibody-drug conjugates; treatments using agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), such as CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, SIRP, CD40, CD47, Siglec 8, Siglec 9, Siglec 15, TIGIT, and VISTA; treatments using bispecific T cell engaging antibodies (BiTEs®), such as blinatumomab; treatments involving the administration of biological response modifiers, such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatments using therapeutic vaccines, such as sipuleucel-T; treatments using Bacillus Calmette-Guerin (BCG); dendritic cell vaccines, and refers to cancer treatments including, but not limited to, treatments using tumor antigen peptide vaccines; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using tumor infiltrating lymphocytes (TILs); treatments using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatments using TALL-104 cells; and treatments using immunostimulants, such as the Toll-like receptor (TLR) agonists CpG and imiquimod.

[0088] "Resistant or refractory cancer" refers to tumor cells or cancers that do not respond to prior anti-cancer treatments, including, for example, chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells can be resistant or refractory at the beginning of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment or that initially respond to treatment for a short period but then become unresponsive. Refractory tumor cells also include tumors that respond to treatment with an anti-cancer therapy but do not respond to subsequent rounds of treatment. For purposes of the present invention, refractory tumor cells also encompass tumors that appear to be inhibited by treatment with an anti-cancer therapy but recur up to five years, and sometimes ten years or more, after treatment has ceased. Anti-cancer treatments can include chemotherapy alone, radiation alone, targeted therapy alone, immunotherapy alone, surgery alone, or a combination thereof. For ease of description and not limitation, it is understood that refractory tumor cells can be interchangeable with resistant tumors.

[0089] As used herein, "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured either through enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology.

[0090] The term "affinity" or "binding affinity," as used herein, refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y is determined by the dissociation constant (K), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). D A particular method for measuring affinity is surface plasmon resonance (SPR).

[0091] The term "reduced binding," as used herein, refers to a decrease in affinity for the respective interaction, e.g., as measured by SPR. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0092] The term "polymer," as used herein, generally includes, but is not limited to, homopolymers; copolymers, such as block, graft, random, and alternating copolymers; and terpolymers; and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term "polymer" includes all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and random symmetries.

[0093] "Polynucleotide" refers to a polymer composed of 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 nucleic acid analogs containing non-naturally occurring bases, nucleotides containing bases that are linked to other nucleotides in linkages other than naturally occurring phosphodiester bonds, or that are attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, without limitation, for example, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally of about 50 nucleotides or less. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it is understood that this also includes RNA sequences in which "U" replaces "T" (i.e., A, U, G, C).

[0094] Conventional notation is used herein to describe polynucleotide sequences: 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 referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to a nascent RNA transcript is referred to as the transcription direction. A DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as the mRNA transcribed from that DNA and which are located 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; and sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0095] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it 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), in which additional DNA segments can be introduced into the viral genome. Certain vectors have the ability to autonomously replicate in 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 upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0096] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can exert its effect, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further 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 (e.g., level, timing, or location of expression) of the nucleotide sequence.

[0097] A "host cell" is a cell that can be used to express a polynucleotide of the present disclosure. A host cell can be a prokaryote, such as E. coli, or 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., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, and the nucleic acid can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express the nucleic acid at a desired level until a regulatory sequence is introduced into the host cell so that the nucleic acid is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the specific subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due, for example, to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0098] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or polynucleotide) refers to a molecule that, by reason of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state; (2) is substantially free from other molecules from the same species; (3) is expressed by cells from a different species; or (4) is not naturally occurring. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule may be assayed by numerous means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, greater resolution may be provided by using HPLC or other means well known in the art for purification.

[0099] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60%-75% of a sample represents a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein typically comprises about 50%, 60%, 70%, 80%, or 90% w / w of a protein sample, more usually about 95% w / w, and is preferably greater than 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by staining the gel with stains well known in the art to visualize a single polypeptide band. For certain purposes, greater resolution may be provided by using HPLC or other means well known in the art for purification.

[0100] The term "heterologous," as used herein, refers to a composition or condition that is not native or not found in nature, e.g., that may be achieved by replacing an existing natural composition or condition with a composition or condition derived from another source. Similarly, expression of a protein in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and heterologous protein.

[0101] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Aspects and embodiments of the disclosure described herein are understood to include aspects and embodiments "consisting of" and / or "consisting essentially of."

[0102] Reference herein to "about" a value or parameter includes (and describes) variations directed to the value or parameter itself. For example, a reference to "about X" includes a description of "X."

[0103] CC chemokine receptor type 8 (CCR8) CCR8 is a G protein-coupled, seven-transmembrane CC chemokine receptor protein expressed in the thymus, spleen, and other organs. 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 as an endogenous ligand of CCR8 (J. Biol. Chem., 1997, Vol. 272, No. 28, pp. 17251-4). Human CCR8 cDNA is composed of the nucleotide sequence represented by GenBank ACC No. NM_005201.3, and mouse CCR8 cDNA is composed of the nucleotide sequence represented by GenBank ACC No. NM_007720.2.

[0104] The term "CCR8" as used herein includes human CCR8 (hCCR8), variants, isoforms, and species homologs of hCCR8, as well as analogs that share at least one epitope with hCCR8. In various embodiments, hCCR8 as used herein may comprise the amino acid sequence set forth in SEQ ID NO: 1: MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL (SEQ ID NO: 1)

[0105] In various embodiments, the CCR8 comprises an amino acid sequence that shares, 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, the 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 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, or at least 3-fold the activity of human CCR8 of SEQ ID NO: 1. Variants of CCR8 are sometimes described herein by reference to additions, deletions, or substitutions of amino acid residues present at given positions in the 360 ​​amino acid sequence of SEQ ID NO: 1. So, for example, the term "T10S" indicates that the "T" (threonine in standard single-letter code) residue at position 10 in SEQ ID NO: 1 has been substituted with an "S" (serine in standard single-letter code).

[0106] antibody Methods for generating novel antibodies that bind to human CCR8 are known to those skilled in the art. For example, a method for generating a monoclonal antibody that specifically binds to CCR8 may include administering to a mouse an immunogenic composition containing an effective amount of CCR8 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. Once a hybridoma is obtained, it can be propagated in a cell culture, optionally under culture conditions that allow hybridoma-derived cells to produce monoclonal antibodies that specifically bind to CCR8. The monoclonal antibody may be purified from the cell culture. A variety of different techniques are then available for testing antigen:antibody interactions to identify particularly desirable antibodies.

[0107] Other suitable methods for producing or isolating antibodies of the required specificity can be used, including, for example, methods that rely on selecting recombinant antibodies from libraries or on immunizing transgenic animals (e.g., mice) 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. Pat. No. 5,545,806; Surani et al., U.S. Pat. No. 5,545,807.

[0108] Antibodies can be engineered in many ways. They can be produced as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs™), Fab and F(ab')2 fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications describe 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.

[0109] 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 restriction enzymes to remove the murine Fc-encoding region and replace it with the equivalent portion of a gene encoding a human Fc constant region (Robinson et al., International Publication No. PCT / US86 / 02269; Akira, et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Publication No. WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application No. 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).

[0110] Methods for humanizing antibodies have been described in the art. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than an intact human variable region has been substituted by the corresponding sequence from a non-human species. To some extent, this can be achieved in conjunction with humanization techniques and display technologies using appropriate libraries. It will be appreciated 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., Immunol Today, 14:43-46, 1993; and Wright et al., Crit. Reviews in Immunol., 12125-168, 1992). The antibody of interest may be engineered by recombinant DNA technology to replace the CH1, CH2, CH3, hinge domain, and / or framework domain with the corresponding human sequence (see International Publication No. 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). The use of Ig cDNA to construct chimeric immunoglobulin genes is also known in the art (Liu et al., PNAS 84:3439, 1987; J. Immunol. 139:3521, 1987). mRNA is isolated from hybridomas or other cells that produce antibodies and used to produce 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, a library can be created and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to a human constant region sequence.The sequences of human constant regions for the genes can be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, NIH publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype is guided by the desired effector function, such as complement fixation or activity in antibody-dependent cellular cytotoxicity. In various embodiments, the isotype is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Either a human light chain constant region, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.

[0111] Queen et al., U.S. Patent No. 5,693,761, discloses the refinements made by Winter et al. for humanizing antibodies and is based on the premise that the loss of avidity is due to problems with structural motifs in the humanized framework that interfere with the folding of CDRs into the binding-competent conformation found in mouse antibodies due to steric or other chemical incompatibility. To address this problem, Queen teaches using human framework sequences that are closely homologous in linear peptide sequence to the framework sequences of the mouse antibody to be humanized. Thus, Queen's method focuses on comparing framework sequences between species. Typically, all available human variable region sequences are compared with a unique mouse sequence, and the percentage of identity between corresponding framework residues is calculated. The human variable region with the highest percentage is selected to provide the framework sequence for the humanization project. Queen also teaches that it is important to retain certain amino acid residues from the mouse framework in the humanized framework that are essential for supporting the CDRs in a binding-competent conformation. Potential essentiality is assessed from molecular models. Candidate residues for retention are typically those that are adjacent to the CDRs in the linear sequence or that are physically within 6 Å of any CDR residue.

[0112] In another approach, the importance of particular framework amino acid residues is determined experimentally, once a low-avidity humanized construct is obtained, by backmutating single residues to the murine sequence and assaying antigen binding as described in Riechmann et al., 1988. Another exemplary approach to identifying important amino acids in framework sequences is disclosed by Carter et al., U.S. Pat. No. 5,821,337, and Adair et al., U.S. Pat. No. 5,859,205. These references disclose specific Kabat residue positions in the framework that may require substitution with the corresponding murine amino acid to preserve avidity in humanized antibodies.

[0113] Another method for humanizing antibodies, referred to as "framework shuffling," relies on generating combinatorial libraries with nonhuman CDR variable regions fused in-frame to a pool of individual human germline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). The libraries are then screened to identify clones encoding humanized antibodies that retain good binding.

[0114] The selection of human variable regions, both light and heavy, to be used in the production of a desired humanized antibody is crucial to reducing antigenicity. According to the so-called "best fit" method, the sequence of the variable region of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence closest 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 unique framework region derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chain variable regions. The same framework may 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).

[0115] The selection of non-human residues for substitution into human variable regions can be influenced by various factors. These factors include, for example, the rarity of the amino acid at a particular position, the probability of interaction with either the CDR or antigen, and the probability of participation in the interface between the light and heavy chain variable domains. (See, e.g., U.S. Pat. Nos. 5,693,761, 6,632,927, and 6,639,055.) One way to analyze these factors is through the use of three-dimensional models of the non-human and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, for example, analysis of residues that affect the ability of the candidate immunoglobulin to bind to an antigen. In this way, non-human residues can be selected and substituted for human variable region residues to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0116] Methods for producing fully human antibodies have been described in the art. For example, a method for producing an anti-CCR8 antibody or its antigen-binding fragment includes the steps of synthesizing a library of human antibodies on phage, screening the library with CCR8 or its antibody-binding portion, isolating phages that bind to CCR8, and obtaining antibodies from the phages. As another example, one method for preparing an antibody library for use in phage display technology includes the steps of immunizing a non-human animal containing a human immunoglobulin locus with CCR8 or its antigenic portion 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 present 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 so that the antibody is expressed on the phage. The recombinant anti-CCR8 antibody of the present invention may be obtained in this way.

[0117] The recombinant human anti-CCR8 antibody of the present invention can also be isolated by screening a recombinant combinatorial antibody library. Preferably, the library is an 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™ Phage Display Kit, catalog number 240612).There are other methods and reagents that can be used in generating and screening antibody display libraries (e.g., U.S. Pat. No. 5,223,409; PCT Publication Nos. 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. 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. al.,Proc.Natl.Acad.Sci.USA 88:7978-7982, 1991; each of which is incorporated herein by reference for the purposes of teaching the preparation and screening of phage display libraries).

[0118] Human antibodies have also been produced by immunizing non-human transgenic animals containing part or all of the human immunoglobulin heavy and light chain loci within their genome, e.g., XenoMouse™ animals (Abgenix, Inc. / Amgen, Inc., Fremont, Calif.), with human IgE antigens. XenoMouse™ mice are engineered mouse strains that contain large fragments of the human immunoglobulin heavy and light chain loci and are deficient 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™ mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human antibodies. In some embodiments, XenoMouse™ mice contain approximately 80% of the human antibody V gene repertoire through the introduction of megabase-sized germline fragments of human heavy chain and kappa light chain loci in yeast artificial chromosomes (YACs). In other embodiments, the XenoMouse™ mouse further contains nearly the entire human lambda 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 of which is incorporated by reference in its entirety for purposes of teaching the preparation of fully human antibodies).In another embodiment, the present invention provides a method for producing 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. Such animals can be produced using the methods described in the above-referenced documents.

[0119] Characterization of antibody binding to antigen Antibodies of the present invention can be tested for binding to human CCR8, for example, by standard ELISA. For example, microtiter plates are coated with purified CCR8 or cells overexpressing human CCR8 in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from CCR8-immunized mice) are added to each well and incubated for 1-2 hours at 37°C or 4°C. The plates are washed with PBS / Tween and then incubated with a secondary reagent conjugated to alkaline phosphatase (e.g., for human antibodies, a goat anti-human IgG Fc-specific polyclonal reagent) for 1 hour at 37°C. After washing, the plates are developed with pNPP substrate (1 mg / ml) and analyzed at OD 405-650 nm. Preferably, mice generating the highest titers are used for fusion. ELISA can also be used to screen for hybridomas that show positive reactivity with CCR8 immunogens. Hybridomas that bind to CCR8 with high avidity are subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parental cells (by ELISA) can be selected to generate a cell bank of 5-10 vials stored at -140°C and for antibody purification.

[0120] To determine whether selected anti-CCR8 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using CCR8-coated ELISA plates as described above. Binding of biotinylated mAbs can be detected with a streptavidin-alkaline phosphatase probe. To determine the isotype of purified antibodies, isotype ELISAs can be performed using reagents specific for antibodies 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 anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plate is reacted with up to 1 μg / ml of the test monoclonal antibody or purified isotype control at ambient temperature for 1-2 hours. The wells can then be reacted with alkaline phosphatase-conjugated probes specific for either human IgG1 or human IgM. The plates are developed and analyzed as described above.

[0121] Anti-CCR8 human IgG can be further tested for reactivity with the CCR8 antigen by Western blotting. 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. Binding of human IgG can be detected using anti-human IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

[0122] Identification of anti-CCR8 antibodies The present invention provides monoclonal antibodies that specifically bind to the CCR8 antigen, and antigen-binding fragments thereof.

[0123] 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 compete for binding to the same epitope as the anti-CCR8 antibody of the present invention, a cross-blocking assay, such as 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 competing antibody, and then a biotin-labeled anti-CCR8 antibody of the present invention is added. The amount of labeled anti-CCR8 antibody bound to the CCR8 antigen in the well is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody can be labeled with a radioactive or fluorescent label or any other detectable and measurable label. The amount of labeled anti-CCR8 antibody bound to the antigen is indirectly correlated to the ability of the candidate competing antibody (test antibody) to compete for binding to the same epitope; that is, the greater the affinity of the test antibody for the same epitope, the less labeled antibody will bind to the well coated with the antigen. A candidate competing antibody is considered to be an antibody that substantially binds to the same epitope as an anti-CCR8 antibody of the present invention or competes for binding to the same epitope if it can block 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 run in parallel in the absence of the candidate competing antibody. It will be understood that variations of this assay can be performed to arrive at the same quantitative value.

[0124] The amino acid sequences of the heavy and light chain variable region CDRs of various murine mAbs, MAbs A1 to A19, generated as described herein, are shown in Table 2 below. TIFF2025534340000003.tif153170TIFF2025534340000004.tif195170 TIFF2025534340000005.tif198170

[0125] In various embodiments of the 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 as set forth in Table 3: TIFF2025534340000006.tif150170

[0126] In various embodiments, the antibodies of the present invention include antibodies that bind to the same epitope as murine antibodies MAb1 to MAb19.

[0127] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO:26: EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYFCVRGGTYGSSSYFDYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26) and the light chain sequence of SEQ ID NO: 27: DIVMTQAAPSVPVTPGESVSIPCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLQIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 27) It is a mouse-human chimeric antibody (derived from mouse antibody A1 ("41E1C2A5") and human IgG1) comprising:

[0128] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO:28: EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKARFTISRDDSESMLYLQMNNLKTEDTAMYFCVRGGTYGSTSYFDYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28) and the light chain sequence of SEQ ID NO: 29: DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29) It is a mouse-human chimeric antibody (derived from mouse antibody A3 ("80E4D1F11") and human IgG1) comprising:

[0129] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 30: EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGAINPNNGGTGYTQKFKGKATLTVDKSSSTAFMELRSLTSEDSAVYYCARRGVYMFAYWGQGTLV TVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30) and the light chain sequence of SEQ ID NO: 31: DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 31) It is a mouse-human chimeric antibody (derived from mouse antibody A4 ("419C7B3B2") and human IgG1) comprising:

[0130] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 78: EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYAASVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDVWG TGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 78) and the light chain sequence of SEQ ID NO: 79: DIVMTQAAPSVFVIPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 79) It is a mouse-human chimeric antibody (derived from mouse antibody A18 ("504E12D8D12") and human IgG1) comprising:

[0131] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 80: EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDSESMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDVWG TGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 80) and the light chain sequence of SEQ ID NO: 81: DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPERFSGSGSGSAFTLRVSRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 81) It is a mouse-human chimeric antibody (derived from mouse antibody A10 ("516D7D12") and human IgG1) comprising:

[0132] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 82: EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSENMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDVWG TGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 82) and the light chain sequence of SEQ ID NO: 83: DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPERFSGSGSGSAFTLRVSRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83) It is a mouse-human chimeric antibody (derived from mouse antibody A11 ("525F2F3F11") and human IgG1) comprising:

[0133] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 84: EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLDWVARIRSKSNNYATYYADSVKDRFTISRDSESMLYLQMNNLKTEDTAMYFCVRQTYGSRDYAMDYWGQ GTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84) and the light chain sequence of SEQ ID NO: 85: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCCQSTHVPPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) It is a mouse-human chimeric antibody (derived from mouse antibody A13 ("531B9B1C9") and human IgG1) comprising:

[0134] The antibody or antigen-binding fragment thereof of the present invention can comprise any constant region known in the art. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as an IgA-, IgD-, IgE-, IgG-, or IgM-type heavy chain constant region. In various embodiments, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region.

[0135] Techniques for deriving antibodies of a different subclass or isotype 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 technology can also be used. Cloned DNA encoding a specific antibody polypeptide, for example, DNA encoding the constant domain of an antibody of the desired isotype, can be used in such procedures. See also Lanitto et al., Methods Mol. Biol. 178:303-16, 2002.

[0136] In various embodiments, the antibodies of the invention further comprise a light chain kappa or lambda constant domain, or fragment thereof, and further comprise a heavy chain constant domain, or fragment thereof. The sequences of the light and heavy chain constant regions used in the exemplary antibodies, and the polynucleotides encoding them, are provided below. Light chain (kappa) constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 32) Light chain (lambda) constant region GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 33) heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34)

[0137] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 86: EVQLVESGGGLVQPGGSLKLSCAASGFSFNAYAMNWVRQASGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYFCVRGGTYGSSSYFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 86) and the light chain sequence of SEQ ID NO: 87: DIVMTQSPLSLPVTPGEPASIPCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 87) and a humanized antibody ("41E1C2A5-HC3-LC4") comprising:

[0138] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 88: EVQLVESGGGLVQPGGSLKLSCAASGFSFNAYAMNWVRQASGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSENTAYLQMNSLKTEDTAVYFCVRGGTYGSSSYFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88) and the light chain sequence of SEQ ID NO: 89: DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) and a humanized antibody ("41E1C2A5-HC4-LC2") comprising:

[0139] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 90: EVQLVESGGGLVQPGGSLKLSCAASGFSFNTYAMNWVRQASGKGLEWVGRIRTKSNNYATYYAASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSGLNYVRYFDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90) and the light chain sequence of SEQ ID NO: 91: DIVMTQTPPSLPVNPGEPASISCRSSKSLLHSNGNTYLYWYLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGSDFTLKISWVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 91) and a humanized antibody ("504E12D8D12-HC1-LC1") comprising:

[0140] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 90 and the light chain sequence of SEQ ID NO: 92: DIVMTQTPPSLPVNPGEPASISCRSSKSLLHSNANTYLYWYLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGSDFTLKISWVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 92) and a humanized antibody ("504E12D8D12-HC1-LC1(G34A)") comprising the compound.

[0141] Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Also included in the present invention are antibodies that bind to CCR8 with 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).

[0142] 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 compete for binding to the same epitope as the anti-CCR8 antibody of the present invention, a cross-blocking assay, such as 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 competing antibody, and then a biotin-labeled anti-CCR8 antibody of the present invention is added. The amount of labeled anti-CCR8 antibody bound to the CCR8 antigen in the well is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody can be labeled with a radioactive or fluorescent label or any other detectable and measurable label. The amount of labeled anti-CCR8 antibody bound to the antigen is indirectly correlated to the ability of the candidate competing antibody (test antibody) to compete for binding to the same epitope; that is, the greater the affinity of the test antibody for the same epitope, the less labeled antibody will bind to the well coated with the antigen. A candidate competing antibody is considered to be an antibody that substantially binds to the same epitope as an anti-CCR8 antibody of the present invention or competes for binding to the same epitope if it is able to block binding of the CCR8 antibody by at least 20%, at least 30%, at least 40%, or at least 50% compared to a control run in parallel in the absence of the candidate competing antibody. It will be understood that variations of this assay can be performed to arrive at the same quantitative value.

[0143] In certain alternative embodiments, the antibodies of the invention comprise one or both variable regions (i.e., V H and / or V L), or by modifying residues within the constant region, e.g., to alter the effector function of the antibody. In various embodiments, the variable region of the antibody is modified by CDR grafting using framework sequences that can be obtained from public DNA databases or published references containing germline antibody gene sequences (e.g., Tomlinson, I.M., et al., J. Mol. Biol. 227:776-798, 1992; and Cox, J.P. Let al., Eur. J. Immunol. 24:827-836, 1994; the contents of each of which are expressly incorporated herein by reference). In various embodiments, antibodies may be modified using site-directed mutagenesis or PCR-mediated mutagenesis to introduce mutations in VH and / or VL that improve binding affinity and / or reduce immunogenicity. In various embodiments, antibodies may be modified in the Fc region for the purpose of altering the serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity of the antibody. In various embodiments, antibodies may be modified for the purpose of altering the glycosylation of the antibody. Methods for making each of the modifications described herein, and others, are well known to those of skill in the art.

[0144] Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition comprising the above-described antibody or antigen-binding fragment thereof. The pharmaceutical compositions, methods and uses of the present invention therefore also encompass embodiments of combinations (co-administration) with other active agents, as detailed below.

[0145] In general, the antibody of the present invention, or its antigen-binding fragment antibody, is suitable for administration as a formulation in association with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any component other than the compound of the present invention. The choice of excipient depends to a large extent on factors such as the specific mode of administration, the excipient's effect on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it is preferable to include an isotonic agent, such as a sugar, a polyalcohol 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 or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. Pharmaceutical compositions of the present invention and methods for their preparation are readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). Pharmaceutical compositions are preferably produced under GMP conditions.

[0146] The pharmaceutical composition of the present invention can be prepared, packaged or sold in bulk, as a single unit dose or as a plurality of single unit doses.As used herein, " unit dose " is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of active ingredient that is administered to subject or the convenient ratio of this dosage, for example, half or one-third of this dosage, etc.

[0147] Any art-recognized method of administering a peptide, protein, or antibody may be suitably used with the antibodies and portions of the present invention.

[0148] The pharmaceutical compositions of the present invention are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route characterized by physical breaching of the target tissue and administration of the pharmaceutical composition through breach in the tissue, generally resulting in direct administration into the bloodstream, muscle, or internal organs. Parenteral administration therefore includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, and application of the composition through a tissue-penetrating non-surgical wound. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial injection or infusion; and kidney dialysis infusion techniques. Various embodiments include intravenous and subcutaneous routes.

[0149] Pharmaceutical compositions suitable for parenteral administration typically contain the active ingredient combined 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 continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, and pastes. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to 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 3 to 9); however, for some applications, they may be more suitably formulated as sterile non-aqueous solutions or as a dry form for use in combination with a suitable vehicle, such as sterile, pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form or in a liposomal preparation. Formulations for parenteral administration may be formulated for immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0150] For example, in one embodiment, sterile injectable solution can be prepared by incorporating the required amount of anti-CCR8 antibody into a suitable solvent, if required, with one or a combination of the ingredients listed above, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed above.For the preparation of sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying, which produces a powder of active ingredient and any additional desired ingredients from the previously sterile-filtered solution.The proper fluidity of the solution can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, such as monostearate salts and gelatin, in the composition.

[0151] Antibodies of the invention may also be administered intranasally or by inhalation, typically in the form of a dry powder (alone, in a mixture, or as mixed component particles, e.g., mixed with suitable pharmaceutically acceptable excipients) from a dry powder inhaler, with or without the use of a suitable propellant, as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer that uses electrohydrodynamics to produce a fine mist), or nebulizer, or as nasal drops.

[0152] The pressurized container, pump, spray, atomizer, or nebulizer will generally contain a solution or suspension of the antibody of the invention, e.g., as a solvent, a suitable agent to disperse, solubilize, or prolong the release of the active agent, a propellant.

[0153] Prior to use in a dry powder or suspension formulation, the drug product is generally micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any suitable comminution method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[0154] Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base and a performance modifier.

[0155] Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled / intranasal administration.

[0156] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0157] In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units according to the invention are typically configured to administer a metered dose or "puff" of an antibody of the invention. The total daily dose is typically administered in a single dose or, more usually, as divided doses throughout the day.

[0158] The antibodies and antibody portions of the invention may also be formulated for oral administration, which may involve swallowing, so that the compound enters the gastrointestinal tract, and / or buccal, lingual, or sublingual administration, by which the compound enters the blood stream directly from the mouth.

[0159] Suitable formulations for oral administration include solid, semi-solid, and liquid systems, such as tablets; soft or hard capsules containing multi- or nanoparticles, liquids, or powders; lozenges (including liquid-filled lozenges); chews; gels; fast-dispersing dosage forms; films; vaginal ovules; sprays; and buccal / mucoadhesive patches.

[0160] Pharmaceutical compositions intended for oral use can be prepared according to any method known in the art for producing pharmaceutical compositions, and such compositions can contain one or more agents selected from the group consisting of sweeteners to provide pharmaceutical-grade and tasty preparations.For example, to prepare orally deliverable tablets, antibody or its antigen-binding fragment is mixed with at least one pharmaceutical excipient, and the solid preparation is compressed according to known methods to form tablets for delivery to the gastrointestinal tract.Tablet compositions are typically formulated with additives such as saccharide or cellulose carriers, binders such as starch paste or methylcellulose, fillers, disintegrants, or other additives commonly used in the production of medical preparations.To prepare orally deliverable capsules, DHEA is mixed with at least one pharmaceutical excipient, and the solid preparation is placed in a capsule container suitable for delivery to the gastrointestinal tract. Compositions containing antibodies or antigen-binding fragments thereof may be prepared as generally described in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042), Chapter 89, which is incorporated herein by reference.

[0161] In various embodiments, the pharmaceutical composition is formulated as an orally deliverable tablet containing an antibody or antigen-binding fragment thereof mixed with non-toxic pharmaceutically acceptable excipients suitable for tablet production. These excipients may be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as maize starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. The tablets may be uncoated, or they may be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate, alone or with a wax, may be used.

[0162] In various embodiments, the pharmaceutical compositions are formulated as hard gelatin capsules 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 soft gelatin capsules in which the antibody or antigen-binding fragment thereof is mixed with an aqueous or oily medium, such as peanut oil, peanut oil, liquid paraffin, or olive oil.

[0163] Liquid preparations include suspensions, solutions, syrups and elixirs.Such preparations can be used as fillers in soft or hard capsules (e.g., made from gelatin or hydroxypropylmethylcellulose), and typically contain carriers such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or suitable oils, and one or more emulsifiers and / or suspending agents.Liquid preparations can also be prepared by reconstituting solids, for example, from sachets.

[0164] therapeutic use In another aspect, the present invention relates to a method for treating a subject suffering from a CCR8-associated disorder, 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-associated 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 previously responded to treatment with an anti-cancer therapy but suffered a relapse upon cessation of the therapy (hereinafter "recurrent cancer"). In various embodiments, the subject has a resistant or refractory cancer. In various embodiments, the cancerous cells are an immunogenic tumor (e.g., a tumor in which vaccination with the tumor itself can lead to immunity against tumor challenge).

[0165] In various embodiments, a method of treating a subject afflicted with cancer comprises administering to the subject a therapeutically effective amount of any one of the Treg-depleting anti-CCR8 Abs, e.g., mAbs, immunoconjugates, or bispecific molecules disclosed herein, or a pharmaceutical composition comprising any one of said Abs, e.g., anti-CCR8 mAbs, immunoconjugates, or bispecific molecules, such that the subject is treated.

[0166] In another aspect, the present invention relates to a combination therapy designed to treat cancer in a subject. In various embodiments, a method for inhibiting tumor cell growth 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 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 immune system inhibition or increases immune system stimulation, such that tumor cell growth is inhibited in the subject. 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, and the combination therapy provides increased cell killing of tumor cells, i.e., synergism exists between the isolated antibody or antigen-binding fragment and the additional therapy when administered in combination.

[0167] In another aspect, the invention relates to a method of enhancing an immune response to cancerous cells in a subject, comprising administering to the subject (as a monotherapy or in a combination treatment regimen) a therapeutically effective amount of an isolated antibody or antigen-binding fragment of the invention. In various embodiments, the invention relates to a method of treating cancerous cells in a subject, comprising administering to the subject (as a monotherapy or in a combination treatment regimen) a therapeutically effective amount of an antibody or antigen-binding fragment thereof 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.

[0168] In various embodiments, the cancer to be treated is a solid tumor, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis cancers, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and combinations of the foregoing cancers. In various embodiments, the cancerous cell is an immunogenic tumor (e.g., a tumor in which vaccination with the tumor itself can lead to immunity against tumor challenge). In various embodiments, the cancer is 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.

[0169] In various embodiments, the solid tumor is a cancer selected from HNSCC, cervical, CRC, NSCLC-SCC, NSCLC-ADC, pancreatic, gastric, bladder, and breast cancer.

[0170] In various embodiments, the cancer is a hematological malignancy, including liquid tumors derived from either of the two major blood cell lineages, i.e., the myeloid lineage (which produces granulocytes, erythrocytes, thrombocytes, macrophages, and mast cells) or the lymphoid lineage (which produces B, T, NK, and plasma cells), including all types of leukemia, lymphoma, and myeloma. Hematological malignancies that may be treated using the therapeutic methods of the present invention include, for example, cancers selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), advanced, metastatic, refractory, and / or recurrent hematological malignancies, and any combination of the foregoing hematological malignancies.

[0171] In various embodiments, the antibodies and antigen-binding fragments thereof of the present invention can be utilized to directly kill or ablate cancerous cells in vivo. Direct killing involves administering the antibody (optionally fused to a cytotoxic drug) to a subject requiring such treatment. In various embodiments, the cancer comprises cancer cells that express CCR8 at higher levels than non-cancerous cells of comparable tissue. Because the antibody recognizes CCR8 on cancer cells, any such cells to which the antibody binds are destroyed. When the antibody is used alone to kill or ablate cancer cells, such killing or ablation can be affected by initiating endogenous host immune functions, such as CDC and / or ADCC. Assays for determining whether an antibody kills cells in this manner are within the purview of those skilled in the art.

[0172] In various embodiments, the antibodies and antigen-binding fragments thereof of the present invention can be used to inhibit and / or promote the growth of cancerous tumor cells. These methods may inhibit or prevent the growth of cancer cells in the subject by, for example, 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%. Consequently, where the cancer is a solid tumor, the modulation may 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%.

[0173] Inhibition of cancer cell proliferation can be measured using 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); or the dye Alamar Blue (available from Biosource International) (Voytik-Harbin et al., In Vitro Cell Dev Biol Anim 34:239-46, 1998). Anchorage-independent growth of cancer cells can be assessed by colony formation assays in soft agar, for example, 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).

[0174] The inhibition of cancer cell proliferation in a subject may be evaluated by monitoring cancer growth in the subject, for example, in an animal model or in a human subject. One exemplary monitoring method is a tumorigenicity assay. In one example, a xenograft comprises human cells from an existing tumor or from a tumor cell line. Tumor xenograft assays are known in the art and are described herein (see, for example, Ogawa et al., Oncogene 19:6043-6052, 2000). In another embodiment, tumorigenicity is monitored using a hollow fiber assay, as described in U.S. Pat. No. 5,698,413, the entirety of which is incorporated herein by reference.

[0175] 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 (typically without modulator treatment). For example, the number of cancer cells or cancer cell colonies (colony formation assay), or PRDU or [ 3 If [H]-thymidine incorporation is A (under treatment with modulator) and C (under negative control conditions), then the percentage of inhibition is (CA) / C×100%.

[0176] Examples of tumor cell lines derived from human tumors and available for use in in vitro and in vivo studies include leukemia cell lines (e.g., CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPM1-8226, 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., DMS 114 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); kidney 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).

[0177] A "therapeutically effective amount" or "therapeutically effective dose" refers to that amount of a therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated.

[0178] The therapeutically effective dose is IC50 The dose can then be estimated from cell culture assays by first determining the IC 50 The compound can be formulated in animal models to achieve a circulating plasma concentration range including the range of 0.1 to 1.5 mg / kg of circulating plasma. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by HPLC. The exact composition, route of administration, and dosage can be chosen by the individual physician in consideration of the subject's condition.

[0179] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, or several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the mammalian subject to be treated; each unit contains a predetermined amount of 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 are primarily dictated by the unique characteristics of the antibody and the specific therapeutic or prophylactic effect to be achieved.

[0180] Therefore, those skilled in the art will understand based on the disclosure provided herein that dosage and dosage regimen will be adjusted according to the method well known in the therapeutic field.That is, the maximum acceptable dosage can be easily established, and the effective amount that provides detectable therapeutic benefit to the subject can also be determined, as well as the time requirement for administering each agent to provide detectable therapeutic benefit to the subject.Therefore, although certain dosage and dosage regimen are exemplified herein, these examples do not limit the dosage and dosage regimen that can be provided to the subject in the implementation of the present disclosure.

[0181] It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated and may include single or multiple doses. For any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and it should be further understood that the dosage ranges described herein are merely exemplary and are not intended to limit the scope or implementation of the claimed compositions. Furthermore, dosage regimens using the compositions of the present disclosure may be based on various factors, including the type of disease, the subject's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibody used. Thus, dosage regimens can vary widely but can be routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, e.g., toxic effects, and / or laboratory values. Therefore, the present disclosure encompasses intra-subject dose escalation as determined by those skilled in the art. It will be appreciated that determination of appropriate dosages and regimens is well known in the relevant art and could be accomplished by one of ordinary skill in the art once provided with the teachings disclosed herein.

[0182] For administration to human subjects, the total monthly dose of the antibodies or antigen-binding fragments thereof of the present disclosure may range, of course, from 0.5 to 1200 mg per subject, 0.5 to 1100 mg per subject, 0.5 to 1000 mg per subject, 0.5 to 900 mg per subject, 0.5 to 800 mg per subject, 0.5 to 700 mg per subject, 0.5 to 600 mg per subject, 0.5 to 500 mg per subject, 0.5 to 400 mg per subject, 0.5 to 300 mg per subject, 0.5 to 1000 mg per subject, 0.5 to 1200 mg per subject, 0.5 to 1100 mg per subject, 0.5 to 1000 mg per subject, 0.5 to 1000 mg per subject, 0.5 to 1200 mg per subject, 0.5 to 1100 mg per subject, 0.5 to 1000 mg per subject, 0.5 to 1200 mg per subject, 0.5 to 15 ... The dose can be within the range of 1-200 mg per subject, 0.5-100 mg per subject, 0.5-50 mg per subject, 1-1200 mg per subject, 1-1100 mg per subject, 1-1000 mg per subject, 1-900 mg per subject, 1-800 mg per subject, 1-700 mg per subject, 1-600 mg per subject, 1-500 mg per subject, 1-400 mg per subject, 1-300 mg per subject, 1-200 mg per subject, 1-100 mg per subject, or 1-50 mg per subject. For example, a monthly intravenous dose may require approximately 1-1000 mg per subject. In various embodiments, an antibody or antigen-binding fragment thereof of the present disclosure may be administered at approximately 1-200 mg per subject, 1-150 mg per subject, or 1-100 mg per subject. The total monthly dose may be administered in single or divided doses and, at the physician's discretion, can fall outside of the typical range given herein.

[0183] In various embodiments, non-limiting daily dosage ranges for a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure include 0.001-100 mg / kg, 0.001-90 mg / kg, 0.001-80 mg / kg, 0.001-70 mg / kg, 0.001-60 mg / kg, 0.001-50 mg / kg, 0.001-40 mg / kg, and the like. g / kg, 0.001~30mg / kg, 0.001~20mg / kg, 0.001~10mg / kg, 0.001~5mg / kg, 0.001~4mg / kg, 0.001 ~3mg / kg, 0.001~2mg / kg, 0.001~1mg / kg, 0.010~50mg / kg, 0.010~40mg / kg, 0.010~30mg / kg, 0.0 10~20mg / kg, 0.010~10mg / kg, 0.010~5mg / kg, 0.010~4mg / kg, 0.010~3mg / kg, 0.010~2mg / kg, 0 .010~1mg / kg, 0.1~50mg / kg, 0.1~40mg / kg, 0.1~30mg / kg, 0.1~20mg / kg, 0.1~10mg / kg, 0.1~5mg / kg, 0.1-4 mg / kg, 0.1-3 mg / kg, 0.1-2 mg / kg, 0.1-1 mg / kg, 1-50 mg / kg, 1-40 mg / kg, 1-30 mg / kg, 1-20 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-4 mg / kg, 1-3 mg / kg, 1-2 mg / kg, or 1-1 mg / kg body weight.

[0184] For repeated administration over a period of several days or longer, depending on the condition, treatment is continued until a desired suppression of symptoms occurs or until a sufficient therapeutic level is achieved, e.g., to reduce pain. An exemplary dosing regimen involves administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of anti-CCR8 antibody, or a maintenance dose of about 1 mg / kg every two weeks. However, other dosage regimens may be useful depending on the pharmacokinetic degradation pattern the practitioner desires to achieve. For example, in some embodiments, dosing 1 to 4 times per week is contemplated. The progress of this treatment is easily monitored by conventional techniques and assays. The dosing regimen (including the use of a CCR8 antagonist) can be varied over time. In various embodiments, the appropriate dosage of the anti-CCR8 antagonist antibody depends 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 administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the agent, and the discretion of the attending physician. Typically, the clinician will administer the anti-CCR8 antagonist antibody until a dosage that achieves the desired result is reached. The dosage and / or frequency can vary over the course of treatment.

[0185] It should be noted that dosage values ​​can vary with the type and severity of the condition to be alleviated.It should be further understood that for any specific subject, specific dosage regimen should be adjusted over time according to individual need and the professional judgment of the person who administers or supervises the composition, and that the dosage ranges described herein are merely illustrative and are not intended to limit the scope or implementation of the claimed compositions.

[0186] In various embodiments, the total dose administered achieves a plasma antibody concentration within the range of, for example, about 1 to 1000 μg / ml, about 1 to 750 μg / ml, about 1 to 500 μg / ml, about 1 to 250 μg / ml, about 10 to 1000 μg / ml, about 10 to 750 μg / ml, about 10 to 500 μg / ml, about 10 to 250 μg / ml, about 20 to 1000 μg / ml, about 20 to 750 μg / ml, about 20 to 500 μg / ml, about 20 to 250 μg / ml, about 30 to 1000 μg / ml, about 30 to 750 μg / ml, about 30 to 500 μg / ml, or about 30 to 250 μg / ml.

[0187] The toxicity and therapeutic index of the pharmaceutical compositions of the present invention can be determined, for example, by the LD 50 (a dose lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutically effective doses is the therapeutic index, which is the ratio LD 50 / ED 50 Compositions that exhibit large therapeutic indices are generally preferred.

[0188] In various embodiments, single or multiple administrations of pharmaceutical compositions are administered, depending on the dosage and frequency required and tolerated by the subject.In any case, the composition should provide at least one of the antibodies or antigen-binding fragments disclosed herein in a sufficient amount to effectively treat the subject.Dosage can be administered once, but can also be applied periodically until either therapeutic results are achieved or side effects warrant the discontinuation of treatment.

[0189] The dosing frequency of the pharmaceutical composition of antibody or its antigen-binding fragment is dependent on the nature of treatment and the specific disease being treated.Subjects can be treated at regular intervals, for example, weekly or monthly, until the desired therapeutic result is achieved.Exemplary dosing frequencies include, but are not limited to, once a week without interruption; once every two weeks; once every two weeks; once every three weeks; once a week for two weeks without interruption, then monthly; once a week for three weeks without interruption, then monthly; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months, or yearly.

[0190] Combination treatment As used herein, the terms "co-administration," "co-administered," and "in combination with," referring to an antibody or antigen-binding fragment thereof of the present disclosure and one or more other therapeutic agents, are intended to mean, and refer to and include, the simultaneous administration of an antibody or antigen-binding fragment thereof of the present disclosure and a combination of therapeutic agents to a subject in need of treatment, where such components are formulated together in a single dosage form that releases said components to said subject at substantially the same time; the substantially simultaneous administration of an antibody or antigen-binding fragment thereof of the present disclosure and a combination of therapeutic agents to a subject in need of treatment, where such components are formulated separately from one another in separate dosage forms that are taken by said subject at substantially the same time and that release said components to said subject at substantially the same time. the sequential administration of a combination of an antibody or antigen-binding fragment thereof of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated apart from one another to be separate dosage forms taken by the subject at successive times with a significant time interval between each administration, and where such components are released to the subject at substantially different times; and the sequential administration of a combination of an antibody or antigen-binding fragment thereof of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated together to be a single dosage form that releases the components in a controlled manner, where they are released to the subject at the same and / or different times in a concurrent, sequential, and / or overlapping manner, where each portion may be administered by the same or different routes.

[0191] In another aspect, the present 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 of the present 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 protocols, and stem cell transplantation, wherein the combination therapy provides increased cell killing of tumor cells, i.e., synergism exists between the isolated antibody or antigen-binding fragment and the additional therapy when administered in combination.

[0192] In various embodiments, immunotherapy includes treatments using agonist, antagonist, or blocking antibodies against costimulatory 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; treatments using bispecific T cell engaging antibodies (BiTEs®), such as blinatumomab; biological response modifiers, such as IL-2, IL-7, IL-12, IL-15, IL-21, GM-CSF, STING agonists, and IFN-α, IFN-β, and IFN-β. 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); treatments using TALL-104 cells; and treatments using immunostimulants, such as the Toll-like receptor (TLR) agonists CpG and imiquimod.

[0193] In various embodiments, the additional therapy comprises an antibody that specifically binds to an immune checkpoint protein antigen from the 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, the PD-1 inhibitor used in the combination treatment method is selected from the group consisting of, but not limited to, pembrolizumab (Merck), nivolumab (Bristol-Myers Squibb), cemiplimab (Regeneron), dostallimab (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 dostallimab. In various embodiments, the PD-1 inhibitor is retifanlimab. In various embodiments, about 0.1 mg / kg to about 10 mg / kg of the PD-1 inhibitor is administered. In various embodiments, about 1 mg / kg to about 15 mg / kg of the PD-1 inhibitor is administered.

[0194] A wide range of conventional compounds have been shown to have anti-neoplastic activity.These compounds have been used as pharmaceutical 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 has been effective in treating various 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 function synergistically and allow for the reduction of the dosage of each treatment, thereby reducing the harmful side effects caused by each compound at higher dosages.In other cases, malignancies that are refractory to treatment may respond to the combination treatment of two or more different treatments.

[0195] When the antibodies or antigen-binding fragments disclosed herein are administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such antibodies or antigen-binding fragments may enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent, allowing for a reduction in the dosage of the anti-neoplastic agent, thereby reducing undesirable side effects or restoring the effectiveness of the anti-neoplastic agent in resistant T cells.

[0196] Pharmaceutical compounds that may be used for combination anti-tumor therapy include, by way of example 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, hydroxybenzoates, benzocaine, benzodiazepines ... Urea, 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 These include acetaminophen, 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.

[0197] These chemotherapeutic antitumor compounds, according to their mechanism of action, can be divided into, for example, the following groups: antimetabolites / anticancer drugs, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products, such as antiproliferative / antimitotic drugs, including vinca alkaloids (vinblastine, vincristine, and vinorelbine). agents, microtubule disrupting agents such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ibuprofen, phosphamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparagine-reducing agents) that are systemically L-asparaginase, which metabolizes and eliminates cells that do not have the ability to synthesize their own asparagine; antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazene-dacarbazine (DTIC);Antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and thoron fibrinolytic agents (e.g., tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (brefeldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic 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 (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, geniposide, epirubicin, etoposide, idarubicin, and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0198] In various embodiments, chemotherapy is selected from the group consisting of daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, 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, carbamazepine, cycloheximide ... In addition to ruboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyurea taxanes (e.g., paclitaxel and docetaxel) and / or anthracycline antibiotics, the combination of agents includes, but is not limited to, DA-EPOCH, CHOP, CVP, or FOLFOX.

[0199] In various embodiments, the small molecule kinase inhibitor targeted therapy comprises a small molecule kinase inhibitor selected from the group consisting of a Bruton's tyrosine kinase (BTK) inhibitor, a phosphatidylinositol-3-kinase (PI3K) inhibitor, a SYK inhibitor (e.g., entospletinib), an AKT inhibitor, an mTOR inhibitor, a Src inhibitor, a JAK / STAT inhibitor, a Ras / Raf / MEK / ERK inhibitor, and an Aurora inhibitor (see D'Cruz et al, Expert Opin Pharmacother, 14(6):707-21, 2013).

[0200] In various embodiments, the combination therapy comprises administering the antibody or antigen-binding fragment thereof and one or more additional therapies simultaneously. In various embodiments, the antibody or antigen-binding fragment thereof composition and the one or more additional therapies are administered sequentially, i.e., the antibody or antigen-binding fragment thereof composition is administered either before or after the administration of the one or more additional therapies.

[0201] In various embodiments, the administration of the antibody or antigen-binding fragment thereof composition and the one or more additional therapies is concurrent, i.e., the administration periods of the antibody or antigen-binding fragment thereof composition and the one or more additional therapies overlap with each other.

[0202] In various embodiments, the administration of the antibody or antigen-binding fragment thereof composition and the one or more additional therapies are non-concurrent. For example, in various embodiments, the administration of the antibody or antigen-binding fragment thereof composition is terminated before the administration of the one or more additional therapies. In various embodiments, the administration of the one or more additional therapies is terminated before the administration of the antibody or antigen-binding fragment thereof composition.

[0203] When the antibodies or antigen-binding fragments thereof disclosed herein are administered in combination with one or more additional therapies, either concomitantly or sequentially, such antibodies or antigen-binding fragments thereof may enhance the therapeutic effect of the one or more additional therapies or overcome cellular resistance to the one or more additional therapies, allowing for a reduction in the dosage or duration of the one or more additional therapies, thereby reducing undesirable side effects or restoring the effectiveness of the one or more additional therapies.

[0204] Diagnostic Use In another embodiment, the present invention provides a method for detecting the presence of human CCR8 peptide in a sample in vitro or in vivo, for example, to diagnose a human CCR8-associated disorder. In some methods, this is achieved by contacting the sample to be tested, along with a control sample, with a human sequence antibody or human monoclonal antibody of the present invention, or an antigen-binding portion thereof (or bispecific or multispecific molecule) under conditions that allow the formation of a complex between the antibody and human CCR8. Complex formation is then detected in both samples (e.g., using ELISA), and any statistically significant difference in complex formation between the samples indicates the presence of human CCR8 antigen in the test sample.

[0205] In various embodiments, a method for detecting or confirming the diagnosis of a CCR8-associated disorder in a subject is provided. The method includes contacting a biological sample from the subject with an isolated antibody or antigen-binding fragment thereof of the present invention and detecting binding of the isolated human monoclonal antibody or antigen-binding fragment thereof to the sample. An increase in binding of the isolated human monoclonal antibody or antigen-binding fragment thereof to the sample compared to binding of the isolated human monoclonal antibody or antigen-binding fragment thereof to a control sample detects or confirms the diagnosis of a CCR8-associated disorder in the subject. The control can be a sample from a subject known not to have a CCR8-associated disorder or a standard value. The sample can be any sample, including, but not limited to, tissue from biopsy, autopsy, and pathology specimens. Biological samples also include tissue sections, such as frozen sections taken for histological purposes. Biological samples further include bodily fluids, such as blood, serum, plasma, sputum, and spinal fluid.

[0206] In one embodiment, a kit is provided for detecting CCR8 in a biological sample, such as a blood sample. The kit for detecting a polypeptide typically comprises a human antibody that specifically binds to CCR8, such as any of the antibodies disclosed herein. In some embodiments, the kit comprises an antibody fragment, such as an Fv fragment. For in vivo use, the antibody can be an scFv fragment. In a further embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzyme label).

[0207] In one embodiment, the kit includes instructional materials disclosing how to use the antibody that specifically binds to CCR8. The instructional materials may be written in electronic form (e.g., a computer disk or compact disk) or visual (e.g., a video file). The kit may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, or an appropriate secondary label, such as a secondary antibody). The kit may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0208] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay may vary with the particular format used, a method for detecting CCR8 in a biological sample generally comprises contacting the biological sample with an antibody that specifically reacts with CCR8 under immunologically reactive conditions. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0209] In various embodiments, the antibody or antigen-binding fragment can be labeled or unlabeled for diagnostic purposes. Typically, diagnostic assays require detecting the formation of a complex resulting from the binding of the antibody to CCR8. The antibody can be directly labeled. A variety of labels can be used, including but not limited to radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens). 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 not labeled, the antibody can be used in assays, such as agglutination assays. An unlabeled antibody can also be used in combination with another suitable reagent(s) that can be used to detect the antibody, such as a labeled antibody (e.g., a second antibody) reactive with the first antibody (e.g., an anti-idiotypic antibody or other antibody specific for the unlabeled immunoglobulin) or other suitable reagent (e.g., labeled Protein A).

[0210] The antibodies or antigen-binding fragments provided herein may also be used in methods for detecting a mammal's susceptibility to a particular disease. For example, the method can be used to detect a mammal's susceptibility to a disease that progresses based on the amount of CCR8 present on cells in the mammal and / or the number of CCR8-positive cells. In one embodiment, the present application provides a method for detecting a mammal's susceptibility to tumors. In this embodiment, the sample to be tested is contacted with an antibody or a portion thereof that binds to CCR8 under conditions suitable for the antibody to bind to the sample, and the sample contains cells that express CCR8 in a normal individual. The amount of antibody binding and / or binding is detected, which indicates the individual's susceptibility to tumors, and a higher level of the receptor correlates with an increased individual's susceptibility to tumors.

[0211] In various embodiments, the antibody or antigen-binding fragment is attached to a detectable label (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 a radioactive heavy metal, e.g., an iron chelate, a radioactive chelate of gadolinium or manganese, a positron emitter 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 The binding agent attached to such a moiety may be Tc. The binding agent attached to such a moiety may be used as an imaging agent and administered in an amount effective for diagnostic use to a mammal, for example, a human, and the localization and accumulation of the imaging agent are then detected. The localization and accumulation of the imaging agent may be detected by radioscintigraphy, nuclear magnetic resonance imaging, computed tomography, or positron emission tomography.

[0212] Immunoscintigraphy using antibodies or antigen-binding fragments directed against CCR8 may be used to detect and / or diagnose cancer and the vasculature. For example, 99 technetium, 111 Indium, or 125Iodine-labeled monoclonal antibodies against the CCR8 marker can be effectively used for such imaging. As will be apparent to those skilled in the art, the amount of radioisotope to be administered will depend on the radioisotope. One skilled in the art can easily formulate the amount of imaging agent to be administered based on the specific activity and energy of a given radionuclide used as the active moiety. Typically, 0.1 to 100 millicuries, 1 to 10 millicuries, or 2 to 5 millicuries are administered per dose of imaging agent. Thus, the disclosed compositions useful as imaging agents comprising a targeting moiety conjugated to a radioactive moiety contain 0.1 to 100 millicuries, in some embodiments 1 to 10 millicuries, in some embodiments 2 to 5 millicuries, and in some embodiments 1 to 5 millicuries.

[0213] Antibody-drug conjugates (ADCs) and immunoconjugates Antibody-drug conjugates (ADCs) combine the efficacy of drugs, such as cytotoxic agents, anticancer drugs, and immunosuppressants, with the binding specificity of antibodies. The use of ADCs allows for target-specific delivery of drugs that, when administered as unconjugated drugs, may result in unacceptable levels of toxicity to normal cells. The mechanism of ADCs is to recognize and bind to specific antigens through antibodies, triggering a series of reactions and then entering the cytoplasm through endocytosis, where the highly cytotoxic drug is released from the antibody after degradation by lysosomal enzymes, killing cancer cells. Compared to traditional chemotherapy, which indiscriminately damages both cancer cells and normal tissues, targeted drug delivery allows drugs to act directly on cancer cells and reduces damage to normal cells.

[0214] The present application further provides ADCs comprising the novel antibodies and antigen-binding fragments of the invention linked to a second molecule selected from the group consisting of a cytotoxic agent, an anti-cancer drug, or an immunosuppressant.

[0215] Cytotoxic compounds contemplated for use in antibody-drug conjugates are directed against a variety of essential cellular targets, such as microtubules (maytansinoids, auristatins, taxanes; U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333,410; 6,441,163; 6,340,701; : Specification No. 6,372,738; Specification No. 6,436,931; Specification No. 6,596,757: Specification No. 7.276,497; Specification No. 7,301,019 Specification No. 7,303,749; Specification No. 7,368,565; Specification No. 7,473,796; Specification No. 7,585,857; Specification No. 7,598,290 Nos. 7,495,114; 7,601,354; U.S. Patent Application Nos. 20100092495, 20100129314, 20090274713, 20090076263, and 20080171865) and DNA (calicheamicin, doxorubicin, CC-106 5 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).

[0216] The present application further provides immunoconjugates or fusion proteins comprising the antibodies or antigen-binding fragments of the present invention conjugated (or linked) directly or indirectly to an effector molecule. In this context, the terms "conjugated" or "linked" refer to the joining of two polypeptides into a single, continuous polypeptide molecule. Linking can be achieved by either chemical or recombinant means. In one embodiment, linking is chemical, where reaction between the antibody moiety and the effector molecule generates a covalent bond between the two molecules to form a single molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the effector molecule. In various embodiments, the antibody or antigen-binding fragment is conjugated to the effector molecule. In other embodiments, the antibody or antigen-binding fragment conjugated to the effector molecule is further conjugated to a lipid, protein, or peptide to increase its half-life in the body. Thus, in various embodiments, the antibodies of the present disclosure can be used to deliver various effector molecules.

[0217] The effector molecule can be a detectable label, an immunotoxin, a cytokine, a chemokine, a therapeutic agent, or a chemotherapeutic agent.

[0218] Specific non-limiting examples of immunotoxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, e.g., PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, cholera toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells.

[0219] "Cytokines" are a class of proteins or peptides released by one cell population that act on another cell as intercellular mediators. Cytokines can act as immunomodulators. Examples of cytokines include lymphokines, monokines, growth factors, and traditional polypeptide hormones. Thus, embodiments include interferons (e.g., IFN-α, IFN-β, and IFN-γ); tumor necrosis factor superfamily (TNFSF) members; human growth hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle-stimulating hormone (FSH); thyroid-stimulating hormone (TSH); luteinizing hormone (LH); hepatic growth factors; prostaglandins, fibroblast growth factors; prolactin; placental lactogen, OB protein; TNF-α; TNF-β; integrins; thrombopoietin (TPO); nerve growth factors, e.g., NGF-β; platelet growth factors; TGF Cytokines include cytokines such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (IL-1 through 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, as well as several others (Sharpe et al., Nat Immunol, 8:239-45, 2007). These cytokines include proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines.

[0220] In various embodiments, the effector molecule is selected from the list provided in Table 4. Each associated reference is incorporated herein by reference for purposes of identifying the referenced tumor marker. TIFF2025534340000007.tif214170TIFF2025534340000008.tif201170 TIFF2025534340000009.tif73170

[0221] In various embodiments, the effector molecule is selected from the list provided in Table 5. These targets may also be applicable for cancer targeting. TIFF2025534340000010.tif99170TIFF2025534340000011.tif181170

[0222] Chemokines can also be conjugated to the antibodies disclosed herein. Chemokines are small (roughly about 4 to about 14 kDa) molecules that primarily act as chemoattractants and activators of specific white blood cell subtypes. D Chemokine receptors (CCRs) are a superfamily of inducible and secreted pro-inflammatory cytokines. Chemokine production is induced by inflammatory cytokines, growth factors, and pathogenic stimuli. Chemokine proteins are divided into subfamilies (alpha, beta, and delta) 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 adjacent to the amino terminus. To date, more than 50 chemokines have been discovered, and there are at least 18 human seven-transmembrane domain (7TM) chemokine receptors. Chemokines used include, but are not limited to, RANTES, MCAF, MCP-1, and fractalkine.

[0223] Therapeutic agent can be a chemotherapeutic agent.Those skilled in the art can easily identify the chemotherapeutic agent used (see, for example, Slapak and Kufe, Principles of Cancer Therapy Chapter 86, Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.17, Abeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc.; Baltzer L., Berkery R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Useful chemotherapeutic agents for the preparation of immunoconjugates include auristatins, dolastatins, MMAE, MMAF, AFP, DM1, AEB, doxorubicin, daunorubicin, methotrexate, melphalan, chlorambucil, vinca alkaloids, 5-fluorouridine, mitomycin-C, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitroso urea, 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, as well as their salts, solvents, and derivatives. In various embodiments, the chemotherapeutic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof, as well as pharmaceutical salts or solvates thereof.Exemplary auristatin derivatives include DM1, AEB, AEVB, AFP, MMAF, and MMAE. The synthesis and structure of auristatin E and its derivatives, as well as linkers, 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-dolaisorownine-dolaproine-phenylalanine (MMAF) or monomethylauristatin E (MMAE). In various embodiments, the therapeutic agent is a maytansinoid or a maytansinol analog. In various embodiments, the maytansinoid is DM1.

[0224] Effector molecules can be linked to antibodies or antigen-binding fragments of the present invention using any number of means known to those skilled in the art. Both covalent and noncovalent attachment means can be used. The procedure for attaching an effector molecule to an antibody varies depending on the chemical structure of the effector molecule. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (--NH), or sulfhydryl (--SH) groups, which are available for reaction with suitable functional groups on the antibody to result in attachment of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization may involve the attachment of any of a number of linker molecules, such as those available from Pierce Chemical Company, Rockford, Ill. The linker can be any molecule used to conjugate an antibody to an effector molecule. The linker is capable of forming covalent bonds to 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 or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecule are polypeptides, the linkers may be attached to the constituent amino acids through their side groups (e.g., to cysteine ​​through a disulfide linkage) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0225] In some situations, it is desirable to release the effector molecule from the antibody when the immunoconjugate reaches its target site. Thus, in these situations, the immunoconjugate includes a cleavable linkage near the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or near the target site.

[0226] Procedures for conjugating effector molecules to antibodies have been previously described and are within the purview of those skilled in the art. For example, procedures for preparing enzymatically active polypeptides of immunotoxins are described in WO 84 / 03508 and WO 85 / 03508, which are incorporated herein by reference for 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. Pat. No. 5,057,313; Shih Cancer Res. 51:4192, WO 02 / 088172; U.S. Pat. No. 6,884,869; WO 2005 / 081711; U.S. Patent Application Publication No. 2003-0130189 A; and U.S. Patent Application No. 20080305044, each of which is incorporated herein by reference for purposes of teaching such techniques.

[0227] The immunoconjugates of the invention retain the immunoreactivity of the antibody or antigen-binding fragment, e.g., the antibody or antigen-binding fragment has about the same, or only slightly reduced, ability to bind to the antigen after conjugation as before conjugation.

[0228] bispecific molecule Bispecific antibodies are antibodies containing two distinct antigen-binding sites of a monoclonal antibody that can bind to two different antigens or two distinct sites on a single antigen. In addition to simultaneously blocking two different signaling pathways and thereby enhancing tumor cell killing, bispecific antibodies can also potentially increase binding specificity by interacting with two different cell surface antigens instead of one. They are now considered the next generation of effective molecules for cancer therapy. They can minimize regulatory and commercial challenges resulting from the administration of multiple therapeutic molecules. They also have the potential for novel activities not present in mixtures of parent antibodies. Several bispecific antibodies are commercially available, and many are in clinical development.

[0229] In another aspect, the present invention features bispecific molecules comprising an anti-CCR8 antibody of the present invention, or an antigen-binding fragment thereof. The antibody of the present invention, or an antigen-binding fragment thereof, can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., a ligand for another antibody or receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the present invention may, 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 a bispecific molecule of the present invention, the antibody of the present invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, resulting in a bispecific molecule. In various embodiments, the invention comprises bispecific molecules capable of binding to both FcγR- or FcαR-expressing effector cells (e.g., monocytes, macrophages, or polymorphonuclear cells (PMNs)) and PD-expressing target cells. In such embodiments, the bispecific molecules target CCR8-expressing cells to effector cells and trigger Fc receptor-mediated effector cell activity, such as phagocytosis of CCR8-expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or superoxide anion generation. Methods for preparing bispecific molecules of the invention are well known in the art.

[0230] In various embodiments, the second functional molecule is an antibody, antibody fragment, or protein or peptide that binds to an immune checkpoint protein antigen present on the surface of immune cells. 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 may comprise an antibody against an immune checkpoint protein antigen present on the surface of tumor cells, selected from the group consisting of, but not limited to, PD-L1, B7-H3, and B7-H4.

[0231] In various embodiments of the invention, the antibody or antigen-binding fragment has the heavy chain sequence of SEQ ID NO: 93: (SEQ ID NO: 93) and a light chain sequence selected from the group consisting of SEQ ID NO: 91 and SEQ ID NO: 92 and a CCR8 / CTLA4 bispecific antibody comprising:

[0232] Polynucleotide and antibody expression The present application further 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, various nucleic acid sequences encode each antibody amino acid sequence. The present application further provides a polynucleotide that hybridizes to a polynucleotide encoding an antibody that binds to human CCR8, for example, under stringent or lower stringency hybridization conditions as defined herein.

[0233] Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6xSSC at about 45°C followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65°C, highly stringent conditions, such as hybridization to filter-bound DNA in 6xSSC at about 45°C followed by one or more washes in 0.1xSSC / 0.2% SDS at about 60°C, or any other stringent hybridization conditions known to those of skill in the art (see, for example, 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, pages 6.3.1-6.3.6 and 2.10.3).

[0234] Polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides may be determined, by any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which briefly involves synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating the oligonucleotides, and then amplifying the ligated oligonucleotides by PCR. In one embodiment, the codons used include those typical for humans or mice (see, e.g., Nakamura, Y., Nucleic Acids Res. 28:292 (2000)).

[0235] Polynucleotides encoding antibodies may also be generated from nucleic acid from a suitable source. If a clone containing nucleic acid encoding a particular antibody is not available but the sequence of the antibody molecule is known, nucleic acid encoding an immunoglobulin may 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 that expresses the antibody, e.g., a hybridoma cell selected to express the antibody, or isolated nucleic acid, preferably polyA+ RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence, e.g., to identify a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

[0236] The present invention is also directed to host cells that express the CCR8 and / or anti-CCR8 antibodies of the present invention. A variety of host expression systems known in the art can be used to express the antibodies of the present invention, including prokaryotic (bacterial) and eukaryotic expression systems (e.g., yeast, baculovirus, plant, mammalian and other animal cells, transgenic animals, and hybridoma cells), as well as phage display expression systems.

[0237] The antibodies of the present invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transformed, transduced, or infected 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 may be independently expressed from different operably linked promoters in one vector, or alternatively, the heavy and light chains may be independently expressed from different operably linked promoters in two vectors, one expressing the heavy chain and one expressing the light chain. Optionally, the heavy and light chains may be expressed in different host cells.

[0238] Additionally, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody light chain and / or heavy chain from the host cell. The antibody light chain and / or heavy chain gene 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 medium in which the host cells are cultured, from which it can be recovered or purified.

[0239] Isolated DNA encoding an HCVR can be converted into a full-length heavy chain gene by operably linking the HCVR-encoding DNA to another DNA molecule encoding a heavy chain constant region. The sequences of heavy chain constant region genes of other mammals, in addition to humans, are known in the art. DNA fragments encompassing these regions can be obtained, for example, by standard PCR amplification. The heavy chain constant region can be a constant region of any type (e.g., IgG, IgA, IgE, IgM, or IgD), class (e.g., IgG1, IgG2, IgG3, and IgG4), or subclass, as well as any allotypic variant thereof, as described in Kabat (supra).

[0240] The isolated DNA encoding the LCVR region may be converted into a full-length light chain gene (and a Fab light chain gene) by operably linking the LCVR-encoding DNA to another DNA molecule encoding a light chain constant region. The sequences of light chain constant region genes of other mammals, as well as humans, are known in the art. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0241] In addition to the antibody heavy and / or light chain genes, the recombinant expression vectors of the invention contain regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) as needed to control the transcription or translation of the antibody chain genes. The design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed and the level of protein expression desired. Preferred regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from viral elements that direct high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and / or polyoma virus.

[0242] Additionally, the recombinant expression vectors of the present invention may contain additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and one or more selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, in host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (dhfr) gene (for use in dhfr-negative host cells with methotrexate selection / amplification), the neo gene (for G418 selection), and glutamine synthetase (GS) in GS-negative cell lines (e.g., NSO) for selection / amplification.

[0243] For expression of the light and / or heavy chains, expression vectors encoding the heavy and / or light chains are introduced into host cells by standard techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, transduction, and infection. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, eukaryotic cells are preferred, and most preferably mammalian host cells, because such cells are more likely to assemble and secrete a properly folded and immunologically active antibody. Preferred mammalian host cells for expressing recombinant antibodies of the invention include Chinese hamster ovary (CHO) cells (including dhfr-negative CHO cells, such as those described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-20, 1980, used with a DHFR selection marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol. 159:601-21, 1982), NSO myeloma cells, COS cells, and SP2 / 0 cells. When a recombinant expression vector encoding an 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 for expression of the antibody in the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown under appropriate conditions known in the art. Antibodies can be recovered from the host cell and / or culture medium using standard purification methods.

[0244] The present invention provides host cells comprising the nucleic acid molecules of the present invention. Preferably, the host cells of the present invention comprise one or more vectors or constructs comprising the nucleic acid molecules of the present invention. For example, the host cells of the present invention are cells into which a vector of the present invention has been introduced, wherein the vector comprises a polynucleotide encoding the LCVR of an antibody of the present invention and / or a polynucleotide encoding an HCVR of the present invention. The present invention also provides host cells into which two vectors of the present invention have been introduced; one vector comprises a polynucleotide encoding the LCVR of an 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 a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element) to drive high levels of gene transcription.

[0245] Once expressed, 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, and gel electrophoresis. Standard procedures for the purification of therapeutic antibodies are described, for example, in the article entitled "Current Therapeutic Antibody Production and Process Optimization" by Feng L1, Joe X. Zhou, Xiaoming Yang, Tim Tressel, and Brian Lee (BioProcessing Journal, September / October 2005), which is incorporated by reference in its entirety for purposes of teaching the purification of therapeutic antibodies. Additionally, standard techniques for removing viruses from recombinantly 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)). It is known that the effectiveness of filtration to remove viruses from therapeutic antibody preparations depends, at least in part, on the protein and / or antibody concentration in the solution being filtered. The purification process for antibodies of the present invention may include a filtration step to remove viruses from the mainstream of one or more chromatography operations. Preferably, prior to filtration through a pharmaceutical-grade nanofilter to remove viruses, the chromatographic mainstream containing the antibody of the present invention is diluted or concentrated to give a total protein and / or antibody concentration of about 1 g / L to about 3 g / L. Even more preferably, the nanofilter is a DV20 nanofilter (e.g., Pall Corporation; East Hills, NY).For pharmaceutical uses, substantially pure immunoglobulins of at least about 90%, about 92%, about 94%, or about 96% homogeneity are preferred, and about 98 to about 99% or more homogeneity are most preferred. Once purified, partially or to homogeneity as desired, the sterile antibodies may then be used therapeutically, as directed herein.

[0246] In view of the above discussion, the present invention is further directed to antibodies obtainable by a method comprising culturing host cells, including but not limited to mammalian, plant, bacterial, transgenic animal, or transgenic plant cells, transformed with a polynucleotide or vector comprising a nucleic acid molecule encoding an antibody of the invention to express the nucleic acid, and optionally recovering the antibody from the host cell culture medium.

[0247] In certain embodiments, the present application provides hybridoma cell lines as well as monoclonal antibodies produced by these hybridoma cell lines. The disclosed cell lines have uses other than producing monoclonal antibodies. For example, the cell lines can be fused with other cells (e.g., preferably drug-marked human myeloma, mouse myeloma, human-mouse heteromyeloma, or human lymphoblastoid cells) to produce additional hybridomas, thereby providing for the transfer of genes encoding monoclonal antibodies. Additionally, 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., by transfer into other cells using any suitable technique (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter, U.S. Pat. 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 cell lines, and cDNA clones encoding anti-CCR8 immunoglobulin chains can be isolated. Thus, nucleic acids encoding the antibody heavy and / or light chains, or portions thereof, can be obtained, and the nucleic acids can be used according to recombinant DNA technology to produce specific immunoglobulins, immunoglobulin chains, or variants thereof (e.g., humanized immunoglobulins) in various host T cells or in in vitro translation systems. For example, nucleic acids containing cDNAs or derivatives thereof encoding variants, such as humanized immunoglobulins or immunoglobulin chains, can be placed into a suitable prokaryotic or eukaryotic vector (e.g., an expression vector) and introduced into a suitable host T cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), so that the nucleic acid is operably linked to one or more expression control elements (e.g., integrated into a vector or into the host T cell genome).For production, host T cells can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), whereby the encoded polypeptide is produced. If desired, the encoded protein can be recovered and / or isolated (e.g., from the host T cells or culture medium). Production methods are understood to encompass expression in host T cells of transgenic animals (see, e.g., WO 92 / 03918, GenPharm International, published March 19, 1992) (incorporated by reference in its entirety).

[0248] Host cells can also be used to produce portions or fragments of intact antibodies, such as Fab fragments or scFv molecules, by conventional techniques. For example, it may be desirable to transfect host cells with DNA encoding either the light chain or the heavy chain of an antibody of the invention. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to human CCR8. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention.

[0249] Methods for the expression of single chain antibodies from bacteria, such as E. coli, and / or their refolding into a suitable active form, including single chain antibodies, have been described and are well known and are applicable to the antibodies disclosed herein (see, e.g., 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).

[0250] In many cases, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants and subsequent refolding. During the solubilization step, a reducing agent must be present to separate disulfide bonds, as is well known in the art. An exemplary buffer containing a reducing agent is 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). As described in Saxena et al., Biochemistry 9:5015-5021, 1970, incorporated herein by reference, and in particular as described in Buchner et al., supra, reoxidation of disulfide bonds can occur in the presence of reduced and oxidized forms of low-molecular-weight thiol reagents.

[0251] Renaturation is typically achieved by dilution (e.g., 100-fold) of the denatured and reduced protein 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.

[0252] As a modification of the two-chain antibody purification protocol, the heavy and light chain regions are solubilized and reduced separately and then combined in a refolding solution. Exemplary yields are obtained when these two proteins are mixed in a molar ratio such that one protein is not more than 5-fold molar excess over the other. Excess oxidized glutathione or other oxidizing low-molecular-weight compounds can be added to the refolding solution after redox shuffling is complete.

[0253] 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 achieved 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 & Merrifield, *The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., *J. Am. Chem. Soc.* 85: 2149-2156, 1963; and Stewart et al., *Solid Phase Peptide Synthesis,* 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Proteins of greater length may also be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods for forming peptide bonds by activation of the carboxyl terminus (eg, by use of the coupling reagent N,N'-dicyclohexylcarbodiimide) are well known in the art.

[0254] The following examples are given to more fully illustrate the present invention, but are not to be construed as limiting the scope thereof. [Example]

[0255] Example 1 Generation of a murine monoclonal antibody that specifically targets human CCR8 A human CCR8 expression plasmid was constructed, and hCCR8-overexpressing CHO-K1 cells were generated and used as an immunogen to generate anti-hCCR8 monoclonal antibodies. BALB / c, C57BL / 6, A / J, and SJL mice were immunized every two weeks (a total of six or more immunizations) with plasmid DNA harboring hCCR8, hCCR8-expressing cells, or membrane protein. The immunogen preparation was injected subcutaneously or intraperitoneally. Serum from immunized mice was collected and tested by flow cytometry on both hCCR8-expressing CHO-K1 cells and parental CHO-K1 cells. Mice with significant levels (titers) of antibodies against hCCR8 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 (American Type Culture Collection, ATCC). Hybridoma cells were obtained using an electrofusion method.

[0256] Hybridoma supernatants were screened for antigen binding by flow cytometry and / or cell-based ELISA. In cell-based ELISA, both CHO-K1 cells and hCCR8-expressing cells were incubated with the supernatant. The cells were washed with PBS and then incubated with goat anti-mouse IgG-HRP secondary antibody. TMB was added after washing the cells. Absorbance was read at 450 nm using a plate reader. The primary hybridoma clone with the highest OD450 ratio for hCCR8-expressing cells to CHO-K1 cells was selected for subcloning by limiting dilution.

[0257] Subclone supernatants were tested by cell-based ELISA to confirm the presence of antibodies that specifically bind to hCCR8. Only subclones with an OD450 ratio higher than 1.9 were selected for further analysis.

[0258] Human CCL1 (hCCL1) is the primary ligand for CCR8. Subclone supernatants were tested in a cell-based CCR8 CHO-K1 β-arrestin bioassay (Eurofins) to determine the antagonist activity of anti-CCR8 antibodies, i.e., blockage of hCCL1-induced CCR8 downstream signaling. Briefly, approximately 10,000 cells were seeded into wells of a 96-well plate and incubated at 37°C and 5% CO2 for 24–48 hours. Supernatants were added and incubated for 30 minutes to allow the antibody to bind to hCCR8 on the cells. After adding 2 nM hCCL1, the plate was incubated for 90 minutes at 37°C and 5% CO2 to stimulate β-arrestin production. Finally, detection solution was added, and the plate was incubated for 1 hour in the dark at room temperature. The plate was read using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Subclones that showed at least 50% inhibition of β-arrestin production in the assay were selected for sequencing (see Table 3).

[0259] Total RNA was isolated from hybridoma cells according to the manufacturer's instructions (Vazyme). Total RNA was then reverse transcribed into cDNA using either isotype-specific antisense primers or universal primers according to the SMARTScribe Reverse Transcriptase (TaKaRa) technical manual. Heavy and light chain antibody fragments were amplified according to ProBio's rapid amplification of cDNA ends (RACE) standard operating procedure. The amplified antibody fragments were separately cloned into standard cloning vectors. Colony PCR was performed to screen for clones with the correct insert 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 set forth in SEQ ID NOS: 3-25 and 35-77.

[0260] Example 2 Anti-hCCR8 antibodies block hCCL1 binding to hCCR8 The murine antibodies 41E1C2A5, 46A5C4B1, and 80E4D1F11, as well as the chimeric antibodies (human IgG1), 504E12D8D12, 516D7D12, 525F2F3F11, and 531B9B1C9, were evaluated in the CCR8 CHO-K1 β-arrestin assay to determine their antagonist activity in blocking hCCL1-induced activation of CCR8 signaling. Data were plotted using GraphPad Prism version 9 (San Diego, CA), and IC50 values ​​were determined by nonlinear regression curve fitting and are summarized in Table 6. TIFF2025534340000012.tif59170

[0261] Example 3 Anti-hCCR8 antibodies do not bind to human CCR4 The mouse antibodies 41E1C2A5, 46A5C4B1, and 80E4D1F11, as well as the chimeric antibodies 504E12D8D12, 516D7D12, 525F2F3F11, and 531B9B1C9, specifically bound to hCCR8-expressing CHO-K1 cells and blocked the binding of hCCL1 to hCCR8, indicating that they were CCR8-specific antibodies. The antibodies were further screened for binding to human CCR4 (hCCR4) by flow cytometry. The antibodies were incubated with CHO-K1 cells and hCCR4-expressing CHO-K1 cells for 60 minutes at 4°C. The cells were thoroughly washed 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 the anti-hCCR8 antibody did not bind to hCCR4. TIFF2025534340000013.tif55170

[0262] Example 4 ADCC activity of anti-hCCR8 chimeric antibodies The ADCC activity of the anti-hCCR8 chimeric antibody was measured using a reporter bioassay (BPS Bioscience). Briefly, hCCR8-expressing CHO-K1 cells were seeded into a 96-well assay plate at a density of 12,000 cells / well in 100 μl of assay medium and incubated overnight at 37°C, 5% CO2. After discarding the medium, 60 μl of serially diluted anti-CCR8 antibody was added and incubated for 1 hour. 40 μl of ADCC / NFAT-reporter Jurkat cells (approximately 75,000 cells) was added. After 5–6 hours of incubation, 100 μl of luciferase substrate was added to each well, and the plate was gently shaken for 15 minutes to 1 hour at room temperature. Luminescence was measured using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Each treatment was performed in triplicate. Data analysis was performed using GraphPad Prism (version 9) to determine EC50.

[0263] The results showed that the mouse-human chimeric (human IgG1) antibodies 41E1C2A5, 504E12D8D12 and 531B9B1C9 had potent ADCC activity (Table 8). TIFF2025534340000014.tif33170

[0264] Example 5 Humanization of mouse anti-hCCR8 antibody Humanization of mouse anti-hCCR8 mAb 41E1C2A5 The murine anti-hCCR8 mAb 41E1C2A5 was humanized by CDR grafting and backmutation. The structure of the parent antibody was modeled using a computer-assisted homology modeling program (MOE). CDRs were grafted into the most closely related human germline framework based on sequence similarity. Human germline IGHV3-73*01 was selected for the heavy chain, and IGKV2-28*01 for the light chain. To preserve the antibody structure, some residues in the heavy and light chain frameworks were backmutated to the corresponding residues in the murine antibody.

[0265] Heavy and light chains were designed and paired with each other to produce antibodies by transient expression for affinity ranking by flow cytometry. Briefly, 50 μl of hCCR8-expressing CHO-K1 cells (1×10 5 Cells (number of cells / well) were loaded onto a 96-well plate. A 3-fold dilution series (11 points) of each antibody was prepared at a final starting concentration of 45 μg / ml. The antibodies were incubated with the cells for 1 hour at 4°C. After extensive washing, the cells were incubated with Alexa Fluor 647-conjugated goat anti-human IgG (H+L) antibody. Geometric means were measured by flow cytometry. A sigmoidal curve was generated using nonlinear regression curve fit (4PL) in GraphPad Prism to generate EC50 values. 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) have binding affinities comparable to the chimeric 41E1C2A5 mAb.

[0266] Humanization of mouse anti-hCCR8 mAb 504E12D8D12 The murine anti-hCCR8 mAb 504E12D8D12 was humanized by grafting the CDRs into the frameworks of the closest human germline, IGHV3-73*01 for the heavy chain and IGKV2-18*01 for the light chain. Back mutations were made in the framework sequences. There is a potential deamidation motif NG in the light chain CDR1 of 504E12D8D12. Mutations N33Q and G34A were made to eliminate potential deamidation problems. The heavy and light chains were designed and paired to produce 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.

[0267] Example 6 Humanized antibodies block hCCR8-mediated calcium flux The humanized antibodies were tested using the FLIPR calcium flux assay to assess their antagonist activity in blocking hCCL1-induced hCCR8-mediated calcium flux. Briefly, hCCR8-expressing CHO-K1 cells (ProBio) were seeded onto a 384-well assay plate and incubated at 37°C, 5% CO2 for 16–20 hours. After incubation, the plate was placed at room temperature. A dye loading solution (FLIPR Calcium 4 assay kit, Molecular Devices) was prepared using GPCR buffer, and 20 μl was transferred into each well. Serial dilutions of the antibody were prepared, and 10 μl was added. After 1 hour of incubation at 37°C, 5% CO2, hCCL1 was added to each well to a final concentration equal to the EC80. Fluorescence signals were monitored using the FLIPR Tetra system. Anti-hCCR8 antibodies (Reference Ab #1 and Reference Ab #2) described in the literature were also tested in the assay. Data were recorded and analyzed using the ScreenWorks (version 3.1) program. A sigmoidal curve was generated using nonlinear regression curve fit (4PL) in GraphPad Prism to calculate IC50.

[0268] The dose response curves for humanized antibodies 41E1C2A5-HC3+LC4 and 41E1C2A5-HC4+LC2 are shown in FIG. 1 and the IC50 values ​​are summarized in Table 9. TIFF2025534340000015.tif39170

[0269] The dose-response curves for humanized antibodies 504E12D8D12-HC1+LC1 and 504E12D8D12-HC1+LC1(G34A) are shown in Figure 2, and the IC50 values ​​are summarized in Table 10. 504E12D8D12-HC1+LC1 and 504E12D8D12-HC1+LC1(G34A) had stronger or similar antagonist activity compared to the reference antibody. TIFF2025534340000016.tif48170

[0270] Example 7 Humanized antibodies bind to hCCR8 with high affinity The humanized antibodies 504E12D8D12-HC1+LC1 and 504E12D8D12-HC1+LC1(G34A) were tested by kinetic exclusion assay (KinExA, Sapidyne Instruments) to determine their binding affinity to hCCR8-overexpressing cells. Briefly, polystyrene particles (#442178, Sapidyne Instruments) were coated with goat anti-human IgG Fc-specific Fab fragment (#109-007-008, Jackson ImmunoResearch Lab) according to the manufacturer's protocol. The antibodies were incubated with serial dilutions of hCCR8-expressing HEK293 cells overnight at room temperature with gentle rotation to achieve equilibrium. The final antibody concentrations were adjusted to 0.05 nM and 1 nM, respectively. The supernatant was collected and run on a KinExA 3200 (Sapidyne Instruments), and the antibody was captured by coated polystyrene particles. The captured antibody was detected with Alexa Fluor 647-conjugated goat anti-human IgG (H+L) antibody (#109-605-003, Jackson ImmunoResearch Lab). Data was recorded and analyzed using KinExA Pro software (version 4.3.20).

[0271] Humanized antibodies 504E12D8D12-HC1+LC1 and 504E12D8D12-HC1+LC1(G34A) were found to have high binding affinity for hCCR8 (Table 11). TIFF2025534340000017.tif33170

[0272] Example 8 Bispecific antibodies targeting CCR8 and CTLA-4 Construction of a bispecific antibody targeting CCR8 and CTLA-4 A heavy chain designated 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-HC1 (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 conjugated with 504E12D8D12-LC1(G34A) (SEQ ID NO: 92) to produce bispecific antibody FP578-01. FP578-HC was also conjugated with 504E12D8D12-LC1 (SEQ ID NO: 91) to produce bispecific antibody FP578-02.

[0273] Bispecific antibodies can bind to CTLA-4 while also binding to CCR8 Bispecific antibodies FP578-01 and FP578-02 and anti-hCCR8 antibodies 504E12D8D12-HC1+LC1(G34A) and 504E12D8D12-HC1+LC1 were incubated with hCCR8-expressing HEK293 cells for 1 hour at 4° C. The cells were washed and then incubated with biotinylated recombinant human CTLA-4-Fc chimera (#786704, BioLegend) for 1 hour at 4° C. After extensive washing of the cells, CTLA-4 was detected by flow cytometry with phycoerythrin (PE)-conjugated streptavidin (#405203, BioLegend).

[0274] The results demonstrate that both bispecific antibodies FP578-01 and FP578-02 can bind to CTLA-4 while binding in parallel to hCCR8 on hCCR8-expressing HEK293 cells. No specific binding of anti-hCCR8 antibodies to CTLA-4 was observed (Figure 3).

[0275] The bispecific antibody has high binding affinity to hCCR8 The affinities of the bispecific antibodies FP578-01 and FP578-02 were determined by kinetic exclusion assay as described above. K D The data showed that the α- and β-glucan-containing compounds were equivalent (Table 12). TIFF2025534340000018.tif32170

[0276] Bispecific antibodies block hCCL1 binding to hCCR8 The bispecific antibodies FP578-01 and FP578-02 were tested in the cell-based CCR8 CHO-K1 β-arrestin assay as described above to determine their antagonist activity. The data showed that the potency of FP578-01 and FP578-02 was comparable to that of anti-hCCR8 antibodies in blocking hCCL1-induced hCCR8 downstream signaling (Table 13). TIFF2025534340000019.tif29170

[0277] All of the articles and methods disclosed and claimed in this application can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the invention. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety for all purposes to the same 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 may suitably be practiced in the absence of any element not specifically disclosed herein. Therefore, while the present invention has been particularly disclosed by preferred embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be undertaken by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

[0278] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and one-letter codes for amino acids, as defined in 37 CFR 1.822. SEQ ID NO: 1 is the amino acid sequence comprising human CCR8. SEQ ID NO: 2 is the amino acid sequence containing human CCL1. SEQ ID NOs: 3, 7, and 9 are the amino acid sequences of heavy chain CDR1 in monoclonal antibodies that specifically bind to CCR8. SEQ ID NOs: 4, 8, and 10 are the amino acid sequences of heavy chain CDR2 in monoclonal antibodies that specifically bind to CCR8. SEQ ID NOs: 5, 6, and 11 are the amino acid sequences of heavy chain CDR3 in monoclonal antibodies that specifically bind to CCR8. SEQ ID NOs: 12 and 15 are the amino acid sequences of the light chain CDR1 in a monoclonal antibody that specifically binds to CCR8. SEQ ID NOs: 13 and 16 are the amino acid sequences of the light chain CDR2 in a monoclonal antibody that specifically binds to CCR8. SEQ ID NOs: 14 and 17 are the amino acid sequences of the light chain CDR3 in a monoclonal antibody that specifically binds to CCR8. SEQ ID NOs: 18, 20, 22, and 24 are the amino acid sequences of the heavy chain variable regions of mouse monoclonal antibodies that specifically bind to CCR8. SEQ ID NOs: 19, 21, 23, and 25 are the amino acid sequences of the light chain variable regions of mouse monoclonal antibodies that specifically bind to CCR8. SEQ ID NOs: 26, 28, and 30 are the amino acid sequences of the heavy chains of mouse-human chimeric antibodies that specifically bind to CCR8. SEQ ID NOs: 27, 29, and 31 are the amino acid sequences of the light chains of mouse-human chimeric antibodies that specifically bind to CCR8. SEQ ID NO: 32 is the amino acid sequence of the light chain constant region amino acid sequence. SEQ ID NO: 33 is the amino acid sequence of the light chain constant region amino acid sequence. SEQ ID NO: 34 is the amino acid sequence of the heavy chain constant region amino acid sequence. SEQ ID NOs: 35 to 38 are the amino acid sequences of heavy chain CDR2 in monoclonal antibodies that specifically bind to CCR8. SEQ ID NOs: 39 to 45 are the amino acid sequences of the heavy chain CDR3 in the monoclonal antibody that specifically binds to CCR8. SEQ ID NOs: 46 to 47 are the amino acid sequences of the light chain CDR1 in a monoclonal antibody that specifically binds to CCR8. SEQ ID NOs: 48 to 49 are the amino acid sequences of the light chain CDR2 in a monoclonal antibody that specifically binds to CCR8. SEQ ID NOs: 50 to 52 are the amino acid sequences of the light chain CDR3 in a monoclonal antibody that specifically binds to CCR8. SEQ ID NOs: 53 to 67 are amino acid sequences of the heavy chain variable regions of mouse monoclonal antibodies that specifically bind to CCR8. SEQ ID NOs: 68 to 77 are the amino acid sequences of the light chain variable regions of mouse monoclonal antibodies that specifically bind to CCR8. SEQ ID NOs: 78, 80, 82 and 84 are the amino acid sequences of the heavy chains of mouse-human chimeric antibodies that specifically bind to CCR8. SEQ ID NOs: 79, 81, 83 and 85 are the amino acid sequences of the light chains of mouse-human chimeric antibodies that specifically bind to CCR8. SEQ ID NOs: 86, 88 and 90 are the amino acid sequences of the heavy chains of humanized antibodies that specifically bind to CCR8. SEQ ID NOs: 87, 89, 91 and 92 are the amino acid sequences of the light chains of humanized antibodies that specifically bind to CCR8. SEQ ID NO: 93 is the amino acid sequence of the heavy chain of a CCR8 / CTLA4 bispecific antibody.

[0279] Sequence Listing SEQ ID NO: 1 - Amino acid sequence of CCR8 MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASE DGVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL SEQ ID NO:2 - Amino acid sequence of CCL1 KSMQVPFSRCCFSFAEQEIPLRAILCYRNTSSICSNEGLIFKLKRGKEACALDTVGWVQRHRKMLRHCPSKRK SEQ ID NO: 3 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR1 AYAMN SEQ ID NO: 4 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2 RIRSKSNNYATYYADSVKD SEQ ID NO: 5 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 GGTYGSSSYFDY SEQ ID NO: 6 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 GGTYGSTSYFDY SEQ ID NO: 7 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR1 TYAMN SEQ ID NO: 8 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2 RIRSKSNNYATYYADSVKA SEQ ID NO: 9 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR1 DYNMD SEQ ID NO: 10 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2 AINPNNGGTGYTQKFKG SEQ ID NO: 11 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 RGVYMFAY SEQ ID NO: 12 - Amino acid sequence of mouse monoclonal antibody light chain CDR1 RSSKSLLHSNGNTYLY SEQ ID NO: 13 - Amino acid sequence of mouse monoclonal antibody light chain CDR2 RMSNLAS SEQ ID NO: 14 - Amino acid sequence of mouse monoclonal antibody light chain CDR3 MQHLEYPFT SEQ ID NO: 15 - Amino acid sequence of mouse monoclonal antibody light chain CDR1 KSSQSLLHSDGKTYLN SEQ ID NO: 16 - Amino acid sequence of mouse monoclonal antibody light chain CDR2 LVSKLDS SEQ ID NO: 17 - Amino acid sequence of mouse monoclonal antibody light chain CDR3 WQGTHFPYT SEQ ID NO: 18 - Amino acid sequence of the heavy chain variable region of a mouse monoclonal antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYFCVRGGTYGSSSYFDYWGQGTTLTVSS SEQ ID NO: 19 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVPVTPGESVSIPCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLQIR SEQ ID NO: 20 - Amino acid sequence of the heavy chain variable region of a mouse monoclonal antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFIISRDDSESMLYLQMNNLKTEDTAMYFCVRGGTYGSTSYFDYWGQGTTLTVSS SEQ ID NO: 21 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK SEQ ID NO: 22 - Amino acid sequence of the heavy chain variable region of a mouse monoclonal antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKARFTISRDDSESMLYLQMNNLKTEDTAMYFCVRGGTYGSTSYFDYWGQGTTLTVSS SEQ ID NO: 23 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK SEQ ID NO: 24 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGAINPNNGGTGYTQKFKGKATLTVDKSSSTAFMELRSLTSEDSAVYYCARRGVYMFAYWGQGTLVTVSA SEQ ID NO: 25 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK SEQ ID NO: 26 - Amino acid sequence of the heavy chain of a mouse-human chimeric antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYFCVRGGTYGSSSYFDYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 27 - Amino acid sequence of the light chain of a mouse-human chimeric antibody DIVMTQAAPSVPVTPGESVSIPCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKL QIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 28 - Amino acid sequence of the heavy chain of a mouse-human chimeric antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKARFTISRDDSESMLYLQMNNLKTEDTAMYFCVRGGTYGSTSYFDYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 29 - Amino acid sequence of the light chain of a mouse-human chimeric antibody DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 30 - Amino acid sequence of the heavy chain of a mouse-human chimeric antibody EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGAINPNNGGTGYTQKFKGKATLTVDKSSSTAFMELRSLTSEDSAVYYCARRGVYMFAYWGQGT LVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 31 - Amino acid sequence of the light chain of a mouse-human chimeric antibody DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32 - Amino acid sequence of the light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 33 - Amino acid sequence of the light chain constant region GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 34 - Amino acid sequence of the heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 35 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2 RIRTKSNNYATYYADSVKD SEQ ID NO: 36 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2 RIRTKSNNYATFYADSVKD SEQ ID NO: 37 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2 RIRTKSNNYATYYAASVKD SEQ ID NO: 38 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR2 RIRSKSNNFATYYADSVKD SEQ ID NO: 39 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 GGSGIKYVRYFDV SEQ ID NO: 40 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 GGSGIRYVKYFDV SEQ ID NO: 41 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 GGSGISYVRYFDV SEQ ID NO: 42 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 GGSGLNYVRYFDV SEQ ID NO: 43 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 GGSGLRYVRYFDV SEQ ID NO: 44 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 QTYGSRDYAMDY SEQ ID NO: 45 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 GGSGIRYVRYFDV SEQ ID NO: 46 - Amino acid sequence of mouse monoclonal antibody light chain CDR1 RSSQSLVHSNGNTYLH SEQ ID NO: 47 - Amino acid sequence of mouse monoclonal antibody light chain CDR1 RSSKSLQHSNGNIYLY SEQ ID NO: 48 - Amino acid sequence of mouse monoclonal antibody light chain CDR2 KVSNRFS SEQ ID NO: 49 - Amino acid sequence of mouse monoclonal antibody light chain CDR2 RMSDLAS SEQ ID NO: 50 - Amino acid sequence of mouse monoclonal antibody light chain CDR3 CQSTHVPPYT SEQ ID NO: 51 - Amino acid sequence of mouse monoclonal antibody heavy chain CDR3 SQSTHVPPYT SEQ ID NO: 52 - Amino acid sequence of mouse monoclonal antibody light chain CDR3 SQNTHVPPYT SEQ ID NO: 53 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSENILYLQMNNLKTEDTAMYYCVRGGSGIKYVRYFDVWGTGTTVTVSS SEQ ID NO: 54 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPRGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSESMLYLQMINLKTEDTAMYYCVRGGSGIRYVKYFDVWGTGTTVTVSS SEQ ID NO: 55 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPGGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSESMLYLQMINLKTEDTAMYYCVRGGSGIRYVKYFDVWGTGTTVTVSS SEQ ID NO: 56 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDSESMLYLQMNNLKTEDTAMYYCVRGGSGISYVRYFDVWGTGTTVTVSS SEQ ID NO: 57 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFKTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDVWGTGTTVTVSS SEQ ID NO: 58 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDSESMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDVWGTGTTVTVSS SEQ ID NO: 59 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSENMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDVWGTGTTVTVSS SEQ ID NO: 60 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLDWVARIRSKSNNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYFCVRQTYGSRDYAMDYWGQGTSVTVSS SEQ ID NO: 61 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLDWVARIRSKSNNYATYYADSVKDRFTISRDSESMLYLQMNNLKTEDTAMYFCVRQTYGSRDYAMDYWGQGTSVTVSS SEQ ID NO: 62 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDSESMLYLQMNNLKTEDTAMYYCVRGGSGIRYVRYFDVWGTGTTVTVSS SEQ ID NO: 63 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPRGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNFATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRQTYGSRDYAMDYWGQGTSVTVSS SEQ ID NO: 64 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFKTYAMNWVRQAPGEGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDVWGPGTTVTVSS SEQ ID NO: 65 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATFYADSVKDRFTISRHDSESMLYLQMNNLKTEDTAMYYCVRGGSGIRYVRYFDVWGTGTTVTVSS SEQ ID NO: 66 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYAASVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDVWGTGTTVTVSS SEQ ID NO: 67 - Amino acid sequence of mouse monoclonal antibody heavy chain variable region EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLEWVARIRSKSNNFATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRQTYGSRDYAMDYWGQGTSVTVSS SEQ ID NO: 68 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPERFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK SEQ ID NO: 69 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK SEQ ID NO: 70 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPERFSGSGSGSAFTLRVSRVEAEEDVGVYYCMQHLEYPFTFGSGTKLEIK SEQ ID NO: 71 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCCQSTHVPPYTFGGGTKLEIK SEQ ID NO: 72 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVPVTPGESVSISCRSSKSLQHSNGNIYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK SEQ ID NO: 73 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DVVMTQTPLSLPVSLGDRASISCRSSQSLVHSNGNTYLHWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPYTFGGGTKLEIK SEQ ID NO: 74 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVLVTPGESVSFSCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIFRMSNLASGVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK SEQ ID NO: 75 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVTVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSDLASGVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK SEQ ID NO: 76 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DIVMTQAAPSVFVIPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK SEQ ID NO: 77 - Amino acid sequence of the light chain variable region of a mouse monoclonal antibody DVVMTQTPLSLPVSLGDRASISCRSSQSLVHSNGNTYLHWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPYTFGGGTKLEIK SEQ ID NO: 78 - Amino acid sequence of the heavy chain of a mouse-human chimeric antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYAASVKDRFTISRDDSETMLYLQMNNLKTEDTAMYYCVRGGSGLNYVRYFDV WGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 79 - Amino acid sequence of the light chain of a mouse-human chimeric antibody DIVMTQAAPSVFVIPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGSAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 80 - Amino acid sequence of the heavy chain of a mouse-human chimeric antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDV WGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 81 - Amino acid sequence of the light chain of a mouse-human chimeric antibody DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPERFSGSGSGSAFTLRVSRVEAEEDVGVYYYCMQHLEYPFTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 82 - Amino acid sequence of the heavy chain of a mouse-human chimeric antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSENMLYLQMNNLKTEDTAMYYCVRGGSGLRYVRYFDV WGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 83 - Amino acid sequence of the light chain of a mouse-human chimeric antibody DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPERFSGSGSGSAFTLRVSRVEAEEDVGVYYYCMQHLEYPFTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 84 - Amino acid sequence of the heavy chain of a mouse-human chimeric antibody EVQLVESGGGLVQPKGSLKLSCAASGFSFNAYAMNWVRQAPGKGLDWVARIRSKSNNYATYYADSVKDRFTISRDSESMLYLQMNNLKTEDTAMYFCVRQTYGSRDYAMDYW GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 85 - Amino acid sequence of the light chain of a mouse-human chimeric antibody DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCCQSTHVPPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 86 - Amino acid sequence of the heavy chain of the humanized antibody EVQLVESGGGLVQPGGSLKLSCAASGFSFNAYAMNWVRQASGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYFCVRGGTYGSSSYFDYW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 87 - Amino acid sequence of the light chain of the humanized antibody DIVMTQSPLSLPVTPGEPASIPCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 88 - Amino acid sequence of the heavy chain of the humanized antibody EVQLVESGGGLVQPGGSLKLSCAASGFSFNAYAMNWVRQASGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSENTAYLQMNSLKTEDTAVYFCVRGGTYGSSSYFDYW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 89 - Amino acid sequence of the light chain of the humanized antibody DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEEDVGVYYCMQHLEYPFTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 90 - Amino acid sequence of the heavy chain of the humanized antibody EVQLVESGGGLVQPGGSLKLSCAASGFSFNTYAMNWVRQASGKGLEWVGRIRTKSNNYATYYAASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSGLNYVRYFDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 91 - Amino acid sequence of the light chain of the humanized antibody DIVMTQTPPSLPVNPGEPASISCRSSKSLLHSNGNTYLYWYLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGSDFTLKISWVEAEDVGVYYCMQHLEYPFTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 92 - Amino acid sequence of the light chain of the humanized antibody DIVMTQTPPSLPVNPGEPASISCRSSKSLLHSNANTYLYWYLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGSDFTLKISWVEAEDVGVYYCMQHLEYPFTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 93 - Amino acid sequence of the heavy chain of the CCR8 / CTLA4 bispecific antibody EVQLVESGGGLVQPGGSLKLSCAASGFSFNTYAMNWVRQASGKGLEWVGRIRTKSNNYATYYAASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSGLNYVRYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKGGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTYSRHCLGWFRQAPGKGREAVSTIDSDGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAIGPNPRYCSGAPNTRGAEHYFGYWGQGTLVTVSS

Claims

1. An isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human CCR8 and comprises: (a) a heavy chain CDR1 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 3, 7, and 9; (b) a heavy chain CDR2 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 4, 8, 10, and 35-38; (c) a heavy chain CDR3 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 5, 6, 11, and 39-45; (d) a light chain CDR1 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 12, 15, and 46-47; (e) a light chain CDR2 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 13, 16, and 48-49; and (f) a light chain CDR3 sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 14, 17, and 50-52.

2. (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 sequence; 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: 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 heavy chain CDR2 sequence of SEQ ID NO:41; light chain CDR1 sequence of SEQ ID NO:12; light chain CDR2 sequence of SEQ ID NO:13; and light chain CDR3 sequence of SEQ ID NO:14; or (9) heavy chain CDR1 sequence of SEQ ID NO:7; heavy chain CDR2 sequence of SEQ ID NO:35; heavy chain CDR3 sequence of SEQ ID NO:42; light chain CDR1 sequence of SEQ ID NO:12; light chain CDR2 sequence of SEQ ID NO:13; and light chain CDR3 sequence of SEQ ID NO:14; or (10) heavy chain CDR1 sequence of SEQ ID NO:7; heavy chain CDR2 sequence of SEQ ID NO:35; heavy chain CDR3 sequence of SEQ ID NO:43; 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 R3 sequence; 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; SEQ ID NO: 3 the heavy chain CDR2 sequence of SEQ ID NO: 8; 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; sequences 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. Human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, antigen-binding antibody fragment, single-chain antibody, diabody, triabody, tetrabody, Fab fragment, Fab' fragment, Fab ２ Fragment, F(ab)' ２ The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment is selected from a fragment, a domain antibody, a non-fucosylated antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgG1 antibody with at least one mutation that enhances ADCC / FcR affinity, or an IgG4 antibody with at least one mutation in the hinge region that reduces the tendency to form intra-H chain disulfide bonds.

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

5. 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 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 an isolated antibody or antigen-binding fragment thereof that specifically binds to human CCR8, comprising any of 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 to 4, further comprising a set of four variable region framework regions from a human immunoglobulin (IgG).

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

85.

8. An isolated humanized antibody or antigen-binding fragment thereof, which specifically binds to human CCR8 and comprises a heavy chain sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 86, 88, and 90; and a light chain sequence selected from the group of amino acid sequences defined by SEQ ID NOs: 87, 89, and 91-92.

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

87.

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

89.

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

91.

12. 9. The isolated humanized or human monoclonal antibody or antigen-binding fragment of 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 comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 12 in admixture with a pharmaceutically acceptable carrier.

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

15. 15. The isolated immunoconjugate of claim 14, wherein the effector molecule is selected from the group consisting of an immunotoxin, a cytokine, a chemokine, a therapeutic agent, and a chemotherapeutic agent.

16. A pharmaceutical composition comprising the immunoconjugate of any one of claims 14-15 in admixture with a pharmaceutically acceptable carrier.

17. 13. An antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12 linked to a second molecule selected from the group consisting of a cytotoxic agent, an anticancer drug, and an immunosuppressant.

18. 20. A pharmaceutical composition comprising the ADC of claim 17 in admixture with a pharmaceutically acceptable carrier.

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

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

92.

21. 20. The bispecific antibody of claim 19, comprising a heavy chain of SEQ ID NO: 93 and a light chain of SEQ ID NO:

91.

22. A pharmaceutical composition comprising the bispecific antibody of any one of claims 19 to 21 in admixture with a pharmaceutically acceptable carrier.

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

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

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

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

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

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