Chemokine receptor 8 (CCR8) antibody

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 treating cancer diseases caused by loss of regulatory T cells. Methods of using the anti-CCR8 antibodies or antigen-binding fragments thereof are also provided herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 417,507, filed October 19, 2022, and U.S. Provisional Patent Application No. 63 / 519,004, filed August 11, 2023, the entire contents of which are incorporated herein by reference.

[0002] This document relates to materials and methods for treating cancer, in particular the use of anti-CCR8 antibodies to reduce or eliminate cells that express CCR8 and treat cancer.

[0003] Incorporation-by-Reference of Material Submitted on a Compact Disc This application contains a Sequence Listing that has been submitted via EFS-Web in XML format and is incorporated herein by reference in its entirety. The XML copy, created on October 19, 2023, is named IBIO:2022WO.xml and is 117,514 bytes in size. [Background technology]

[0004] Without limiting the scope of the present invention, its background is described in relation to the elimination of cells expressing CCR8.

[0005] One such patent is U.S. Patent No. 11,427,640, issued to Berndt et al. and entitled "CCR8 Antibodies for Therapeutic Applications." The inventors are described to teach the generation of antibodies that specifically bind to chemokine receptors, such as CC or CXC chemokine receptors. The isolated sulfated polypeptides and conjugates thereof can be used as antigens or in off-target panning to facilitate the generation of anti-human, anti-cynomolgus, and / or anti-mouse chemokine receptor antibodies, e.g., to generate antibodies with fully human CDRs and / or other properties favorable for therapeutic use. The inventors also teach antibodies that specifically bind to human, cynomolgus, and / or mouse CCR8 with properties for therapeutic use, such as cross-reactive antibodies, fully human antibodies, low internalization (including non-internalization) antibodies, and antibodies that efficiently induce ADCC and / or ADCP in Treg cells.

[0006] Another such invention is taught in U.S. Patent Application Publication No. 20220195057A1, filed by Li and Huang and entitled "Anti-CCR8 Monoclonal Antibodies and Uses Thereof." This application is directed to providing antibodies or fragments thereof having binding specificity for the human chemokine (CC motif) receptor 8 (CCR8) protein. These antibodies are capable of binding to CCR8 with high affinity and mediating antibody-dependent cellular cytotoxicity (ADCC). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 11,427,640 [Patent Document 2] U.S. Patent Application Publication No. 20220195057A1 Summary of the Invention [Problem to be solved by the invention]

[0008] Despite these advances, what is needed are novel antibodies, methods and uses that specifically bind to CCR8 without binding to closely related members of the chemokine receptor family, such as the CC or CXC chemokine receptors. [Means for solving the problem]

[0009] As exemplified and broadly described herein, embodiments of the disclosure relate to an anti-CCR8 antibody or 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 NOs: 1, 49, 67, 73, 79, or 85; and a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 50, 68, 74, 80, or 86; and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 6, 54, 57, 60, 63, 66, 72, 78, 84, or 90. In one embodiment, the antibody comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 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 NOs: 16, 17, 18, 19, 20, 21, 22, 23, or 24, respectively. In another embodiment, 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 NOs: 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 NOs: 34, 35, 36, 37, 18, 29, 40, 41, or 42. In another embodiment, the antibody is a monoclonal, bispecific, multivalent, multispecific, diabody, chimeric, scFv antibody, or fragment thereof. In another embodiment, the antibody is fused to the Fc domain of any one of human IgG1, human IgG2, human IgG3, and human IgG4. In another embodiment, the antibody is a defucosylated full-length antibody. In another embodiment, the nucleic acid sequence is optimized for expression in bacterial, fungal, mammalian, insect, or plant cells.In another embodiment, the antibody or antigen-binding fragment does not bind to CCR4. In another embodiment, the heavy chain CDRs are SEQ ID NOs: 1, 2 and 3, and the light chain CDRs are SEQ ID NOs: 4, 5 and 6.

[0010] As exemplified and broadly described herein, aspects of the present disclosure relate to a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody described above. In another aspect, the disease is cancer. In another aspect, the cancer is infiltrated with regulatory T cells. In another aspect, the subject is human.

[0011] As exemplified and broadly described herein, an aspect of the disclosure is a method of making 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 a heavy chain variable domain (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 49, 67, 73, 79, or 85; and a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 50, 68, 74, 80, or 86; and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 6, 54, 57, 60, 63, 66, 72, 78, 84, or 90. In one embodiment, the cell is a bacterial, fungal, human, plant, or insect cell. In another embodiment, the antibody is a monoclonal, bispecific, multivalent, multispecific, diabody, chimeric, scFv antibody, or fragment thereof. In another embodiment, the antibody or antigen-binding fragment is defucosylated. In another embodiment, the antibody or antigen-binding fragment does not bind to CCR4.

[0012] As exemplified and broadly described herein, an embodiment of the present disclosure relates to a nucleic acid comprising an anti-CCR8 antibody or antigen-binding fragment, the nucleic acid comprising a heavy chain variable domain encoding a polynucleotide having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, or 33; and a light chain variable domain encoding a polynucleotide having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or 42. In one embodiment, the antibody is a monoclonal, bispecific, multivalent, multispecific, diabody, chimeric, scFv antibody, or fragment thereof. In another embodiment, 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 embodiment, the nucleic acid sequence is optimized for expression in bacterial, fungal, mammalian, insect, or plant cells. In another embodiment, the antibody or antigen-binding fragment does not bind to CCR4.

[0013] As exemplified and broadly described herein, aspects of the present disclosure relate to vectors comprising the nucleic acids disclosed above. As exemplified and broadly described herein, aspects of the present disclosure relate to host cells comprising nucleic acids that are vectors disclosed above. [Brief explanation of the drawings]

[0014] For a more complete understanding of the features and advantages of the present invention, reference is now made to the following detailed description of the invention taken in conjunction with the accompanying drawings. [Figure 1A-1B] Figure 1A shows flow cytometry graphs depicting cell binding of hybridoma supernatants from immunized successful clone 1E6, identified from a CCR8 MEM-ELISA binding and cell-binding hybridoma screen. Figure 1A shows that clone 1E6 binds to CCR8-expressing cells. Figure 1B shows that clone 1E6 specifically binds to CCR8 versus the most closely related CCR4 GPCR. [Figure 2]FIG. 1 shows that the 1E6 hybridoma clone competes with the GS-1811 benchmark for binding to CCR8-expressing cells. [Figure 3A-3C] FIG. 1 shows that anti-CCR8 antibodies derived from chimeric or humanized hybridoma clone 1E6 specifically bind to human CCR8, but do not bind to human CCR4 or mouse CCR8. [Figure 4A-4B] FIG. 1 shows the human CCR8 binding potency of anti-CCR8 antibodies. [Figure 5A-5B] FIG. 1 shows the antagonist activity of anti-CCR8 antibodies. [Figures 6A-6C] FIG. 1 shows 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). [Figure 7] 1 is a graph showing the thermal stability of anti-CCR8 antibodies. [Figures 8A-8D] Figures 8A and 8B show the study design and results of an in vivo efficacy study using the anti-CCR8 antibody SD-171467-afuc as monotherapy. Figures 8B and 8C show tumor volume and percent tumor volume change after drug treatment. Figure 8D shows the change in mouse body weight from drug administration. [Figures 9A-9C] Figure 9 shows the results of a pharmacokinetic study. Figure 9A shows the pharmacokinetic study design for SD-171467-afuc. Figure 9B shows the pharmacokinetic profile of SD-171467-afuc. Figure 9C summarizes the calculated pharmacokinetic parameters. [Figures 10A-10F]Figures 10A and 10B 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 mechanism of action study on the depletion of CCR8+ Tregs. Figure 10A shows the in vivo study design of the combination treatment of anti-PD1 antibody and SD-171467-afuc and the depletion of CCR8+ Tregs. Figures 10B and 10C show the tumor volume and percent tumor volume change after drug treatment. Figure 10D shows the change in mouse body weight from drug administration. Figures 10E and 10F show Treg-CCR8+ levels in the tumor and spleen after PBS or SD-171467-afuc treatment. DETAILED DESCRIPTION OF THE INVENTION

[0015] While the making and use of various embodiments of the invention are described in detail below, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a variety of specific contexts. The specific embodiments described herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0016] To facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings as commonly understood by one of ordinary skill in the art in the areas relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer to a singular entity only, but include a general class of which a particular example may be used for illustration. While the terms herein are used to describe particular embodiments of the present invention, their usage does not limit the present invention except as outlined in the claims.

[0017] Unless expressly indicated, in any method described or disclosed herein that includes two or more acts, the order of the acts is not necessarily limited to the order in which the acts of the method are recited, although it should be understood that the present disclosure encompasses example embodiments in which the order of the acts is very limited.

[0018] The present invention is directed to novel anti-CCR8 antibodies and antigen-binding fragments thereof that are used to bind to human CCR8 without binding to other members of the chemokine receptor family, including the closely related CCR4.

[0019] Chemokine (C-C motif) receptor 8, also known as CCR8, is a protein encoded by the CCR8 gene in humans and is also designated CDw198 (cluster of differentiation w198). CCR8 is a member of the beta-chemokine receptor family and a seven-transmembrane G protein-coupled receptor (GPCR) protein. The ligand for CCR8 is CCL1. Human CCR8 is UniProt P51685, RefSeq (mRNA) NM_005201, and RefSeq (protein) NP_005192.

[0020] CCR8 is primarily expressed in regulatory T cells (Tregs), and CCR8 + Regulatory T cells (Tregs) are important for immune suppression. Recent studies have shown that CCR8 is involved in the regulation of human tumor-resident Tregs and CCR8 in cancer patients. + It is upregulated in myeloid cells, both of which have been shown to be expanded in cancer patients.

[0021] The antibodies of the present invention can be used to treat or inhibit cancer by depleting Tregs, which inhibit anti-tumor T cells. In some cases, the patient's cancer cells express or overexpress CCR8. Cancers that can be treated by depleting Tregs (sometimes called tumor-infiltrating lymphocytes (TILs)) include, for example, gastric, pancreatic, esophageal, ovarian, and lung tumors.

[0022] Throughout this specification, the term "antibody" is used in the 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.

[0023] The antibody fragments of the present disclosure retain CCR8 antigen-binding specificity and include antigen-binding fragments (Fab), variable fragments (Fv) containing VH and VL sequences, single-chain variable fragments (scFv) containing VH and VL sequences linked in a single chain, single-chain antibody fragments (scAb), or other antibody variable region fragments that retain antigen-binding specificity.

[0024] Tandem scFv-Fc antibodies of the present disclosure consist of two or more scFv binding sites in tandem on each antibody arm, which may be linked by a linker or may be linked by a flexible linker, resulting in a total of four or five or more scFv binding sites within one scFv-Fc format antibody.

[0025] Throughout this specification, the term "mesoscale molecules (MEMs)" includes modified peptides and polypeptides of about 1 kDa to about 10 kDa. Throughout this specification, the term "MEMs-nanoparticles" includes MEMs conjugated to nanoparticles (e.g., ferritin nanoparticles).

[0026] As used herein, a "subject" may be a mammalian subject. Mammalian subjects include humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cattle, sheep, pigs, horses, goats, etc.), etc. 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., cat, dog).

[0027] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0028] antibody As used herein, the term "antibody" refers to an intact antibody or a binding fragment thereof that specifically binds to a target antigen. Binding fragments are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain variable fragment (scFv) antibodies. An antibody substantially inhibits receptor attachment to a counter-receptor if the excess antibody reduces the amount of receptor bound to the counter-receptor by at least about 20%, 40%, 60%, or 80%, more usually by more than about 85%, as measured in an in vitro competitive binding assay. The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length antibodies or other bivalent, Fc region-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 monoclonal antibodies (and binding fragments thereof) that are fully recombinant, i.e., have complementary determining regions (CDRs) genetically inserted into a human antibody framework, often referred to as veneered antibodies. Thus, in certain aspects, the monoclonal antibodies are fully synthetic antibodies. In certain embodiments, the monoclonal antibodies (and binding fragments thereof) may be produced in bacterial, fungal, mammalian, insect, or plant cells.

[0029] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen-binding or variable region, and includes Fab, Fab', F(ab')2, Fv, and scFv fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each containing one antigen-binding site and a residual "Fc" fragment, so named because of its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment with two antigen-binding fragments capable of cross-linking antigen, and another residual fragment (termed pFc'). As used herein, "functional fragment" with respect to antibodies refers to Fv, F(ab) and F(ab')2 fragments.

[0030] As used herein, an "Fv" fragment is the minimum antibody fragment containing a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). In this configuration, the three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0031] Fab fragments, also designated F(ab), also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. As used herein, Fab'-SH is the designation for Fab' in which the cysteine ​​residues of the constant domains bear free thiol groups. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art.

[0032] Natural antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by at least one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains 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 a constant domain. 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 is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596 (1985), relevant portions of which are incorporated herein by reference.

[0033] As used herein, an "isolated" antibody is one that has been identified and separated and / or recovered from components of the environment in which it is produced. Contaminant components of its production environment are materials that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be purified measurably by at least three different methods: 1) greater than 50% antibody by weight as determined by the Lowry method, such as greater than 75%, or greater than 85%, or greater than 95%, or greater than 99% by weight; 2) sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence, such as at least 15 residues of sequence, by use of a spinning cup sequenator; or 3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody will be free of at least one component of the antibody's natural environment and thus includes the antibody in situ within recombinant cells. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0034] As used herein, the term "antibody mutant" or "antibody variant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues have been modified. Such mutants necessarily have less than 100% sequence identity or similarity with an amino acid sequence having at least 75%, such as at least 80%, or at least 85%, or at least 90%, or at least 95, 96, 97, 98, or 99%, amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody.

[0035] As used herein, the term "variable" in the context of antibody variable domains refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its specific antigen. However, the variation is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments, called complementarity-determining regions (CDRs), also known as hypervariable regions, found in both the light- and heavy-chain variable domains. There are at least two techniques for determining CDRs: (1) methods based on interspecies sequence variation [i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)]; and (2) methods based on crystallographic studies of antigen-antibody complexes (Chothia, C. et al. (1989), Nature 342: 877), or both Chothia and Kabat. The more highly conserved portions of variable domains are called framework regions (FRs). The variable domains of naturally occurring heavy and light chains each contain four FR regions that are primarily arranged in a β-sheet configuration, connected by three CDRs that form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs within each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al.). The constant domains are not directly involved in binding the antibody to its cognate antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0036] The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly specific types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the heavy chain constant domain, "immunoglobulins" can be assigned to different classes. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which 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.

[0037] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in the presently disclosed and claimed inventions can be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), relevant portions of which are incorporated herein by reference.

[0038] All monoclonal antibodies used in accordance with the presently disclosed and claimed inventions will be either (1) the result of a deliberate immunization protocol, as described in more detail below; or (2) the result of an immune response that naturally leads to the production of antibodies during the course of disease or cancer.

[0039] Use of the monoclonal antibodies of the presently disclosed and claimed inventions may require the administration of such or similar monoclonal antibodies to subjects, such as humans. However, when monoclonal antibodies are produced in non-human animals, such as rodents or chickens, administration of such antibodies to human patients typically provokes an immune response that is directed against the antibody itself. Such a response limits the duration and effectiveness of such therapy. To address this problem, the monoclonal antibodies of the presently disclosed and claimed inventions can be "humanized," i.e., modified so that their antigenic portions are removed and analogous portions of human antibodies are replaced, thus preserving the antibody's affinity for CCR8. This modification may involve only a few amino acids or may involve the entire framework region of the antibody, leaving only the complementarity-determining regions of the antibody intact. Several methods for humanizing antibodies are known to those skilled in the art and are disclosed in U.S. Pat. No. 6,180,370, issued to Queen et al. on January 30, 2001; U.S. Pat. No. 6,054,927, issued to Brickell on April 25, 2000; U.S. Pat. No. 5,869,619, issued to Studnicka on February 9, 1999; U.S. Pat. No. 5,861,155, issued to Lin on January 19, 1999; U.S. Pat. No. 5,712,120, issued to Rodriquez et al. on January 27, 1998; and U.S. Pat. No. 4,816,567, issued to Cabilly et al. on March 28, 1989, the relevant portions of which are incorporated herein by reference.

[0040] Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies) composed primarily of human immunoglobulin sequences, containing minimal sequences derived from non-human immunoglobulins. Humanization can be performed by substituting non-human (i.e., rodent, chicken) CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988); see, e.g., U.S. Patent No. 5,225,539. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues from the donor antibody. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all, at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin variable domain and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0041] The presently disclosed and claimed invention further includes the use of fully human monoclonal antibodies against CCR8. Fully human antibodies essentially refer to antibody molecules in which the entire sequences of both the light and heavy chains, including the CDRs, arise from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be prepared, for example, by trioma technology; human B-cell hybridoma technology [see Kozbor, et al., Hybridoma, 2:7 (1983)]; and EBV hybridoma technology for producing human monoclonal antibodies [see Cole, et al., PNAS 82:859 (1985)], or by the techniques taught herein. Human monoclonal antibodies may be utilized in the practice of the presently disclosed and claimed invention, and may be produced by using human hybridomas [see Cote, et al., PNAS 80:2026 (1983)] or by transforming human B cells in vitro with Epstein-Barr virus (see Cole et al., 1985), relevant portions of which are incorporated herein by reference.

[0042] Additionally, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This technique is described, for example, but not by way of limitation, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, as well as 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), relevant portions of which are incorporated herein by reference.

[0043] A method for producing a desired antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771, issued June 29, 1999 to Hori et al., which is incorporated herein by reference. The method comprises introducing into one mammalian host cell in culture an expression vector containing a nucleotide sequence encoding the heavy chain and into another mammalian host cell an expression vector containing a nucleotide sequence encoding the light chain, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy and light chains.

[0044] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0045] As used herein, the term "disorder" refers to any condition that would benefit from treatment with a polypeptide, including chronic and acute disorders or diseases, including infectious or pathological conditions that predispose a mammal to the disorder in question.

[0046] Antibodies or antibody fragments can be produced with altered sequences or glycosylation states to confer desired levels of activity in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP) or antibody-dependent complement deposition (ADCD) function as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0047] Alternatively, or in addition, it may be useful to combine an amino acid modification with one or more additional amino acid modifications that alter the complement component Clq binding and / or complement-dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19-binding molecule. Binding polypeptides of particular interest may be those that bind Clq and exhibit complement-dependent cytotoxicity. Polypeptides with pre-existing Clq-binding activity, and optionally, the additional ability to mediate CDC, may be modified to enhance one or both of these activities. Amino acid modifications that alter Clq and / or modify its complement-dependent cytotoxicity function are described, for example, in WO / 0042072, incorporated herein by reference.

[0048] The Fc region of an antibody can be engineered to alter effector function, for example, by modifying Clq binding and / or FcγR binding, thereby altering complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. These "effector functions" are involved in activating or attenuating 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; and down-regulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0049] For example, a variant Fc region of an antibody can be generated that has improved Clq binding and improved FcγRIII binding (e.g., both improved ADCC activity and improved CDC activity). Alternatively, if reduced or eliminated effector function is desired, the variant Fc region can be engineered to have reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities may be increased, optionally with a decrease in the other activity (e.g., generating an Fc region variant that has improved ADCC activity but reduced CDC activity, and vice versa).

[0050] Single-chain variable fragments (scFvs) are fusions of the variable regions of immunoglobulin heavy and light chains, linked together by a short (usually serine or glycine) linker. These chimeric molecules retain the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker peptide. This modification usually leaves specificity unaltered. These molecules were historically created to facilitate phage display, where it is very convenient to express the antigen-binding domain as a single peptide. Alternatively, scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore lack the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0051] Flexible linkers are generally composed of amino acid residues that promote helices and turns, such as alanine, serine, and glycine. However, other residues may function as well. Using phage display, tailored linkers for single-chain antibodies (scFv) can be rapidly selected from protein linker libraries. A random linker library was constructed in which heavy and light chain variable domain genes were linked by segments encoding 18-amino acid polypeptides of various compositions. The scFv repertoire (approximately 5 × 10 6 The antibody fragments (different members of the species) were displayed on filamentous phage and subjected to affinity selection with haptens. The population of selected variants exhibited significantly increased binding activity while retaining considerable sequence diversity. Sequence analysis revealed a conserved proline in the linker two residues after the VH C-terminus and abundant arginine and proline at other positions as the only common features of the selected chains. In certain embodiments, the antibody fragments are further modified to increase their serum half-life by modifying the Fc region or using mutations to various constant regions, as known to those skilled in the art.

[0052] In certain embodiments, the antibodies of the invention are formulated for administration to humans. For example, the antibodies of the invention can be included in a pharmaceutical composition formulated for administration intranasally, intrapulmonary, intrabronchial, intravenous, oral, intraadipose, intraarterial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intrapericardial, intraperitoneal, intrapleural, intravesical, topical, mucosal, parenteral, enteral, subcutaneous, sublingual, local, buccal, transdermal, by inhalation, by injection, in a cream, in a lipid composition, by catheter, by gastric lavage, by continuous infusion, by injection, by local delivery, or by regional perfusion, and the composition is a serum, drop, gel, ointment, spray, container, or atomizer.

[0053] As used herein, the term "antigen" refers to a molecule containing one or more epitopes (either linear, conformational, or both) that stimulate the host's immune system to elicit a humoral and / or cellular antigen-specific response. The term is used interchangeably with the term "immunogen." Typically, a B-cell epitope will contain at least about five amino acids, but may be as small as three or four amino acids. A T-cell epitope, such as a CTL epitope, will contain at least about seven to nine amino acids, and a helper T-cell epitope will contain at least about 12 to 20 amino acids. Typically, an epitope will contain about 7 to 15 amino acids, such as 9, 10, 12, or 15 amino acids. The term includes polypeptides containing modifications, such as deletions, additions, and substitutions (generally conservative substitutions in nature), relative to the native sequence, so long as the protein retains its ability to elicit an immune response as defined herein. These modifications may be deliberate, such as by site-directed mutagenesis, or accidental, such as by mutation of hosts producing the antigen.

[0054] As used herein, the term "epitope" refers to a particular amino acid sequence or molecule (e.g., carbohydrate, small molecule, lipid, etc.) that, when present in the appropriate form, provides a reactive site for an antibody (e.g., a B cell epitope) or, in the case of a peptide, a reactive site for a T cell receptor (e.g., a T cell epitope).

[0055] Portions of a given polypeptide containing B-cell epitopes can be identified using a number of epitope mapping techniques known to those skilled in the art (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed., 1996, Humana Press, Totowa, NJ). For example, linear epitopes can be determined by, for example, simultaneously synthesizing a large number of peptides corresponding to portions of a protein molecule on a solid support and reacting the peptides with an antibody while they are still bound to the support. Such techniques are known to those skilled in the art and are described, for example, in U.S. Pat. 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.

[0056] 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 the majority percentage of the sample in which it is present. Generally, a substantially purified component in a sample comprises 50%, preferably 80%-85%, and more preferably 90-95% of the sample. Techniques for purifying desired polynucleotides and polypeptides are well known to those of skill in the art and include, for example, ion exchange chromatography, affinity chromatography, and density-based precipitation.

[0057] As used herein, the term "treatment" refers to any of the following: (i) prevention of infection or reinfection, as in a conventional vaccine; (ii) reduction or elimination of disease symptoms; and (iii) substantial or complete elimination of the cancer in question.

[0058] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, which are within the skill of the art. Such techniques are explained fully in the literature, e.g., Remington's Pharmaceutical Sciences, 18 th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2 nd Edition, 1989);Short Protocols in Molecular Biology, 4 th ed. (Ausubel et al. eds., 1999, John Wiley & Sons);Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press);PCR (Introduction to Biotechniques Series), 2 nd ed. (Newton & Graham eds., 1997, Springer Verlag); Fundamental Virology, Second Edition (Fields & Knipe eds., 1991, Raven Press, New York), relevant portions are incorporated herein by reference.

[0059] Conservative amino acid substitutions include replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small amino acids Ala, Ser, Thr, Met, and Gly.

[0060] Methods for expressing DNA sequences containing 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 cells). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora crassa, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for growing mammalian cells in culture are well known [see Jakoby and Pastan (eds), 1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, NY]. 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 cells engineered to provide higher expression of desired glycosylation patterns or other characteristics can also be used. As mentioned above, techniques for transforming yeast cells, such as polyethylene glycol transformation, protoplast transformation, and gene guns, are also known in the art (see Gietz and Woods, Meth Enzymol 350: 87-96, 2002).

[0061] Transformation of host cells 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, E. coli, competent cells capable of incorporating DNA can be prepared from cells harvested after the exponential growth phase using procedures well known in the art and subsequently treated by the CaCl method. Alternatively, MgCl or RbCl can be used. Transformation can be carried out after forming a protoplast of the host cell, if desired, or by electroporation.

[0062] When the host is a eukaryotic organism, methods of DNA transfection, such as calcium phosphate coprecipitation, conventional mechanical procedures such as microinjection, electroporation, insertion of plasmids in liposomes, or viral vectors, can be used. Eukaryotic cells can also be cotransformed with a polynucleotide sequence encoding an antibody or portion thereof and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method uses eukaryotic viral vectors, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express proteins (see, e.g., Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

[0063] Antibodies or portions and variants thereof can also be produced in plants. For example, polypeptides can be expressed in plants using the IBIOLAUNCH™ gene expression platform (iBio, Inc., Newark, DE), described in, for example, U.S. Patent No. 7,491,509 (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 may have advantages over methods that utilize animal cells or microorganisms, and over systems that require transgenic plants.

[0064] Therapeutic methods and pharmaceutical compositions The antibodies or portions thereof disclosed herein, or nucleic acids encoding the antibodies or portions thereof, can be used to treat cancer. In some examples, the antibodies or portions thereof, or nucleic acids encoding these polypeptides, are useful for reducing cancer, such as in a subject. Thus, in some embodiments, the method includes administering to a subject a therapeutically effective amount of one or more of the antibodies or portions thereof disclosed herein, or polynucleotides 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.

[0065] A therapeutically effective amount of an antibody or portion thereof, or a polynucleotide encoding a peptide, can be administered in a pharmaceutically acceptable carrier. Pharmacologically 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., about pH 7.0 to about 8.0, or about pH 7.4). One specific, non-limiting example of a physiologically compatible buffer solution is phosphate-buffered saline. Other pharmacologically acceptable carriers include penetrating agents, which are particularly suitable for pharmaceutical formulations intended for topical application (e.g., to promote healing of surgical wounds).

[0066] The pharmaceutical compositions disclosed herein facilitate the use of at least one antibody or portion thereof, or a polynucleotide encoding an antibody or portion thereof, to reduce cancer either in vivo or ex vivo. Such compositions may be suitable for delivery of 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 lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmacologically (e.g., physiologically or pharmaceutically) acceptable carriers, and optionally, adjuvants that facilitate processing of the active compound into pharmaceutically usable preparations. Appropriate formulations depend on the chosen route of administration. Thus, for injection, the active ingredient may be formulated in an aqueous solution. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0067] For oral administration, a therapeutically effective amount of at least one antibody or portion thereof or nucleic acid encoding a peptide may be combined with a carrier suitable for incorporation into a tablet, pill, capsule, liquid, gel, syrup, slurry, suspension, or the like.

[0068] Protein drugs are subject to protease-mediated degradation in the gastrointestinal tract by the action of enzymes such as trypsin, chymotrypsin, and brush border peptidases, and oral administration of large protein molecules often does not produce the intended therapeutic effect (Soltero and Ekwruibe, 2001 Innovations in Pharmaceutical Technology, 1:106-110).

[0069] In some embodiments, for parenteral administration, a therapeutically effective amount of an antibody or portion thereof or nucleic acid encoding a peptide may be administered by injection, such as a bolus injection or continuous infusion. Such compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Other pharmacological excipients are known in the art.

[0070] Optionally, the polynucleotide encoding the antibody or portion thereof, or peptide, can be contained in or conjugated to a heterologous protein, carbohydrate, or lipid, whether administered in vitro or in vivo. Concurrent administration can be such that the polynucleotide encoding the antibody or portion thereof, or peptide, is administered before, substantially simultaneously with, or after the protein, carbohydrate, or lipid. In some embodiments, the polynucleotide encoding the antibody or portion thereof, or peptide, is administered substantially simultaneously with the protein, carbohydrate, or lipid.

[0071] Other delivery systems may include sustained-release, delayed-release, or sustained-release delivery systems. Such systems can prevent repeated administration of the compositions of the present invention, increasing convenience for patients and physicians. Many types of release delivery systems are available and known to those skilled in the art. These include systems based on polymers such as poly(lactide glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Drug-containing microcapsules of the aforementioned polymers are described, for example, in U.S. Pat. No. 5,075,109. Delivery systems also include non-polymeric systems such as lipids containing sterols, such as cholesterol, cholesterol esters, and fatty acids or neutral lipids, such as mono-, di-, and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; tableting using conventional binders and excipients; and partially fused implants. Specific examples include, but are not limited to, (a) erosion systems in which a polynucleotide encoding an antibody or portion thereof or peptide is contained within a matrix, such as those described in U.S. Patent 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 ingredient permeates at a controlled rate through a polymer, such as those described in U.S. Patent Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems may also be used, some of which are adapted for implantation.

[0072] A therapeutically effective amount of a polynucleotide encoding an antibody or portion thereof, or a peptide will depend on the antibody or portion thereof, or the polynucleotide encoding the peptide employed, 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 per kilogram (kg) to about 1 g per kg of body weight, such as about 1 μg to about 5 mg per kg of body weight, or about 5 μg to about 1 mg per kg of body weight. The exact dose can be readily determined by one of ordinary skill in the art based on the potency of the particular compound and the age, weight, sex, and physiological condition of the subject.

[0073] In relation to administration of nucleic acids, one approach for administering nucleic acids is direct administration using plasmid DNA, such as using a mammalian expression plasmid. As described above, the nucleotide sequence encoding the antibody or portion thereof can be placed under the control of a promoter to increase expression of the molecule.

[0074] When viral vectors are used for in vivo administration, the dosage of each recombinant virus in the composition is about 10 5 ~about 10 10It is desirable to provide the recipient with a dose in the range of plaque-forming units / mg mammal, although lower or higher doses can be administered. The recombinant viral vector composition can be introduced into a mammal prior to any evidence of cancer, or to mediate disease regression in a mammal afflicted with cancer. Examples of methods for administering the composition to a mammal include, but are not limited to, exposing cells to the recombinant virus ex vivo, or injecting the composition or virus into the affected tissue via intravenous, subcutaneous, intradermal, or intramuscular administration. Alternatively, a recombinant viral vector or combination of recombinant viral vectors in a pharmaceutically acceptable carrier may be administered locally by direct injection into a cancerous lesion. Generally, the amount of 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 immunogen to be administered is 10 viral particles per mammal, such as a human. 5 ~10 10 There are individuals.

[0075] In one specific, non-limiting example, a pharmaceutical composition for intravenous administration will contain about 0.1 μg to 10 mg of antibody or portion thereof per patient per day. Dosages of 0.1 to about 100 mg per patient per day may be used, particularly if the agent is administered to an isolated site and not into the circulatory or lymphatic system, such as a body cavity or organ lumen. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in Remington's Pharmaceutical Sciences, 1999. th Ed., Mack Publishing Company, Easton, Pa., 1995.

[0076] Single or multiple administrations of the composition are administered at dosages and frequencies depending on the need and tolerance of the subject. In some embodiments, the dosage is administered once as a bolus, while in other embodiments, it can be applied periodically until a therapeutic result is achieved. Generally, the dosage is sufficient to treat or improve the symptoms or signs of the disease without causing unacceptable toxicity to the subject. Systemic or local administration can be used.

[0077] In another method, an additional agent is administered. In one example, the administration is sequential. In another example, the additional agent is administered simultaneously with the antibody or portion thereof.

[0078] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0079] Antibodies that bind to CCR8 are provided herein. These antibodies are referred to herein as anti-CCR8 antibodies. A number of discovery strategies have been used to obtain exemplary antibodies of the present disclosure, which are discussed further below.

[0080] In some embodiments, MEM-nanoparticles and full-length anti-CCR8 are used together to immunize a subject, thereby producing antibodies specific to the MEM epitope. Monoclonal hybridomas are then created to produce epitope-specific anti-CCR8 antibodies. In other embodiments, mouse serum is collected and used to generate an in vitro scFv library. Epitope-specific clones are then isolated by phage panning against full-length anti-CCR8 and MEM-nanoparticles.

[0081] In some embodiments, the half maximal effective concentration (EC 50 ) is about 1.6, 1.8, 1.9, 2.0, 2.2, 2.3, 2.6 or 2.7 nM.

[0082] Those skilled in the art will recognize that binding specificity can be determined by a series of competitive binding paradigms, in which a desired antibody demonstrates its ability to interfere with the binding of a known reference antibody to its target epitope at various concentrations. In some embodiments, the reference anti-CCR8 antibody is GS-1811.

[0083] In some embodiments, the anti-CCR8 antibody is a full-length antibody (referring to an antibody having two heavy chains and two light chains linked to an Fc domain, forming a Y-shape). In some embodiments, the Fc domain (or simply referred to as Fc) is a human Fc domain. In some embodiments, the Fc domain of the anti-CCR8 antibody is derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0084] Exemplary Anti-CCR8 Antibody-CDR Sequences The sequences of exemplary anti-CCR8 antibodies of the present disclosure are provided herein, including the complementarity determining region (CDR) sequences and variable heavy and light domain sequences (VH, VL) that constitute the anti-CCR8 antigen-binding domain of the present disclosure. The discovery of these antibodies is described in detail in the Examples section.

[0085] As referred to 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 for the antibodies of the disclosure.

[0086] [Table 1]

[0087] Tables 2A and 2B show the heavy and light chain anti-CCR8 humanized antibody amino acid sequences, respectively. All heavy and light chain CDRs are in Table 1.

[0088] Table 2

[0089] Table 3

[0090] Table 4 JPEG2026505138000006.jpg255169 JPEG2026505138000007.jpg117170

[0091] Table 5 JPEG2026505138000009.jpg255170 JPEG2026505138000010.jpg122170

[0092] Table 6 JPEG2026505138000012.jpg250170

[0093] In some embodiments, an anti-CCR8 antibody is provided herein, the antibody comprising a heavy chain variable domain (VH) complementarity determining region (CDR)1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 49, 67, 73, 79, or 85; and a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 50, 68, 74, 80, or 86; and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 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 NOs: 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 NOs: 5, 53, 56, 59, 62, 65, 71, 77, 83, or 89. CDR2; and a VL CDR3 amino acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 6, 54, 57, 60, 63, 66, 72, 78, 84 or 90.

[0094] Exemplary Tandem scFv-Fc Anti-CCR8 Antibodies In some embodiments, the present disclosure provides tandem scFv antibodies with multiple CCR8-binding sites. The tandem scFv-Fc antibodies of the present disclosure are composed of two or more scFv-binding sites in tandem on each antibody arm, which may be linked by a linker or a flexible linker. In some embodiments, the tandem scFv antibodies have a total of four or five or more scFv-binding sites within a single scFv-Fc format antibody.

[0095] In some embodiments, the scFv1 of each antibody arm comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and the 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 connected by a linker, e.g., a flexible linker. The VH2 and VL2 of each scFv2 may be connected by a linker, e.g., a flexible linker. The scFvs on each antibody arm may be connected by a linker, e.g., a flexible linker.

[0096] Therapeutic Uses of Anti-CCR8 Antibodies In some embodiments, the anti-CCR8 antibodies provided herein are useful for treating diseases or conditions associated with an immune response.

[0097] Administration of therapeutic anti-CCR8 antibodies In vivo administration of the therapeutic anti-CCR8 antibodies described herein may be performed intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intrathecally, intraventricularly, intranasally, transmucosally, by implantation, or by inhalation. Intravenous administration may be performed by 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 performed with any suitable excipient, carrier, or other agent to provide suitable or improved tolerance, transfer, delivery, etc. [Example]

[0098] Example 1: Discovery of anti-CCR8 based on modified MEM-nanoparticle immunization MEMs were designed to exemplify the structure and dynamics of CCR8 extracellular loop 1 (DQWVFGT) (SEQ ID NO: 91) and extracellular loop 2 (VASEDGVLQC) (SEQ ID NO: 92). While keeping the epitope residues of extracellular loops 1 and 2 fixed, a protein scaffold was created using the Protein Design AI algorithm, thereby displaying the epitope structure. The scaffold sequence was optimized to meet three criteria: [1] structural identity with the predicted epitope structure of CCR8, [2] stability of the MEM molecule, and [3] water solubility. The length of the protein scaffold was optimized to be as short as possible without sacrificing these three criteria. The amino acid sequence of the final CCR8 MEM design is SPEIKKLIEQIKSDQWVFGTEACNEIQTLLKEESGPAKIEVASEDGVLQCRIVF (SEQ ID NO: 93). The structure of this MEM design was verified by nuclear magnetic resonance (NMR) to be within 3.2 Å RMSD of the predicted structure. When only the epitope residues were considered, the RMSD between the experimental and predicted structures was 1.6 Å.

[0099] CCR8 MEM was expressed and purified as a nanoparticle fusion protein with ferritin from Helicobacter pylori. MEM was attached at 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.

[0100] BALB / c mice were immunized in two separate cohorts. In one cohort, immunizations were repeated every two weeks with MEM immunizations and CCR8 virus-like particles (VLPs) (Acro Biosystems, catalog number CC8-H52P4). In another cohort, mice were immunized with CCR8-expressing HEK293 and / or CHO cells for one week and with CCR8 VLPs every two weeks. MEM immunizations were 15-25 μg per injection, and CCR8 VLPs were 6 μg per injection.

[0101] Example 2: The antibodies produced by the top monoclonal hybridomas exhibited potent anti-CCR8 binding, did not bind to CCR4, and competed with the GS-1811 benchmark The antibodies produced by the hybridomas were incubated with cells transiently expressing either 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, the antibodies produced by the hybridomas were co-incubated with GS-1811 as 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 once and measured by flow cytometry. All measurements were performed on ice using chilled buffers.

[0102] Figures 1A and 1B show that the successful anti-CCR8 hybridoma clone 1E6 specifically binds to CCR8-expressing cells but not to CCR4-expressing cells. Figure 2 shows that the 1E6 hybridoma clone competes with the GS-1811 benchmark in binding to CCR8-expressing cells.

[0103] 3A-3C show that anti-CCR8 antibodies derived from chimeric or humanized hybridoma clone 1E6 specifically bind to human CCR8 and not to human CCR4 or mouse CCR8. FACS analysis showing 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 plotted values ​​are the percentage of antibody bound to target cells.

[0104] Figures 4A and 4B show the binding potency of anti-human CCR8 antibodies to a CHO-K1 cell line overexpressing human CCR8. FACS analysis showing the binding of anti-CCR8 antibodies or control antibodies (hIgG1 isotype, anti-mouse CCR8, and anti-human CCR4) to CHO-K1 cells overexpressing human CCR8. Plotted values ​​are median fluorescence intensity values. EC 50 Values ​​are the average of n=2 experiments. n / a: no activity.

[0105] Figures 5A and 5B show the antagonist activity of anti-human CCR8 antibodies in a CHO-K1 reporter cell line overexpressing human CCR8. Luminescence readout showing inhibition of activation of human CCR8 with the agonist CCL1 by pre-treating reporter cells with anti-CCR8 antibodies. Plotted values ​​are relative luminescence units. EC 50 Values ​​are the average of n=2 experiments. n / a: no activity.

[0106] 6A-6C show the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-CCR8 antibodies using human peripheral blood mononuclear cells (PBMC). FACS analysis showing the ADCC efficacy of anti-CCR8 antibodies from two different PBMC donors in a dose-dependent manner. The plotted values ​​are the percentage of killed target cells relative to the total target cells (% cytolysis). EC 50 Values ​​are the average of n=1 to 2 experiments. n / a: no activity.

[0107] Figure 7 is a graph showing the thermal stability of anti-CCR8 antibodies. The melting temperature (T M ) was determined by monitoring the change in fluorescence intensity of the antibody loaded into the capillary using a Prometheus Panta instrument.

[0108] Figures 8A-8D show the study design and results of an in vivo efficacy study using the anti-CCR8 antibody SD-171467-afuc as monotherapy. Figures 8B and 8C show tumor volume and percent tumor volume change after drug treatment. Tumor measurements and drug administration were on days 0, 3, 7, 10, and 17. Figure 8D shows the change in mouse weight on days 0, 3, 7, 10, and 17 after drug administration. For all regimens, no significant decrease in mouse weight was observed after drug treatment.

[0109] Figures 9A-9C show the results of the pharmacokinetic study. Figure 9A shows the pharmacokinetic study design for SD-171467-afuc. Figure 9B shows the pharmacokinetic profile of SD-171467-afuc. After a single dose of 10 mg / kg, serum was collected at the indicated time points. Human IgG levels were quantified using the MSD Human IgG kit. Figure 9C summarizes the pharmacokinetic parameters calculated by Phoenix WinNonLin.

[0110] Figures 10A-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 treatment, and a mechanism of action study on the reduction of CCR8+ Tregs. Figure 10A shows the in vivo study design of the combination treatment of anti-PD1 antibody and SD-171467-afuc and the reduction of CCR8+ Tregs. Figures 10B and 10C show the tumor volume and percent tumor volume change after drug treatment. Tumor measurements and drug administration were on days 0, 4, 7, 11, and 18. Figure 10D shows the change in mouse weight on days 0, 4, 7, 11, and 18 after drug administration. For all regimens, no significant reduction in mouse weight was observed after drug treatment. Figures 10E and 10F show that treatment with SD-171467-afuc significantly reduced CCR8+ Tregs in the tumor, but not in the spleen. Tumor (Figure 10E) and spleen (Figure 10F) samples from the PBS and SD-171467-afuc (1 mg / kg) treatment groups were analyzed for the reduction of Treg-CCR8+ using flow cytometry. Graphs show the percentage of Treg-CCR8+ (CD4+Foxp3+CCR8+) among live CD45+CD3+CD4+Foxp3+ cells in tumors and spleens. A two-tailed Student's t-test was used to compare the two treatment groups. p values ​​are indicated within the graphs ( * p less than 0.05; NS p > 0.05).

[0111] Hybridomas were generated from immunized mouse B cells using standard electrofusion techniques. Spleens were isolated from immunized mice and disrupted in 15–20 mL of DMEM (no supplements) on ice. Splenocytes and Ag8.653 mouse myeloma cells were mixed at a 1:1 ratio in DMEM on ice. The cells were cultured at a density of 2 × 10 before electrofusion. 6 The cells were resuspended in a volume of ECF buffer to a concentration of 100 cells / mL. Electrofusion was performed using the fusion cycle parameters (Nepagene / ECFG21). Seven days after fusion, the cells were seeded into 96-well plates containing selective medium to verify fusion efficiency.

[0112] Antibody Expression and Expression Antibody expression plasmids were transiently introduced into animal cell lines using the ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific, catalog number A29129) to generate transformants producing anti-CCR8 chimeric or humanized antibodies. ExpiCHO-S (Thermo Fisher Scientific, catalog number A29127) or a CHO suspension cell line with a knockout of the α1,6 fucosyltransferase (FUT8) gene (referred to as "WT CHO" and "FUT8 CHO" in other references) was used as the host cell line. After 6-12 days of growth after DNA transfection, the WT CHO or FUT8 CHO cell suspension was harvested by centrifugation at 4,000 × g for 20 minutes and then filtered using a 0.2 μm disposable PES filter unit (Fisher Scientific, catalog number FB12566504). Anti-CCR8 antibodies were recovered from the filtrate using Protein A purification (HiTrap MabSelect SuRe; Cytiva, catalog no. GE11-0034-93). WT CHO was used to express antibodies with standard glycosylation, and FUT8 CHO was used to express defucosylated antibodies with enhanced effector function (indicated by "-afuc").

[0113] Antibody Humanization Humanization was achieved by multiple approaches. In some cases, CDRs were directly grafted into the human germline using publicly available tools (DOI: 10.1080 / 19420862.2021.2020203). To potentially improve expression and performance, one amino acid was added back to the parental mouse sequence. In other cases, two or more amino acids were added back to the parental mouse sequence. In all cases, the CDRs were left unchanged. The humanized variants were then tested, and efficacy was compared to the parent chimera.

[0114] thermal stability Antibodies were prepared at a concentration of 1 mg / mL in PBS (pH 7.4). Ten microliters of each sample was loaded by pipette into the appropriate capillary (Nanotemper, Cat. No. PR-C002). The capillary was loaded into the Nanotemper Prometheus Panta instrument and the protocol was defined. Thermal unfolding was performed from 25 to 95°C at a rate of 1°C / min. Fluorescence was monitored and T m was calculated by the instrument software.

[0115] Cell binding assay PathHunter CHO-K1β-arrestin cells (DiscoverX, Catalog No. 93-0196C2) were cultured in AssayComplete Cell Culture Kit-107 (DiscoverX, Catalog No. 92-3107G) supplemented with G418 (Gibco, Catalog No. 10131-035) and hygromycin B (Invitrogen, Catalog No. 10687010) according to the manufacturer's instructions. ExpiCHO-S cells (Thermo Fisher, Catalog No. A29127) were cultured in ExpiCHO Expression Medium (Gibco, Catalog No. A29100-01).

[0116] To generate human CCR8, ExpiCHO cells, which express human CCR4 or mouse CCR8, were cultured at 6 × 10 6 The cells were diluted to 1000 cells / mL and transfected with the corresponding expression plasmids using the ExpiFectamine CHO transfection kit (Gibco, Cat. No. A29129) according to the manufacturer's instructions. The transfected cells were used for cell binding assays within 5 days after transfection. To detect human CCR4 or mouse CCR8 in the transfected cells, anti-hCCR4 (BioLegend, Cat. No. 359402) or anti-moCCR8 (BioLegend, Cat. No. 150302) were used for staining, respectively.

[0117] For cell binding assays, PBS (Corning, Cat. No. 21-040-CV) supplemented with 2% FBS (MilliporeSigma, Cat. No. F4135) and 2 mM EDTA (Quality Biological, Cat. No. 351-027-721) was used as the assay buffer. Cells were counted and then resuspended in assay buffer at 1 × 10 5 Cells were then seeded at 1000 cells / well into a 96-well plate (VWR, catalog number 89089-826). The plate was centrifuged, the supernatant removed, and then kept on ice for the remainder of the assay. After the supernatant was removed, the indicated antibodies were diluted in assay buffer and added to the cells at increasing concentrations (0.004-66.66 nM) for 20 minutes on ice. After incubation, the plate was centrifuged, the supernatant removed, and the cells were then washed once with assay buffer, followed by centrifugation and subsequent washing. After washing, rat anti-human IgG Fc Alexa Fluor 647 (BioLegend, Catalog No. 410714), rat anti-mouse IgG Alexa Fluor 647 (BioLegend, Catalog No. 406618), or goat anti-rat IgG Alexa Fluor 647 secondary (BioLegend, Catalog No. 405416) was diluted 1:200 in assay buffer and added to the cells for 20 minutes on ice. After incubation, the plate was centrifuged, the supernatant removed, and the cells were then washed once with assay buffer, followed by centrifugation and washing. After washing, DAPI (BioLegend, Catalog No. 422801) was diluted 1:5000 in assay buffer and added to the cells. Cell binding was analyzed using a Miltenyi MACSQuant 16 flow cytometer. Flow cytometry data were analyzed using FlowJo flow cytometry analysis software. Graphs were generated and EC 50 GraphPad Prism 9.3.0 was used to calculate values.

[0118] CCR8 antagonist assay PathHunter CHO-K1 β-arrestin cells (DiscoverX, Catalog No. 93-0196C2) were cultured in AssayComplete Cell Culture Kit-107 (DiscoverX, Catalog No. 92-3107G) supplemented with G418 (Gibco, Catalog No. 10131-035) and hygromycin B (Invitrogen, Catalog No. 10687010) according to the manufacturer's instructions.

[0119] For the CCR8 antagonist assay, PathHunter CHO-K1β-arrestin cells were detached using AssayComplete Cell Detachment Reagent (DiscoverX, Cat. No. 92-0009), then counted and plated at 2.5 × 10 cells per well into 96-well white opaque microplates (Corning, Cat. No. 3917) using AssayComplete Cell Plating 2 Reagent. 4 Cells were seeded at 1000 cells / well overnight at 37°C and 5% CO2. The indicated antibodies were then 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). Cells were incubated with the antibodies for 30 min at 37°C and 5% CO2, followed by EC200 determined as agonist. 80 A CCL1 (PeproTech, Cat. No. 300-37) agonist was added to the cells at 10x the initial concentration. Cells were incubated with the antibody and agonist for 90 minutes at 37°C and 5% CO2 (both at 1x final concentration). After incubation with the agonist, a detection substrate was generated and added to the cells using a PathHunter Detection Kit (DiscoverX, Cat. No. 93-0001) according to the manufacturer's instructions. Cells were incubated with the detection substrate for 1 hour at room temperature and then analyzed using an Agilent Cytation 5 Cell Imaging / Multimode Reader with the following settings: read speed: 1 sec / well, read height: 1 mm, gain: 135. IC50 Values ​​were calculated using Graphpad Prism 9.3.0.

[0120] ADCC / PBMC PathHunter CHO-K1 β-arrestin cells (DiscoverX, Catalog No. 93-0196C2) were cultured in AssayComplete Cell Culture Kit-107 (DiscoverX, Catalog No. 92-3107G) supplemented with G418 (Gibco, Catalog No. 10131-035) and hygromycin B (Invitrogen, Catalog No. 10687010) according to the manufacturer's instructions.

[0121] For the ADCC / PBMC assay, PathHunter CHO-K1 β-arrestin cells were counted to assess cell number and viability. Cells were stained with Cell-trace CFSE (Thermo Fisher, Cat. No. C345554A). Cells were centrifuged and collected at 1 × 10 5 Resuspend in growth medium at 1 x 10 cells / mL per well 4 Cells were seeded at 1000p per well into a 96-well plate (VWR, catalog no. 29442-056) and incubated overnight in a cell culture incubator at 37°C and 5% CO2. After incubation, increasing concentrations of the indicated antibodies (0.00000667-6.666667 nM or 0.0000333-33.33333 nM) in assay medium (RPMI 1640 containing 10% FBS, 1% penicillin / streptomycin, and 5 ng / mL IL2) were added to the cells at 37°C and 5% CO2 for 20 minutes. Subsequently, 2 x 10 peripheral blood mononuclear cells (PBMCs) (Stemcell, catalog no. 70025.1) were added to the cells. 5100 mg of FITC+ Live / Dead+ IgG was added to each well of a 96-well plate. Cells and antibodies were incubated for 18-24 hours at 37°C in a 5% CO2 incubator. Samples were then collected, stained with a LIVE / DEAD fixable Aqua Dead Cell Stain Kit (Thermo Fisher, Cat. No. L34957), and analyzed on a Miltenyi MACSQuant 16 flow cytometer. % dead target cells were gated using FITC+ Live / dead+. EC 50 Values ​​were calculated using Graphpad Prism 9.3.0.

[0122] In vivo efficacy and mechanistic studies. Eight-week-old female transgenic B-hCCR8 mice (Biocytogen, Catalog No. 110096) were used. MC-38 cells (Shanghai Shunran Biotechnology, Catalog No. M023) were cultured at 0.5 × 10 in 100 μl of PBS. 6 Each mouse was inoculated with a single tumor by subcutaneous injection into the upper left side after local shaving. Tumor growth and mouse body weight were monitored twice weekly. For each individual tumor, the longest longitudinal diameter (length) and widest transverse diameter (width) were measured using a Traceable Digital Caliper (VWR, Cat. No. 62379-531). Tumor volume (TV) was calculated using the formula TV = [length × (width)]. 2 The mean tumor volume was 87–89 mm in the monotherapy and combination therapy / mechanism studies, respectively. 3 and 109-111mm 3Once the mice reached 1 mg / mL, they were randomized. Each group contained eight mice. For monotherapy studies, the human IgG1 negative control, GS-1811, and SD-171467-afuc were prepared in stock solutions at 1 mg / mL. For combination therapy / mechanism studies, anti-mouse PD-1 and SD-171467-afuc were prepared in stock solutions at 0.03 and 0.1 mg / mL, respectively. In addition, a mixed solution containing both anti-mouse PD-1 (0.03 mg / mL) and SD-171467-afuc (0.1 mg / mL) was used for combination therapy / mechanism studies. The volume of vehicle control (PBS) or antibody administered to each mouse was calculated using the formula: volume (μl) = mouse body weight (g) × 10 μl / g. The antibody drug was administered via the intraperitoneal (ip) route twice weekly for the first two weeks, with the fifth dose administered one week after the fourth dose, for a total of five doses. Tumor growth was monitored for 14 days after the first drug treatment.

[0123] At the end of the combination therapy / mechanism study (22 days after treatment), tumor and spleen samples were collected from the human PBS and 1 mg / kg SD-171467-afuc treated groups and minced into small pieces in digestion buffer [DMEM medium (Corning, catalog number MT10013CM) supplemented with 0.36 mg / ml collagenase type IV (MP Biomedicals, catalog number IC19511090) and 10 μg / ml DNase I (STEMCELL Technologies, catalog number 07900)]. The samples were then incubated at 37°C for 30–45 minutes and then filtered through a cell strainer (Corning, catalog number 352340). The filtered cell suspension was centrifuged, and the supernatant was then discarded. The cell pellet was resuspended in red blood cell lysis buffer (BioLegend, catalog number 420301) to remove red blood cells. The remaining cell suspension was then counted and the total cell number recorded using an automated cell counter. 100 ul of the cell suspension was used for FAC analysis. Cell samples were stained with Live / Dead Marker (Thermo Fisher, Cat. No. L34957) for 20 minutes at room temperature, treated with mouse Fc-blocking antibody (BioLegend, Cat. No. 101302), and then stained with an antibody cocktail [anti-mouse CD45.1-APC / Cy7 (BioLegend, Cat. No. 103116), anti-mouse CD3-BV-605 (BioLegend, Cat. No. 100237), anti-mouse CD4-FITC (BioLegend, Cat. No. 130308), anti-CD8-mouse-APC (BioLegend, Cat. No. 162305), and anti-human CCR8-PE (BioLegend, Cat. No. 365704)] for 30 minutes on ice. Samples were then fixed with FoxP3 fix / perm buffer (BioLegend, Cat. No. 421401) and permeabilized with FoxP3 perm buffer (BioLegend, Cat. No. 421402). Samples were then stained with anti-Foxp3-BV421 antibody (BioLegend, Cat. No. 126419) and analyzed on a Miltenyi MACSQuant 16 flow cytometer.The CCR8+ Treg cell population was gated (single cells / live cells / CD45.1+ / CD4+ / Foxp3+ / CCR8+).

[0124] Pharmacokinetic studies Eight-week-old female transgenic B-hCCR8 mice (Biocytogen, catalog number 110096) were used. 0.5 × 10 MC-38 (Shanghai Shunran Biotechnology, catalog number M023) cells were cultured in 100 μl of PBS. 6 After shaving the area, each mouse was inoculated with 10 mg / kg SD-171467-afuc via subcutaneous injection into the upper left flank. Fourteen days after tumor cell inoculation, tumor-bearing mice were intraperitoneally administered 10 mg / kg SD-171467-afuc. Blood samples were collected from the facial vein of treated mice using a Goldenrod Animal Lancet (3 mm, Medipoint) at 0 h (pretreatment), 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 18 h, 24 h, 48 h, 96 h, 192 h, 384 h, and 576 h (endpoint). 60 mL of blood was collected using heparin-coated BD Microtainer Tubes. After 30–60 min on ice, serum was separated by centrifugation at 5000 rpm for 5 min at 4°C, transferred to 2 mL cryovials (Heathraw Scientific, catalog no. HS23202A), flash-frozen in ethanol on dry ice, and stored at −80°C for downstream analysis. Serum drug concentrations were measured using the Meso Scale Diagnostics Human / NHP IgG quantification kit (MSD, catalog no. K150JLD) according to the kit's instructions. Briefly, dilutions of animal serum were applied to plates precoated with human / NHP capture antibody and detected with an anti-IgG secondary antibody. Serum IgG concentrations were calculated by interpolation from a standard curve using MSD Discovery Workbench software. These data were then used to perform noncompartmental PK analysis using Certara's Phoenix™ WinNonLin software.

[0125] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to achieve the methods of the invention.

[0126] It will be understood that the specific embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalent procedures are considered to be within the scope of the invention and are covered by the claims.

[0127] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0128] The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or," unless expressly stated to refer to alternatives only or that the alternatives are mutually exclusive. However, this disclosure supports definitions and "and / or" that refer to alternatives only. Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error that exists among devices, the method utilized to determine the value, or the study subject.

[0129] As used in this specification and claims, the words "comprising" (and all forms of "comprising", such as "comprise" and "comprises"), "having" (and all forms of "having", such as "have" and "has"), "including" (and all forms of "including", such as "includes" and "include"), or "containing" (and all forms of "containing", such as "contains" and "contain") are all inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any of the embodiments of the compositions and methods provided herein, "comprising" can be replaced with "consisting essentially of" or "consisting of." As used herein, the phrase "consisting essentially of" requires the specified integers or steps and those that do not have a material effect on the nature or function of the claimed invention. As used herein, the term "consisting of" is used to indicate that only the enumerated integer (e.g., feature, element, attribute, property, method / method step, or limitation) or group of integers (e.g., feature, element, attribute, property, method / method step, or limitation) is present.

[0130] 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 A, B, C, AB, AC, BC, or ABC, and, where order is important in the particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Following this example, combinations containing one or more repeats of an item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those skilled in the art will understand that generally, there is no limitation on the number of items or terms in any combination unless otherwise clear from the context.

[0131] As used herein, without limitation, approximation words such as "about," "substantially," or "substantially," when so modified, are understood to be not necessarily absolute or complete, but refer to a state that would be considered close enough to a person skilled in the art to justify specifying the state as it exists. The extent to which deviations from this specification may occur will depend on how significant a change can be made and still enable a person skilled in the art to recognize that the modified feature still possesses the required properties and capabilities of the unmodified feature. Generally, however, in light of the preceding considerations, numerical values ​​herein modified by approximation words such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15%.

[0132] Furthermore, the headings herein are provided to provide organizational guidance otherwise consistent with the suggestions of 37 CFR 1.77. These headings are not intended to limit or characterize the invention(s) set forth in any claims that may arise from this disclosure. In particular and by way of example, headings may refer to "Field of the Invention," but such claims should not be limited by the language of these headings to describe the so-called technical field. Furthermore, the description of a technology in the "Background" section should not be construed as an admission that that technology is prior art to any invention(s) in this disclosure. Neither should the "Summary of the Invention" be deemed a characterization of the invention(s) set forth in the issued claims. Furthermore, any reference to the singular "invention" in this disclosure should not be used to assert that there is only one novelty in this disclosure. Multiple inventions may be set forth pursuant to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the present invention and any equivalents thereof protected thereby. In all instances, such claims should be considered on their own merits in light of this disclosure, but should not be bound by the headings set forth herein.

[0133] For each claim, each dependent claim may depend on both the independent claim and each preceding dependent claim, so long as the preceding claim provides appropriate antecedent for the claim term or element.

[0134] In order to assist the Patent Office, and any reader of any patent that may issue on this application, in interpreting the claims attached hereto, applicants are advised that they do not intend to invoke paragraph 6 of 35 U.S.C. § 112, paragraph (f) of 35 U.S.C. § 112, or the equivalent, as they existed on the filing date of this document, in any of the appended claims, unless the words "means" or "step" are expressly used in a particular claim.

[0135] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. a heavy chain variable domain (VH) complementarity determining region (CDR) 1 comprising any one of the amino acid sequences of SEQ ID NOs: 1, 49, 67, 73, 79, or 85; and a VH CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 2, 50, 68, 74, 80, or 86; and a VH CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 6, 54, 57, 60, 63, 66, 72, 78, 84, or 90; An anti-CCR8 antibody or an antigen-binding fragment thereof comprising:

2. The antibody, VH each comprising the amino acid sequence of any one of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, or 15; and VL each comprising any one of the amino acid sequences of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, or 24; 2. The antibody or antigen-binding fragment of claim 1, comprising:

3. The antibody, 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 NOs: 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 NOs: 34, 35, 36, 37, 18, 29, 40, 41, or 42; 3. The antibody or antigen-binding fragment of claim 1 or 2, comprising:

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

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

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

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

9. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the heavy chain CDRs are SEQ ID NOs: 1, 2 and 3, and the light chain CDRs are SEQ ID NOs: 4, 5 and 6.

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

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

12. 12. The method of claim 11, wherein the cancer is infiltrated with regulatory T cells.

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

14. 1. A method of making an anti-CCR8 antibody or antigen-binding fragment, comprising expressing an 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 comprising any one of the amino acid sequences of SEQ ID NOs: 1, 49, 67, 73, 79, or 85; and a VH CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 2, 50, 68, 74, 80, or 86; and a VH CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 6, 54, 57, 60, 63, 66, 72, 78, 84, or 90; The method comprising:

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

16. 16. The method of claim 14 or 15, wherein the antibody is a monoclonal, bispecific, multivalent, multispecific, diabody, chimeric, scFv antibody or fragment thereof.

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

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

19. a heavy chain variable domain encoding polynucleotide having at least 95, 96, 97, 98, 99, or 100% sequence identity to 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 to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or 42. A nucleic acid comprising an anti-CCR8 antibody or antigen-binding fragment comprising:

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

21. 21. The nucleic acid of claim 20, wherein the antibody binding domain is fused to an 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 of any one of claims 19 to 22, wherein the antibody or antigen-binding fragment does not bind to CCR4.

24. A vector comprising the nucleic acid of claim 19.

25. 25. A host cell comprising a nucleic acid that is the vector of claim 24.

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