Anti-CCR8 antibodies

Abstract

To provide a monoclonal, humanized antibody that specifically binds to CCR8 and mediates ADCC of CCR8 expressing Tregs.SOLUTION: An anti-CCR8 antibody structurally consists of a variable heavy chain and a variable light chain having a specific sequence and comprising complementary determining regions (CDRs) that specifically bind to CCR8. The anti-CCR8 antibody also includes a human heavy chain constant region comprising a fragment crystallizable region (Fc), and a light chain constant region. Such structural elements, as coded by an amino acid sequence of the anti-CCR8 antibody, comprise a pharmaceutical composition effective at treating solid tumors in a patient, either as a monotherapy or in combination with other therapeutics.SELECTED DRAWING: Figure 4B

Description

Technical Field

[0001] This application includes a sequence listing that was electronically submitted as an xml file named SL-ANTI-CCR8 ANTIBODIES, created on July 25, 2022, and having a size of 17 kilobytes. The sequence listing is incorporated herein by reference.

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 226,118, filed July 27, 2021, under 35 U.S.C. § 119(e), the disclosure of which is incorporated herein by reference in its entirety.

[0003] This application relates, inter alia, to novel anti-CCR8 antibodies and methods of making and using the same.

Background Art

[0004] In tumors, regulatory T cells (Tregs) are a key suppressive population known to prevent anti-tumor immune responses. Increased presence of intratumoral Tregs has been associated with poor patient outcomes in several cancers (Shang et al., Nature Sci. Reports, 2015; Fridman et al., Nat Rev Clin Oncol. 2017; Bruni et al., Nat Rev Cancer. 2020). Chemokine receptor 8 (CCR8) is a cell surface protein uniquely expressed by intratumoral Tregs in several human cancers (Plitas et al., Immunity 2016; DeSimone et al., Immunity 2016). Thus, CCR8 is an attractive target for mediating the selective depletion of intratumoral Tregs via antibody-dependent cell-mediated cytotoxicity (ADCC) and enhancing anti-tumor immunity.

[0005] Treg depletion has been studied for a long time, but most of such treatments have limited effectiveness because they target expression on tumor-infiltrating effector T cell populations and / or Tregs outside the tumor environment. There remains a need in the art for monoclonal antibody therapeutics that cause the death of immunosuppressive Tregs within tumors without depleting other key effector T cell populations or peripheral Tregs in the tumor microenvironment.

Prior Art Documents

Non-Patent Documents

[0006]

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Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0007] (Summary of the Invention) Anti-CCR8 monoclonal antibodies have been shown to mediate the selective depletion of intratumoral Tregs via antibody-dependent cellular cytotoxicity (ADCC). Since CCR8 is preferentially expressed by tumor-infiltrating Tregs and not highly expressed in peripheral blood Tregs or by beneficial effector T cell populations, anti-CCR8 antibodies deplete intratumoral Tregs and enhance antitumor immunity. Anti-CCR8 antibody monotherapy is independently effective, but the specific removal of Tregs in tumors creates an environment suitable for means of combination therapy aimed at co-stimulating the antitumor immune response. For example, a combination with a checkpoint inhibitor such as anti-PD-1 may be required to fully drive the potential antitumor immunity upon Treg depletion.

Means for Solving the Problem

[0008] Accordingly, provided is an amino acid sequence for a monoclonal humanized antibody that specifically binds to CCR8 and mediates ADCC of CCR8-expressing Tregs. Structurally, the antibody consists of a variable heavy chain and a variable light chain that contain complementarity-determining regions (CDRs) that specifically bind to CCR8. The antibody also contains a human heavy chain constant region that includes a crystallizable fragment region (Fc), and a light chain constant region. Such structural elements encoded by the amino acid sequence of the antibody are included in a pharmaceutical composition effective in the treatment of solid tumors in a patient, either as monotherapy or in combination with other therapeutic agents.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] Tregs are a subset of CD4+ T cells with immunosuppressive effects within tumors. Tregs suppress the activation, proliferation, and cytokine production of CD4+ T cells and CD8+ T cells to prevent harmful autoimmune responses. However, Tregs also suppress tumor immunity, and high levels of intratumoral Tregs are associated with negative outcomes in some cancers.

[0011] CCR8 is a C-C motif chemokine receptor consisting of a seven-transmembrane protein that mediates chemotaxis and cell / cell interactions in the context of type 2 helper T (Th2) lymphocyte immune responses and T cell trafficking to the skin. CCR8-deficient mice are viable, fertile, and mostly normal, except that they are unable to initiate robust Th2 responses in certain Th2-related preclinical models (Chensue et al., J Exp Med 5, 2001). The ligand that binds mainly to CCR8 is CCL1, although CCL18 (human) and CCL8 (mouse) are also ligands for this receptor.

[0012] CCR8-expressing Tregs infiltrating tumors are highly activated and exhibit an immunosuppressive phenotype. Tumor studies in CCR8 knockout mice have shown that a decrease in CCR8 expression does not affect the recruitment, activation status, or suppressive ability of Tregs to the tumor microenvironment (VanDamme et al., J Immunother Cancer. 9(2), 2021). Rather, CCR8 expression is a marker of highly suppressive Tregs. Thus, depletion of CCR8-expressing Tregs confers antitumor benefits.

[0013] CCR8-specific surrogate antibodies mediate the selective depletion of intratumoral Tregs via ADCC. In addition, anti-CCR8 surrogate antibodies significantly enhance the frequency of circulation of tumor-specific CD8+ effector T lymphocytes. Such effects correlate with efficacy in syngeneic tumor mouse models (Campbell et al., Cancer Research 81, 2021).

[0014] The inventors have developed, for example, a monoclonal therapeutic antibody that specifically binds to CCR8 expressed on the cell surface, such as intratumoral Tregs. In embodiments, the antibody consists of two variable chains, one heavy chain and one light chain. There are three CDRs on each variable chain that enable the antibody to bind to CCR8. There are a total of six different CDRs on both variable chains. In addition, the antibody includes a human heavy chain constant region that includes the human Fc of immunoglobulin class G1 (IgG1). The anti-CCR8 antibodies described herein can be fucosylated or defucosylated to demonstrate in vitro functionality, immune safety, and drug-like properties.

[0015] In some embodiments, the antibody includes a defucosylated IgG Fc constant region. In embodiments, the defucosylated Fc constant region is IgG1. Defucosylation can be performed by techniques known in the art. See, for example, Mol Cancer Ther (2020) 19(5):1102-1109 and PNAS (2013) 110(14)5404-5409. For example, due to the deficiency of GDP-mannose 4,6-dehydratase, such as the production of antibodies in cell lines lacking GDP-fucose formation, the production of antibodies in cells with reduced levels of fucosyltransferase, the production of antibodies in cells with reduced levels of GDP-fucose transporter, the production of antibodies in cells highly expressing β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (GnT-III), or the production of antibodies in cells expressing bacterial GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). In embodiments, the cells used for the production of the defucosylated anti-CCR8 antibody of the present invention are CHO cells modified to express Pseudomonas RMD. The degree of defucosylation of the antibody can be quantified by techniques known in the art.

[0016] To obtain anti-CCR8 antibodies that cross-react with human and cynomolgus monkey CCR8, the ability of mouse-human (rat / hIgG1) chimeric antibodies to bind to Jurkat cells that highly express CCR8 of either human or cynomolgus monkey was evaluated. The results were analyzed by flow cytometry performed by techniques known in the art. As a result, the final anti-CCR8 antibodies cross-react with human and cynomolgus monkey CCR8 and mediate ADCC in both species, but do not bind to mouse, rat or rabbit CCR8.

[0017] In certain embodiments, the defucosylated antibodies of the invention have a high affinity for the activation of IgG receptors and an enhanced activity in a purified natural killer cell or peripheral blood mononuclear cell (PBMC) ADCC assay as compared to the fucosylated form of this antibody. The ADCC activity of the anti-CCR8 antibodies can be demonstrated using ADCC bioassay techniques known in the art. For example, in human FcγRIIIa V158 or F158 allele variant reporter strains cultured with human or cynomolgus monkey CCR8-expressing Jurkat cells, the anti-CCR8 antibodies induced ADCC as measured by luminescence induction using techniques known in the art.

[0018] In addition, the antibodies described herein also bind to CCR8, but interfere with the efficacy of other naturally occurring ligands such as CCL1 that bind only at substantially higher EC50s than for binding or ADCC activity. This assay was performed by techniques known in the art, such as a beta-arrestin reporter assay.

[0019] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to a particular antigen, e.g., CCR8. The anti-CCR8 antibodies of the present disclosure bind to human CCR8 on Tregs and thereby modulate the immune system. The anti-CCR8 antibodies of the present disclosure include complementarity determining regions (CDRs), also known as hypervariable regions, within both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called frameworks (FRs). As is known in the art, the amino acid portions / boundaries delineating the hypervariable regions of an antibody can vary according to these and various definitions known in the art. Some positions within the variable domain can be considered hybrid high-frequency variable positions because such positions appear to be within the hypervariable region under one set of criteria but outside the hypervariable region under another set of criteria. Also, one or more of such positions can be found in extended hypervariable regions. The present disclosure provides antibodies that include modifications at such hybrid hypervariable positions. The variable domains of the native heavy and light chains each typically incorporate four FR regions linked by three CDRs that form loops connecting, and in some cases forming part of, a β-sheet structure. The CDRs of each chain are joined in close proximity by the FR regions and together with the CDRs of the other chain contribute to the formation of the target binding site of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987).

[0020] The antibodies of the present disclosure are polyclonal, monoclonal, genetically modified and / or otherwise naturally modified antibodies, and may include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies, single-chain antibodies, etc. In various embodiments, the antibody includes all or a portion of the constant region of the antibody. In some embodiments, the constant region is an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), and IgM. In certain embodiments, the anti-CCR8 antibodies described herein include IgG1. In other embodiments, the anti-CCR8 antibody includes IgG2. In still other embodiments, the anti-CCR8 antibody includes IgG4. As used herein, the "constant region" of an antibody includes the native constant region, allotype or variant.

[0021] The light chain constant region of the anti-CCR8 antibody can be a kappa (κ) light chain region or a lambda (λ) region. The λ light chain region can be any one of the known subtypes, e.g., λ1, λ2, λ3 or λ4. In some embodiments, the anti-CCR8 antibody includes a kappa (κ) light chain region.

[0022] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies are derived from a single clone and include any eukaryotic, prokaryotic or phage clone by any available means or known in the art. Monoclonal antibodies useful according to the present disclosure can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant and phage display technologies or combinations thereof.

[0023] The term "chimeric" antibody as used herein refers to an antibody having a variable sequence derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region typically selected from a human immunoglobulin template.

[0024] The "humanized" form of a non-human (e.g., mouse) antibody contains substantially all of at least one and typically two variable domains, wherein all or substantially all of these CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of these FR regions are the FR regions of a human immunoglobulin sequence. Also, a humanized antibody can contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin consensus sequence.

[0025] A "human antibody" includes an antibody having the amino acid sequence of a human immunoglobulin, including antibodies isolated from a human immunoglobulin library or from an animal into which one or more human immunoglobulins have been introduced and which does not express a functional endogenous immunoglobulin. Human antibodies can be made by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0026] The anti-CCR8 antibodies of the present disclosure include full-length (intact) antibody molecules.

[0027] An anti-CCR8 antibody can be an antibody whose sequence is modified to alter at least one biological effector function mediated by the constant region. For example, the anti-CCR8 antibodies described herein can acquire or improve at least one biological effector function mediated by the constant region as compared to an unmodified antibody, for example, by enhancing FcγR interaction (see, e.g., U.S. Patent Application No. 2006 / 0134709) or modifying the antibody to enhance its ability to mediate ADCC. For example, the anti-CCR8 antibodies of the present disclosure can have a constant region that binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB with a higher affinity than the corresponding unmodified constant region. The anti-CCR8 antibodies of the present disclosure can have a modified Fc region and can be antibodies that mediate enhanced ADCC responses, in which case the ADCC response is enhanced with respect to an antibody having the same variable regions (i.e., VH and VL) and a wild-type IgG1 Fc region (i.e., wild-type CL, CH1, CH2, and CH3). Fc modifications capable of enhancing ADCC, such as amino acid sequence mutations, are known in the art and can include the following sets of mutations: S239D / I332E; F243L / R292P / Y300L / V305I / P396L; S239D / I332E / A330L; and S298A / E333A / K334A.

[0028] Anti-CCR8 antibodies containing a human IgG4 constant region can include the S228P mutation, which has been reported to prevent Fab arm exchange. See, for example, Silva, JP et al., Journal of Biological Chemistry, 290(9), 5462-5469 (2015).

[0029] In some embodiments, the anti-CCR8 antibody comprises modifications that increase or decrease the binding affinity of these antibodies to the neonatal Fc receptor, FcRn, for example, by mutating immunoglobulin constant region segments in specific regions involved in FcRn interaction. In certain embodiments, an IgG class anti-CCR8 antibody is mutated to substitute at least one of amino acid residues 250, 314, and 428 in the heavy chain constant region, either alone or in any combination thereof. At position 250, the amino acid residue to be substituted may be any amino acid residue other than threonine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine. At position 314, the amino acid residue to be substituted may be any amino acid residue other than leucine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. At position 428, the amino acid residue to be substituted may be any amino acid residue other than methionine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. An example substitution known to modify Fc effector function is Fc substitution M428L, which can occur in combination with Fc substitution T250Q. Further specific combinations of suitable amino acid substitutions are identified in Table 1 of U.S. Patent No. 7,217,797. Such mutations improve binding to FcRn, thereby protecting the antibody from degradation and improving its half-life.

[0030] Anti-CCR8 antibodies with high affinity for human CCR8 may be desirable for therapeutic and diagnostic uses. Accordingly, the present disclosure contemplates antibodies having high binding affinity for human CCR8. In certain embodiments, the anti-CCR8 antibody binds to human CCR8 with an affinity of at least about 100 nM, but may exhibit a high affinity of, for example, at least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM or even higher than these. In some embodiments, the antibody binds to human CCR8 with an affinity in the range of about 1 pM to about 10 nM, about 100 pM to about 10 nM, about 100 pM to about 1 nM, or in a range spanning between any of the aforementioned values.

[0031] In some embodiments, the present invention provides monoclonal anti-CCR8 antibodies comprising two sets of six different complementarity-determining regions (CDRs), two sets of two different variable regions, two complete heavy chains, two complete light chains, and a human heavy chain constant region.

[0032] In some embodiments, the antibody is a recombinant, defucosylated, humanized, IgG1 kappa monoclonal antibody that binds to chemokine receptor 8.

[0033] In embodiments, the antibody comprises six CDRs having the following sequences.

[0034]

Chemical formula

[0035] In some embodiments, the antibodies of the present disclosure comprise CDR-H1 having the amino acid sequence shown as SEQ ID NO: 1, CDR-H2 having the amino acid sequence shown as SEQ ID NO: 2, CDR-H3 having the amino acid sequence shown as SEQ ID NO: 3, CDR-L1 having the amino acid sequence shown as SEQ ID NO: 4, CDR-L2 having the amino acid sequence shown as SEQ ID NO: 5, and CDR-L3 having the amino acid sequence shown as SEQ ID NO: 6.

[0036] In some embodiments, the antibody of the present disclosure has an amino acid sequence shown as SEQ ID NO: 7:

[0037]

Chemical formula

[0038]

Chemical formula

[0039] In some embodiments, the antibody of the present disclosure has an amino acid sequence shown as SEQ ID NO: 9 (the constant region is in bold, the variable heavy chain domain is underlined, and the CDRs are in bold italic with underline (disclosed as SEQ ID NOs: 1-3 in the order of display)):

[0040]

Chemical formula

[0041]

Chemical formula

[0042] In an embodiment, the antibody of the present disclosure has a light chain according to SEQ ID NO: 10 and a heavy chain with the C-terminal lysine cleaved, for example, a heavy chain according to SEQ ID NO: 9 with the C-terminal lysine cleaved:

[0043]

Chemical formula

[0044] In one embodiment, the heavy chain of the antibody of the present disclosure is encoded by the following nucleotide sequence (the full-length sequence disclosed as SEQ ID NO: 12).

[0045]

Chemical formula

[0046] In one embodiment, the light chain of the antibody of the present disclosure is encoded by the following nucleotide sequence (the full-length sequence disclosed as SEQ ID NO: 13).

[0047]

Chemical formula

[0048] In some embodiments, the antibody includes a human heavy chain constant region that includes human CH1, human hinge, human CH2, and human CH3 domains. In some embodiments, the encoded heavy chain constant region includes an Fc portion, where the Fc portion is of human IgG1, IgG2, IgG3, IgG4, or IgM isotype. In an embodiment, the Fc is IgG1 and the allotype is z non a. In an embodiment, the light chain is a kappa light chain.

[0049] In some embodiments, the antibody comprises a defucosylated IgG1 Fc constant region. Defucosylation can be carried out by techniques known in the art. For example, due to the lack of GDP-mannose 4,6-dehydratase, such as the production of antibodies in cell lines lacking GDP-fucose formation, the production of antibodies in cells with reduced levels of fucosyltransferase, the production of antibodies in cells with reduced levels of GDP-fucose transporter, the production of antibodies in cells highly expressing β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (GnT-III), or the production of antibodies in cells expressing bacterial GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). The cells used for the production of the defucosylated anti-CCR8 antibody of the present invention are CHO cells modified to express Pseudomonas RMD. The degree of defucosylation of the antibody can be quantified by techniques known in the art. Typically, the antibody is 70% or more, 80% or more, 90% or more, or about 99% or about 100% defucosylated. Preferably, the degree of defucosylation is 80% or more. In some embodiments, the antibody is 70% or more, 80% or more, 90% or more, or about 100% defucosylated at the position of ASN-300 (EU: ASN-297). Defucosylation can be quantified by hydrophilic interaction chromatography (HILIC) assay technology, and the degree of this defucosylation is quantified by the polarity-dependent separation of fragmented antibodies.

[0050] In embodiments, the total amount of defucosylated glycan species is quantified by analysis of free N-linked glycans by HILIC using fluorescence detection. The glycans are released using peptide N-glycosidase F (PNGaseF) and then labeled with a fluorescent tag. The fluorescently labeled N-linked glycans are analyzed by HILIC using fluorescence detection. The percent defucosylated glycan species is quantified by comparing to the total peak area of all glycan peaks in the chromatogram based on the sum of the peak areas of all defucosylated glycan peaks. All peaks with a relative abundance of 0.5% or more are included in the quantification of the percent defucosylated glycan species.

[0051] 7.1. Polynucleotides Encoding Anti-CCR8 Antibodies, Expression Systems, and Methods for Producing Antibodies The present disclosure encompasses polynucleotide molecules encoding the immunoglobulin light and heavy chain genes of anti-CCR8 antibodies, vectors containing such polynucleotides, and host cells capable of producing the anti-CCR8 antibodies of the present disclosure.

[0052] The anti-CCR8 antibodies of the present disclosure can be prepared by recombinant expression of the immunoglobulin light and heavy chain genes in host cells. To recombinantly express the antibodies, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cells and, optionally, secreted into the medium in which the host cells are cultured, and the antibodies can be recovered from this medium.

[0053] To generate polynucleotides encoding such anti-CCR8 antibodies, DNA fragments encoding the light and heavy chain variable regions are first obtained. Such DNA can be obtained, for example, by amplification and modification of germline DNA or cDNA encoding the light and heavy chain variable sequences using polymerase chain reaction (PCR).

[0054] Once DNA fragments encoding anti-CCR8 antibody-related VH and VL segments are obtained, such DNA fragments can be further manipulated by standard recombinant DNA techniques to, for example, convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In such manipulations, the VL or VH coding DNA fragments are operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked," as used in this context, is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0055] By operably linking VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (CH1, CH2, CH3 and optionally CH4), the isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing such regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but in certain embodiments, it is IgG1 or IgG4. In the Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0056] By operably linking VL-encoding DNA to another DNA molecule encoding the light chain constant region CL, the isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as the Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing such regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but in certain embodiments, it is the kappa constant region.

[0057] To express the anti-CCR8 antibodies of the present disclosure, the DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector to operably link the gene to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean ligating the antibody gene into the vector such that the transcriptional and translational regulatory sequences within the vector serve their intended function of controlling the transcription and translation of the antibody gene. The expression vector and expression regulatory sequences are selected to be compatible with the expression host cell to be used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector.

[0058] The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of antibody gene fragments and complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present). Prior to insertion of the anti-CCR8 antibody-related light or heavy chain sequence, the expression vector may already possess the antibody constant region sequence. For example, one approach for the conversion of VH and VL sequences related to an anti-CCR8 monoclonal antibody into a full-length antibody gene is to insert them into an expression vector that already encodes the heavy chain constant and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment(s) within the vector and the VL segment is operably linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates the secretion of the antibody chain from the host cell. By cloning the antibody chain gene into the vector, the signal peptide can be ligated in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0059] In addition to the antibody chain gene, the recombinant expression vector of the present disclosure carries a control sequence that regulates the expression of the antibody chain gene in a host cell. The term "control sequence" is intended to include promoters, enhancers and other expression regulatory elements (e.g., polyadenylation signals) that regulate the transcription or translation of the antibody chain gene.

[0060] In addition to the antibody chain gene and the control sequence, the recombinant expression vector of the present disclosure may carry additional sequences such as a sequence (e.g., origin of replication) that controls the replication of the vector in a host cell and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced. For the expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0061] It is possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells. In certain embodiments, antibody expression is carried out in eukaryotic cells, such as mammalian host cells, that are optimal for the secretion of properly folded and immunologically active antibodies. Examples of mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells (e.g., DHFR-CHO cells described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selectable marker described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When introducing a recombinant expression vector encoding an antibody gene into a mammalian host cell, the antibody is produced by culturing the host cell for a period sufficient to allow for expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods. Also, intact antibody moieties, such as Fab fragments or scFv molecules, can be produced using the host cells. It is understood that variations of the above procedures are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding either (but not both) the light or heavy chain of the anti-CCR8 antibody of the present disclosure.

[0062] Furthermore, recombinant DNA techniques can be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are unnecessary for binding to human CCR8. Also, molecules expressed from such truncated DNA molecules are encompassed by the antibodies of the present disclosure.

[0063] In the recombinant expression of the anti-CCR8 antibodies of the present disclosure, host cells can be co-transfected with two expression vectors of the present disclosure, where the first vector encodes a polypeptide derived from the heavy chain and the second vector encodes a polypeptide derived from the light chain. The two vectors may contain the same selectable marker or they may contain separate selectable markers. Alternatively, a single vector encoding both the heavy chain and light chain polypeptides can be used.

[0064] Once a polynucleotide encoding one or more portions of the anti-CCR8 antibody is obtained, further changes or mutations can be introduced into the coding sequence to generate, for example, polynucleotides encoding antibodies with various CDR sequences, antibodies with reduced affinity for Fc receptors, or antibodies of various subclasses.

[0065] Also, the anti-CCR8 antibodies of the present disclosure can be produced by chemical synthesis or by using a cell-free platform.

[0066] 7.2. Purification of Anti-CCR8 Antibodies When the polypeptides of the present disclosure are produced by recombinant expression, they can be purified by any method known in the art for protein purification. Once isolated, the anti-CCR8 antibodies can be further purified.

[0067] 7.3. Compositions The antibodies of the present disclosure can be provided as compositions suitable for administration to a subject. In some embodiments, the antibody composition is a pharmaceutical composition comprising the antibody of the present disclosure and a pharmaceutically acceptable carrier.

[0068] In an embodiment, a pharmaceutical composition comprising a plurality of anti-CCR8 antibodies and a pharmaceutically acceptable carrier is provided. In an embodiment, the plurality of anti-CCR8 antibodies of the pharmaceutical composition are defucosylated. Typically, the plurality of antibodies are 70% or more, 80% or more, 90% or more, or about 99% or about 100% defucosylated. Preferably, the degree of defucosylation is 90% or more. In some embodiments, the plurality of antibodies are 70% or more, 80% or more, 90% or more, or about 100% defucosylated at the position of ASN-300 (EU: ASN-297). The degree of defucosylation of the antibody can be quantified by techniques known in the art. Defucosylation can be quantified by hydrophilic interaction chromatography (HILIC) assay technology, and the degree of this defucosylation is quantified by polarity-dependent separation of fragmented antibodies. In an embodiment, the total amount of defucosylated glycan species is quantified by analysis of free N-linked glycans by HILIC using fluorescence detection. The glycan is released using peptide N-glycosidase F (PNGaseF) and then labeled with a fluorescent tag. The fluorescently labeled N-linked glycans are analyzed by HILIC using fluorescence detection. The percent defucosylated glycan species is quantified by comparing the sum of the peak areas of all defucosylated glycan peaks to the total peak area of all glycan peaks in the chromatogram, based on the sum of the peak areas of all defucosylated glycan peaks. All peaks with a relative abundance of 0.5% or more are included in the quantification of the percent defucosylated glycan species.

[0069] 7.4. Summary of the properties of the antibodies of the subject matter The properties of the antibodies of the subject matter, illustrated by, but not limited to, ABBV-514, include the following.

[0070] High affinity binding to CCR8, for example, when quantified by CCR8 endogenously expressed on TALL-1 cells (human adult T-ALL; RRID: CVCL 1736), FACS binding mean EC 50is 2 μg / mL or less, 1 μg / mL or less, 0.3 - 0.7 μg / mL, 0.4 - 0.6 μg / mL, about 0.5 μg / mL, or 0.55 μg / mL; or the mean FACS binding EC for human CCR8 highly expressed on Jurkat cells 50 is 2 μg / mL or less, 1 μg / mL or less, 0.3 - 0.8 μg / mL, 0.4 - 0.7 μg / mL, about 0.5 μg / mL, or 0.6 μg / mL; or the mean FACS binding EC for CCR8 expressed on human blood CD45RA low Treg 50 is 2 μg / mL or less, 1 μg / mL or less, 0.2 - 0.6 μg / mL, 0.3 - 0.5 μg / mL, about 0.5 μg / mL, or 0.48 μg / mL.

[0071] High specific binding to human CCR8, for example, non-specific FACS binding to TALL-1 CCR8 knockout cells or parental Jurkat cells.

[0072] Cross-reactivity with cynomolgus CCR8, for example, the mean FACS binding EC for cynoCCR8 highly expressed on Jurkat cells 50 is 5 μg / mL or less, 3 μg / mL or less, 2 μg / mL or less, approximately 1.5 μg / mL, or 1.82 μg / mL; or the mean FACS binding EC for CCR8 expressed on cyno blood CD45RA low Treg 50 is 5 μg / mL or less, 3 μg / mL or less, 2 μg / mL or less, approximately 1.5 μg / mL, or 1.62 μg / mL.

[0073] Weak ability to block the CCL1 / CCR8 interaction, for example, the EC in CCL1 / CCR8 blocking activity 50 is at least 30× higher, at least 40× higher, 30× - 70× higher, at least 50× higher, or at least about 50× higher than the EC in the binding to human CCR8 50

[0074] Enhanced ability to induce ADCC for antibodies having the same variable region and wild-type fucosylated IgG1. ​

[0075] Enhanced binding to Fcγ receptors for antibodies having the same variable region and wild-type fucosylated IgG1.

[0076] Good immunological safety as quantified by cytokine release assay.

[0077] 7.5. Method of Use In embodiments, the methods described herein include treating a patient having a solid tumor with an anti-CCR8 antibody of the invention. In embodiments, a composition comprising an anti-CCR8 antibody is administered to a subject in need thereof.

[0078] In a PD-1 resistant Pan02 mouse model, a combination of CCR8 and PD-1 targeting antibodies showed improved in vivo efficacy over monotherapy with either antibody. Depletion of immunosuppressive CCR8+ Tregs can cooperate with PD-1 / PD ligand 1 (PD-L1) blockade to further enhance CD8+ effector T cell responses and anti-tumor immunity. In embodiments, a composition comprising an anti-CCR8 antibody is administered as part of a combination therapy comprising administration of a PD-1 or PD-L1 targeting antibody. In embodiments, a composition comprising an anti-CCR8 antibody is administered as combination therapy with pembrolizumab, budigalimab, nivolumab, semiprimab or dostarlimab. In embodiments, a composition comprising an anti-CCR8 antibody is administered as combination therapy with atezolizumab, avelumab, durvalumab.

Examples

[0079] The following examples are provided for illustrative purposes to highlight specific features and properties of embodiments that are examples of the antibodies and binding fragments described herein.

[0080] 8.1. [Example 1] Production of Rat Hybridomas Rats were immunized with two different full-length human CCR8c DNA vectors (6 rats per vector). Lymph node cells were isolated and fused to NS0 to generate hybridomas. After growth, the hybridoma supernatants were screened for binding to human or cynomolgus monkey ("cyno" or "cy") CCR8 highly expressed on HEK293 cells. Forty-six rat / hIgG1 chimeric mAbs were expressed by high-throughput antibody production, and 21 were confirmed to bind to human CCR8 (Figure 1). Four chimeric mAbs, AC-254290, AC-254532, AC-254546, and AC-254259, were selected for full humanization based on the highest cyno cross-reactivity in a Jurkat CCR8 high-expressing strain.

[0081] 8.2. [Example 2] Epitope mapping by competitive binding to huCCR8 The selected chimeric antibodies were examined by a cell epitope mapping assay for binding to human CCR8 on Jurkat cells together with human CCR8 antibodies from BD Biosciences (clone 433H) and Biolegend (clone L263G8). Both commercially available antibodies were reported to be generated by immunization of mice with human CCR8 transfectants and neither was cross-reactive with cynoCCR8. Antibodies were tested in pairs with cells stained with antibody 1 at saturating concentration, washed, then stained with antibody 2, or vice versa.

[0082] The mAbs of AC-254290 and AC-254532 interfere with binding in either order, suggesting that they bind to the same epitope (epitope A) (Figure 2). AC-254546 shows moderate epitope binding interference with AC-254290 and AC-254532, but there is no evidence of binding to the same epitope. Thus, AC-254546 binds to epitope B, which is unique but close to epitope A. AC-254259 has no binding interference with any other antibody and may be a unique epitope (epitope C). Clone 433H from BD Biosciences has the same epitope binding as AC-254290 and some interfering binding with AC-254532, AC-254546, and clone L263G8 from Biolegend, and thus is binned to epitope A. Based on the interference data by clone 433H from BD Biosciences and other antibodies, clone L263G8 from Biolegend is binned as epitope D, which is unique but close to epitope A rather than epitope B.

[0083] 8.3. [Example 3] Humanization of Rat Variable Domain / Human IgG1 Fc Chimera Four chimeric antibodies, AC-254290, AC-254532, AC-254546, and AC-254259, were humanized by (i) identifying rodent antibody sequences, (ii) identifying CDRs and antibody frameworks, (iii) creating VH-VL structural models, (iv) identifying framework residues for back mutations to maintain the function of the rodent antibody, (v) selecting human germline with high identity, the most similar CDR canonical structure, and the required minimum back mutations, and (vi) generating VH / VL sequences by CDR transplantation and incorporation of the selected back mutations.

[0084] Four humanized antibodies were generated for each of the selected antibodies. AC-254290 required a humanization approach based on both sequence and structure. Straightforward CDR grafting resulted in antibodies with weak binding to human CCR8-expressing Jurkat cells. Putative rodent VH / VL structure / interface-based revertant mutations were examined. The triplet of residues preceding HCDR3 was CTA, which was noncanonical. The 94th residue (A of CTA) was most often arginine (R). Two humanized versions with CTR showed weak binding to human CCR8-expressing Jurkat cells, and EC 50 was 3.96 and 5.61 μg / mL. In contrast, two humanized versions with CTA preceding HCDR3 (CTAGDRNKPFAY (SEQ ID NO: 14)), one of which was AC-264700, demonstrated strong binding, and EC 50 was 0.2559 and 0.2302 μg / mL, not inferior to the AC-254290 parental antibody (EC 50 was 0.4538 μg / mL).

[0085] The humanized antibodies were evaluated for their ability to bind to human or cynomolgus CCR8. EC 50 was quantified by examining the binding of antibodies at eight concentrations starting at 30 μg / mL at 5× dilutions to human or cynomolgus CCR8 high-expressing Jurkat cells. After incubation with the CCR8 test antibody, the cells were washed, incubated with a fluorochrome-labeled secondary antibody, washed, and analyzed by flow cytometry.

[0086] AC-254259 and AC-254532 showed a decrease in binding to cynoCCR8 and high non-specific binding to HEK293 cells, respectively. AC-264711 (humanized AC-254546) and AC-264700 (humanized AC-254290) were advanced due to excellent target binding, absence of non-specific binding, and limited requirements for liability modification.

[0087] 8.4. [Example 4] Liability Modification of AC-264700 AC-264700 has a DS motif, which is a risk of isomerization, at amino acids 61-62 and 27-28 (kabat) of HCDR2 and LCDR1, respectively, and has a DP motif that potentially fragments at amino acids 94-95 (kabat) of LCDR3. The DS liability of HCDR2 was mutated to DA based on the experience of previous antibodies. The binding was disrupted by the removal of the DP motif, but the fragmentation of the antibody retaining the DP motif did not appear under stress testing. Twenty-two LCDR1 DS variant antibodies were generated and their binding properties were evaluated using flow cytometry against huCCR8 or cyCCR8 overexpressing Jurkat cells. The non-specific binding to Jurkat parental cells was evaluated by flow cytometry. Ten variant antibodies were selected for further characterization based on favorable function and drug-like properties. Two were selected for advanced benefits based on the full combination of properties, including binding, reduced self-interaction, lack of non-specificity, cyno cross-reactivity, and activity in a cyno cross-reactivity and hFcγRIIIa V variant ADCC reporter bioassay (Promega).

[0088] Candidate molecules were evaluated for binding ability to highly expressed huCCR8 or cyCCR8 on Jurkat cell surfaces using flow cytometry by known methods. Example antibodies were tested for the ability to self-interact, bind to huCCR8 and cyCCR8, the ability of ADCC using a reporter bioassay, and non-specific binding to HEK293 cells. AC-277357 had excellent binding ability for both huCCR8 and cyCCR8 Jurkat cells when compared to the parent and other candidate antibodies (Table 1). The AC-SINS self-interaction score for each candidate was <1. At 100 μg / mL, the maximum non-specific binding values ranged from 97 to 144 GMFI. At 10 μg / mL, the maximum non-specific binding values ranged from 94 to 111 GMFI. At 1 μg / mL, the maximum non-specific binding values ranged from 87 to 102 GMFI. AC-277357 had the lowest maximum non-specific binding value at 100 μg / mL to HEK293 cells, the strongest binding to huCCR8 and cyCCR8 Jurkat cells, and the maximum fold signal induction by both huCCR8 and cyCCR8 Jurkat cells in the ADCC reporter bioassay. In addition, AC-277357 had one of the lowest self-interaction scores when compared to the parent antibody.

[0089]

Table 1

[0090] 8.5. [Example 5] Liability modification of AC-264711 AC-264711 had an NS motif presenting a risk of deamination at amino acids 60 - 61 (kabat) in the HCDR2 region. In addition, the M100c residue of HCDR3 showed high levels of oxidation during stress testing.

[0091] The AC-264711 candidate variant with an NS mutation that removes liability self-interacts, binds non-specifically to HEK293 cells, binds to huCCR8 and cyCCR8 jackett cells, and was tested for its ability to induce a signal in an ADCC reporter bioassay by huCCR8-expressing jackett cells as a target. Removal of the NS motif showed tolerance. Twenty-nine NS variants were generated and their binding properties were evaluated using flow cytometry against jackett parental cells and high-expressing huCCR8 or cyCCR8 jackett cells. Five antibodies with the best binding and drug-like property profiles were tested in an ADCC reporter bioassay, and three antibodies, AC-275889, AC-275896, and AC-275898, with the best combination of properties were advanced due to the M100c mutation that prevents oxidation.

[0092] Variants of AC-275889, AC-275896, and AC-275898 with the M100c mutation were generated and tested in the same assay as NS variants. Using the data from such assays, eight priorities for antibodies were established for drug-like property testing. AC-291774 and AC-291790 had the highest self-interaction AC-SINS scores of 8.38 and 6.75, respectively. The remaining candidates had comparable AC-SINS scores ranging from 1.73 to 2.79. AC-275896 and AC-275898 had the highest non-specific binding to HEK293 cells of 1780 and 1065 GFMI, respectively. AC-291790, AC-275889, and AC-275898 had non-specific binding maxima of 335, 148, and 141, respectively. At 10 and 1 μg / mL, all candidate molecules had comparable non-specific binding maxima ranging from 61 - 67 and 57 - 60, respectively. AC-291774 and AC-291790 had the lowest non-specific binding compared to AC-275889, AC-275896, and AC-275898 as shown by binding to Jurkat parental cells and hydrophobic interaction chromatography, but strong binding to huCCR8- and cyCCR8-expressing Jurkat cells, and comparable EC 50 and fold induction were maintained (Table 2).

[0093]

Table 2

[0094] 8.6. [Example 6] Selection of Final Candidate Antibodies AC-277357, AC-277371, AC-291774, and AC-291790 were selected and further evaluated in additional drug-like property and in vitro immune safety experiments. To generate defucosylated variants for property determination, cells expressing AC-277357 were transfected with a plasmid to express the Pseudomonas enzyme GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). This converts the intermediate to the final product in the de novo synthesis of fucose, resulting in the production of defucosylated AC-277357.

[0095] Candidate molecules were evaluated for binding to Jurkat huCCR8, Jurkat cyCCR8 and TALL-1 binding activity, hPBMC ADCC activity by TALL-1 as a target, ADCC activity in F158 effector cells by huCCR8 high-expressing Jurkat cells as a target and ADCC activity in V158 effector cells by huCCR8 high-expressing Jurkat cells as a target. AC-277357 (also called PR-1925514) showed improved binding to the endogenous level of CCR8 expression on TALL-1 cells, and the defucosylated version was even more active in the primary cell ADCC assay (data not shown). AC-277357 / PR-1925514 bound more potently to CCR8 on human Tregs than AC-291774 (also called PR-1928444) and had a better profile in the cytokine release assay (data not shown). AC-277357 / PR-1925514 with defucosylated human IgG1 Fc was selected as the lead antibody due to its best in vitro functionality and in vitro immune safety (INIS) profile, as well as excellent drug-like properties.

[0096] 8.7. [Example 7] Binding and Functional Ability Assays Antibodies were screened by single-point flow cytometry to evaluate binding to Jurkat cell lines highly expressing human or cynomolgus CCR8. EC 50Eight concentrations of the antibody starting at 30 μg / mL at 5× dilution were quantified by flow cytometry for binding to Jurkat human CCR8, Jurkat cynoCCR8, and parental Jurkat cells. After incubation with the test CCR8 antibody, the cells were washed and incubated with a fluorochrome-labeled secondary antibody, washed, and analyzed by flow cytometry.

[0097] The human FcγRIIIa V variant core kit or human FcγRIIIa F variant cell proliferation model was purchased from Promega and used as directed. Jurkat target cell lines highly expressing human or cynomolgus monkey CCR8 or parental Jurkat control cells were washed once in ADCC assay buffer and diluted to 6×10 6 cells per mL in ADCC assay buffer and seeded at 25 μL per well into white-walled 96-well assay plates (Costar). The antibody was diluted at 3× the final concentration in ADCC assay buffer and 25 μL per well was added (a 1:5-fold 7-point dilution ranging from 1 μg / mL to 0.000064 μg / mL was tested). Effector cells (either human FcγRIIIa V158 variant, human FcγRIIIa F158 variant, or mouse FcγRIV, and stably expressing the NFAT response element driving the expression of firefly luciferase) were thawed and added at a final effector-to-target ratio of 1:2:5. After incubation at 37 °C, 5% CO2 for 6 hours, BioGlo reagent (Promega) was mixed and added as directed, and the luminescence signal was measured.

[0098] 8.8. [Example 8] Dose-response curve of ABBV-514 The fucosylated and defucosylated (renamed ABBV-514) versions of AC-277357 / PR-1925514 were evaluated for binding to cells expressing high levels of human or cyno CCR8 in Jurkat cell lines and TALL-1 cells expressing endogenous levels of CCR8. Cell binding was evaluated by flow cytometry using a similar method as described above. In addition, binding was evaluated for parental Jurkat (CCR8-negative) and CCR8 knockout (KO) TALL-1 control cell lines. ABBV-514 showed binding to both highly expressed human and cyno CCR8 in Jurkat cells, but not in parental Jurkat or CCR8 KO TALL-1 cells (Figure 4A). Also, ABBV-514 bound to endogenous CCR8 on TALL-1 cells, similar to fucosylated (WT) AC-277357 / PR-1925514 (Figure 4B).

[0099] Both fucosylated WT PR-1925514 and ABBV-514 mediated ADCC as measured by luminescence induction in a human FcγRIIIa reporter cell-based bioassay using either the FcγRIIIa V158 (Figure 5A, 5C) or F158 (Figure 5B, 5D) allele variant reporter cell lines co-cultured with human (Figure 5A, 5B) or cyno (Figure 5C, 5D) CCR8-high expressing Jurkat cells. The fold change in luminescence compared to untreated co-cultures is shown as the mean ± SEM of 2 - 5 independent experiments. ABBV-514 showed an increase in ADCC activity as shown by the ADCC reporter bioassay compared to fucosylated WT PR-1925514. For comparison purposes with concentrations expressed in μg / mL, a concentration of 1 μg / mL of antibody is equal to 6.76 nM.

[0100] 8.9. [Example 9] Primary Cell ADCC Assay Data for ABBV-514 The activity of ABBV-514 was further evaluated in an ADCC assay based on the cytotoxicity of cells using TALL-1 cells as targets and human primary NK cells as effectors. Six donor sources of NK cells were used as target cells, and cell death of TALL-1 target cells was evaluated by flow cytometry using a similar method as described above. ABBV-514 mediated ADCC of CCR8-expressing TALL-1 cells by all six donor NK cell specimens (Figure 6). ADCC activity in human whole blood cultures was evaluated by flow cytometry using the adaptation procedure described above. After 48 hours, cultures of human whole blood with ABBV-514 were analyzed to evaluate the percentage of CD45RA low Treg in the total amount of CD45+ hematopoietic cells. CD45RA low Treg includes CD45+, CD3+, CD4+, CD25+, CD127 low, Foxp3+, CD45RA low live cells, which are known to be enriched in CCR8 expression. Human whole blood cell data from 11 healthy donors were evaluated. In each donor, ABBV-514 decreased the percentage of the amount of CD45RA low Treg in CD45+ live cells (data not shown).

[0101] 8.10. [Example 10] Evaluation of CCL1 Ligand Blockade by ABBV-514 The CCR8 beta-arrestin reporter assay was used to determine whether ABBV-514 blocks the interaction of CCR8 with its ligand CCL1 (Figure 7). Inhibition of CCL1-mediated reporter activity in the presence of ABBV-514 was observed only at concentrations of antibody substantially higher than those at which binding or ADCC activity was observed, and the EC 50 was observed to be 23.1 μg / mL.

[0102] Exemplary Embodiments Various specific embodiments are illustrated and disclosed, some of which are described below, and it is understood that various changes can be made without departing from the spirit and scope of the invention(s).

[0103] 1. An anti-CCR8 antibody comprising (i) a VH chain comprising three CDRs and (ii) a VL chain comprising three CDRs, wherein VH CDR#1 is GFIFSNAVMY (SEQ ID NO: 1), VH CDR#2 is RIKTKFNNYATYYADAVKG (SEQ ID NO: 2), VH CDR#3 is GDRNKPFAY (SEQ ID NO: 3), VL CDR#1 is RASTSVITLLH (SEQ ID NO: 4), VL CDR#2 is GASNLES (SEQ ID NO: 5), VL CDR#3 is QQSWNDPYT (SEQ ID NO: 6). 2. The anti-CCR8 antibody according to embodiment 1, comprising the amino acid sequence CTA immediately before VH CDR#3, thereby comprising the amino acid sequence CTAGDRNKPFAY (SEQ ID NO: 14). 3. The anti-CCR8 antibody according to embodiment 1, comprising a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 8. 4. The anti-CCR8 antibody according to embodiment 1, comprising a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth as SEQ ID NO: 10. 5. The anti-CCR8 antibody according to embodiment 3, which is defucosylated. 6. A composition comprising a plurality of anti-CCR8 antibodies according to embodiment 1. 7. The composition according to embodiment 6, wherein more than 80% of the anti-CCR8 antibody in the composition is defucosylated. 8. The anti-CCR8 antibody according to embodiment 1, which is IgG. 9. The anti-CCR8 antibody according to embodiment 1, comprising a human heavy chain constant region containing an Fc portion, wherein the Fc portion is of human IgG1, IgG2, IgG3, IgG4 or IgM isotype. 10. The anti-CCR8 antibody according to embodiment 9, comprising a kappa light chain constant region. 11. The anti-CCR8 antibody according to embodiment 4, wherein the lysine at the C-terminus of the heavy chain is cleaved. 12. The anti-CCR8 antibody according to embodiment 1, which is a humanized antibody. 13. A method for treating solid tumors, comprising the step of administering the composition according to embodiment 6 to a patient in need thereof. 14. A polynucleotide comprising a nucleotide sequence encoding an anti-CCR8 antibody, wherein the antibody comprises (i) a VH chain comprising three CDRs and (ii) a VL chain comprising three CDRs, VH CDR#1 is GFIFSNAVMY (SEQ ID NO: 1), VH CDR#2 is RIKTKFNNYATYYADAVKG (SEQ ID NO: 2), VH CDR#3 is GDRNKPFAY (SEQ ID NO: 3), VL CDR#1 is RASTSVITLLH (SEQ ID NO: 4), VL CDR#2 is GASNLES (SEQ ID NO: 5), VL CDR#3 is QQSWNDPYT (SEQ ID NO: 6), the polynucleotide. 15. An expression vector comprising the polynucleotide according to embodiment 14. 16. A eukaryotic host cell transfected with the vector according to embodiment 15. 17. The eukaryotic host cell according to embodiment 16, which is a mammalian host cell. 18. A method for producing an anti-CCR8 antibody, comprising (a) culturing the eukaryotic host cell according to embodiment 15 and (b) recovering the anti-CCR8 antibody. 19. A method for treating solid tumors, comprising the step of administering the composition according to embodiment 6 to a patient in need thereof. 20. The following characteristics: (a) Cross-reactivity with cynomolgus monkey CCR8, (b) Weak ability to block CCL1 / CCR8 interaction, (c) Enhanced ability to induce ADCC, (d) Enhanced binding to Fcγ receptor, and (e) Good immune safety as quantified by cytokine release assay An anti-CCR8 antibody having one or more of the above. 21. The following characteristics: (a) Cross-reactivity with cynomolgus CCR8, (b) Weak ability to block CCL1 / CCR8 interaction, (c) Enhanced ability to induce ADCC, (d) Enhanced binding to Fcγ receptor, and (e) Good immune safety as quantified by cytokine release assay An anti-CCR8 antibody having the above characteristics. 22. The following characteristics: (a) Cross-reactivity with cynomolgus CCR8, (b) Weak ability to block CCL1 / CCR8 interaction, (c) Enhanced ability to induce ADCC An anti-CCR8 antibody having the above characteristics. 23. An anti-CCR8 antibody according to any one of embodiments 19 to 21, comprising (i) a VH chain comprising three CDRs and (ii) a VL chain comprising three CDRs, wherein VH CDR#1 is GFIFSNAVMY (SEQ ID NO: 1), VH CDR#2 is RIKTKFNNYATYYADAVKG (SEQ ID NO: 2), VH CDR#3 is GDRNKPFAY (SEQ ID NO: 3), VL CDR#1 is RASTSVITLLH (SEQ ID NO: 4), VL CDR#2 is GASNLES (SEQ ID NO: 5), VL CDR#3 is QQSWNDPYT (SEQ ID NO: 6). 24. An anti-CCR8 antibody according to any one of embodiments 20 to 22, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. 25. An anti-CCR8 antibody according to any one of embodiments 20 to 22, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. 26. The anti-CCR8 antibody according to any one of embodiments 20 to 22, which is defucosylated. 27. An anti-CCR8 antibody according to any one of embodiments 20 to 22, comprising (i) a VH chain comprising three CDRs and (ii) a VL chain comprising three CDRs, wherein VH CDR#1 is GFIFSNAVMY (SEQ ID NO: 1), VH CDR#2 is RIKTKFNNYATYYADAVKG (SEQ ID NO: 2), VH CDR#3 is GDRNKPFAY (SEQ ID NO: 3), VL CDR#1 is RASTSVITLLH (SEQ ID NO: 4), VL CDR#2 is GASNLES (SEQ ID NO: 5), VL CDR#3 is QQSWNDPYT (SEQ ID NO: 6), and the antibody is defucosylated, the anti-CCR8 antibody. 28. A method for treating solid tumors, comprising administering to a patient in need thereof a combination of the composition according to embodiment 6 and a composition comprising an anti-PD-1 antibody. 29. The method according to embodiment 28, wherein the anti-PD-1 antibody is selected from the group consisting of budigalimab, pembrolizumab, nivolumab, semaprilimab, and dostarlimab. 30. The method according to embodiment 29, wherein the anti-PD-1 antibody is budigalimab. 31. The method according to embodiment 29, wherein the anti-PD-1 antibody is pembrolizumab. 32. A method for treating solid tumors, comprising administering to a patient in need thereof a combination of the composition according to embodiment 6 and a composition comprising an anti-PD-L1 antibody. 33. The method according to embodiment 32, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab. 34. The method according to embodiment 33, wherein the anti-PD-L1 antibody is atezolizumab. 35. The composition according to embodiment 6, wherein about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the anti-CCR8 antibody in the composition is defucosylated. 36. The anti-CCR8 antibody according to any one of Embodiments 2 to 4, which is defucosylated. 37. A composition comprising a plurality of the anti-CCR8 antibodies according to Embodiment 36. 38. The composition according to Embodiment 37, wherein about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the anti-CCR8 antibodies in the composition are defucosylated. 39. An anti-CCR8 antibody comprising two heavy chains each comprising the amino acid sequence set forth as SEQ ID NO: 9 and two light chains each comprising the amino acid sequence set forth as SEQ ID NO: 10, which is defucosylated. 40. A composition comprising a plurality of the anti-CCR8 antibodies according to Embodiment 39. 41. The composition according to Embodiment 40, wherein about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the anti-CCR8 antibodies in the composition are defucosylated.

Claims

1. (i) a VH chain comprising three CDRs; and (ii) a VL chain comprising three CDRs, VH CDR#1 is GFIFSNAVMY (SEQ ID NO:1); VH CDR#2 is RIKTKFNNYATYYADAVKG (SEQ ID NO:2); VH CDR#3 is GDRNKPFAY (SEQ ID NO:3); VL CDR#1 is RASTSVITLLH (SEQ ID NO:4); VL CDR#2 is GASNLES (SEQ ID NO:5); An anti-CCR8 antibody in which VL CDR#3 is QQSWNDPYT (SEQ ID NO:6).

2. 2. The anti-CCR8 antibody of claim 1, comprising a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO:

8.

3. 2. The anti-CCR8 antibody of claim 1, comprising a heavy chain comprising the amino acid sequence set forth as SEQ ID NO:9 and a light chain comprising the amino acid sequence set forth as SEQ ID NO:

10.

4. The anti-CCR8 antibody of claim 3, which is defucosylated.

5. A composition comprising a plurality of anti-CCR8 antibodies according to claim 1.

6. The composition of claim 5, wherein greater than 80% of the anti-CCR8 antibody in the composition is defucosylated.

7. The anti-CCR8 antibody of claim 1 which is an IgG.

8. 8. The anti-CCR8 antibody of claim 7, comprising a human heavy chain constant region comprising an Fc portion, said Fc portion being of human IgG1, IgG2, IgG3, IgG4 or IgM isotype.

9. The anti-CCR8 antibody of claim 8, comprising a kappa light chain constant region.

10. 10. A method of treating a solid tumor comprising administering the antibody of claim 4 to a patient in need thereof.

11. A polynucleotide comprising a nucleotide sequence encoding an anti-CCR8 antibody, the antibody comprising (i) a VH chain comprising three CDRs and (ii) a VL chain comprising three CDRs; VH CDR#1 is GFIFSNAVMY (SEQ ID NO:1); VH CDR#2 is RIKTKFNNYATYYADAVKG (SEQ ID NO:2); VH CDR#3 is GDRNKPFAY (SEQ ID NO:3); VL CDR#1 is RASTSVITLLH (SEQ ID NO:4); VL CDR#2 is GASNLES (SEQ ID NO:5); A polynucleotide in which VL CDR#3 is QQSWNDPYT (SEQ ID NO:6).

12. An expression vector comprising the polynucleotide of claim 11.

13. A eukaryotic host cell transfected with the vector of claim 12.

14. The eukaryotic host cell of claim 13 which is a mammalian host cell.

15. 15. A method for producing an anti-CCR8 antibody, comprising the steps of: (a) culturing the eukaryotic host cell of claim 14; and (b) recovering the anti-CCR8 antibody.

16. 10. A method of treating a solid tumor comprising administering to a patient in need thereof the composition of claim 5.

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