Chemokine receptor 8 (CCR8) antibodies

Abstract

Provided herein are anti-CCR8 antibodies, or antigen-binding fragments thereof, that bind to CCR8 wherein the CCR8 is human CCR8 and the antibody does not bind to human CCR4. The anti-CCR8 antibodies or antigen-binding fragments of the present disclosure are useful for the treatment of cancer diseases by elimination of regulatory T cells. Also provided herein are methods of use of anti-CCR8 antibodies or antigen-binding fragments thereof.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 417,507, filed on October 19, 2022, and U.S. Provisional Application No. 63 / 519,004, filed on August 11, 2023, the entire contents of which are incorporated herein by reference.

[0003] Technical field of the invention

[0004] This document relates to materials and methods for treating cancer, and particularly to the use of anti - CCR8 antibodies to reduce or eliminate cells expressing CCR8 and to treat cancer.

[0005] Materials incorporated by reference submitted on compact disc

[0006] This application includes a sequence listing that has been submitted via EFS - Web in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on October 19, 2023, is named IBIO:2022WO.xml, and is 117,514 bytes in size.

[0007] Background of the invention

[0008] Without limiting the scope of the present invention, its background is described in connection with eliminating cells expressing CCR8.

[0009] One such patent is U.S. Patent No. 11,427,640, titled "CCR8 Antibodies for Therapeutic Applications," granted to Berndt et al. These inventors are alleged to have taught the generation of antibodies that specifically bind chemokine receptors, such as CC or CXC chemokine receptors. Isolated sulfated polypeptides and their conjugates can be used as antigens or for off - target panning to facilitate the generation of anti - human, anti - cynomolgus monkey, and / or anti - mouse chemokine receptor antibodies, for example, to generate antibodies with fully human CDRs and / or other properties favorable for therapeutic use. These inventors also taught antibodies that specifically bind human, cynomolgus monkey, and / or murine CCR8 and have properties for therapeutic use, such as cross - reactive antibodies, fully human antibodies, low - internalization (including non - internalization) antibodies, and antibodies that effectively induce ADCC and / or ADCP in Treg cells.

[0010] Another such invention is taught in the US patent application with publication number US20220195057A1, titled "Anti-CCR8 Monoclonal Antibodies and Uses Thereof", filed by Li and Huang. It is alleged that this application provides antibodies or fragments thereof that have binding specificity for the human chemokine (C-C motif) receptor 8 (CCR8) protein. These antibodies are capable of binding CCR8 with high affinity and can mediate antibody-dependent cell cytotoxicity (ADCC).

[0011] Despite these advances, there remains a need for novel antibodies, methods, and uses that specifically bind CCR8 without binding closely related members of the chemokine receptor family, such as CC or CXC chemokine receptors.

[0012] Summary of the Invention

[0013] As embodied and broadly described herein, one aspect of the present disclosure relates to an anti-CCR8 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 49, 67, 73, 79 or 85; and a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 2, 50, 68, 74, 80 or 86; and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 3, 51, 69, 75, 81 or 87; and a light chain variable domain (VL) CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 52, 55, 58, 61, 64, 70, 76, 82 or 88; and a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 53, 56, 59, 62, 65, 71, 77, 83 or 89; and a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 6, 54, 57, 60, 63, 66, 72, 78, 84 or 90. In one aspect, the antibody comprises: a VH comprising the amino acid sequence of any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 or 15; and a VL comprising the amino acid sequence of any one of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23 or 24. In another aspect, the antibody comprises: a VH encoded by a nucleic acid sequence having at least 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32 or 33; and a VL encoded by a nucleic acid sequence having at least 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID NO: 34, 35, 36, 37, 18, 29, 40, 41 or 42. In another aspect, the antibody is a monoclonal antibody, bispecific antibody, multivalent antibody, multispecific antibody, diabody, chimeric antibody, scFv antibody or a fragment thereof. In another aspect, the antibody is fused to the Fc domain of any one of: human IgG1, human IgG2, human IgG3 and human IgG4. In another aspect, the antibody is a non-fucosylated full-length antibody. In another aspect, the nucleic acid sequence is optimized for expression in bacterial, fungal, mammalian, insect or plant cells. In another aspect, the antibody or antigen-binding fragment does not bind CCR4. In another aspect, the heavy chain CDRs are SEQ ID NO: 1, 2 and 3, and the light chain CDRs are SEQ ID NO: 4, 5 and 6.

[0014] As embodied and broadly described herein, one aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the antibody described above. In another aspect, the disease is cancer. In another aspect, the cancer is infiltrated by regulatory T cells. In another aspect, the subject is a human.

[0015] As embodied and broadly described herein, one aspect of the present disclosure relates to a method of preparing an anti-CCR8 antibody or antigen-binding fragment, which comprises expressing the antibody or antigen-binding fragment in a cell or in vitro, wherein the antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) complementarity determining region (CDR) 1, which comprises the amino acid sequence of any one of the following SEQ ID NO: 1, 49, 67, 73, 79 or 85; and VH CDR2, which comprises the amino acid sequence of any one of the following SEQ ID NO: 2, 50, 68, 74, 80 or 86; and VH CDR3, which comprises the amino acid sequence of any one of the following SEQ ID NO: 3, 51, 69, 75, 81 or 87; and a light chain variable domain (VL) CDR1, which comprises the amino acid sequence of any one of the following SEQ ID NO: 4, 52, 55, 58, 61, 64, 70, 76, 82 or 88; and VL CDR2, which comprises the amino acid sequence of any one of the following SEQ ID NO: 5, 53, 56, 59, 62, 65, 71, 77, 83 or 89; and VL CDR3, which comprises the amino acid sequence of any one of the following SEQ ID NO: 6, 54, 57, 60, 63, 66, 72, 78, 84 or 90. In one aspect, the cell is a bacterial, fungal, human, plant or insect cell. In another aspect, the antibody is a monoclonal antibody, bispecific antibody, multivalent antibody, multispecific antibody, diabody, chimeric antibody, scFv antibody or a fragment thereof. In another aspect, the antibody or antigen-binding fragment is non-fucosylated. In another aspect, the antibody or antigen-binding fragment does not bind CCR4.

[0016] As embodied and broadly described herein, one aspect of the present disclosure relates to a nucleic acid comprising an anti-CCR8 antibody or antigen-binding fragment thereof, which comprises: a polynucleotide encoding a heavy chain variable domain having at least 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32 or 33; and a polynucleotide encoding a light chain variable domain having at least 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41 or 42. In one aspect, the antibody is a monoclonal antibody, bispecific antibody, multivalent antibody, multispecific antibody, diabody, chimeric antibody, scFv antibody or fragment thereof. In another aspect, the antibody binding domain is fused to the Fc domain of any one of: human IgG1, human IgG2, human IgG3 and human IgG4. In another aspect, the nucleic acid sequence is optimized for expression in bacterial, fungal, mammalian, insect or plant cells. In another aspect, the antibody or antigen-binding fragment does not bind CCR4.

[0017] As embodied and broadly described herein, one aspect of the present disclosure relates to a vector comprising the nucleic acid disclosed above. As embodied and broadly described herein, one aspect of the present disclosure relates to a host cell comprising the nucleic acid vector disclosed above.

[0018] Brief Description of the Drawings

[0019] To more fully understand the features and advantages of the present invention, reference is now made to the detailed description of the invention and the accompanying drawings, in which:

[0020] Figure 1A and Figure 1B are flow cytometry plots showing the cell binding of the hybridoma supernatant of immunopositive clone 1E6, which was identified from CCR8 MEM-ELISA binding and cell binding hybridoma screening. Figure 1A Show that clone 1E6 binds to cells expressing CCR8. Figure 1B Show that clone 1E6 specifically binds CCR8 compared to the most closely related CCR4 GPCR. Figure 2 Show that the 1E6 hybridoma clone competes with the GS-1811 benchmark for binding to cells expressing CCR8.

[0021] Figures 3A to 3C Show that the chimeric or humanized anti-CCR8 antibody derived from the hybridoma clone 1E6 specifically binds human CCR8 without binding human CCR4 or mouse CCR8.

[0022] Figure 4A andFigure 4B Show the human CCR8 binding potency of anti-CCR8 antibodies.

[0023] Figure 5A and Figure 5B Show the antagonist activity of anti-CCR8 antibodies.

[0024] Figures 6A to 6C Show the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-CCR8 antibodies against CHO-K1 cells overexpressing human CCR8 using human peripheral blood mononuclear cells (PBMC).

[0025] Figure 7 Is a graph showing the thermal stability of anti-CCR8 antibodies.

[0026] Figure 8A Figures 8A to 8D show the study design and results of an in vivo efficacy study using the anti-CCR8 antibody SD-171467-afuc as a single therapy. Figures 8B and 8C show the tumor volume and the percentage change in tumor volume after drug treatment. Figure 8D shows the change in mouse body weight since drug administration.

[0027] Figures 9A to 9C Show the results of a pharmacokinetic study. Figure 9A Show the pharmacokinetic study design of SD-171467-afuc. Figure 9B Show the pharmacokinetic profile of SD-171467-afuc. Figure 9C Summarize the calculated pharmacokinetic parameters.

[0028] Figure 10A Figures 10A to 10F show the study design and results of an in vivo efficacy study using SD-171467-afuc and an anti-PD1 antibody as a combination therapy, as well as a study on the mechanism of action of CCR8+ Treg depletion. Figure 10A Show the in vivo study design of combination treatment of SD-171467-afuc and an anti-PD1 antibody and CCR8+ Treg depletion. Figures 10B and 10C show the tumor volume and the percentage change in tumor volume after drug treatment. Figure 10D shows the change in mouse body weight since drug administration. Figures 10E and 10F show the Treg-CCR8+ levels in tumors and spleens after PBS or SD-171467-afuc treatment.

[0029] Elaborate

[0030] Although the formation and use of various embodiments of the present invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be embodied in many specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways of forming and using the present invention and do not define the scope of the present invention.

[0031] For the purpose of facilitating the understanding of the present invention, a number of terms are defined below. The terms defined herein have the meanings as commonly understood by those of ordinary skill in the relevant art of the present invention. Terms such as "a", "an", and "the" are not intended to refer only to a singular entity, but include general categories for which specific instances can be used for illustration. The terms herein are used to describe specific embodiments of the present invention, but their use does not delimit the present invention, unless outlined in the claims.

[0032] It should be understood that, unless explicitly stated, in any method described or disclosed herein that includes more than one action, the order of the actions is not necessarily limited to the order of the actions of the method, but the present disclosure covers exemplary embodiments in which the order of the actions is so limited.

[0033] The present invention relates to novel anti-CCR8 antibodies and antigen-binding fragments thereof, which are used for binding to human CCR8 without binding to other members of the chemokine receptor family, including closely related CCR4.

[0034] Chemokine (C-C motif) receptor 8, also known as CCR8, is a protein encoded by the CCR8 gene in humans, also known as CDw198 (cluster of differentiation w198). CCR8 is a member of the beta chemokine receptor family and is a seven-transmembrane G protein-coupled receptor (GPCR) protein. The ligand of CCR8 is CCL1. Human CCR8 is UniProt P51685, RefSeq (mRNA) NM_005201 and RefSeq (protein) NP_005192.

[0035] CCR8 is mainly expressed on regulatory T cells (Tregs), and is important for CCR8 + regulatory T cell (Treg)-mediated immunosuppression. Recent studies have shown that CCR8 is upregulated in human tumor-resident Tregs and CCR8 + myeloid cells in cancer patients, and both of these cell types are expanded in cancer patients.

[0036] The antibodies of the present invention can be used to treat cancer by eliminating Tregs that inhibit anti-tumor cell T cells, thereby treating or inhibiting cancer. In some cases, the cancer cells of a patient express or overexpress CCR8. Cancers that can be treated by eliminating Tregs (sometimes referred to as tumor-infiltrating lymphocytes (TILs)) include, for example, gastric tumors, pancreatic tumors, esophageal tumors, ovarian tumors, and lung tumors.

[0037] As used throughout this text, the term "antibody" is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, non-human antibodies, chimeric antibodies, monovalent antibodies, antibody fragments, and tandem scFv-Fc antibodies.

[0038] Antibody fragments of the present disclosure retain CCR8 antigen-binding specificity. Antibody fragments include antigen-binding fragments (Fab), variable fragments (Fv) containing VH and VL sequences, single-chain variable fragments (scFv) containing VH and VL sequences linked together in one chain, single-chain antibody fragments (scAb), or other antibody variable region fragments, such as other antibody variable region fragments that retain antigen-binding specificity.

[0039] The tandem scFv-Fc antibodies of the present disclosure consist of two or more scFv binding sites tandemly arranged on each antibody arm, optionally linked by a linker, optionally by a flexible linker, such that a total of four or more scFv binding sites are generated in an antibody in a single scFv-Fc form.

[0040] As used throughout this text, the term "mesoscale molecule (MEM)" includes engineered peptides and polypeptides from about 1 kDa to about 10 kDa. As used throughout this text, the term "MEM nanoparticle" includes MEM that has been conjugated to a nanoparticle (e.g., a ferritin nanoparticle).

[0041] As used herein, a "subject" can be a mammalian subject. Mammalian subjects include humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus monkey. In some embodiments, the subject is a companion animal (e.g., cats, dogs).

[0042] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference.

[0043] Antibody

[0044] As used herein, the term "antibody" refers to a full-length antibody or a binding fragment thereof that specifically binds to a target antigen. The binding fragments are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of a full-length antibody. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain variable fragment (scFv) antibodies. An antibody is considered to substantially inhibit the adhesion of a receptor to a counter-receptor when an excess of the antibody reduces the amount of receptor bound to the counter-receptor by at least about 20%, 40%, 60%, or 80%, more typically greater than about 85% (as measured in an in vitro competitive binding assay). The term "antibody" is used in the broadest sense and specifically includes monoclonal antibodies (including full-length antibodies or other bivalent Fc-containing antibodies, such as bivalent scFv Fc fusion antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, scFv), so long as they exhibit the desired biological activity. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. The present invention includes fully recombinant monoclonal antibodies (and their binding fragments), in other words, where the complementarity-determining regions (CDRs) are genetically spliced into a human antibody framework, which is commonly referred to as antibody surface modification. Thus, in certain aspects, monoclonal antibodies are fully synthetic antibodies. In certain embodiments, monoclonal antibodies (and their binding fragments) can be prepared in bacterial, fungal, mammalian, insect, or plant cells.

[0045] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, usually the antigen-binding region or variable region, and includes Fab, Fab', F(ab')2, Fv, and scFv fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called Fab fragments, each having a single antigen-binding site, and also produces a residual "Fc" fragment, so named because of its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment having two antigen-binding fragments capable of cross-linking antigens, and a residual other fragment (called pFc'). As used herein, a "functional fragment" with respect to an antibody refers to Fv, F(ab), and F(ab')2 fragments.

[0046] As used herein, an "Fv" fragment is the smallest antibody fragment that contains the complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain (VH-VL dimer) that are tightly and non-covalently associated. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. The six CDRs together confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0047] The Fab fragment, also known as F(ab), also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH1). The Fab' fragment differs from the Fab fragment in that several residues, including one or more cysteines from the antibody hinge region, are added at the carboxyl terminus of the heavy-chain CH1 domain. Fab'-SH herein refers to a Fab' in which the (multiple) cysteine residues of the constant domain have free thiol groups. The F(ab') fragment is produced by cleaving the disulfide bond at the hinge cysteine of the F(ab')2 pepsin digestion product. Additional chemical conjugations of antibody fragments are known to those of ordinary skill in the art.

[0048] Natural antibodies and immunoglobulins are generally heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by at least one covalent disulfide bond. However, the number of disulfide bonds varies with the heavy chain of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the light chain variable domain aligns with the heavy chain variable domain. Specific amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain (Clothia et al., J. Mol. Biol. 186, 651-66, 1985; Novotny and Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4596 (1985)), the relevant portions of which are incorporated herein by reference. As used herein, an "isolated" antibody is one that has been identified and separated and / or recovered from the components of its production environment. Contaminant components of its production environment are materials that would interfere with the diagnostic or therapeutic use of the antibody and can include enzymes, hormones, and other protein or non-protein solutes. In certain embodiments, the antibody will be purified, as measurable by at least three different methods: 1) to greater than 50% by weight, such as greater than 75% by weight, or greater than 85% by weight, or greater than 95% by weight, or greater than 99% by weight, of the antibody, as determined by the Lowry method; 2) to an extent sufficient to obtain at least 10 residues, such as at least 15 residues of the N-terminal or internal amino acid sequence, by using a spinning cup sequencer; or 3) to show homogeneity by SDS-PAGE using Coomassie blue staining or preferably silver staining under reducing or non-reducing conditions. Isolated antibodies include in situ antibodies within recombinant cells, since at least one component of the antibody's natural environment will be absent. However, generally isolated antibodies will be prepared by at least one purification step.

[0049] As used herein, the terms “antibody mutant” or “antibody variant” refer to an amino acid sequence variant of an antibody in which one or more amino acid residues have been modified. Such a mutant necessarily has a sequence identity or similarity of less than 100% but at least 75% amino acid sequence identity or similarity, such as at least 80%, or at least 85%, or at least 90%, or at least 95, 96, 97, 98 or 99% with the amino acid sequence of the heavy chain variable domain or the light chain variable domain of the antibody. As used herein, in the context of the variable domain of an antibody, the term “variable” refers to the fact that certain portions of the variable domain vary extensively in sequence among antibodies and are used for binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) (also called hypervariable regions) in both the light chain variable domain and the heavy chain variable domain. There are at least two techniques for determining CDRs: (1) a method based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)); and (2) a method based on crystallographic studies of antigen-antibody complexes (Chothia, C. et al. (1989), Nature 342: 877), or both, i.e., Chothia plus Kabat. The more highly conserved portions of the variable domain are called framework (FR). The variable domains of the native heavy and light chains each contain four FR regions, predominantly adopting a β-sheet configuration, connected by three CDRs, which form loops connecting the β-sheet structures and in some cases form part of the β-sheet structure. The CDRs in each chain are held in close proximity to each other by the FR regions and, together with the CDRs from the other chain, contribute to form the antigen-binding site of the antibody (see Kabat et al.). The constant domains do not directly participate in the binding of the antibody to its cognate antigen but rather exhibit various effector functions, such as antibody-dependent cell cytotoxicity in which the antibody participates. Based on the amino acid sequence of the constant domain, the light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called κ and λ. Depending on the amino acid sequence of the constant domain of its heavy chain, an “immunoglobulin” can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3 and IgG4; IgA-1 and IgA-2. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0050] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies comprising the group are identical except for possible minor variations that may occur naturally. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Besides their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma cultures and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies used in accordance with the presently disclosed and claimed invention may be prepared by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), the relevant portions of which are incorporated herein by reference.

[0051] All monoclonal antibodies used in accordance with the presently disclosed and claimed invention will be (1) the result of a purposeful immunization protocol as described in more detail below; or (2) the result of an immune response that naturally leads to antibody production during the course of a disease or cancer.

[0052] The use of monoclonal antibodies of the presently disclosed and claimed inventions may require the administration of such or similar monoclonal antibodies to a subject, such as a human. However, when monoclonal antibodies are produced in non-human animals, such as rodents or chickens, the administration of such antibodies to a human patient will generally elicit an immune response, where the immune response is directed against the antibody itself. Such a reaction limits the duration and effectiveness of such therapies. To overcome such problems, the monoclonal antibodies of the presently disclosed and claimed inventions can be "humanized," i.e., the antibody is engineered such that its antigenic portions are removed and thus replaced with analogous portions of a human antibody, while maintaining the antibody's affinity for CCR8. Such engineering may involve only a few amino acids, or may include the entire framework region of the antibody, leaving only the intact antibody complementarity determining regions. Several methods of humanizing antibodies are known in the art and are disclosed in U.S. Patent No. 6,180,370, issued January 30, 2001 to Queen et al.; U.S. Patent No. 6,054,927, issued April 25, 2000 to Brickell; U.S. Patent No. 5,869,619, issued February 9, 1999 to Studnicka; U.S. Patent No. 5,861,155, issued January 19, 1999 to Lin; U.S. Patent No. 5,712,120, issued January 27, 1998 to Rodriquez et al.; and U.S. Patent No. 4,816,567, issued March 28, 1989 to Cabilly et al., the relevant portions of which are incorporated herein by reference.

[0053] A humanized form of an antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of an antibody), which consists predominantly of the sequences of human immunoglobulins and contains a minimal sequence derived from a non-human immunoglobulin. Humanization can be performed according to the methods of Winter and co-workers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988), i.e., by replacing the corresponding sequences of a human antibody with the CDR or CDR sequences of a non-human (i.e., rodent, chicken) antibody, see, for example, U.S. Patent No. 5,225,539. In some cases, the Fv framework residues of the human immunoglobulin are replaced with the corresponding non-human residues from the donor antibody. A humanized antibody may also contain residues that are neither present in the recipient antibody nor in the introduced CDR or framework sequences. Generally, a humanized antibody will contain substantially all, at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the framework regions are framework regions of human immunoglobulin consensus sequences. A humanized antibody preferably also contains at least a portion of the immunoglobulin constant region (Fc), usually the constant region of a human immunoglobulin.

[0054] The presently disclosed and claimed invention also includes the use of fully human monoclonal antibodies against CCR8. A fully human antibody essentially refers to an antibody molecule in which the entire sequences of both the light and heavy chains, including the CDRs, are derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared by, for example, the trioma technique; the human B-cell hybridoma technique (see Kozbor et al., Hybridoma, 2:7(1983)) and the EBV hybridoma technique for generating human monoclonal antibodies (see Cole et al., PNAS 82:859(1985)), or as taught herein. Human monoclonal antibodies can be used in the practice of the presently disclosed and claimed invention and can be produced by using human hybridomas (see Cote et al., PNAS 80:2026 (1983)) or by in vitro transformation of human B cells with Epstein-Barr virus (see Cole et al., 1985), relevant portions of which are incorporated herein by reference.

[0055] Alternatively, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, human antibody production is observed, which is very similar in all respects to that observed in humans, including gene rearrangement, assembly, and antibody repertoire. This method is described in, for example but not limited to, U.S. Patents Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and Marks et al., J Biol. Chem. 267:16007, (1992); Lonberg et al., Nature, 368:856 (1994); Morrison, 1994; Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger, Nat. Biotechnol. 14:826 (1996); and Lonberg and Huszar, Int Rev Immunol. 13:65 (1995), the relevant portions of which are incorporated herein by reference.

[0056] A method for generating antibodies of interest, such as human antibodies, is disclosed in U.S. Patent No. 5,916,771, issued to Hori et al. on June 29, 1999, which is incorporated herein by reference. It includes introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing a heavy chain and a light chain.

[0057] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Persons in need of treatment include those already suffering from a disorder as well as those in need of preventing a disorder.

[0058] As used herein, the term "disorder" refers to any condition that would benefit from treatment with a polypeptide. This includes chronic and acute disorders or diseases, including those infectious or pathological conditions that render a mammal susceptible to the disorder in question.

[0059] Antibodies or antibody fragments can be generated with engineered sequences or glycosylation states to confer a preferred level of activity in antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions, as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0060] Alternatively, or additionally, combining the amino acid modification with one or more other amino acid modifications that alter the complement-dependent cytotoxicity (CDC) function of the complement component Clq-binding and / or IL-23p19-binding molecule Fc region may be useful. Particularly interesting binding polypeptides may be binding polypeptides that bind Clq and exhibit complement-dependent cytotoxicity. Polypeptides with pre-existing Clq-binding activity, optionally further having the ability to mediate CDC, can be modified such that one or both of these activities are enhanced. Amino acid modifications that alter Clq and / or modify its complement-dependent cytotoxicity function are described, for example, in W0 / 0042072, which is incorporated herein by reference.

[0061] The Fc region of an antibody can be designed to alter effector functions, for example, by modifying Clq-binding and / or FcγR-binding, thereby altering complement-dependent cytotoxicity (CDC) activity and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. These "effector functions" are responsible for activating or reducing biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: Clq-binding; CDC; Fc receptor-binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to bind to a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays (e.g., Fc-binding assay, ADCC assay, CDC assay, etc.).

[0062] For example, variant Fc regions of antibodies with improved Clq-binding and improved FcγRIII-binding (e.g., with both improved ADCC activity and improved CDC activity) can be generated. Alternatively, if it is desired to reduce or eliminate effector functions, variant Fc regions can be engineered to have reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities can be increased, and optionally, the other activity can also be reduced (e.g., to generate a variant Fc region with improved ADCC activity but reduced CDC activity, and vice versa).

[0063] A single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, joined together by a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and introduction of the linker peptide. This modification generally leaves the specificity unchanged. These molecules were historically generated to facilitate phage display, in which it is convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be directly generated from the heavy and light chains of subclones derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region present in the intact antibody molecule and thus lack the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can generally be purified / immobilized using protein L, since protein L interacts with the variable region of the κ light chain.

[0064] Flexible linkers typically consist of amino acid residues that promote helices and turns, such as alanine, serine, and glycine. However, other residues can also play a role. Phage display can be used to rapidly select tailor-made linkers for single-chain antibodies (scFvs) from a library of protein linkers. A random linker library is constructed in which the genes for the heavy-chain variable domain and the light-chain variable domain are joined by a segment encoding a 18-amino acid polypeptide of variable composition. The scFv repertoire (≈5×10 6 different members) is displayed on filamentous phage and affinity selected with a hapten. The selected variant population exhibits a significant increase in binding activity but retains substantial sequence diversity. Sequence analysis reveals a conserved proline in two residues of the linker following the C terminus of VH and abundant arginine and proline at other positions, which is the only common feature of the selected tethers. In certain embodiments, as known in the art, antibody fragments are further modified to increase their serum half-life by using a modified Fc region or mutating various constant regions.

[0065] In certain embodiments, the antibodies of the invention are formulated for administration to a human. For example, the antibodies of the invention can be included in a pharmaceutical composition that is formulated for administration, i.e.: intranasally, intrapulmonary, intrabronchially, intravenously, orally, intraadipally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intrapericardially, intraperitoneally, intrapleurally, intravesically, topically, mucosally, parenterally, enterally, subcutaneously, sublingually, locally, transmucosally, transdermally, via inhalation, via injection, via a paste, via a lipid composition, via a catheter, via lavage, via continuous infusion, via infusion, via local delivery, or via local perfusion, and wherein the composition is a serum, a drop, a gel, an ointment, a spray, a depot, or an aerosol. As used herein, the term "antigen" refers to a molecule that contains one or more epitopes (linear, conformational, or both) that will stimulate the host's immune system to produce a humoral and / or cell antigen-specific response. The term can be used interchangeably with the term "immunogen". Generally, a B cell epitope will include at least about 5 amino acids, but can be as small as 3 - 4 amino acids. T cell epitopes, such as CTL epitopes, will include at least about 7 - 9 amino acids, and helper T cell epitopes will include at least about 12 - 20 amino acids. Generally, an epitope will include about 7 to 15 amino acids, such as 9, 10, 12, or 15 amino acids. The term includes polypeptides that include modifications as compared to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature), so long as the protein maintains the ability to elicit an immune response as defined herein. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host that produces the antigen.

[0066] As used herein, the term "epitope" refers to a specific amino acid sequence or molecule (such as a carbohydrate, a small molecule, a lipid, etc.) that, when present in the appropriate conformation, provides a reaction site for an antibody (e.g., a B cell epitope) or in the case of a peptide for a T cell receptor (e.g., a T cell epitope).

[0067] Portions of a given polypeptide that include B cell epitopes can be identified using any number of epitope mapping techniques known in the art (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, edited by Glenn E. Morris, 1996, Humana Press, Totowa, N.J.). For example, linear epitopes can be determined by, for example, simultaneously synthesizing a large number of peptides on a solid support, the peptides corresponding to a portion of a protein molecule, and reacting the peptides with an antibody while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715.

[0068] As used herein, the term "substantially purified" refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises a major percentage of its sample. Typically, in a sample, a substantially purified component comprises 50% of the sample, preferably 80%-85%, more preferably 90-95%. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation according to density.

[0069] As used herein, the term "treatment" refers to (i) preventing infection or reinfection (as with traditional vaccines), (ii) reducing or eliminating symptoms, and (iii) substantially or completely eliminating any of the cancers under discussion.

[0070] Unless otherwise indicated, the practice of the present invention employs conventional methods of chemistry, biochemistry, molecular biology, immunology, and pharmacology within the skill of the art. Such techniques are well explained in the literature. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (ed. S. Colowick and N. Kaplan, Academic Press, Inc.); and Handbook of Experimental Immunology, Volumes I-IV (ed. D. M. Weir and C. C. Blackwell, 1986, Blackwell Scientific Publications); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Short Protocols in Molecular Biology, 4th Edition (ed. Ausubel et al., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course, (ed. Ream et al., 1998, Academic Press); PCR (Introduction to Biotechniques Series), 2nd Edition (ed. Newton & Graham, 1997, Springer Verlag); Fundamental Virology, Second Edition (ed. Fields & Knipe, 1991, Raven Press, New York), the relevant portions of which are incorporated herein by reference.

[0071] Conservative amino acid substitutions involve the replacement of an amino acid with an aliphatic or hydrophobic amino acid such as Ala, Val, Leu, and Ile; the replacement of a hydroxyl residue with Ser and Thr; the replacement of an acidic residue with Asp and Glu; the replacement of an amide residue with Asn and Gln; the replacement of a basic residue with Lys, Arg, and His; the replacement of an aromatic residue with Phe, Tyr, and Trp; and the replacement of a small-sized amino acid with Ala, Ser, Thr, Met, and Gly.

[0072] Methods for expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archaea, insects, fungi (e.g., yeast), plants, and animal cells (e.g., mammalian cells such as human). Exemplary cells used include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the proliferation of mammalian cells in culture are well known (see Jakoby and Pastan (eds.), 1979, Cell Culture. Methods in Enzymology, Vol. 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalian host cell lines are VERO and Hela cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines such as those designed to provide higher expression, desired glycosylation patterns, or other characteristics can also be used. As noted above, techniques for transforming yeast cells, such as polyethylene glycol transformation, protoplast transformation, and gene gun, are also known in the art (see Gietz and Woods, Meth Enzymol 350: 87-96, 2002).

[0073] Transformation of a host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as but not limited to Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and then prepared by treatment of the cells with a process well known in the art using CaCl 2 Alternatively, MgCl 2 or RbCl can be used. If desired, transformation can also be carried out after formation of protoplasts of the host cell, or by electroporation.

[0074] When the host is a eukaryote, DNA transfection methods such as calcium phosphate co - precipitation can be used, as well as conventional mechanical processes such as microinjection, electroporation, inserting plasmids or viral vectors encapsulated in liposomes. Eukaryotic cells can also be co - transformed with a polynucleotide sequence encoding an (an) antibody or an (a) portion thereof and a second foreign DNA molecule encoding an alternative phenotype, such as the herpes simplex thymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells using eukaryotic viral vectors such as simian virus 40 (SV40) or bovine papillomavirus and express the protein (see, for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, edited by Gluzman, 1982).

[0075] An (an) antibody or an (a) portion thereof and variants can also be produced in plants. For example, polypeptides can be expressed in plants using the IBIOLAUNCH™ gene expression platform (iBio, Inc., Newark, Delaware), as described, for example, in U.S. Patent No. 7,491,509, which is incorporated herein by reference in its entirety. The IBIOLAUNCH™ platform can be used to produce high levels of target proteins in non - transgenic plants. This platform can have advantages compared to methods using animal cells or microorganisms and compared to systems that require transgenic plants.

[0076] Therapeutic methods and pharmaceutical compositions.

[0077] The antibodies or portions thereof disclosed herein or nucleic acids encoding the antibodies or portions thereof can be used to treat cancer. In several instances, the antibodies or portions thereof or nucleic acids encoding these polypeptides can be used to reduce cancer in a subject, such as. Thus, in several embodiments, the method comprises administering to a subject a therapeutically effective amount of one or more of the antibodies or portions thereof disclosed herein or a polynucleotide encoding these polypeptides to reduce cancer. However, any of the antibodies or portions thereof disclosed herein can be used to reduce cancer. In some embodiments, the peptide can be administered as a unit dose. In some embodiments, the polypeptide is administered as a multimer.

[0078] A therapeutically effective amount of an (an) antibody or an (a) portion thereof or a polynucleotide encoding the peptide can be administered in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers (e.g., physiologically or pharmaceutically acceptable carriers) are well - known in the art and include, but are not limited to, buffer solutions at physiological pH (e.g., a pH of about 7.0 to about 8.0, or a pH of about 7.4). A specific non - limiting example of a physiologically compatible buffer solution is phosphate - buffered saline. Other pharmaceutically acceptable carriers include osmotic agents, which are particularly suitable for pharmaceutical formulations intended for topical administration (e.g., administration at a surgical wound to promote healing).

[0079] The pharmacological compositions disclosed herein facilitate the use of at least one (or more) antibody or its (or their) portion(s) or polynucleotide encoding an (or the) antibody or its (or their) portion(s) in vivo or ex vivo to reduce cancer. Such compositions may be suitable for delivering the active ingredient to any suitable subject and may be prepared in a manner known per se, for example, by conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilization processes. One or more pharmaceutically (e.g., physiologically or pharmacologically) acceptable carriers and optionally auxiliaries may be used to formulate the pharmacological composition in a conventional manner, the auxiliaries facilitating the processing of the active compound into a pharmaceutically usable preparation. Suitable formulations depend on the chosen route of administration. Thus, for injection, the active ingredient may be formulated in an aqueous solution. For transmucosal administration, permeating agents suitable for the barrier to be penetrated are used in the formulation. Such permeating agents are generally known in the art.

[0080] For oral administration, a therapeutically effective amount of at least one (or more) antibody or its (or their) portion(s) or nucleic acid encoding a peptide may be combined with a carrier suitable for incorporation into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc.

[0081] Protein drugs are subject to protease-mediated degradation in the digestive tract by the action of enzymes such as trypsin, chymotrypsin and brush border peptidases, such that oral administration of large protein molecules generally does not produce the desired therapeutic effect (Soltero and Ekwruibe, 2001 Innovations in Pharmaceutical Technology, 1:106-110).

[0082] In some embodiments, for parenteral administration, a therapeutically effective amount of an (or the) antibody or its (or their) portion(s) or nucleic acid encoding a peptide may be administered by injection, such as by bolus injection or continuous infusion. Such compositions may take the form of suspensions, solutions or emulsions in oily or aqueous carriers and may contain formulating agents such as suspending agents, stabilizers and / or dispersing agents. Other pharmaceutical excipients are known in the art.

[0083] Optionally, an (or the) antibody or its (or their) portion(s) or polynucleotide encoding a peptide may be incorporated within or conjugated to a heterologous protein, hydrocarbon or lipid for in vitro or in vivo administration. Co-administration may be such that the an (or the) antibody or its (or their) portion(s) or polynucleotide encoding a peptide is administered before, substantially simultaneously with or after the protein, hydrocarbon or lipid. In some embodiments, the an (or the) antibody or its (or their) portion(s) or polynucleotide encoding a peptide is administered substantially simultaneously with the protein, hydrocarbon or lipid.

[0084] Other delivery systems can include time-release, delayed-release, or sustained-release delivery systems. Such systems can avoid repeated administration of the compositions of the present invention described above, increasing convenience for the subject and the physician. Many types of release delivery systems are available and are known to those of ordinary skill in the art. They include polymer-based systems such as poly(lactide-co-glycolide), poly(orthoesters), polycaprolactone, polyesters amides, polyorthoesters, polyhydroxybutyrates, and polyanhydrides. Microcapsules of the aforementioned polymers containing a drug are described, for example, in U.S. Patent No. 5,075,109. Delivery systems also include non-polymer systems such as lipids, including sterols such as cholesterol and cholesterol esters, and fatty acids or neutral fats such as monoglycerides, diglycerides, and triglycerides; hydrogel release systems; silicone rubber systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosion systems in which the antibody or its portion(s) or polynucleotide encoding a peptide is / are contained within a matrix in one form, such as those described in U.S. Patents Nos. 4,452,775, 4,667,014, 4,748,034, 5,239,660, and 6,218,371, and (b) diffusion systems in which the active component(s) permeate(s) from the polymer at a controlled rate, such as those described in U.S. Patents Nos. 3,832,253 and 3,854,480. Additionally, pump-based hardware delivery systems can be used, some of which are suitable for implantation.

[0085] A therapeutically effective amount of the antibody or its portion(s) or polynucleotide encoding a peptide will depend on the antibody or its portion(s) or polynucleotide encoding a peptide utilized, the subject being treated, the severity and type of affliction, and the mode of administration. For example, a therapeutically effective amount of a polynucleotide encoding a peptide can vary from about 0.01 μg / kg body weight to about 1 g / kg body weight, such as from about 1 μg to about 5 mg / kg body weight, or from about 5 μg to about 1 mg / kg body weight. Those skilled in the art can readily determine the precise dosage based on the potency of the specific compound and the age, weight, gender, and physiological condition of the subject.

[0086] Regarding the administration of nucleic acids, one method of administering nucleic acids is direct treatment with plasmid DNA, such as direct treatment with a mammalian expression plasmid. As described above, the nucleotide sequence encoding the antibody or its portion(s) can be under the control of a promoter to increase the expression of the molecule.

[0087] When using viral vectors for in vivo administration, the dosage range of each recombinant virus in the composition to be provided to the recipient is desirably from about 10 5 to about 1010 Plaque forming units / mg of mammal, although lower or higher doses may be administered. The composition of the recombinant viral vector can be introduced into a mammal before any signs of cancer are present or mediate regression of the disease in a mammal suffering from cancer. Examples of methods of administering the composition to a mammal include, but are not limited to, ex vivo exposure of cells to the recombinant virus, or injection of the composition into the affected tissue, or intravenous, subcutaneous, intradermal, or intramuscular administration of the virus. Alternatively, the recombinant viral vector or combination of recombinant viral vectors can be administered locally by direct injection into the cancerous lesion with a pharmaceutically acceptable carrier. Generally, the amount of the recombinant viral vector carrying the nucleic acid sequence of one or more antibodies or portions thereof to be administered is based on the titer of the viral particles. An exemplary range of the immunogen to be administered is 10 5 to 10 10 viral particles / mammal such as a human.

[0088] In one specific non-limiting example, a pharmaceutical composition for intravenous administration will contain from about 0.1 μg to 10 mg of the antibody or portion(s) thereof / patient / day. Doses from 0.1 to up to about 100 mg / day / patient can be used, especially if the agent is administered to a secluded site without entering the circulatory or lymphatic system, such as into a body cavity or into the lumen of an organ. The actual methods of preparing the administrable composition will be known or apparent to those skilled in the art and are described in more detail in publications such as Remington’s Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pa., 1995.

[0089] The pharmaceutical composition is administered in a single dose or multiple doses depending on the dose and frequency required and tolerated by the subject. In some embodiments, the dose is administered as a single large dose, but in another embodiment, it may be administered periodically until a therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate the symptoms or signs of the disease without causing unacceptable toxicity to the subject. Systemic or local administration can be utilized.

[0090] In another method, additional agents are administered. In one example, the administration is sequential. In other examples, the additional agents are administered concurrently with the antibody or portion(s) thereof.

[0091] The invention is further described in the following examples, which do not limit the scope of the invention as set forth in the claims.

[0092] The present disclosure provides antibodies that bind to CCR8. These antibodies are referred to herein as anti-CCR8 antibodies. A number of discovery strategies have been employed to obtain the exemplary antibodies of the present disclosure, which are further discussed below.

[0093] In some embodiments, MEM nanoparticles are used together with full-length anti-CCR8 to immunize a subject to generate antibodies specific for the MEM epitope. Monoclonal hybridomas are then prepared to generate epitope-specific anti-CCR8 antibodies. In other embodiments, mouse sera are collected and used to generate a scFv library in vitro. Phage panning is then performed against full-length anti-CCR8 and MEM nanoparticles to isolate epitope-specific clones.

[0094] In some embodiments, the half-maximal effective concentration (EC 50 ) of the anti-CCR8 antibody for CCR8 is about 1.6, 1.8, 1.9, 2.0, 2.2, 2.3, 2.6, or 2.7 nM.

[0095] Those skilled in the art will recognize that binding specificity can be determined by a series of competitive binding paradigms, in which the desired antibody demonstrates its ability to block the binding of a known reference antibody to its target epitope at different concentrations. In some embodiments, the reference anti-CCR8 antibody is GS-1811.

[0096] In some embodiments, the anti-CCR8 antibody is a full-length antibody (an antibody having two heavy chains and two light chains linked to an Fc domain, in a "Y" shape). In some embodiments, the Fc domain (or simply Fc) is a human Fc domain. In some embodiments, the Fc domain of the anti-CCR8 antibody is from human IgG1, human IgG2, human IgG3, or human IgG4.

[0097] Exemplary anti-CCR8 antibody - CDR sequences

[0098] The present disclosure provides the sequences of exemplary anti-CCR8 antibodies of the present disclosure. Complementary determining region (CDR) sequences are included, as well as variable heavy and light chain domain sequences (VH, VL), which constitute the anti-CCR8 antigen-binding domain of the present disclosure. The discovery of these antibodies is described in detail in the Examples section.

[0099] As mentioned below, the light chain variable (VL) domain CDR1 region is referred to as CDR-L1; the VL CDR2 region is referred to as CDR-L2; the VL CDR3 region is referred to as CDR-L3; the heavy chain variable (VH) domain CDR1 region is referred to as CDR-H1; the VH CDR2 region is referred to as CDR-H2; and the VH CDR3 region is referred to as CDR-H3. Table 1 provides exemplary CDR combinations of the antibodies of the present disclosure.

[0100] Table 1: Antibody CDRs

[0101] Antibody CDR1 CDR2 CDR3 1E6H2K - HC GFTFNTNA IRSKSNNYAT VRGKDTSGSYYAMDY SEQ ID NO: 1 2 3 1E6H2K - LC KSLLHSNGNTY RMS MQHLEYPFT SEQ ID NO: 4 5 6

[0102] Tables 2A and 2B show the amino acid sequences of the anti-CCR8 humanized heavy and light chain antibodies, respectively. The CDRs of each heavy and light chain are in Table 1.

[0103] Table 2A. Amino Acid Sequence of Anti-CCR8 Humanized Antibody - Heavy Chain

[0104] Antibody ID VH Seq SEQ IDNO: SD-356253_VH QVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWLARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMKNLKSEDTAMYYCVRGKDTSGSYYAMDYWGQGTSVTVSS 7 SD-171467_VH QVQLVESGGGLVQPGGSLKLSCAASGFTFNTNAMNWVRQASGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRGKDTSGSYYAMDYWGQGTTVTVSS 8 SD-962141_VH EVQLVESGGGLVQPGGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLKTEDTAVYYCVRGKDTSGSYYAMDYWGQGTLVTVSS 9 SD-611534_VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSTNAMNWVRQAPGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRGKDTSGSYYAMDYWGQGTLVTVSS 10 SD-580600_VH QVQLVESGGGLVQPGGSLKLSCAASGFTFNTNAMNWVRQASGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRGKDTSGSYYAMDYWGQGTTVTVSS 11 SD-388950_VH QVQLVESGGGLVQPGGSLKLSCAASGFTFNTNAMNWVRQASGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNSLKTEDTAVYYCVRGKDTSGSYYAMDYWGQGTTVTVSS 12 SD-117248_VH QVQLVESGGGLVQPGGSLKLSCAASGFTFNTNAMNWVRQASGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRGKDTSGSYYAMDYWGQGTTVTVSS 13 SD-166154_VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSTNAMNWVRQASGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGKDTSGSYYAMDYWGQGTMVTVSS 14 SD-969683_VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSTNAMNWVRQASGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGKDTSGSYYAMDYWGQGTMVTVSS 15

[0105] Table 2B. Amino Acid Sequence of Anti-CCR8 Humanized Antibody - Light Chain

[0106] Seq ID VL Seq SEQ IDNO: SD-356253_VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIR 16 SD-171467_VL DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK 17 SD-962141_VL DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK 18 SD-611534_VL DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK 19 SD-580600_VL DIVMTQSALSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK 20 SD-388950_VL DIVMTQSALSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK 21 SD-117248_VL DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK 22 SD-166154_VL DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK 23 SD-969683_VL DIVMTQSPLSLPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK 24

[0107] Table 3A. Nucleic Acid Sequence of Anti-CCR8 Humanized Antibody - Heavy Chain

[0108] Seq ID VH Seq SEQ IDNO: SD-356253_VH CAAGTGCAGCTCGTGGAAACTGGCGGCGGACTGGTGCAGCCGAAGGGTAGCCTGAAGCTGTCCTGTGCGGCCTCCGGTTTCACTTTTAACACCAACGCCATGAATTGGGTCCGCCAGGCACCCGGGAAAGGGCTGGAATGGCTCGCGAGAATCCGGTCCAAGTCGAACAACTACGCCACCTACTACGCTGACTCGGTCAAGGACAGGTTCACGATTTCCCGCGACGATTCACAGAGCATGCTGTACCTTCAAATGAAGAACTTGAAGTCAGAGGATACCGCCATGTACTATTGCGTGCGGGGAAAGGACACCTCCGGATCCTACTACGCCATGGACTACTGGGGACAGGGCACCAGCGTGACTGTGTCCTCG 25 SD-171467_VH CAGGTCCAGCTGGTAGAGTCCGGCGGCGGACTGGTCCAGCCTGGCGGAAGTCTGAAATTGTCTTGCGCTGCTAGCGGGTTCACTTTCAATACAAATGCAATGAACTGGGTGCGCCAGGCAAGTGGCAAAGGACTGGAGTGGGTCGGAAGAATCAGATCCAAGAGCAATAACTACGCCACTTACTATGCCGACTCCGTTAAGGATCGCTTCACGATATCCCGCGACGACAGTAAGAATACTCTCTACCTGCAGATGAACTCCTTAAAAACAGAAGACACTGCCGTATACTACTGTGTAAGAGGTAAAGACACCTCAGGCTCCTATTATGCCATGGACTATTGGGGACAGGGTACAACCGTCACAGTGTCCAGC 26 SD-962141_VH GAAGTGCAGCTGGTTGAATCCGGCGGGGGGCTGGTGCAGCCTGGAGGCTCCCTCAAGCTCTCTTGCGCTGCCTCCGGATTTACCTTCAATACAAATGCGATGAACTGGGTGCGCCAGGCACCTGGGAAAGGTTTGGAGTGGGTAGGCCGCATACGCAGCAAGAGCAATAACTATGCCACCTATTATGCAGATAGCGTAAAAGACAGATTCACTATCAGTCGGGACGACTCTAAATCCACCCTCTATCTGCAGATGAACTCACTGAAGACTGAGGACACCGCTGTCTATTACTGCGTGCGGGGCAAGGATACGTCTGGTAGCTATTATGCTATGGACTATTGGGGCCAGGGGACACTGGTCACGGTTTCTTCT 27 SD-611534_VH GAGGTGCAGCTGGTAGAGTCTGGGGGAGGGTTAGTCCAGCCGGGGGGTTCCCTGAAGCTCAGCTGTGCCGCCTCCGGGTTTACATTTTCCACCAACGCTATGAACTGGGTAAGACAGGCTCCCGGAAAGGGACTCGAATGGGTGGGCCGCATTCGATCAAAGAGCAACAACTATGCCACGTACTACGCGGACTCAGTAAAAGATCGCTTCACTATATCCAGAGATGACTCTAAAAACACCTTGTACCTGCAGATGAACTCACTTAAAACTGAAGACACTGCTGTCTACTACTGCGTAAGGGGCAAGGACACATCCGGCAGCTATTATGCCATGGATTACTGGGGTCAAGGTACCCTCGTTACAGTGAGCAGC 28 SD-580600_VH CAGGTCCAGCTGGTAGAGTCCGGCGGCGGACTGGTCCAGCCTGGCGGAAGTCTGAAATTGTCTTGCGCTGCTAGCGGGTTCACTTTCAATACAAATGCAATGAACTGGGTGCGCCAGGCAAGTGGCAAAGGACTGGAGTGGGTCGGAAGAATCAGATCCAAGAGCAATAACTACGCCACTTACTATGCCGACTCCGTTAAGGATCGCTTCACGATATCCCGCGACGACAGTAAGAATACTCTCTACCTGCAGATGAACTCCTTAAAAACAGAAGACACTGCCGTATACTACTGTGTAAGAGGTAAAGACACCTCAGGCTCCTATTATGCCATGGACTATTGGGGACAGGGTACAACCGTCACAGTGTCCAGC 29 SD-388950_VH CAAGTGCAATTGGTCGAGTCCGGCGGGGGCCTGGTGCAACCTGGCGGGAGCCTGAAGCTCTCCTGCGCGGCAAGCGGGTTTACTTTTAACACCAATGCCATGAATTGGGTTAGGCAAGCGTCCGGTAAGGGATTGGAGTGGGTGGGGAGGATCCGATCAAAATCTAATAATTACGCCACCTATTACGCAGATTCTGTCAAAGATCGCTTTACTATCTCAAGAGATGACAGTCAGAGTATGCTCTATCTGCAAATGAACTCCCTGAAGACGGAAGACACAGCTGTTTACTATTGTGTGCGGGGGAAGGACACTAGTGGTAGCTATTACGCCATGGACTACTGGGGTCAGGGAACCACAGTCACCGTGAGCTCT 30 SD-117248_VH CAGGTCCAGCTGGTAGAGTCCGGCGGCGGACTGGTCCAGCCTGGCGGAAGTCTGAAATTGTCTTGCGCTGCTAGCGGGTTCACTTTCAATACAAATGCAATGAACTGGGTGCGCCAGGCAAGTGGCAAAGGACTGGAGTGGGTCGGAAGAATCAGATCCAAGAGCAATAACTACGCCACTTACTATGCCGACTCCGTTAAGGATCGCTTCACGATATCCCGCGACGACAGTAAGAATACTCTCTACCTGCAGATGAACTCCTTAAAAACAGAAGACACTGCCGTATACTACTGTGTAAGAGGTAAAGACACCTCAGGCTCCTATTATGCCATGGACTATTGGGGACAGGGTACAACCGTCACAGTGTCCAGC 31 SD-166154_VH GAGGTGCAGTTGGTCGAGAGTGGTGGGGGACTTGTACAGCCTGGGGGCTCACTGAAATTATCCTGTGCGGCGAGTGGATTTACTTTTTCTACTAATGCTATGAATTGGGTTCGCCAGGCGAGCGGAAAAGGCCTGGAATGGGTCGGGAGGATTCGCTCTAAATCTAACAACTATGCAACCTACTATGCCGACTCCGTTAAGGATCGCTTTACTATAAGCAGAGATGACAGCAAAAACACAGCGTATCTGCAAATGAACAGCCTCAAAACAGAGGACACAGCCGTTTACTACTGTACTAGAGGCAAAGACACTAGCGGTAGTTACTATGCCATGGATTACTGGGGTCAGGGGACGATGGTTACCGTGAGTTCC 32 SD-969683_VH GAGGTGCAGTTGGTCGAGAGTGGTGGGGGACTTGTACAGCCTGGGGGCTCACTGAAATTATCCTGTGCGGCGAGTGGATTTACTTTTTCTACTAATGCTATGAATTGGGTTCGCCAGGCGAGCGGAAAAGGCCTGGAATGGGTCGGGAGGATTCGCTCTAAATCTAACAACTATGCAACCTACTATGCCGACTCCGTTAAGGATCGCTTTACTATAAGCAGAGATGACAGCAAAAACACAGCGTATCTGCAAATGAACAGCCTCAAAACAGAGGACACAGCCGTTTACTACTGTACTAGAGGCAAAGACACTAGCGGTAGTTACTATGCCATGGATTACTGGGGTCAGGGGACGATGGTTACCGTGAGTTCC 33

[0109] Table 3B. Nucleic Acid Sequence of Anti-CCR8 Humanized Antibody - Light Chain

[0110] Seq ID VL Seq SEQ ID NO: SD-356253_VL GACATCGTGATGACTCAGGCCGCCCCTTCCGTGCCCGTCACCCCGGGAGAATCCGTGTCAATCTCGTGTCGGTCCTCCAAGTCGCTGCTTCATTCCAACGGGAACACCTATCTGTACTGGTTCTTGCAAAGACCCGGACAGAGCCCGCAGCTGCTGATCTACCGCATGTCTAACCTCGCTAGCGGAGTGCCAGATCGGTTTTCGGGATCCGGCAGCGGCACCGCGTTCACTCTGCGCATTTCAAGGGTCGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGTACCCTTTCACCTTCGGGTCCGGTACCAAGCTCGAAATTCGG 34 SD-171467_VL GATATTGTGATGACTCAGTCGCCGCTGTCATTGCCCGTGACCCCTGGAGAACCCGCGTCGATTTCCTGTCGGTCCTCCAAGTCCCTCCTGCACTCCAACGGGAACACCTACCTCTACTGGTTTCTGCAAAAGCCGGGACAGAGCCCACAGCTGCTGATCTATCGGATGAGCAACCTTGCCTCGGGAGTGCCTGACCGCTTCTCCGGAAGCGGCTCAGGGACCGACTTCACCCTGAAAATCTCCAGAGTGGAGGCCGAAGATGTCGGCGTGTACTACTGCATGCAGCATCTGGAGTACCCGTTCACTTTCGGCGGTGGTACCAAGGTCGAAATCAAG 35 SD-962141_VL GATATTGTGATGACTCAGTCGCCGCTGTCATTGCCCGTGACCCCTGGAGAACCCGCGTCGATTTCCTGTCGGTCCTCCAAGTCCCTCCTGCACTCCAACGGGAACACCTACCTCTACTGGTTTCTGCAAAAGCCGGGACAGAGCCCACAGCTGCTGATCTATCGGATGAGCAACCTTGCCTCGGGAGTGCCTGACCGCTTCTCCGGAAGCGGCTCAGGGACCGACTTCACCCTGAAAATCTCCAGAGTGGAGGCCGAAGATGTCGGCGTGTACTACTGCATGCAGCATCTGGAGTACCCGTTCACTTTCGGCGGTGGTACCAAGGTCGAAATCAAG 36 SD-611534_VL GATATTGTGATGACTCAGTCGCCGCTGTCATTGCCCGTGACCCCTGGAGAACCCGCGTCGATTTCCTGTCGGTCCTCCAAGTCCCTCCTGCACTCCAACGGGAACACCTACCTCTACTGGTTTCTGCAAAAGCCGGGACAGAGCCCACAGCTGCTGATCTATCGGATGAGCAACCTTGCCTCGGGAGTGCCTGACCGCTTCTCCGGAAGCGGCTCAGGGACCGACTTCACCCTGAAAATCTCCAGAGTGGAGGCCGAAGATGTCGGCGTGTACTACTGCATGCAGCATCTGGAGTACCCGTTCACTTTCGGCGGTGGTACCAAGGTCGAAATCAAG 37 SD-580600_VL GACATTGTTATGACCCAGTCTGCCTTATCTCTGCCCGTAACCCCTGGGGAGCCCGCGAGCATAAGTTGCAGGTCCTCAAAAAGCCTTTTGCATAGTAATGGCAATACATACTTGTATTGGTTTCTGCAGAAGCCTGGACAGAGCCCTCAGCTGCTGATTTATAGAATGAGCAACCTCGCTTCTGGAGTGCCCGACCGCTTTAGCGGCTCTGGATCAGGGACTGATTTTACACTGAA39GATAAGCAGAGTGGAAGCTGAGGATGTTGGCGTGTATTACTGTATGCAGCATCTCGAGTATCCATTTACATTTGGTGGGGGCACTAAGGTGGAAATCAAG 38 SD-388950_VL GACATTGTTATGACCCAGTCTGCCTTATCTCTGCCCGTAACCCCTGGGGAGCCCGCGAGCATAAGTTGCAGGTCCTCAAAAAGCCTTTTGCATAGTAATGGCAATACATACTTGTATTGGTTTCTGCAGAAGCCTGGACAGAGCCCTCAGCTGCTGATTTATAGAATGAGCAACCTCGCTTCTGGAGTGCCCGACCGCTTTAGCGGCTCTGGATCAGGGACTGATTTTACACTGAAGATAAGCAGAGTGGAAGCTGAGGATGTTGGCGTGTATTACTGTATGCAGCATCTCGAGTATCCATTTACATTTGGTGGGGGCACTAAGGTGGAAATCAAG 39 SD-117248_VL GATATCGTCATGACTCAGAGCCCCCTGTCCCTGCCGGTGACCCCCGGCGAGCCAGCGTCCATTTCCTGTCGCAGCTCTAAATCACTGCTGCATTCTAATGGCAATACCTACTTGTACTGGTTCCTTCAGAAACCAGGCCAGTCACCACAGCTGCTGATATATCGTATGTCTAATTTGGCGAGCGGCGTGCCTGACCGATTCTCAGGCTCTGGCAGCGGCACTGCTTTTACCCTGAAGATTAGTCGGGTCGAGGCCGAAGATGTGGGAGTCTACTACTGTATGCAGCATCTGGAATACCCTTTTACTTTTGGGGGCGGCACTAAGGTAGAGATTAAA 40 SD-166154_VL GATATCGTCATGACTCAGAGCCCCCTGTCCCTGCCGGTGACCCCCGGCGAGCCAGCGTCCATTTCCTGTCGCAGCTCTAAATCACTGCTGCATTCTAATGGCAATACCTACTTGTACTGGTTCCTTCAGAAACCAGGCCAGTCACCACAGCTGCTGATATATCGTATGTCTAATTTGGCGAGCGGCGTGCCTGACCGATTCTCAGGCTCTGGCAGCGGCACTGCTTTTACCCTGAAGATTAGTCGGGTCGAGGCCGAAGATGTGGGAGTCTACTACTGTATGCAGCATCTGGAATACCCTTTTACTTTTGGGGGCGGCACTAAGGTAGAGATTAAA 41 SD-969683_VL GACATAGTGATGACCCAGAGTCCATTATCCCTGCCAGTCACGCCTGGGGAGTCAGTGAGCATTTCCTGCCGATCCAGTAAAAGCCTCCTTCATTCCAATGGTAATACATACCTGTACTGGTTCCTCCAGAAACCCGGCCAATCTCCTCAGTTACTTATTTACAGAATGTCCAACCTCGCATCTGGGGTGCCCGACAGGTTTTCCGGAAGTGGGTCAGGCACTGACTTCACCCTGAGGATCAGTAGGGTGGAGGCCGAGGATGTGGGCGTGTACTACTGCATGCAGCATCTCGAGTACCCTTTCACCTTCGGGGGAGGAACAAAAGTAGAGATAAAA 42

[0111] Table 4. Additional CDR Sequences

[0112] Antibody CDR1 CDR2 CDR3 1E6H3K-HC GFTFNTNA IRSKSNNYAT VRGKDTSGSYYAMDY SEQ ID NO: 43 44 45 1E6H3K-LC KSLLHSNGNTY RMS MQHLEYPFT SEQ ID NO: 46 47 48 7B_600_030-HC GFIFETYA IRSKSNNYAT VRGLLRYRFFDV SEQ ID NO: 49 50 51 7B_600_030-LC KSLLHSNGNTY KVE MQHLEYPFT SEQ ID NO: 52 53 54 7B_000_032 GFTFNTYA IRSKSNNYAT VRGLLRYRFFDV SEQ ID NO: 55 56 57 7B_000_032 KSLLHSNGNTY KVE QQHTNWPFT SEQ ID NO: 58 59 60 7B_600_022 GFIFETYA IRSKSNNYAT VRGLLRYRFFDV SEQ ID NO: 61 62 63 7B_600_022 KSLLHSNGNTY KAN QQHTNWPFT SEQ ID NO: 64 65 66 7B_600_032-HC GFIFETYA IRSKSNNYAT VRGLLRYRFFDV SEQ ID NO: 67 68 69 7B_600_032-LC KSLLHSNGNTY KVE QQHTNWPFT SEQ ID NO: 70 71 72 7B_600_012-HC GFIFETYA IRSKSNNYAT VRGLLRYRFFDV SEQ ID NO: 73 74 75 7B_600_012-LC KSLLHSNGNTY GLT QQHTNWPFT SEQ ID NO: 76 77 78 7B_600_020-HC GFIFETYA IRSKSNNYAT VRGLLRYRFFDV SEQ ID NO: 79 80 81 7B_600_020-LC KSLLHSNGNTY KAN MQHLEYPFT SEQ ID NO: 82 83 84 K17_050_050-HC GYTFTSYN VHPGSGST ARKGGTPFAY SEQ ID NO: 85 86 87 K17_050_050-LC QSLVHSNAATY QVK SQSTHVPYT SEQ ID NO: 88 89 90

[0113] In some embodiments, provided herein is an anti-CCR8 antibody, wherein the antibody comprises a heavy chain variable domain (VH) complementarity determining region (CDR) 1 having an amino acid sequence comprising any one of the following SEQ ID NOs: 1, 49, 67, 73, 79, or 85; and a VH CDR2 having an amino acid sequence comprising any one of the following SEQ ID NOs: 2, 50, 68, 74, 80, or 86; and a VH CDR3 having an amino acid sequence comprising any one of the following SEQ ID NOs: 3, 51, 69, 75, 81, or 87; and a light chain variable domain (VL) CDR1 having an amino acid sequence comprising any one of the following SEQ ID NOs: 4, 52, 55, 58, 61, 64, 70, 76, 82, or 88; and a VL CDR2 having an amino acid sequence comprising any one of the following SEQ ID NOs: 5, 53, 56, 59, 62, 65, 71, 77, 83, or 89; and a VL CDR3 having an amino acid sequence comprising any one of the following SEQ ID NOs: 6, 54, 57, 60, 63, 66, 72, 78, 84, or 90.

[0114] Exemplary tandem scFv-Fc anti-CCR8 antibodies. In some embodiments, the present disclosure provides tandem scFv antibodies having multiple CCR8 binding sites. The tandem scFv-Fc antibodies of the present disclosure are composed of two or more scFv binding sites tandemly arranged on each antibody arm, optionally linked by a linker, optionally by a flexible linker. In some embodiments, the tandem scFV antibodies have a total of four or more scFv binding sites in a single scFv-Fc form of the antibody.

[0115] In some embodiments, scFv1 of each antibody arm comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and scFv2 of each antibody arm comprises a first heavy chain variable domain (VH2) and a first light chain variable domain (VL2). The VH1 and VL1 of each scFV1 may be linked by a linker, e.g., a flexible linker. The VH2 and VL2 of each scFV2 may be linked by a linker, e.g., a flexible linker. The scFvs on each antibody arm may be linked by a linker, e.g., a flexible linker.

[0116] Therapeutic uses of anti-CCR8 antibodies

[0117] In some embodiments, the anti-CCR8 antibodies provided herein can be used to treat diseases or disorders involving an immune response.

[0118] Administration of Therapeutic Anti-CCR8 Antibodies. In vivo administration of the therapeutic anti-CCR8 antibodies described herein can be carried out by intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, intrathecal, intracerebroventricular, intranasal, transmucosal, by implantation or by inhalation. Intravenous administration can be carried out via injection or infusion. In some embodiments, the anti-CCR8 antibodies of the present disclosure are administered intravenously. In some embodiments, the anti-CCR8 antibodies of the present disclosure are administered subcutaneously. Administration of the therapeutic anti-CCR8 antibodies can be carried out with any suitable excipient, carrier or other reagent to provide suitable or improved tolerance, transfer, delivery, etc. Examples

[0119] Example 1: Discovery of Anti-CCR8 Based on Immunization with Engineered MEM Nanoparticles.

[0120] MEM was designed to embody the structure and dynamics of extracellular loop 1 (DQWVFGT) (SEQ ID NO: 91) and extracellular loop 2 (VASEDGVLQC) (SEQ ID NO: 92) of CCR8. Keeping the epitope residues of extracellular loop 1 and extracellular loop 2 fixed, a protein backbone was constructed using a protein design AI algorithm to present the epitope structure. The backbone sequence was optimized to meet three criteria: [1] the structure matches the predicted epitope structure in CCR8, [2] the stability of the MEM molecule, and [3] water solubility. Without sacrificing these three criteria, the length of the protein backbone was optimized to be as small as possible. The amino acid sequence of the final CCR8 MEM design is SPEIKKLIEQIKSDQWVFGTEACNEIQTLLKEESGPAKIEVASEDGVLQCRIVF (SEQ ID NO:93). The structure of this MEM design was confirmed by nuclear magnetic resonance (NMR) to be within 3.2 Å RMSD of the predicted structure. When only considering the epitope residues, the RMSD between the experimental structure and the predicted structure was 1.6 Å.

[0121] CCR8 MEM was expressed and purified as a nanoparticle fusion protein fused to ferritin from Helicobacter pylori. MEM was linked to the N-terminus of each ferritin subunit using the linker sequence GGGGS (SEQ ID NO: 94). These nanoparticles were expressed in Escherichia coli and purified by nickel-NTA affinity chromatography.

[0122] BALB / c mice were divided into two separate groups for immunization. In one group, immunization was alternated between: MEM immunization for one week and CCR8 virus-like particles (VLP) (Acro Biosystems, Cat# #CC8-H52P4) immunization every other week. In the other group, mice were immunized with HEK293 and / or CHO cells expressing CCR8 for one week and with CCR8 VLP every other week. MEM immunization was 15 - 25 μg per injection, and CCR8 VLP was 6 μg per injection.

[0123] Example 2: Antibodies produced by Top monoclonal hybridomas showed strong anti-CCR8 binding, did not bind CCR4, and competed with the GS-1811 benchmark.

[0124] Hybridoma-produced antibodies were incubated with cells transiently expressing CCR8 or CCR4 for 1 hour, washed once with DPBS + 1% FBS, and then labeled with anti-mouse IgG secondary antibody for 1 hour. The cells were then washed again and measured by flow cytometry. For cross-blocking studies, hybridoma-produced antibodies were co-incubated with GS-1811, which is a human IgG1, for 1 hour, washed once with DPBS + 1% FBS, and then labeled with anti-mouse IgG secondary antibody for 1 hour. The cells were then washed 1 time and measured by flow cytometry. All measurements were performed on ice and using freezing buffer.

[0125] Figure 1A and Figure 1B Showed that anti-CCR8 hybridoma positive clone 1E6 specifically bound to cells expressing CCR8, but not to cells expressing CCR4. Figure 2 Showed that 1E6 hybridoma clone competed with the GS-1811 benchmark for binding to cells expressing CCR8.

[0126] Figures 3A to 3C Showed that chimeric or humanized anti-CCR8 antibodies derived from hybridoma clone 1E6 specifically bound to human CCR8, but not to human CCR4 or mouse CCR8. FACS analysis showed the binding of anti-CCR8 antibodies or control antibodies (hIgG1 isotype, anti-mouse CCR8, and anti-human CCR4) to ExpiCHO cells expressing human CCR8, human CCR4, or mouse CCR8. The values plotted are the percentages of antibodies binding to the target cells.

[0127] Figure 4A and Figure 4BShow the binding potency of anti-human CCR8 antibody to CHO-K1 cell line overexpressing human CCR8. FACS analysis shows the binding of anti-CCR8 antibody or control antibodies (hIgG1 isotype, anti-mouse CCR8 and anti-human CCR4) to CHO-K1 cells overexpressing human CCR8. The values plotted are median fluorescence intensity. EC 50 Value is the average of n = 2 experiments. n / a: Inactive.

[0128] Figure 5A and Figure 5B Show the antagonistic activity of anti-human CCR8 antibody in CHO-K1 reporter cell line overexpressing human CCR8. Luminescence readings show that the activation of human CCR8 by agonist CCL1 was inhibited by pre-treating the reporter cells with anti-CCR8 antibody. The values plotted are relative luminescence units. EC 50 Value is the average of n = 2 experiments. n / a: Inactive.

[0129] Figures 6A to 6C Show the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-CCR8 antibody using human peripheral blood mononuclear cells (PBMC). FACS analysis shows the ADCC potency of anti-CCR8 antibody from two different PBMC donors in a dose-dependent manner. The values plotted are the percentage of dead target cells (cell lysis %) out of the total target cells. EC 50 Value is the average of n = 1 - 2 experiments. n / a: Inactive.

[0130] Figure 7 Is a graph showing the thermal stability of anti-CCR8 antibody. Using a Prometheus Panta instrument, the melting temperature (T M ) of the CCR8-binding antibody was determined by monitoring the change in fluorescence intensity of the antibody loaded into a capillary as the temperature was slowly increased.

[0131] Figure 8A Figures 8B to 8D show the study design and results of an in vivo efficacy study using anti-CCR8 antibody SD-171467-afuc as a single therapy. Figures 8B and 8C show the tumor volume and percentage change in tumor volume after drug treatment. Tumor measurements and drug administrations were performed on day 0, day 3, day 7, day 10, and day 17. Figure 8D shows the change in body weight of mice derived from drug administrations on day 0, day 3, day 7, day 10, and day 17. For all protocols, no significant loss of body weight in mice was observed after drug treatment.

[0132] Figures 9A to 9C Show the results of the pharmacokinetic study. Figure 9A Show the pharmacokinetic study design of SD-171467-afuc. Figure 9BShow the pharmacokinetic curve of SD-171467-afuc. After a single administration of 10 mg / kg, sera were collected at the indicated time points. Human IgG levels were quantified using the MSD human IgG kit. Figure 9C Summarize the pharmacokinetic parameters calculated by Phoenix WinNonLin.

[0133] Figure 10A Figures 10A to 10F show the study design and results of an in vivo efficacy study using SD-171467-afuc and anti-PD1 antibody as combination treatment, as well as the mechanism of action study of CCR8+ Treg depletion. Figure 10A Show the in vivo study design of combination treatment of SD-171467-afuc and anti-PD1 antibody and CCR8+ Treg depletion. Figures 10B and 10C show the tumor volume and percentage change in tumor volume after drug treatment. Tumor measurements and drug administrations were performed on days 0, 4, 7, 11, and 18. Figure 10D shows the change in body weight of mice derived from drug administration on days 0, 4, 7, 11, and 18. For all protocols, no significant body weight loss in mice was observed after drug treatment. Figures 10E and 10F show that treatment with SD-171467-afuc significantly reduced Treg-CCR8+ in tumors, but not in the spleen. Flow cytometry was used to analyze Treg-CCR8+ depletion in tumor (Figure 10E) and spleen (Figure 10F) samples from the PBS and SD-171467-afuc (1 mg / kg) treatment groups. The figure shows the percentage of Treg-CCR8+ (CD4+Foxp3+CCR8+) in live CD45+CD3+CD4+Foxp3+ cells in tumors and spleens. The student t-test (two-tailed) was used to compare the two treatment groups. p-values are shown in the figure (*p < 0.05; NS p > 0.05).

[0134] Hybridomas were generated from immune murine B cells using standard electrocell fusion methods. Spleens were isolated from immune mice and ground on ice in 15 - 20 mL of DMEM (without additives). Splenocytes and Ag8.653 murine myeloma cells were mixed in DMEM on ice at a ratio of 1:1. Prior to electrofusion, the cells were resuspended in a certain volume of ECF buffer such that their density was 2×10 6 cells / mL. Electrocell fusion was performed using the fusion cycle parameters (Nepagene / ECFG21). The cells were plated in 96-well plates containing selection medium, and the fusion efficiency was confirmed 7 days after fusion.

[0135] Antibody expression and expression level: The antibody expression plasmid was transiently introduced into an animal cell line using the ExpiFectamine CHO transfection kit (Thermo Fisher, Cat# A29129) to obtain transfectants that produce anti-CCR8 chimeric or humanized antibodies. For the host cell line, ExpiCHO-S (Thermo Fisher, Cat# A29127) or a suspension CHO cell line with the α1,6-fucosyltransferase (FUT8) gene knocked out (referred to as "WT CHO" and "FUT8 CHO" hereafter when mentioned again) was used. After growing for 6 - 12 days after DNA introduction, the cell suspension of WT CHO or FUT8 CHO was harvested by centrifugation at 4000×g for 20 min, and then filtered using a 0.2 μm disposable PES filter unit (Fisher Scientific, Cat# FB12566504). The anti-CCR8 antibody was recovered from the filtrate using protein A purification (HiTrap MabSelect SuRe; Cytiva, Cat# GE11-0034-93). WT CHO was used to express antibodies with standard glycosylation, and FUT8 CHO was used to express afucosylated antibodies with enhanced effector function (denoted as "-afuc").

[0136] Antibody humanization: Humanization was achieved via multiple methods. In some cases, the CDRs were directly transplanted onto the human germline using publicly available tools (DOI: 10.1080 / 19420862.2021.2020203). To potentially improve expression and performance, single amino acids were reverted to the parental mouse sequence. In other cases, two or more amino acids were reverted to the parental mouse sequence. In all cases, the CDRs remained unchanged. Then the humanized variants were tested and their potencies were compared with the parental chimeras.

[0137] Thermal stability: Antibodies were prepared at a concentration of 1 mg / mL in PBS (pH 7.4). 10 μL of each sample was loaded into a suitable capillary (Nanotemper, Cat# PR-C002) via a pipette. The capillary was loaded into a Nanotemper Prometheus Panta instrument and a protocol was defined. Thermal unfolding was carried out from 25 - 95 °C at a rate of 1 °C / min. Fluorescence was monitored and T m .

[0138] Cell binding assay: PathHunter CHO-K1 β-arrestin cells (DiscoverX, Cat# 93-0196C2) were cultured in AssayComplete Cell Culture Kit-107 (DiscoverX, Cat# 92-3107G) supplemented with G418 (Gibco, Cat# 10131-035) and hygromycin B (Invitrogen, Cat# 10687010) according to the manufacturer's instructions. ExpiCHO-S cells (Thermo Fisher, Cat# A29127) were cultured in ExpiCHO Expression Medium (Gibco, Cat# A29100-01).

[0139] To generate cells expressing human CCR8, human CCR4, or mouse CCR8, ExpiCHO cells were diluted to 6×10 6 cells / mL and transfected with the corresponding expression plasmid using the ExpiFectamine CHO transfection kit (Gibco, Cat# A29129) according to the manufacturer's instructions. Within 5 days after transfection, the transfected cells were used for the cell binding assay. To detect human CCR4 or mouse CCR8 in the transfected cells, they were stained with anti-hCCR4 (BioLegend, Cat# 359402) or anti-moCCR8 (BioLegend, Cat# 150302), respectively.

[0140] For the cell binding assay, PBS (Corning, Cat# 21-040-CV) supplemented with 2% FBS (MilliporeSigma, Cat# F4135) and 2 mM EDTA (Quality Biological, Cat# 351-027-721) was used as the assay buffer. The cells were counted and then resuspended in the assay buffer, and then at 1×10 5Cells were seeded at [number] cells / well in a 96-well plate (VWR, Cat# 89089-826). After removing the supernatant, the plate was centrifuged and kept on ice for the remaining assays. After removing the supernatant, the indicated antibodies were diluted in assay buffer and added to the cells at increasing concentrations (0.004 - 66.66 nM) and kept on ice for 20 minutes. After incubation, the plate was centrifuged and the supernatant removed, then the cells were washed once with assay buffer, followed by centrifugation and removal of the wash. After washing, rat anti-human IgG Fc Alexa Fluor 647 (BioLegend, Cat# 410714), rat anti-mouse IgG Fc Alexa Fluor 647 (BioLegend, Cat# 406618), or goat anti-rat IgG Fc Alexa Fluor 647 secondary antibody (BioLegend, Cat# 405416) diluted 1:200 in assay buffer was added to the cells and kept on ice for 20 minutes. After incubation, the plate was centrifuged and the supernatant removed, then the cells were washed once with assay buffer, followed by centrifugation and removal of the wash. After washing, DAPI (BioLegend, Cat# 422801) diluted 1:5000 in assay buffer was added to the cells. Cells were analyzed for binding on a Miltenyi MACSQuant 16 flow cytometer. Flow cytometry data were analyzed using FlowJo flow cytometry analysis software. Graphs were made and EC 50 values were calculated using GraphPad Prism 9.3.0.

[0141] CCR8 antagonist assay: PathHunter CHO-K1 β-arrestin cells (DiscoverX, Cat# 93-0196C2) were cultured according to the manufacturer's instructions in AssayComplete Cell Culture Kit-107 (DiscoverX, Cat# 92-3107G) supplemented with G418 (Gibco, Cat# 10131-035) and hygromycin B (Invitrogen, Cat# 10687010).

[0142] For the CCR8 antagonist assay, PathHunter CHO-K1 β-arrestin cells were detached using AssayComplete Cell Detachment Reagent (DiscoverX, Cat# 92-0009), then counted and seeded at 2.5×10 4 cells / well in a 96-well white opaque microplate (Corning, Cat# 3917) and incubated at 37°C and 5% CO 2Overnight. Next, the indicated antibody was diluted in AssayComplete Cell Plating 2 reagent to a final concentration of 10X and added to the cells at increasing concentrations (10X final concentration range: 0.04 - 666.6 nM). The cells were incubated with the antibody at 37 °C and 5% CO 2 for 30 minutes, followed by adding the CCL1 (PeproTech, Cat# 300 - 37) agonist to the cells at 10 times the EC 80 value determined by the agonist. The cells were incubated with the antibody and agonist (now both at a final concentration of 1X) at 37 °C and 5% CO 2 for 90 minutes. After agonist incubation, according to the manufacturer's instructions, a detection substrate was generated using the PathHunter Detection Kit (DiscoverX, Cat# 93 - 0001) and added to the cells. The cells were incubated with the detection substrate at room temperature for 1 hour and then analyzed on an Agilent Cytation 5 Cell Imaging / Multi - mode Reader with the following settings: read speed: 1 second / well, read height: 1 mm, gain: 135. The IC 50 value was calculated using Graphpad Prism 9.3.0.

[0143] ADCC / PBMC: According to the manufacturer's instructions, PathHunter CHO - K1 β - arrestin cells (DiscoverX, Cat# 93 - 0196C2) were cultured in AssayComplete Cell Culture Kit - 107 (DiscoverX, Cat# 92 - 3107G) supplemented with G418 (Gibco, Cat# 10131 - 035) and hygromycin B (Invitrogen, Cat# 10687010).

[0144] For the ADCC / PBMC assay, the PathHunter CHO - K1 β - arrestin cells were counted to assess cell number and viability. The cells were stained with cell - trace CFSE (Thermo Fisher, Cat# C345554A). The cells were centrifuged and resuspended in growth medium at 1×10 5 cells / mL and seeded at 1×10 4 cells / well into a 96 - well plate (VWR, Cat# 29442 - 056) and incubated in a cell culture incubator at 37 °C and 5% CO 2Incubate overnight. After incubation, add the indicated antibodies in the assay medium (RPMI 1640, containing 10% FBS, 1% penicillin / streptomycin and 5 ng / mL IL2) at increasing concentrations (0.00000667 - 6.666667 nM or 0.0000333 - 33.33333 nM) to the cells and incubate at 37 °C, 5% CO 2 for 20 minutes. Then, add 2×10 5 peripheral blood mononuclear cells (PBMCs) (Stemcell, Cat# 70025.1) to each well of a 96-well plate. Incubate the cells and antibodies at 37 °C in a 5% CO 2 incubator for 18 - 24 hours. Collect the samples, then stain with the LIVE / DEAD Fixable Aqua Dead Cell Staining Kit (Thermo Fisher, Cat# L34957) and analyze on a Miltenyi MACSQuant 16 flow cytometer. Gate on % dead target cells using FITC+ Live / Dead+. Calculate the EC 50 values using Graphpad Prism 9.3.0.

[0145] In Vivo Efficacy and Mechanism Study: Use 8-week-old female transgenic B-hCCR8 mice (Biocytogen, Cat#110096). After local hair shaving, inoculate 0.5×10 6 MC-38 (ShanghaiShunran Biotechnology Co., Ltd., Cat# M023) cells in 100 μl PBS subcutaneously into the upper left side of each mouse. Monitor tumor growth and mouse body weight twice a week. For each individual tumor, measure the longest longitudinal diameter as the length and the widest transverse diameter as the width using a Traceable digital caliper (VWR, Cat#62379-531). Then calculate the tumor volume (TV) using the formula: TV = [length × (width) 2 / 2. When the average tumor volume reaches 87 - 89 mm 3 and 109 - 111 mm 3At that time, the mice were randomly grouped. Each group contained 8 mice. In the monotherapy study, human IgG1 negative control, GS-1811, and SD-171467-afuc were prepared as stock solutions at 1 mg / mL. In the combination therapy / mechanism study, anti-mouse PD-1 and SD-171467-afuc were prepared as stock solutions at 0.03 and 0.1 mg / mL, respectively. Additionally, a mixed solution containing both anti-mouse PD-1 (0.03 mg / mL) and SD-171467-afuc (0.1 mg / mL) was used for the combination therapy / mechanism study. For each mouse, the volume of vehicle control (PBS) or antibody to be administered was calculated using the formula: volume (μl) = mouse body weight (g) × 10 μl / g. During the first two weeks, the antibody drugs were administered twice a week via the intraperitoneal (i.p.) route, and the fifth dose was administered one week after the fourth dose, for a total of 5 doses. After the initial drug treatment, tumor growth was monitored for 14 days.

[0146] At the end of the combination therapy / mechanism study (22 days post-treatment), tumor and spleen samples were collected from human PBS and the 1 mg / kg Sd-171467-afuc treatment group and minced into small pieces in digestion buffer [DMEM medium (Corning, Cat# MT10013CM) supplemented with 0.36 mg / mL type IV collagenase (MPBiomedicals, Cat# Ic19511090) and 10 μg / mL DNase I (STEMCELL Technologies Inc., Cat# 07900)]. The samples were then incubated at 37 °C for 30 - 45 minutes and then filtered through a cell strainer (Corning, Cat# 352340). The filtered cell suspension was centrifuged and the supernatant was discarded. The cell pellet was resuspended in red blood cell lysis buffer (BioLegend, Cat# 420301) to remove red blood cells. The remaining cell suspension was then counted using an automated cell counter and the total cell number was recorded. 100 ul of the cell suspension was used for FACS analysis. The cell samples were stained with a live / dead marker (Thermo Fisher, Cat#L34957) for 20 minutes at room temperature and treated with a mouse Fc blocker (BioLegend, Cat# 101302), then stained with an antibody mixture [anti-mouse CD45.1-APC / Cy7 (BioLegend, Cat# 103116), anti-mouse CD3-BV-605 (BioLegend, Cat# 100237), anti-mouse CD4-FITC (BioLegend, Cat# 130308), anti-CD8-mouse-APC (BioLegend, Cat# 162305), anti-human CCR8-PE (BioLegend, Cat# 365704 antibody)] and kept on ice for 30 minutes. The samples were then fixed with FoxP3 fix / perm buffer (BioLegend, Cat# 421401) and permeabilized with FoxP3 perm buffer (BioLegend, Cat# 421402). The samples were then stained with an anti-Foxp3-BV421 (BioLegend, Cat# 126419) antibody and analyzed using a Miltenyi MACSQuant 16 flow cytometer. The CCR8+ Treg cell population was gated (single cell / live cell / CD45.1+ / CD4+ / Foxp3+ / CCR8+).

[0147] Pharmacokinetic study: 8-week-old female transgenic B-hCCR8 mice (Biocytogen, Cat#110096) were used. After local hair shaving, 0.5×10 6 MC-38 cells (ShanghaiShunran Biotechnology Co., Ltd., Cat# M023) in 100 μl PBS were inoculated subcutaneously into the upper left side of each mouse. Fourteen days after tumor cell inoculation, SD-171467-afuc was administered intraperitoneally to the tumor-bearing mice at 10 mg / kg. At 0 hours (pre-treatment), 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 18 hours, 24 hours, 48 hours, 96 hours, 192 hours, 384 hours, and 576 hours (endpoint), blood samples were collected from the facial vein of the treated mice using a Goldenrod Animal Lancet (3 mm, Medipoint). 60 μL of blood was collected using a BD microcollection tube with a heparin coating. After standing on ice for 30 - 60 min, the serum was separated by centrifugation at 5,000 rpm × 5 min at 4°C, transferred to a 2 mL cryovial (Heathraw Scientific, Cat# HS23202A), rapidly frozen in dry ice in ethanol, and stored at -80°C for downstream analysis. According to the kit instructions, the concentration of the drug in the serum was measured using a Human / NHP IgG Quantification Kit from Meso Scale Diagnostics (MSD, Cat# K150JLD). Briefly, a dilution of the animal serum was applied to a plate pre-coated with a human / NHP capture antibody and detected with an anti-IgG secondary antibody. Using MSD DiscoveryWorkbench software, the serum concentration of IgG was calculated by interpolation from a standard curve. These data were then used for non-compartmental PK analysis using Certara Phoenix™ WinNonLin.

[0148] It is expected that for any method, kit, reagent, or composition of the present invention, any embodiment discussed in this specification can be implemented, and vice versa. In addition, the compositions of the present invention can be used to implement the methods of the present invention.

[0149] It should be understood that the specific embodiments described herein are shown by way of illustration and not as a limitation of the present invention. The main features of the present invention can be adopted in various embodiments without departing from the scope of the present invention. Those skilled in the art will recognize or be able to determine many equivalents of the specific processes described herein using only routine experimentation. Such equivalents are considered to be within the scope of the present invention and are covered by the claims.

[0150] All publications and patent applications mentioned in this specification represent the technical level of those skilled in the art to which the present invention pertains. All publications and patent applications are incorporated herein by reference to the extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0151] When used in the claims and / or the specification in conjunction with the term "comprising", the use of the word "a" or "an" can mean "one", but it also conforms to the meanings of "one or more", "at least one", and "one or more than one". The term "or" used in the claims is used to mean "and / or", unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the present disclosure supports both the definition of referring only to alternatives and "and / or". Throughout this application, the term "about" is used to indicate that a value includes the inherent variations of the device error, the method for determining the value, or the variations present in the subject of study.

[0152] As used in this specification and the claims, the word "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any embodiment of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of". As used herein, the phrase "consisting essentially of" requires the specified integer or step and those that do not materially affect the characteristics or functions of the claimed invention. As used herein, the term "consisting" is only used to indicate the presence of the listed integers (e.g., features, elements, characteristics, properties, method / process steps, or limitations) or groups of integers (e.g., features, elements, characteristics, properties, method / process steps, or limitations).

[0153] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations that contain one or more repetitions of an item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of ordinary skill in the art will understand that there is generally no limit to the number of items or terms in any combination, unless it is apparent from the context.

[0154] As used herein, approximate words such as, but not limited to, "about", "substantial", or "substantially" are meant to be understood as not necessarily being absolute or perfect when so modified, but will be considered close enough by one of ordinary skill in the art to warrant the situation being indicated as existing. The degree to which the description may vary will depend on how much change can be made and still have one of ordinary skill in the art recognize that the modified feature still possesses the desired characteristics and capabilities of the unmodified feature. Generally, but subject to the foregoing discussion, a numerical value modified by an approximate word such as "about" in this disclosure may vary from the stated value by at least ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 10%, 12%, or 15%.

[0155] In addition, the section headings provided herein are for compliance with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings should not limit or characterize the (multiple) inventions set forth in any claim that may issue from this disclosure. Specifically, for example, although the heading is called "Field of the Invention", such a claim should not be limited by the language describing the so-called technical field under that heading. Additionally, the technical description in the "Background of the Invention" section should not be construed as an admission that the technology is prior art to any (multiple) invention in this disclosure. Nor should the "Summary" be considered a characterization of the (multiple) inventions set forth in the claims that may issue. Additionally, any reference to the "invention" in the singular in this disclosure should not be used to question the existence of only a single novel point in this disclosure. Multiple inventions may be set forth in the limitations of multiple claims that may issue from this disclosure, and such claims accordingly define the (multiple) inventions so protected and their equivalents. In all cases, the scope of such claims should be considered on its own merits in light of this disclosure, but should not be limited by the headings set forth herein.

[0156] For each item in the claims, each dependent claim can depend on an independent claim and on each item of the prior dependent claims that depend on each claim, provided that the prior claim provides an appropriate prior basis for the claim item or element.

[0157] To assist the Patent Office and any readers of any patent issued on the basis of this application in interpreting the claims appended hereto, the Applicants wish to note that they do not intend for any of the claims appended hereto to be invoked against them by virtue of paragraph 6 of 35 U.S.C. § 112, paragraph (f) of U.S.C. § 112, or equivalent provisions as they existed on the date of filing of this application, unless the words "means for" or "step for" are expressly used in a particular claim.

[0158] According to the present disclosure, all of the compositions and / or methods disclosed and claimed herein can be prepared and implemented without undue experimentation. While the compositions 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 changes can be made in the compositions and / or methods described herein and in the steps of the methods described herein or in the order of the steps of the methods described herein without departing from the spirit, scope, and concept of the present invention. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

Claims

1. An anti-CCR8 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: A heavy chain variable domain (VH) complementarity-determining region (CDR) 1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 49, 67, 73, 79 or 85; and a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 2, 50, 68, 74, 80 or 86; and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 3, 51, 69, 75, 81 or 87; and A light chain variable domain (VL) CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 4, 52, 55, 58, 61, 64, 70, 76, 82 or 88; and a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 5, 53, 56, 59, 62, 65, 71, 77, 83 or 89; and a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 6, 54, 57, 60, 63, 66, 72, 78, 84 or 90.

2. The antibody or antigen-binding fragment according to claim 1, wherein the antibody comprises: A VH comprising the amino acid sequence of any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 or 15 respectively; and A VL comprising the amino acid sequence of any one of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23 or 24 respectively.

3. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody comprises: A VH encoded by a nucleic acid sequence having at least 95, 96, 97, 98, 99 or 100% sequence identity with any one of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32 or 33; and A VL encoded by a nucleic acid sequence having at least 95, 96, 97, 98, 99 or 100% sequence identity with any one of SEQ ID NO: 34, 35, 36, 37, 18, 29, 40, 41 or 42.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody, bispecific antibody, multivalent antibody, multispecific antibody, diabody, chimeric antibody, scFv antibody or a binding fragment thereof.

5. The antibody or antigen-binding fragment according to claim 4, wherein the antibody is fused to the Fc domain of any one of: human IgG1, human IgG2, human IgG3 and human IgG4.

6. The antibody or binding fragment according to any one of claims 1 to 5, wherein the antibody is a non-fucosylated full-length antibody.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the nucleic acid sequence is optimized for expression in bacterial, fungal, mammalian, insect or plant cells.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment does not bind to CCR4.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the heavy chain CDRs are SEQ ID NO: 1, 2 and 3, and the light chain CDRs are SEQ ID NO: 4, 5 and 6.

10. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody according to any one of claims 1 to 9.

11. The method according to claim 10, wherein the disease is cancer.

12. The method according to claim 11, wherein the cancer is infiltrated by regulatory T cells.

13. The method according to any one of claims 10 to 12, wherein the subject is a human.

14. A method of preparing an anti-CCR8 antibody or antigen-binding fragment, comprising expressing the antibody or antigen-binding fragment in a cell or in vitro, wherein the antibody or antigen-binding fragment comprises: Heavy chain variable domain (VH) complementarity determining region (CDR) 1, which comprises the amino acid sequence of any one of the following SEQ ID NO: 1, 49, 67, 73, 79 or 85; and VH CDR2, which comprises the amino acid sequence of any one of the following SEQ ID NO: 2, 50, 68, 74, 80 or 86; and VH CDR3, which comprises the amino acid sequence of any one of the following SEQ ID NO: 3, 51, 69, 75, 81 or 87; and Light chain variable domain (VL) CDR1, which comprises the amino acid sequence of any one of the following SEQ ID NO: 4, 52, 55, 58, 61, 64, 70, 76, 82 or 88; and VL CDR2, which comprises the amino acid sequence of any one of the following SEQ ID NO: 5, 53, 56, 59, 62, 65, 71, 77, 83 or 89; and VL CDR3, which comprises the amino acid sequence of any one of the following SEQ ID NO: 6, 54, 57, 60, 63, 66, 72, 78, 84 or 90.

15. The method according to claim 14, wherein the cell is a bacterial, fungal, human, plant or insect cell.

16. The method according to any one of claims 14 to 15, wherein the antibody is a monoclonal antibody, bispecific antibody, multivalent antibody, multispecific antibody, diabody, chimeric antibody, scFv antibody or a fragment thereof.

17. The method according to any one of claims 14 to 16, wherein the antibody or antigen-binding fragment is non-fucosylated.

18. The method according to any one of claims 14 to 17, wherein the antibody or antigen-binding fragment does not bind to CCR4.

19. A nucleic acid comprising an anti-CCR8 antibody or antigen-binding fragment thereof, comprising: A polynucleotide encoding a heavy chain variable domain, which has at least 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32 or 33; and A polynucleotide encoding a light chain variable domain, which has at least 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41 or 42.

20. The nucleic acid according to claim 19, wherein the antibody is a monoclonal antibody, bispecific antibody, multivalent antibody, multispecific antibody, diabody, chimeric antibody, scFv antibody or a fragment thereof.

21. The nucleic acid according to claim 20, wherein the antibody-binding domain is fused to the Fc domain of any one of: human IgG1, human IgG2, human IgG3 and human IgG4.

22. The nucleic acid according to any one of claims 19 to 21, wherein the nucleic acid sequence is optimized for expression in bacterial, fungal, mammalian, insect or plant cells.

23. The nucleic acid according to any one of claims 19 to 22, wherein the antibody or antigen-binding fragment does not bind CCR4.

24. A vector comprising the nucleic acid according to claim 19.

25. A host cell comprising the nucleic acid vector according to claim 24.

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