Anti-crr8 antibodies and uses thereof

Abstract

The present invention relates to an isolated antibody or antigen binding fragment thereof, which binds to C-C chemokine receptor type 8 (CCR8), wherein said antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising at least one, preferably at least two, more preferably the following three complementarity determining regions (CDRs): - VH-CDR1 : SEQ ID NO: 10, - VH-CDR2: GINPKX1GVTRYNQKFKG, wherein X1 is any amino acid (SEQ ID NO: 38) and - VH-CDR3: SEQ ID NO: 12, said heavy chain variable region (VH) having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NOs 21 to 26; and further comprising a light chain variable region (VL) comprising at least one, preferably at least two, more preferably the following three complementarity determining regions (CDRs): - VL-CDR1 : SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14 and - VL-CDR3: SEQ ID NO: 15, said light chain variable region (VL) having at least 80% sequence identity to a polypeptide sequence selected from the group of SEQ ID NOs 27 to 31.

Description

Technical Field

[0001] This invention relates to antibodies specific to chemokine (CC motif) receptor 8 (CCR8) and their use in depleting or inhibiting regulatory T cells in tumors. Background Technology

[0002] Chemokine (CC motif) receptor 8 (CCR8) is a G protein-coupled receptor (GPCR) chemokine receptor. In humans, CCR8 ligands are chemokine (CC motif) ligands 1, 16, and 18 (CCL1, CCL16, and CCL18).

[0003] CCR8 is primarily expressed by Treg cells, and sometimes by T-lymphoma or leukemia cells. When an immune response is triggered, cells involved in the immune response (such as macrophages) produce CCL1. CCL1-CCR8 binding allows Tregs to home. This causes activation of the cytoskeleton, enabling Treg cells to migrate along the CCL1 gradient to sites of the immune response, where they negatively regulate the immune response by secreting cytokines such as IL-10.

[0004] In the specific context of the immune response to tumors, Treg cells have been shown to infiltrate tumors and promote tumor progression by suppressing the activity of immune cells, such as cytotoxic T lymphocytes.

[0005] Therefore, antibodies targeting CCR8 can be used to treat cancer, and more generally to treat diseases associated with impaired immune response.

[0006] WO2021194942, WO2021194942, and WO2021163064 recently reported new clues about the use of anti-CCR8 antibodies to treat cancer.

[0007] WO2021194942 discloses an anti-CCR8 antibody that exerts antibody-dependent cell-mediated cytotoxicity (ADCC)-mediated depletion of CCR8-expressing cells (e.g., Treg cells) and its use alone or in combination with a mouse anti-mPD-1 antibody in a mouse model of colon cancer.

[0008] WO2021163064 discloses an anti-CCR8 antibody that may include a mutation and / or modification (e.g., defucosylation) or may be chimeric, which may facilitate Treg depletion and reduce tumor growth in MC38 syngeneic mouse models. WO2021163064 discloses that the anti-CCR8 antibody can be used as a monotherapy or in combination with anticancer agents such as immune checkpoint inhibitors (e.g., PD-1).

[0009] WO2021142002 discloses human or cynomolgus monkey antibodies that specifically bind to the N-terminal extracellular domain of human CCR8 and induce ADCC in 293T cells, and their therapeutic uses.

[0010] Despite the progress made in recent years, the current clinical benefits remain unsatisfactory, and there is still a need to improve this strategy to enhance responses in patients who need it. Therefore, there is an unmet medical need to improve these anticancer therapies.

[0011] Here, the inventors provide a novel antibody targeting CCR8, which has a specific CDR sequence and the ability to inhibit the binding of CCL1 to CCR8 and induce CCR8 internalization. This novel anti-CCR8 antibody will target tumor-infiltrating immune cells, thereby reducing tumor progression and thus treating subjects with cancer. Summary of the Invention

[0012] This invention relates to an isolated antibody or antigen-binding fragment thereof that binds to chemokine (CC motif) receptor 8 (CCR8), wherein the antibody or antigen-binding fragment thereof includes a heavy chain variable region (VH), the heavy chain variable region including at least one, preferably at least two, and more preferably the following three complementarity-determining regions (CDRs): - VH-CDR1: SEQ ID NO: 10, - VH-CDR2: GINPKX1GVTRYNQKFKG, where X1 is any amino acid (SEQ ID NO: 38), and X1 is particularly selected from N or D, and - VH-CDR3: SEQ ID NO: 12, The heavy chain variable region (VH) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 21 to 26; It also includes a light chain variable region (VL), which comprises at least one, preferably at least two, and more preferably the following three complementary determinant regions (CDRs): - VL-CDR1: SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14, and - VL-CDR3: SEQ ID NO: 15 The light chain variable region (VL) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

[0013] In some embodiments, the isolated antibody or its antigen-binding fragment is monoclonal.

[0014] In some embodiments, the isolated antibody or its antigen-binding fragment binds to human CCR8 and rabbit CCR8.

[0015] In some embodiments, the isolated antibody or its antigen-binding fragment recognizes an epitope having or consisting of an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0016] In some implementations, the epitope is post-transcriptionally modified.

[0017] In some embodiments, the epitope is sulfonated.

[0018] In some embodiments, the epitope is sulfonated at amino acid position 7 and / or 9, wherein the position number refers to SEQ ID NO: 1 or SEQ ID NO: 2.

[0019] In some embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a heavy chain variable region (VH), said heavy chain variable region comprising the following three complementarity-determining regions (CDRs): - VH-CDR1: SEQ ID NO: 10, - VH-CDR2: SEQ ID NO: 11, and / or - VH-CDR3: SEQ ID NO: 12.

[0020] In some embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a light chain variable region (VL), which comprises three complementarity-determining regions (CDRs): - VL-CDR1: SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14, and / or - VL-CDR3: SEQ ID NO: 15.

[0021] In some embodiments, the antibody or antigen-binding fragment according to the invention includes a heavy chain variable region (VH), said heavy chain variable region comprising the following three complementarity-determining regions (CDRs): - VH-CDR1: SEQ ID NO: 10, - VH-CDR2: SEQ ID NO: 38, especially SEQ ID NO: 11, and - VH-CDR3: SEQ ID NO: 12, The heavy chain variable region (VH) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 21 to 26; It also includes a light chain variable region (VL), which comprises the following three complementary determination regions (CDRs): - VL-CDR1: SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14, and / or - VL-CDR3: SEQ ID NO: 15; The light chain variable region (VL) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

[0022] In some embodiments, the isolated antibody against CCR8 or its antigen-binding fragment inhibits the binding of chemokine (CC motif) ligand 1 (CCL1) and / or chemokine (CC motif) ligand 18 (CCL18) to CCR8.

[0023] The present invention also relates to a nucleic acid encoding an isolated antibody or an antigen-binding fragment thereof according to the present invention.

[0024] Therefore, the present invention also relates to a nucleic acid encoding an isolated antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to chemokine (CC motif) receptor 8 (CCR8), wherein said antibody or antigen-binding fragment thereof includes a heavy chain variable region (VH), said heavy chain variable region including the following three complementarity-determining regions (CDRs): - VH-CDR1: SEQ ID NO: 10, - VH-CDR2: GINPKX1GVTRYNQKFKG, where X1 is any amino acid (SEQ ID NO: 38), and X1 is particularly selected from N or D, and - VH-CDR3: SEQ ID NO: 12, The heavy chain variable region (VH) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 21 to 26; It also includes a light chain variable region (VL), which comprises the following three complementary determination regions (CDRs): - VL-CDR1: SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14, and - VL-CDR3: SEQ ID NO: 15 The light chain variable region (VL) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

[0025] The present invention also relates to a vector containing nucleic acids according to the present invention.

[0026] Therefore, the present invention also relates to a vector containing nucleic acid, wherein the nucleic acid encodes an isolated antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof binding to chemokine (CC motif) receptor 8 (CCR8), wherein the antibody or the antigen-binding fragment thereof includes a heavy chain variable region (VH), the heavy chain variable region including the following three complementarity-determining regions (CDRs): - VH-CDR1: SEQ ID NO: 10, - VH-CDR2: GINPKX1GVTRYNQKFKG, where X1 is any amino acid (SEQ ID NO: 38), and X1 is particularly selected from N or D, and - VH-CDR3: SEQ ID NO: 12, The heavy chain variable region (VH) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 21 to 26; It also includes a light chain variable region (VL), which comprises the following three complementary determination regions (CDRs): - VL-CDR1: SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14, and - VL-CDR3: SEQ ID NO: 15 The light chain variable region (VL) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

[0027] The present invention also relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid or a carrier, and at least one pharmaceutically acceptable excipient.

[0028] Therefore, the present invention also relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to chemokine (CC motif) receptor 8 (CCR8), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), the heavy chain variable region comprising the following three complementarity-determining regions (CDRs): - VH-CDR1: SEQ ID NO: 10, - VH-CDR2: GINPKX1GVTRYNQKFKG, where X1 is any amino acid (SEQ ID NO: 38), and X1 is particularly selected from N or D, and - VH-CDR3: SEQ ID NO: 12, The heavy chain variable region (VH) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 21 to 26; It also includes a light chain variable region (VL), which comprises the following three complementary determination regions (CDRs): - VL-CDR1: SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14, and - VL-CDR3: SEQ ID NO: 15 The light chain variable region (VL) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

[0029] In some embodiments, the pharmaceutical composition further includes at least one additional therapeutic agent.

[0030] In some embodiments, the additional therapeutic agent is a programmed cell death protein 1 (PD-1) inhibitor.

[0031] The present invention also relates to the use of isolated antibodies or antigen-binding fragments thereof, nucleic acids, carriers or pharmaceutical compositions according to the present invention as pharmaceuticals.

[0032] The present invention also relates to isolated antibodies or antigen-binding fragments thereof, nucleic acids, carriers or pharmaceutical compositions according to the present invention for use in treating and / or preventing and / or reducing the likelihood of CCR8-related disease in subjects in need.

[0033] In some implementations, the disease is cancer.

[0034] In some implementations, the cancer is caused by cells expressing CCR8, regardless of whether the cells are cancerous or non-cancer cells.

[0035] The present invention also relates to a method for treating and / or preventing diseases, preferably CCR8-related diseases, comprising administering to a subject in need a therapeutically effective amount of an isolated antibody or an antigen-binding fragment thereof according to the present invention.

[0036] The present invention also relates to the use of isolated antibodies or antigen-binding fragments thereof, nucleic acids, carriers or pharmaceutical compositions according to the present invention in the preparation of medicaments, preferably for the treatment and / or prevention and / or reduction of the likelihood of the occurrence of CCR8-related diseases.

[0037] definition

[0038] In this invention, the following terms have the following meanings: When the term "about" precedes a number, it covers plus or minus 10% or less of the numerical value. It should be understood that the value referred to by the term "about" is itself specifically and preferably disclosed.

[0039] The terms “affinity” and “cohesion” are well known in the art and are used to define the strength of antibody-antigen complexes. Affinity measures the strength of the interaction between an epitope and an antigen-binding site on an antibody. It can be expressed by an affinity constant KA or a dissociation constant KD. Cohesion (or functional affinity) gives a measure of the overall strength of an antibody-antigen complex. It can depend on various parameters, particularly including (ii) the affinity of the antibody or its antigen-binding fragment for the epitope, (iii) the valence of both the antibody and the antigen, and (iii) the structural arrangement of the interacting parts.

[0040] The terms “antibody” and “immunoglobulin” are used interchangeably and refer to a protein having a combination of two heavy chains and two light chains, regardless of whether it possesses any associated specific immunoreactivity. An “antibody” is an assembly that has significant, known specific immunoreactive activity against a target antigen, such as human CCR8. The term “anti-hCCR8 antibody” is used herein to refer to an antibody that exhibits immunospecificity against the human CCR8 protein. As explained elsewhere herein, “specificity” against human CCR8 does not exclude cross-reactivity with species homologs of hCCR8, such as with simian CCR8. Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent linkages. The basic immunoglobulin structures in vertebrate systems are relatively well known. The general term “immunoglobulin” includes five distinct classes of antibodies that can be distinguished biochemically. Although the following discussion will generally refer to IgG class immunoglobulin molecules, all five classes of antibodies are within the scope of this invention. Regarding IgG, immunoglobulins consist of two identical light polypeptide chains with a molecular weight of approximately 23 kDa and two identical heavy chains with a molecular weight of approximately 53-70 kDa. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chain beginning at the opening of the "Y" and continuing through a variable region to surround the heavy chain. The light chains of antibodies are classified as κ or λ. Each heavy chain class can be bonded to either κ or λ light chains. Generally, the light and heavy chains are covalently bonded to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tail" regions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will understand that the heavy chains are classified as γ, μ, α, δ, or ε, with some subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses or "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) have been well characterized and are known to confer functional specialization. In view of this disclosure, modifications of each of these classes and isotypes are readily identifiable to those skilled in the art and are therefore within the scope of this invention. As described above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to epitopes on antigens. That is, the combination of the light chain variable domain (VL domain) and the heavy chain variable domain (VH domain) of the antibody forms the variable region, which defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites present at the ends of each arm of the "Y".

[0041] As used herein, the term "antigen-binding fragment" refers to a portion or region of an antibody according to the invention, comprising fewer amino acid residues than the intact antibody. An "antigen-binding fragment" binds to an antigen and / or competes with the intact antibody from which it is derived for antigen binding (e.g., specific binding to human CCR8). Antibody antigen-binding fragments encompass, but are not limited to, single-chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, biantibodies, triantibodies, and tetraantibodies.

[0042] The term "effective T cells" refers to a group of cells that includes several T cell types (such as CD4+ and CD8+ T cells). These include helper T cells (Th cells) that assist other white blood cells in the immune process, including B cells maturing into plasma cells and memory B cells, as well as cytotoxic T cells (Tc cells, CTLs, killer T cells, cytotoxic T cells) that destroy virus-infected cells and tumor cells, and are also associated with transplant rejection.

[0043] The term "epitope" refers to a specific arrangement of amino acids located on one or more proteins to which an antibody, its antigen-binding fragment, or an antibody mimic binds. Epitopes typically consist of chemically active surface molecular groups such as amino acids or sugar side chains and have specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be linear (or sequential) or conformational, i.e., two or more amino acid sequences involving different regions of an antigen, and these sequences do not necessarily have to be sequential.

[0044] The terms “Fc domain”, “Fc moiety”, and “Fc region” refer to the C-terminal segment of an antibody heavy chain, such as about amino acid (aa) 230 to about aa 450 of the human γ heavy chain or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε, and μ for human antibodies), or its naturally occurring allotypes.

[0045] As used herein, the term "Fv" refers to the smallest antibody fragment that includes both complete antigen recognition and binding sites. This fragment consists of a dimer of one HCVR and one LCVR bound together in a tight, non-covalent bond. Six hypervariable rings (three rings each from the heavy and light chains) are generated from the folding of these two domains, which facilitate antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain can recognize and bind antigens, albeit with a lower affinity than the entire binding site.

[0046] The term "heavy chain region" includes an amino acid sequence derived from a constant domain of the immunoglobulin heavy chain. Proteins including a heavy chain region include at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge) domain, a CH2 domain, and a CH3 domain, or variants or fragments thereof. In one embodiment, an antibody or antigen-binding fragment according to the invention may include an Fc region of the immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, an antibody or antigen-binding fragment according to the invention lacks at least one region of a constant domain (e.g., all or part of the CH2 domain). In some embodiments, at least one, preferably all, of the constant domains are derived from the human immunoglobulin heavy chain. For example, in a preferred embodiment, the heavy chain region includes a fully human hinge domain. In other preferred embodiments, the heavy chain region includes a fully human Fc region (e.g., a sequence of hinge, CH2, and CH3 domains from human immunoglobulins). In some embodiments, the constituent constant domains of the heavy chain region are derived from different immunoglobulin molecules. For example, the heavy chain region of a protein may include a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domain is a chimeric domain comprising regions of different immunoglobulin molecules. For example, the hinge may include a first region from an IgG1 molecule and a second region from an IgG3 or IgG4 molecule. As will be understood by those skilled in the art as described above, the constant domains of the heavy chain region can be modified such that their amino acid sequences differ from those of naturally occurring (wild-type) immunoglobulin molecules. That is, the antibody or antigen-binding fragment thereof according to the invention may include alterations or modifications to one or more heavy chain constant domains (CH1, hinge, CH2, or CH3) and / or to the light chain constant domain (CL). Exemplary modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0047] The term "hinge region" refers to the region in a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding domains to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., 1998. J Immunol. 161(8):4083-90).

[0048] The term "identity" or "identical" when used to refer to the relationship between two or more amino acid sequences or two or more nucleic acid sequences refers to the degree of sequence correlation between amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences after gap alignment (if any) has been processed by a specific mathematical model or computer program (i.e., an "algorithm").

[0049] The identity of related amino acid sequences or nucleic acid sequences can be readily calculated using known methods. Such methods include, but are not limited to, those described in the following literature: Lesk AM (1988). Computational molecularbiology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith DW (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin AM & Griffin HG (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove ortrivial pursuit. San Diego, CA: Academic press; Gribskov MR & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5):1073-82. The preferred method for determining identity is designed to give the maximum match between the tested sequences. The method for determining identity is described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG package, including GAP (GeneticsComputer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10).The BLASTX procedure is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity.

[0050] As used herein, the phrase “characterized by having … amino acids substituted with different amino acids” with respect to a given sequence refers to conserved amino acid modifications occurring in the sequence.

[0051] As used herein, the term "conservative amino acid modification" refers to a modification that does not significantly affect or alter the binding characteristics of an antibody or its antigen-binding fragment containing that amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into antibodies or their antigen-binding fragments using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are generally those in which amino acid residues are replaced by amino acid residues of a side chain having similar physicochemical properties. The specified variable region and CDR sequence may include one, two, three, or more amino acid insertions, deletions, and / or substitutions. When substitution is performed, the substitution is preferably a conserved modification. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR and / or variable region of an antibody or antigen-binding fragment according to the invention can be replaced by other amino acid residues from the same side chain family, and the retained function (i.e., the properties described herein, such as binding to CCR8) of the altered antibody can be tested using the assays described herein. In another embodiment, the amino acid string in the CDR and / or variable region of an antibody or antigen-binding fragment according to the invention can be replaced by a string with a similar structure but different in the order and / or composition of the side chain family members.

[0052] The term "immunotoxin" refers to a bifunctional molecule that comprises a targeting portion (ligand) for delivery and a toxic portion (toxin) for cytotoxicity. Immunotoxins can be used to kill cells that express the ligand receptor.

[0053] The term "mammal" refers to any mammal, including humans, domesticated and farm animals, as well as zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a human.

[0054] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, a benefit of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies or antigen-binding fragments thereof according to the invention can be prepared by the hybridoma method first described by Kohler et al., 1975. Nature. 256(5517):495-7, or can be prepared in bacterial, eukaryotic, or plant cells using recombinant DNA methods (Patent US4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al., 1991. Nature. 352(6336):624-8 and Marks et al., 1991. J Mol Biol. 222(3):581-97.

[0055] "Nanobodies" are well known in the art and refer to antibody-derived therapeutic proteins containing the unique structural and functional properties of naturally occurring heavy chain antibodies (Muyldermans, 2013. Annu Rev Biochem. 82:775-97). These heavy chain antibodies may include a single variable domain (VHH) and two constant domains (CH2 and CH3).

[0056] The term "nucleic acid" or "polynucleotide" refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Nucleic acid" or "polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA of a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA, which may be single-stranded, or more generally double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, "nucleic acid" or "polynucleotide" refers to a triple-stranded region comprising RNA or DNA, or both RNA and DNA. The term "nucleic acid" or "polynucleotide" also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose backbone is modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and rare bases such as inosine. DNA and RNA have undergone various modifications; therefore, "nucleic acid" or "polynucleotide" encompasses chemical, enzymatic, or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA specific to viruses and cells. "Polynucleotide" also includes relatively short polynucleotides, often referred to as oligonucleotides.

[0057] The terms “prevention,” “taking preventative measures,” and “prevention” refer to preventative and preventative measures aimed at reducing the likelihood that a subject will develop a pathological condition or symptom within a given time period. This reduction may be reflected, for example, in the delayed onset of at least one symptom of the pathological condition or symptom in the subject.

[0058] The terms "regulatory T cells" or "Treg cells" refer to a specialized type of T cell, particularly CD4+ T cells, that can suppress the responses of other T cells. Treg cells play a crucial role in inducing and maintaining peripheral self-tolerance to antigens, including those expressed by tumors.

[0059] The term "single-chain Fv," also abbreviated as "sFv" or "scFv," refers to an antibody fragment comprising VH and VL antibody domains linked together as a single amino acid chain. Preferably, the scFv amino acid sequence also includes a peptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding (Plückthun, 1994. Antibodies from Escherichia coli. In Rosenberg & Moore (Eds.), The pharmacology of monoclonal antibodies. Handbook of Experimental Pharmacology, 113:269-315. Springer: Berlin, Heidelberg).

[0060] The term "subject" refers to a mammal, preferably a human. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, who is awaiting or receiving medical care, or has been / is / will be the subject of a medical procedure, or is being monitored for the development of a disease. The term "mammal" herein refers to any mammal, including humans, domesticated and farm animals, as well as zoo, sporting, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.

[0061] The term “therapeutic effective dose” refers to a level or amount of antibody as described herein, intended to, without causing significant negative or adverse side effects on the target, (1) delay or prevent the onset of a disease, disorder, or condition; (2) slow or stop the progression, aggravation, or worsening of one or more symptoms of a disease, disorder, or condition; (3) improve the symptoms of a disease, disorder, or condition; (4) reduce the severity or incidence of a disease, disorder, or condition; or (5) cure a disease, disorder, or condition. Therapeutic effective doses may be administered before the onset of a disease, disorder, or condition for a preventative or preventative effect. Alternatively or additionally, therapeutic effective doses may be administered after the onset of a disease, disorder, or condition for a therapeutic effect.

[0062] The terms “treatment,” “management,” or “mitigation” refer to both therapeutic treatment and preventative or preventive measures; wherein the aim is to prevent or mitigate (mitigate) a targeted pathological condition or disorder. Those requiring treatment include those already suffering from the disorder, those susceptible to it, or those with a disorder to be prevented. A subject or mammal is considered successfully “treated” for cancer or infection if, after receiving a therapeutic dose of antibody according to the method of the invention, the patient exhibits an observable and / or measurable reduction or absence of one or more of the following: a reduction in the number of cancer cells (or tumor size) or pathogenic cells; a decrease in the percentage of cancerous or pathogenic cells in the total cell count; and / or a degree of relief of one or more symptoms associated with the specific disease or condition; a reduction in morbidity and mortality; and an improvement in quality of life. The parameters described above used to assess successful treatment and disease improvement can be readily measured using routine procedures familiar to physicians.

[0063] As used herein, the term “variable” refers to the fact that certain regions of the variable domains VH and VL are sequence-widely different between antibodies and contribute to the binding and specificity of each particular antibody to its target antigen. However, variability is not uniformly distributed within the variable domains of the antibody. It is concentrated in three segments in each of the VL and VH domains, referred to as “hypervariant loops,” which form part of the antigen-binding site. The first, second, and third hypervariant loops of the Vλ light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), and can be defined as comprising residues 24–33 (L1(λ), ​​consisting of 9, 10, or 11 amino acid residues), 49–53 (L2(λ), consisting of 3 residues), and 90–96 (L3(λ), consisting of 6 residues) in the VL domain, respectively (Morea et al., 2000. Methods. 20(3):267–79). The first, second, and third hypervariable rings of the Vκ light chain domain are referred to as L1(κ), L2(κ), and L3(κ) in this paper, and can be defined as including residues 25-33 (L1(κ, consisting of 6, 7, 8, 11, 12, or 13 residues), 49-53 (L2(κ, consisting of 3 residues), and 90-97 (L3(κ, consisting of 6 residues)) in the VL domain, respectively (Morea et al., 2000. Methods. 20(3):267-79). The first, second, and third hypervariable rings of the VH domain are referred to herein as H1, H2, and H3, and can be defined as comprising residues 25-33 (H1, consisting of 7, 8, or 9 residues), 52-56 (H2, consisting of 3 or 4 residues), and 91-105 (H3, highly variable in length) within the VH domain, respectively (Morea et al., 2000. Methods. 20(3):267-79). Unless otherwise stated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable rings of the VL domain, respectively, and encompass hypervariable rings obtained from both Vκ and Vλ isoforms. The terms H1, H2, and H3 refer to the first, second, and third hypervariable rings of the VH domain, respectively, and encompass hypervariable rings obtained from any known heavy chain isoform, including γ, μ, α, δ, or ε.

[0064] As used herein, “isolated antibody” is intended to refer to an antibody that is substantially free of other proteins or antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CCR8 is substantially free of proteins or antibodies that specifically bind to antigens other than CCR8). However, isolated antibodies that specifically bind to CCR8 may be cross-reactive with other antigens, such as CCR8 molecules from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals, particularly those that could interfere with the diagnostic or therapeutic use of the protein or antibody, including but not limited to enzymes, hormones, and other protein- or non-protein-like components.

[0065] If an antibody or its antigen-binding fragment reacts with the antigen (e.g., CCR8) at a detectable level, it is described as "specific," "immune-specific," or "specifically binds" to the antigen, preferably having a value greater than or equal to about 10. 6 M -1 Preferably greater than or equal to about 10 7 M -1 10 8 M -1 5×10 8 M -1 10 9 M -1 5×10 9 M -1 Or a higher affinity constant (KA). The affinity of an antibody or its antigen-binding fragment for its homologous antigen is also typically expressed as an equilibrium dissociation constant (KD). If an antibody or its antigen-binding fragment reacts with the antigen (e.g., CCR8) at a detectable level, it is described as “immune-specific,” “specific,” or “specifically binds” to the antigen, preferably having an affinity of less than or equal to 10. -6 M, preferably less than or equal to 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M or smaller KD. The affinity of an antibody or its antigen-binding fragment can be readily determined using conventional techniques, such as those described in Scatchard, 1949. Ann NYAcad Sci. 51:660-672. The binding properties of an antibody or its antigen-binding fragment to an antigen, cell, or tissue can generally be determined and evaluated using immunoassay methods, including, for example, ELISA, immunofluorescence-based assays (e.g., immunohistochemistry (IHC) and / or fluorescence-activated cell sorting (FACS)), or by surface plasmon resonance (SPR, e.g., using BIAcore®).

[0066] As used herein, a "chimeric antibody" refers to an antibody or antigen-binding fragment thereof comprising a first amino acid sequence linked to a second amino acid sequence, wherein the first and second amino acid sequences are not naturally linked in nature. The amino acid sequences may be normally present in individual proteins, which are combined in a fusion protein, or they may be normally present in the same protein but arranged in a new arrangement in the fusion protein. Chimeric proteins can be produced, for example, by chemical synthesis, or by creating and translating polynucleotides in which peptide regions are encoded in a desired relationship. The term "chimeric antibody" herein encompasses antibodies and their antigen-binding fragments in which a constant region or a portion thereof is altered, replaced, or exchanged such that a variable region is linked to a constant region of a different or altered class, effector function, and / or species, or to a completely different molecule that confers novel properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or the variable region or a portion thereof is altered, replaced, or exchanged by a variable region or a portion thereof with different or altered antigen specificity; or replaced by a corresponding sequence from another species or from another antibody class or subclass.

[0067] As used herein, a “humanized antibody” refers to a chimeric antibody or antigen-binding fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. This includes antibodies produced by non-human cells that have been modified to more closely resemble variable and constant regions of antibodies produced by human cells, for example, by altering the amino acid sequence of the non-human antibody to incorporate amino acids found in human immunoglobulin sequences. Humanized antibodies or antigen-binding fragments thereof according to the invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). “Humanized antibodies” retain antigen specificity similar to the original antibody. However, using certain humanization methods, the affinity and / or binding specificity of the antibody can be increased.

[0068] Regarding CCR8-expressing cells or Tregs, the term "depletion" or "performing depletion" refers to killing, eliminating, lysing, or inducing such killing, elimination, or lysing to negatively affect the number of CCR8-expressing cells present in a sample or subject. In one embodiment, the antibody or its antigen-binding fragment according to the invention allows targeting, blocking proliferation, and / or depletion of CCR8-expressing cells or Tregs. In one embodiment, the depletion is performed via ADCC.

[0069] As used herein, the term “CCR8-related disease” encompasses diseases in which CCR8 is expressed or overexpressed by at least one cell type, and / or in which CCR8-expressing cells are excessively enriched and / or abnormally localized. Non-limiting examples of CCR8-related diseases include cancers, particularly CCR8-expressing T-cell lymphomas or leukemias, or cancers in which CCR8-expressing cells have infiltrated and promoted cancer development; and diseases associated with or related to dysfunction of the immune response.

[0070] "Therapeutic effective dose" refers to a certain level or amount of the isolated antibody or its antigen-binding fragment, nucleic acid, carrier, pharmaceutical composition or drug according to the present invention, which is depleted of CCR8-expressing T cells in infiltrating tumors in individuals requiring treatment. reg Therapeutic effective dose is necessary and sufficient for the cells without causing significant negative or adverse side effects on the individual. In particular, the term "therapeutic effective dose" also refers to a level or amount of the isolated antibody or its antigen-binding fragment, nucleic acid, carrier, pharmaceutical composition or drug according to the invention, which is necessary and sufficient to slow or stop the progression, aggravation or worsening (tumor progression) of one or more symptoms of cancer, particularly solid cancer; or to alleviate the symptoms of cancer, particularly solid cancer; or to cure cancer, particularly solid cancer, without causing significant negative or adverse side effects on the individual.

[0071] As used herein, the term "adjuvant" refers to a compound or combination of compounds that enhances a therapy, such as a cancer therapy. Adjuvants can increase an effective immune response against low-immunogenic or non-immunogenic tumor cells. In one embodiment, an adjuvant is used in conjunction with a known cancer therapeutic agent to treat cancer and thus enhance the immune response against cancer cells. For example, adjuvants can enhance the immune response during cancer therapy, reduce T cell exhaustion (without reducing T cell activation), increase T cell survival, enhance NK cell cytotoxicity, reduce tumor growth and / or tumor size, and / or increase survival, treat or prevent cancer metastasis. In one embodiment, the enhancement of cancer therapy in the presence of an adjuvant is defined by comparison with cancer therapy administered alone.

[0072] As used herein, "immune response" refers to the response of cells of the immune system, such as B cells, T cells (CD4 or CD8), regulatory T cells, antigen-presenting cells, dendritic cells, monocytes, macrophages, NKT cells, NK cells, basophils, eosinophils, or neutrophils, to stress. In some embodiments of any aspect, the response is specific to a particular antigen ("antigen-specific response") and refers to the response of CD4 T cells, CD8 T cells, or B cells through their antigen-specific receptors. In some embodiments of any aspect, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such cellular responses may include, for example, cytotoxicity, proliferation, cytokine or chemokine production, transport, or phagocytosis, and may depend on the nature of the immune cells undergoing the response.

[0073] As used herein, the terms “radiotherapy” or “radiotherapy” refer to the medical use of ionizing radiation, typically as part of cancer treatment to control or kill malignant cells or normal cells infiltrating a tumor. Ionizing radiation is radiation composed of particles that individually carry sufficient kinetic energy to release electrons from atoms or molecules, thereby ionizing them. Sources of ionizing radiation can include external radiation sources such as X-rays, gamma rays, beta rays, neutral or charged particle beams, Auger electron sources, internal radiation sources (brachytherapy or sealed-source radiotherapy), and radioisotope sources (whole-body radioisotope therapy or unsealed-source radiotherapy). Unit radiation doses are typically measured in grays (Gy). Antibodies or their antigen-binding fragments may be used in combination with radiotherapy. Radiotherapy may include the administration of radiation or radiopharmaceuticals to a patient.

[0074] As used herein, the term "radiosensitizer," such as "radiosensitizing compound," can refer to any compound / agent that readily enhances the sensitivity of a given tissue or cellular material (e.g., tumor cells) to radiotherapy. A radiosensitizing compound can be used in combination with any suitable ionizing radiation source that provides electrons capable of reacting with the compound.

[0075] As used herein, the terms "detectable portion" or "detectable label" can refer to any portion or label that is readily detectable as an immunoconjugate; in a non-exhaustive manner, this includes radioactive isotopes, fluorescent labels, chemiluminescent labels, enzyme labels, bioluminescent labels, or colloidal gold. Detectable labels can be radioactive isotopes that can be detected by autoradiography or by any imaging technique such as PET scan. Isotopes particularly useful for the purposes of this invention are... 3 H, 125 I, 131 I, 35 S and 14C. Immunoconjugates can also be labeled with fluorescent compounds. The presence of a fluorescently labeled antibody or its antigen-binding fragment can be determined by exposing the immunoconjugate to light of an appropriate wavelength and detecting the resulting fluorescence. Fluorescently labeled compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescein. Alternatively, immunoconjugates can be detectably labeled by conjugating an antibody to a chemiluminescent compound. The presence of a chemiluminescently labeled immunoconjugate can be determined by detecting the presence of light emission during the chemical reaction. Examples of chemiluminescently labeled compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates. Similarly, bioluminescent compounds can be used to label immunoconjugates. Bioluminescence is a type of chemiluminescence found in biological systems where catalytic proteins enhance the efficiency of the chemiluminescent reaction. The presence of bioluminescent proteins is determined by detecting the presence of light emission. Bioluminescent compounds that can be used for labeling include luciferin, luciferase, and jellyfish luminescent proteins. Alternatively, immunoconjugates can be detectably labeled by linking an antibody or its antigen-binding fragment to an enzyme. When the conjugate is incubated in the presence of a suitable substrate, the enzyme portion reacts with the substrate to produce a chemical moiety, which can be detected, for example, by spectrophotometry, fluorescence, or visual means. Examples of enzymes that can be used for the detectable labeling of multispecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

[0076] As used herein, the term "biological sample" encompasses a variety of sample types, such as those obtained from a subject and applicable to diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. For example, biological samples include cells obtained from tissue samples collected from individuals suspected of having a disease associated with CCR8 expression. Such biological samples can encompass clinical samples, cultured cells, cell supernatants, cell lysates, serum, plasma, biofluids, and tissue samples.

[0077] Within the scope of this invention, the expression "at least one nucleic acid" is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleic acid molecules. Detailed Implementation

[0078] This invention relates to an isolated antibody or antigen-binding fragment thereof that binds to chemokine (CC motif) receptor 8 (CCR8), wherein the antibody or antigen-binding fragment thereof includes a heavy chain variable region (VH), the heavy chain variable region including at least one, preferably at least two, and more preferably the following three complementarity-determining regions (CDRs): - VH-CDR1: SEQ ID NO: 10, - VH-CDR2: GINPKX1GVTRYNQKFKG, where X1 is any amino acid (SEQ ID NO: 38), and X1 is particularly selected from N or D, and - VH-CDR3: SEQ ID NO: 12, The heavy chain variable region (VH) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 21 to 26; It also includes a light chain variable region (VL), which comprises at least one, preferably at least two, and more preferably the following three complementary determinant regions (CDRs): - VL-CDR1: SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14, and - VL-CDR3: SEQ ID NO: 15 The light chain variable region (VL) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

[0079] Advantageously, the antibody and its antigen-binding fragment according to the invention are: (i) specific to both the sulfonated and unsulfonated forms of the N-terminal region of CCR8; (ii) sufficient to inhibit CCL1 binding; and (iii) sufficient to induce internalization of the antibody-GPCR complex and participate in the desensitization mechanism.

[0080] Advantageously, the antibodies and their antigen-binding fragments according to the invention are therefore particularly suitable for both therapeutic applications and diagnostic / imaging applications; including, non-exhaustively, radiotherapy and radiographic imaging.

[0081] In some embodiments, the isolated antibody or its antigen-binding fragment binds to CCR8 from any animal species. In some embodiments, the isolated antibody or its antigen-binding fragment binds to CCR8 from any mammal. In some embodiments, the isolated antibody or its antigen-binding fragment binds to both human and rabbit CCR8. In some embodiments, the isolated antibody or its antigen-binding fragment binds to human CCR8, wherein the amino acid sequence of human CCR8 corresponds to SEQ ID NO: 3. In some embodiments, the isolated antibody or its antigen-binding fragment binds to rabbit CCR8, wherein the amino acid sequence of rabbit CCR8 corresponds to SEQ ID NO: 18. In some embodiments, the isolated antibody or its antigen-binding fragment binds to mouse CCR8, wherein the amino acid sequence of mouse CCR8 corresponds to SEQ ID NO: 19. In some embodiments, the isolated antibody or its antigen-binding fragment binds to rat CCR8, wherein the amino acid sequence of rat CCR8 corresponds to SEQ ID NO: 20.

[0082] In some embodiments, the isolated antibody or its antigen-binding fragment recognizes an epitope having at least 75% sequence identity with the amino acid sequences SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0083] Within the scope of this invention, at least 75% sequence identity refers to 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9, 99.99, 99.999, or 100% sequence identity. Within the scope of this invention, at least 80% sequence identity refers to 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9, 99.99, 99.999, or 100% sequence identity.

[0084] In some embodiments, the isolated antibody or its antigen-binding fragment recognizes an epitope having the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0085] In some embodiments, the isolated antibody or its antigen-binding fragment recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0086] In some embodiments, the isolated antibody or its antigen-binding fragment recognizes an epitope having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated antibody or its antigen-binding fragment recognizes an epitope having the amino acid sequence of SEQ ID NO: 2.

[0087] In some embodiments, the isolated antibody or its antigen-binding fragment recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated antibody or its antigen-binding fragment recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 2.

[0088] In some embodiments, the epitope is post-translational modified. In some embodiments, the epitope is sulfonated.

[0089] As used herein, the term "sulfonated epitope" refers to the addition of at least one sulfonic acid molecule (SO3H) to at least one portion (e.g., an amine or thiol) of the side chain of at least one amino acid (e.g., tyrosine or cysteine) within the amino acid sequence of the epitope.

[0090] In some embodiments, the epitope is sulfonated at amino acid position 7 and / or 9, wherein the position number refers to SEQ ID NO: 1 or SEQ ID NO: 2.

[0091] In some embodiments, the amino acid sequence of the sulfonated epitope is VTTVTDY(SO3H)YY(SO3H)PDIFSSPC or VTAVTDY(SO3H)YY(SO3H)PDIFSSPC.

[0092] In the following text, unless otherwise expressly stated, CDR numbers and definitions are based on the IMGT® numbering system.

[0093] In some embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a heavy chain variable region comprising three complementarity-determining regions (CDRs): SEQ ID NO: 10, SEQ ID NO: 38 and SEQ ID NO: 12.

[0094] In some embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a heavy chain variable region, said heavy chain variable region including the following three complementarity-determining regions (CDRs): SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0095] In some embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a heavy chain variable region comprising three complementarity-determining regions (CDRs): SEQ ID NO: 10, SEQ ID NO: 39 and SEQ ID NO: 12.

[0096] In some embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a heavy chain variable region, which includes three complementarity-determining regions (CDRs): SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.

[0097] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a heavy chain variable region, said heavy chain variable region including VH-CDR1 of sequence SEQ ID NO 10 or SEQ ID NO 32.

[0098] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a heavy chain variable region, said heavy chain variable region including VH-CDR2 of sequence SEQ ID NO 11 or SEQ ID NO 33.

[0099] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the present invention includes a heavy chain variable region comprising VH-CDR2 of sequence SEQ ID NO 38 or SEQ ID NO 39, wherein X1 is selected from N or D, and X2 is selected from I or M. In some specific embodiments, VH-CDR2 is sequence SEQ ID NO 38 or SEQ ID NO 39, wherein X1 is N and X2 is I. In some specific embodiments, VH-CDR2 is sequence SEQ ID NO 38 or SEQ ID NO 39, wherein X1 is N and X2 is M. In some specific embodiments, VH-CDR2 is sequence SEQ ID NO 38 or SEQ ID NO 39, wherein X1 is D and X2 is I. In some specific embodiments, VH-CDR2 is sequence SEQ ID NO 38 or SEQ ID NO 39, wherein X1 is D and X2 is M.

[0100] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a heavy chain variable region, said heavy chain variable region including VH-CDR3 of sequence SEQ ID NO 12 or SEQ ID NO 34.

[0101] In some embodiments, the antibody or antigen-binding fragment according to the invention includes a light chain variable region, said light chain variable region including the following three complementarity-determining regions (CDRs): SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

[0102] In some embodiments, the antibody or antigen-binding fragment according to the invention includes a light chain variable region comprising three complementarity-determining regions (CDRs): SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 15.

[0103] In some embodiments, the antibody or antigen-binding fragment of the present invention includes a light chain variable region comprising three complementarity-determining regions (CDRs): SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 37.

[0104] In some embodiments, the antibody or antigen-binding fragment according to the invention includes a light chain variable region comprising three complementarity-determining regions (CDRs): SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 37.

[0105] In some embodiments, the antibody or antigen-binding fragment according to the invention includes a light chain variable region comprising three complementarity-determining regions (CDRs): SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37.

[0106] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a light chain variable region, said light chain variable region including VL-CDR1 of sequence SEQ ID NO 40; wherein X1 is selected from L or Y.

[0107] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a light chain variable region, said light chain variable region including VL-CDR1 of sequence SEQ ID NO 13.

[0108] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a light chain variable region, said light chain variable region including VL-CDR1 of sequence SEQ ID NO 35.

[0109] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a light chain variable region, said light chain variable region including VL-CDR1 of sequence SEQ ID NO 40.

[0110] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a light chain variable region, said light chain variable region including VL-CDR2 of sequence SEQ ID NO 14 or SEQ ID NO 36.

[0111] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a light chain variable region, said light chain variable region including VL-CDR2 of sequence SEQ ID NO 41; wherein X1 is selected from R or L.

[0112] In some specific embodiments, the isolated antibody or antigen-binding fragment thereof according to the present invention includes a light chain variable region comprising VL-CDR1 of sequence SEQ ID NO 40, wherein X1 is selected from L or Y, and VL-CDR2 of sequence SEQ ID NO 41, wherein X1 is selected from R or L. In some specific embodiments, the isolated antibody or antigen-binding fragment thereof according to the present invention includes a light chain variable region comprising VL-CDR1 of sequence SEQ ID NO 40, wherein X1 is selected from L, and VL-CDR2 of sequence SEQ ID NO 41, wherein X1 is R. In some specific embodiments, the isolated antibody or antigen-binding fragment thereof according to the present invention includes a light chain variable region comprising VL-CDR1 of sequence SEQ ID NO 40, wherein X1 is L, and VL-CDR2 of sequence SEQ ID NO 41, wherein X1 is L. In some specific embodiments, the isolated antibody or antigen-binding fragment thereof according to the present invention includes a light chain variable region comprising VL-CDR1 of sequence SEQ ID NO 40, wherein X1 is Y, and VL-CDR2 of sequence SEQ ID NO 41, wherein X1 is R. In some specific embodiments, the isolated antibody or antigen-binding fragment thereof according to the present invention includes a light chain variable region comprising VL-CDR1 of sequence SEQ ID NO 40, wherein X1 is Y, and VL-CDR2 of sequence SEQ ID NO 41, wherein X1 is L.

[0113] In some specific embodiments, the isolated antibody or its antigen-binding fragment according to the invention includes a light chain variable region, said light chain variable region including VL-CDR3 of sequence SEQ ID NO 15 or SEQ ID NO 37.

[0114] In one embodiment, any one of the CDR1, CDR2 and / or CDR3 of the HCVR having SEQ ID NO 10-12 can be characterized as having one, two, three or more amino acids substituted with different amino acids.

[0115] In one embodiment, any one of the CDR1, CDR2 and / or CDR3 of the LCVR having SEQ ID NO 13-15 can be characterized as having one, two, three or more amino acids substituted with different amino acids.

[0116] In one embodiment, any one of the CDR1, CDR2 and / or CDR3 of the HCVR having SEQ ID NO 10-12 and / or the LCVR having SEQ ID NO 13-15 can be characterized as having one, two, three or more amino acids substituted with different amino acids.

[0117] In one embodiment, any one of the CDR1, CDR2 and / or CDR3 of the HCVR having SEQ ID NO 10-12 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or higher identity with a specific CDR or group of CDRs listed in the corresponding SEQ ID NO.

[0118] In one embodiment, any one of the CDR1, CDR2 and / or CDR3 of the LCVR having SEQ ID NO 13-15 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or higher identity with a specific CDR or group of CDRs listed in the corresponding SEQ ID NO.

[0119] In one embodiment, any one of the CDR1, CDR2, and / or CDR3 of the HCVR having SEQ ID NO 10-12 and / or the LCVR having SEQ ID NO 13-15 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher identity with the specific CDR or CDR group listed in the corresponding SEQ ID NO.

[0120] In one embodiment, LCVR and / or HCVR further include a leader sequence, preferably located at the N-terminus of the LCVR amino acid sequence or the N-terminus of the HCVR amino acid sequence, respectively. Examples of leader sequences include, but are not limited to, SEQ ID NO: 16 and 17.

[0121] SEQ ID NO: 16

[0122] MGWSWIFLFLLSGTAGVLS

[0123] SEQ ID NO: 17

[0124] MDMRVPAHVFGFLLLWFPGTRC

[0125] In one embodiment, LCVR includes an amino acid leader sequence SEQ ID NO: 16 located at the N-terminus of the HCVR amino acid sequence.

[0126] In one embodiment, the LCVR includes an amino acid leader sequence SEQ ID NO: 17 located at the N-terminus of the LCVR amino acid sequence.

[0127] In one embodiment, the antibody or antigen-binding fragment according to the invention comprises a complete or substantially complete human heavy chain constant region (hereinafter abbreviated as HCCR or C). H ) and / or the light chain constant region (abbreviated as LCCR or C in this article) L ).

[0128] In some embodiments, the isolated antibody or its antigen-binding fragment binds to CCR8, inhibiting the binding of chemokine (CC motif) ligand 1 (CCL1) and / or chemokine (CC motif) ligand 18 (CCL18) to CCR8.

[0129] In one embodiment, the isolated antibody or its antigen-binding fragment is purified.

[0130] In one embodiment, the antibody or its antigen-binding fragment is purified to: (1) The weight of the protein or antibody or its antigen-binding fragment as determined by the Lowry method is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher, preferably greater than 96%, 97%, 98% or 99% by weight. (2) Sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues using a rotary cup sequencer; or (3) Homogeneity was demonstrated by SDS-PAGE using Coomassie Brilliant Blue or preferred silver staining under reducing or non-reducing conditions.

[0131] In a preferred embodiment, the isolated antibody or its antigen-binding fragment is monoclonal. In another embodiment, the isolated antibody or its antigen-binding fragment is polyclonal.

[0132] In some embodiments, the isolated antibody is full-length. In some embodiments, the isolated antibody is truncated. In some embodiments, the truncated antibody is an antigen-binding fragment; in practice, the antigen-binding fragment retains the ability to bind to epitopes of the antigen but lacks portions of its amino acid sequence that do not participate in epitope binding, such as the crystallizable fragment region (Fc).

[0133] In some embodiments, the isolated antibody or its antigen-binding fragment is a recombinant antibody. In some embodiments, the isolated antibody or its antigen-binding fragment is a modified antibody, such as an antibody with optimized Fc, which induces increased Fc receptor binding and improves isolated antibody-dependent cell-mediated cytotoxicity (ADCC).

[0134] In some embodiments, the antibody or its antigen-binding fragment or portion thereof is included in the fusion protein.

[0135] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a molecule selected from the group consisting of: intact antibody, humanized antibody, single-chain antibody, dimerized single-chain antibody, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibody, bispecific antibody, biantibody, triantibody, and tetraantibody.

[0136] Antigen-binding fragments of antibodies can be obtained using standard methods. For example, Fab or F(ab')2 fragments can be produced by protease digestion of isolated antibodies using conventional techniques.

[0137] It should also be understood that the antibodies or antigen-binding fragments thereof according to the present invention can be modified using known methods. For example, to slow in vivo clearance and obtain more desired pharmacokinetic characteristics, the antibodies or antigen-binding fragments thereof can be modified with polyethylene glycol (PEG). Methods for PEG conjugation and site-specific conjugation to antibodies or antigen-binding fragments thereof are described, for example, in Leong et al., 2001. Cytokine. 16(3):106-19; Delgado et al., 1996. Br J Cancer. 73(2):175-82.

[0138] In some embodiments, the antibody or antigen-binding fragment of the present invention is conjugated to a therapeutic and / or detectable portion, such as a radioactive portion and / or a radiodetectable portion and / or a drug. The portion may be, for example, a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a cleaved peptide, a radioisotope, and / or a radiosensitizer. Such conjugates are referred to herein as “antibody-drug conjugates” or “ADCs”.

[0139] In some embodiments, the antibody or its antigen-binding fragment is conjugated to a radioisotope or a chelate containing a radioisotope. For example, the antibody may be conjugated to a chelating linker, such as DOTA, DTPA, or tiuxetan, which allows the antibody to complex with the radioisotope. The antibody or its antigen-binding fragment may also, or alternatively, comprise or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Non-limiting examples of radioisotopes include... 3 H, 14 C 15 N、 19 F, 35 S, 90 Y、 99 Tc, 125 I, 131 I, 186 Re、 213 Bi、 225 Ac and 227 For therapeutic or imaging purposes, radioactive isotopes that emit beta or alpha particle radiation, such as... 131 I, 90 Y、 211 At、 212 Bi、 67 Cu、 186 Re、 188 Re and 212 Pb. After the antibody or its antigen-binding fragment has been administered to a patient / subject, the distribution of the antibody or its antigen-binding fragment in the patient's body is detected. Methods for detecting the distribution of any particular marker are known to those skilled in the art, and any suitable method may be used. Some non-limiting examples include computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and ultrasound.

[0140] The antibody and antigen-binding fragments according to the present invention can be used for the detection, diagnosis, and staging of diseases associated with CCR8 expression.

[0141] Typically, the diagnostic method involves using samples obtained from the patient, such as biological samples or fractions thereof.

[0142] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a molecule selected from the group consisting of: single-arm antibodies, domain antibodies, and nanobodies.

[0143] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a complete or substantially complete human heavy chain constant region (hereinafter abbreviated as HCCR or CH) and / or a light chain constant region (hereinafter abbreviated as LCCR or CL).

[0144] In one implementation, the constant region is of human origin.

[0145] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises a complete or substantially complete rabbit HCCR and / or LCCR.

[0146] In one implementation, the constant region is rabbit-derived.

[0147] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a rabbit antibody or fragment thereof.

[0148] In one embodiment, the antibody or its antigen-binding fragment according to the invention comprises a complete or substantially complete mouse HCCR and / or LCCR.

[0149] In one implementation, the constant region is mouse-derived.

[0150] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a mouse antibody or fragment thereof.

[0151] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a chimeric antibody or fragment thereof.

[0152] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a humanized antibody or fragment thereof.

[0153] Methods for humanizing antibodies or antigen-binding fragments according to the present invention are well known in the art. The selection of both the light and heavy chain human variable domains for preparing humanized antibodies or antigen-binding fragments is crucial for reducing antigenicity. According to the so-called "best fit" method, the sequence of the variable domain of the antibody or antigen-binding fragment according to the present invention is screened against a library of known human variable domain sequences. The human sequence that is closest to the mouse sequence is then accepted as the human frame (FR) for the humanized antibody (Sims et al., 1993. J Immunol. 151(4):2296-308; Chothia & Lesk, 1987. J Mol Biol. 196(4):901-17).

[0154] A frame region (FR) refers to an amino acid sequence between CDRs. The amino acid sequence and structure of an antibody or its antigen-binding fragment (e.g., a single-domain antibody) can therefore be considered to include four frame regions, or "FRs," referred to in the art and herein as "Frame Region 1" or "FR1"; "Frame Region 2" or "FR2"; "Frame Region 3" or "FR3"; and "Frame Region 4" or "FR4," respectively; these frame regions are interrupted by three complementarity-determining regions, or "CDRs." Therefore, the antibody or its antigen-binding fragment can be characterized by variable regions (e.g., VH or VL) having the following general structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, where FR1 to FR4 refer to frame regions 1 to 4, respectively, and where CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0155] One or more frame regions (FRs) of the antibody or its antigen-binding fragment may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 42 to SEQ ID NO 67. Specifically, the frame region (FR) may include or consist of a polypeptide sequence having at least 80% or even 90% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 42 to SEQ ID NO 67. Specifically, the frame region (FR) may include or consist of a polypeptide sequence having at least 95% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 42 to SEQ ID NO 67. Specifically, the frame region (FR) may include or consist of a polypeptide sequence selected from the group consisting of SEQ ID NO 42 to SEQ ID NO 67.

[0156] In some embodiments, the antibody or its antigen-binding fragment according to the invention is characterized by comprising a heavy chain variable region (VH): HFR1 – HCDR1 – HFR2 – HCDR2 – HFR3 – HCDR3 – HFR4.

[0157] In some embodiments, the antibody or its antigen-binding fragment according to the invention is characterized in that it includes a light chain variable region (VL): LFR1 – LCDR1 – LFR2 – LCDR2 – LFR3 – LCDR3 – LFR4.

[0158] Heavy chain frame region 1 (HFR1) may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 42, SEQ ID NO 50 and SEQ ID NO 51.

[0159] Heavy chain frame region 2 (HFR2) may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 43 and SEQ ID NO 52 to SEQ ID NO 55.

[0160] Heavy chain frame region 3 (HFR3) may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 44 and SEQ ID NO 56 to SEQ ID NO 58.

[0161] Heavy chain frame region 4 (HFR4) may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 45 and SEQ ID NO 59.

[0162] The light chain framework region 1 (LFR1) may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 46 and SEQ ID NO 60.

[0163] The light chain frame region 2 (LFR2) may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 47, SEQ ID NO 61 and SEQ ID NO 62.

[0164] The light chain frame region 3 (LFR3) may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 48 and SEQ ID NO 63 to SEQ ID NO 66.

[0165] Light chain frame region 4 (LFR4) may include or consist of a polypeptide sequence having at least 80% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 49 or SEQ ID NO 67.

[0166] The frame region (FR) may include or consist of a polypeptide sequence having at least 90% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 42 to SEQ ID NO 67.

[0167] The frame region (FR) may include or consist of a polypeptide sequence having at least 95% sequence identity with a polypeptide sequence selected from the group consisting of SEQ ID NO 42 to SEQ ID NO 67.

[0168] The frame region (FR) may be selected from a list including or consisting of SEQ ID NO 42 to SEQ ID NO 67.

[0169] Another method for humanizing antibodies or antigen-binding fragments according to the invention uses a specific framework of a common sequence from a particular light chain or heavy chain subgroup of all human antibodies. The same framework can be used for several different humanized antibodies (Carter et al., 1992. Proc Natl Acad Sci USA. 89(10):4285-9; Presta et al., 1993. J Immunol. 151(5):2623-32). It is also important that the antibody is humanized while retaining its high affinity for hCCR8 and other favorable biological properties. To achieve this, according to a preferred method, humanized antibodies and their antigen-binding fragments are prepared by analyzing parental sequences and various conceptual humanized products using a three-dimensional model of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs illustrating and demonstrating the possible three-dimensional structures of selected candidate immunoglobulin sequences are available. Examining these displays allows for the analysis of the potential roles of residues in the function of candidate immunoglobulin sequences, i.e., analyzing residues that affect the ability of candidate immunoglobulins to bind to their epitopes.

[0170] Another method for humanizing the antibodies or antigen-binding fragments thereof according to the invention is immunization using transgenic or transchromosomal animals carrying a portion of the human immune system. As hosts, the immunoglobulin genes of these animals have been replaced with functional human immunoglobulin genes. Therefore, the antibodies produced by these animals or in hybridomas prepared from the B cells of these animals are already humanized. Examples of such transgenic or transchromosomal animals include, but are not limited to: XenoMouse (Abgenix, Fremont, CA), described in patents US5,939,598, US6,075,181, US6,114,598, US6,150,584 and US6,162,963; HuMAb Mouse® (Medarex, Inc.), described in Lonberg et al., 1994. Nature. 368(6474):856-859; Lonberg & Huszar, 1995. Int RevImmunol. 13(1):65-93; Harding & Lonberg, 1995. Ann NY Acad Sci. 764:536-46; Taylor et al., 1992. Nucleic Acids Res. 20(23):6287-95; Chen et al., 1993.Int Immunol. 5(6):647-56; Tuaillon et al., 1993. Proc Natl Acad Sci USA. 90(8):3720-4; Choi et al., 1993. Nat Genet. 4(2):117-23; Chen et al., 1993.EMBO J. 12(3):821-30; Tuaillon et al., 1994. J Immunol. 152(6):2912-20; Taylor et al., 1994. Int Immunol. 6(4):579-91; Fishwild et al., 1996. Nat Biotechnol. 14(7):845-51; KM Mouse®, described in patent application WO2002043478; TC mouse, described in Tomizuka et al. al., 2000. Proc Natl Acad Sci USA. 97(2):722-7; and OmniRat™ (OMT, Inc.), described in patent application WO2008151081; Geurts et al., 2009. Science. 325(5939):433; Menoret et al., 2010. Eur J Immunol. 40(10):2932-41.

[0171] Humanized antibodies and their antigen-binding fragments can also be generated using various other techniques, such as immunization with other transgenic animals engineered to express human antibody libraries (Jakobovitz et al., 1993. Nature. 362(6417):255-8), or by selecting antibody libraries using phage display methods. Such techniques are known to those skilled in the art and can be implemented starting from the monoclonal antibodies or their antigen-binding fragments disclosed in this application.

[0172] In some embodiments, the antibody or antigen-binding fragment according to the present invention is characterized in that it comprises: The heavy chain variable region (VH) having at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NOs 21 to 26; and / or The light chain variable region (VL) has at least 80% sequence identity with the polypeptide sequence comprising the group consisting of SEQ ID NO 27 to 31.

[0173] In some embodiments, the antibody or antigen-binding fragment according to the present invention is characterized in that it comprises: The heavy chain variable region (VH) having at least 90% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NOs 21 to 26; and / or The light chain variable region (VL) has at least 90% sequence identity with the polypeptide sequence comprising the group consisting of SEQ ID NO 27 to 31.

[0174] In some embodiments, the antibody or antigen-binding fragment according to the present invention is characterized in that it comprises: The heavy chain variable region (VH) having at least 95% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NOs 21 to 26; and / or The light chain variable region (VL) has at least 95% sequence identity with the polypeptide sequence comprising the group consisting of SEQ ID NO 27 to 31.

[0175] In some embodiments, the antibody or antigen-binding fragment according to the present invention is characterized in that it comprises: Including the heavy chain variable region (VH) of a polypeptide sequence selected from the group consisting of SEQ ID NOs 21 to 26; and / or Includes the light chain variable region (VL) of the polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

[0176] In some embodiments, the antibody or antigen-binding fragment according to the present invention is characterized in that it comprises: - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF).

[0177] In some embodiments, the antibody or antigen-binding fragment according to the present invention is characterized in that it comprises: - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE).

[0178] In some embodiments, the antibody or antigen-binding fragment according to the present invention is characterized in that it comprises: - The heavy chain variable region (VH) having at least 90% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 90% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 90% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 90% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 90% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 90% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 90% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 90% sequence identity with SEQ ID NO 30 (LE).

[0179] In some embodiments, the antibody or antigen-binding fragment according to the present invention is characterized in that it comprises: - The heavy chain variable region (VH) having at least 95% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 95% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 95% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 95% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 95% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 95% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 95% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 95% sequence identity with SEQ ID NO 30 (LE).

[0180] In some embodiments, the antibody or antigen-binding fragment according to the invention is produced in a mammalian species. In some embodiments, the mammalian species is selected from the group consisting of: mice, rats, goats, horses, pigs, dogs, cats, and non-human primates such as chimpanzees. In some embodiments, the mammalian species is a mouse (e.g., a house mouse).

[0181] In one embodiment, the antibody or its antigen-binding fragment according to the invention is derived from the IgG class.

[0182] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is derived from the human IgG1 subclass. In another embodiment, the antibody or antigen-binding fragment thereof according to the invention is derived from the human IgG2 subclass.

[0183] The Fc region of IgG antibodies interacts with cellular Fcγ receptors (FcγRs) to stimulate and regulate downstream effector mechanisms. Five activating receptors are present: FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), as well as one inhibitory receptor, FcγRIIb (CD32b). Communication between IgG antibodies and the immune system is controlled and mediated by FcγRs, which transmit information sensed and collected by antibodies to the immune system, providing a link between the innate and adaptive immune systems, particularly in the context of biotherapeutic agents (Hayes J et al., 2016. JInflamm Res 9: 209-219).

[0184] IgG subclasses differ in their ability to bind to FcγR, and this differential binding determines their ability to elicit a range of functional responses. For example, in humans, FcγRIIIa is the major receptor involved in activating antibody-dependent cell-mediated cytotoxicity (ADCC), and IgG3 (followed by IgG1) exhibits the highest affinity for this receptor, reflecting their ability to effectively induce ADCC. IgG2 has been shown to bind weakly to this receptor.

[0185] In one embodiment, the antibody or its antigen-binding fragment according to the invention binds to FcγR with high affinity, preferably to the activating receptor with high affinity.

[0186] In one embodiment, the antibody or its antigen-binding fragment according to the invention binds to FcγRI and / or FcγRIIa and / or FcγRIIc and / or FcγRIIIa and / or FcγRIIIb with high affinity.

[0187] In one embodiment, the IgG1 antibody binds to at least one Fc activating receptor. For example, the antibody may bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In one embodiment, the antibody is capable of binding FcγRIIIa. In one embodiment, the antibody is capable of binding FcγRIIa. In one embodiment, the antibody is capable of binding FcγRIIIa, FcγRIIc, and optionally FcγRI. In one embodiment, the antibody is capable of binding FcγRIIIa, FcγRIIa, and optionally FcγRI.

[0188] In one embodiment, the antibody or antigen-binding fragment according to the invention is used at a concentration of less than about 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 The dissociation constant of M binds to at least one activating Fcγ receptor.

[0189] In one embodiment, the IgG1 antibody binds FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and / or FcγRIIIb with a higher affinity than it binds to FcγRIIb, and binds to FcγRIIb with a lower affinity.

[0190] In one embodiment, the antibody or antigen-binding fragment of the present invention is an IgG1 antibody, preferably a human IgG1 antibody.

[0191] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is an IgG1κ antibody.

[0192] In one embodiment, the antibody or its antigen-binding fragment according to the invention may include a human heavy chain constant region sequence and allow targeting, blocking, and / or depletion of CCR8-expressing cells to which they bind.

[0193] In one embodiment, the proteins according to the invention deplete the CCR8-expressing cells to which they are bound. In one embodiment, the proteins according to the invention deplete the Tregs to which they are bound. In one embodiment, the proteins according to the invention also deplete or reduce the tumor-infiltrating regulatory T cells to which they are bound.

[0194] In one embodiment, the antibody or its antigen-binding fragment according to the invention depletes the CCR8-expressing cells it binds to. In one embodiment, the antibody or its antigen-binding fragment according to the invention depletes the Tregs it binds to. In one embodiment, the antibody or its antigen-binding fragment according to the invention also depletes or reduces the number of tumor-infiltrating regulatory T cells it binds to.

[0195] In one embodiment, the exhaustion is achieved via ADCP. In another embodiment, the exhaustion is achieved via CDC.

[0196] Therefore, in one embodiment, the antibody of the present invention directly or indirectly causes the depletion of CCR8-expressing cells (e.g., causing the elimination or reduction of 10%, 20%, 50%, 60%, 70%, 80%, 85% or higher of the number of CCR8-expressing cells).

[0197] In one embodiment, the antibody or its antigen-binding fragment according to the invention does not inhibit the binding of interleukin-2 (IL-2) to CCR8 and depletes the Tregs bound to it.

[0198] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention is a polyethylene glycol-modified antibody or a fragment thereof.

[0199] Antibodies can be PEGylated to, for example, increase their biological (e.g., serum) half-life. To PEGylate antibodies, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG) (e.g., a reactive ester or aldehyde derivative of PEG) under conditions that link one or more PEG groups to the antibody or antibody fragment. PEGylation can be carried out by acylation or alkylation reactions with reactive PEG molecules (or similar reactive water-soluble polymers). As used herein, the term "polyethylene glycol" is intended to cover any form of PEG that has been used to derivatize other proteins, such as mono(C1) PEG. 12 -C1 120 Alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be PEGylated is a glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention, such as those described in EP0154316 (Nishimura et al.) and EP0401384 (Ishikawa et al.).

[0200] In one embodiment, the isolated antibody or its antigen-binding fragment, as described above, is used as a drug; or for the preparation of a drug.

[0201] In one embodiment, the isolated antibody or its antigen-binding fragment, as described above, is used as a diagnostic reagent; or is used to prepare a diagnostic reagent.

[0202] According to some embodiments, the present invention also relates to the in vitro or ex vivo or in vivo use of the antibody or antigen-binding fragment thereof as described above for the detection of CCR8-expressing cells; particularly in biological samples or fractions thereof.

[0203] According to some embodiments, the present invention also relates to an in vitro or ex vivo or in vivo method for detecting CCR8-expressing cells, comprising contacting cells or cell populations with an antibody or antigen-binding fragment thereof as described above.

[0204] Therefore, according to some embodiments, the present invention also relates to an in vivo method for detecting CCR8-expressing cells, comprising: a) Expose the antibody or antigen-binding fragment thereof described above to a human or non-human individual, for example, administer the antibody or antigen-binding fragment thereof described above to a human or non-human individual. b) Detect the antibody or its antigen-binding fragment in the individual to detect CCR8-expressing cells.

[0205] According to some embodiments, the present invention also relates to an in vitro or ex vivo method for detecting CCR8-expressing cells, comprising: a) Provide samples, especially biological samples or fractions thereof; b) Contact the sample with the antibody or its antigen-binding fragment as described above; c) Detect the antibody or its antigen-binding fragment in the sample to detect CCR8-expressing cells.

[0206] Specifically, the step of detecting CCR8-expressing cells may include step c) detecting the presence of the antibody or its antigen-binding fragment in the sample and comparing it with a reference value; thereby detecting the binding of the antibody or its antigen-binding fragment to the sample, thereby detecting CCR8-expressing cells.

[0207] The present invention also relates to a nucleic acid encoding an isolated antibody or an antigen-binding fragment thereof according to the present invention.

[0208] In one embodiment, the nucleic acid encoding the isolated antibody or antigen-binding fragment thereof of the present invention may be expressed as a single nucleic acid molecule encoding said isolated antibody or antigen-binding fragment thereof, or as multiple (e.g., two or more) co-expressed nucleic acid molecules. The polypeptides encoded by the co-expressed nucleic acid molecules may bind by, for example, disulfide bonds or other means to form the isolated antibody or antigen-binding fragment thereof as described above.

[0209] In one embodiment, the nucleic acid molecule is DNA. In another embodiment, the nucleic acid molecule is RNA, such as messenger RNA (mRNA).

[0210] In one embodiment, the nucleic acid is linear. In another embodiment, the nucleic acid is circular.

[0211] In one embodiment, the nucleic acid as described above is used as a drug. In another embodiment, the nucleic acid as described above is used to prepare a drug.

[0212] In one embodiment, the nucleic acid as described above is used as a diagnostic reagent. In another embodiment, the nucleic acid as described above is used to prepare a diagnostic reagent.

[0213] Another object of the present invention is a vector comprising a nucleic acid according to the invention. In some embodiments, at least one nucleic acid molecule is included in said vector.

[0214] In some embodiments, the vector allows for the controlled expression of at least one isolated antibody or its antigen-binding fragment. As used herein, the term "controlled expression" is intended to refer to expression that is controlled in time and / or space. In other words, the controlled expression of the isolated antibody or its antigen-binding fragment according to the invention can occur at a specific location in the body, such as a specific organ, and / or within a specific time period.

[0215] In some embodiments, the vector is a viral vector. Non-limiting examples of viral vectors include adenovirus, adeno-associated virus (AAV), alphavirus, herpesvirus, lentivirus, non-integrating lentivirus, retrovirus, vaccinia virus, and baculovirus.

[0216] In some embodiments, the carrier is a non-viral carrier. Non-limiting examples of non-viral carriers include inorganic particles (e.g., gold phosphate, calcium), lipid emulsions, lipid nanoparticles (e.g., liposomes), DNA-binding proteins, or peptides.

[0217] In one implementation, the carrier as described above is used as a drug.

[0218] The present invention also relates to an immunotoxin fusion protein for CCR8-expressing cells (e.g., Treg cells or cancer cells), comprising an antibody or antigen-binding fragment thereof isolated according to the present invention and a toxin or fragment thereof.

[0219] Immunotoxins are ligands combined with toxins that can be used to kill cells expressing the receptor for that ligand. Immunotoxin therapy is also known as ligand-targeted therapy. Therefore, immunotoxins consist of a targeting portion (ligand) for delivery and a toxic portion for cytotoxicity. Currently used ligands include monoclonal antibodies, cytokines / growth factors, and soluble receptors.

[0220] In one embodiment, the immunotoxin fusion protein comprises an antigen-binding fragment of the antibody of the present invention. In some embodiments, the antigen-binding fragment comprises two single-chain Fvs of the variable region of the antibody of the present invention. In some embodiments, the antigen-binding fragment further comprises one or more linkers.

[0221] In some embodiments, the immunotoxin fusion protein comprises a truncated toxin.

[0222] Optionally, the immunotoxin fusion protein may further include a modified N-terminus. Optionally, the immunotoxin fusion protein may also include a signal peptide at its N-terminus, post-translational but pre-secretion.

[0223] The present invention also relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid or a carrier, and at least one other pharmaceutically acceptable excipient.

[0224] In some embodiments, the pharmaceutically acceptable medium is selected from the group consisting of solvents, diluents, carriers, excipients, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delay agents, and any combination thereof. The carrier, diluent, solvent, or excipient must be "acceptable," meaning compatible with the isolated antibody or fragment thereof, nucleic acid, or carrier or derivative thereof according to the invention, and not harmful when administered to an individual. Generally, the medium does not produce adverse, allergic, or other inappropriate reactions when administered to an individual (preferably a human individual).

[0225] For the specific purpose of human administration, the pharmaceutical composition shall meet the sterility, pyrogenicity, general safety and purity standards required by regulatory agencies such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0226] In some embodiments, the pharmaceutical composition further includes at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of anticancer agents and checkpoint inhibitors.

[0227] In some embodiments, the at least one additional therapeutic agent is an anticancer agent.

[0228] Anticancer agents are known from the prior art. Non-limiting examples of anticancer agents include acalatinib, alectinib, alemtuzumab, anastrozole, avapratinib, avelumumab, belitusta, bevacizumab, bleomycin, bonatumab, bosutinib, brigatinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, curapannixicin, cytarabine, daunorubicin, decitabine, dexamethasone, docetaxel, doxorubicin, encofenib, erdatinib, etoposide, and others. Vimulus, exemestane, fludarabine, 5-fluorouracil, gemcitabine, ifosfamide, imatinib mesylate, leuprorelin, lomustine, nitrogen mustard, melphalan, methotrexate, mitomycin, nerabine, paclitaxel, pamidronate, prambistrol, prambistrol, prednisolone, oflambumab, rituximab, temozolomide, topotecan, tosimomab, trastuzumab, vandetanib, vincristine, vorinostat, zanubrutinib, etc.

[0229] In some embodiments, the anticancer agent is administered in combination with, simultaneously with or sequentially with an isolated antibody or its antigen-binding fragment, nucleic acid, carrier, or pharmaceutical composition according to the invention.

[0230] In some embodiments, the at least one additional therapeutic agent is a checkpoint inhibitor therapy.

[0231] As used in this article, "checkpoint inhibitor therapy" is defined as the administration of at least one checkpoint inhibitor to a subject.

[0232] Checkpoint inhibitors (CPIs, also known as immune checkpoint inhibitors or ICIs) are molecules, typically antibodies, that block the interaction between inhibitory receptors (IRs) expressed on T cells and their ligands. As a cancer treatment, checkpoint inhibitor therapy aims to prevent the activation of inhibitory receptors expressed on T cells by ligands expressed by tumor cells. Therefore, checkpoint inhibitor therapy aims to prevent the suppression of T cells present in the tumor (i.e., tumor-infiltrating T cells) and thus enhance the subject's immune response against tumor cells.

[0233] Examples of checkpoint inhibitors include, but are not limited to, inhibitors of the cell surface receptor PD-1 (programmed cell death protein 1), also known as CD279 (differentiation cluster 279); inhibitors of the ligand PD-L1 (programmed death ligand 1), also known as CD274 (differentiation cluster 274) or B7-H1 (B7 homolog 1); inhibitors of the cell surface receptor CTLA4 or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152 (differentiation cluster 152); inhibitors of LAG-3 (lymphocyte activation gene 3), also known as CD223 (differentiation cluster 223); and TIM-3 (containing T cell immunoglobulin). Inhibitors of CD366 (differentiation cluster 366) are also known as HAVCR2 (hepatitis A virus cell receptor 2) or CD366 (differentiation cluster 366); inhibitors of TIGIT (T cell immune receptor with Ig and ITIM domains) are also known as VSIG9 (protein 9 containing V-Set and immunoglobulin domains) or VSTM3 (protein 3 containing V-Set and transmembrane domains); inhibitors of BTLA (B and T lymphocyte attenuator) are also known as CD272 (differentiation cluster 272); and inhibitors of CEACAM-1 (carcinoembryonic antigen-associated cell adhesion molecule 1) are also known as CD66a (differentiation cluster 66a).

[0234] In one embodiment, the at least one checkpoint inhibitor is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA 4 inhibitors, and any mixture thereof.

[0235] In some embodiments, the therapeutic agent is a programmed cell death protein 1 (PD-1) inhibitor. In a preferred embodiment, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that selectively isolates PD-1, preferably a monoclonal antibody. Non-limiting examples of FDA-approved exemplary anti-PD-1 therapeutic antibodies include pembrolizumab (Keytruda®, Merck), nivolumab (Opdivo®, Bristol Myers Squibb), cimiprimab (Libtayo®, Sanofi Genzyme), and dostalimumab (Jemperli®, GlaxoSmithKline). In another embodiment, the PD-1 inhibitor is a small molecule or nucleic acid, such as interfering RNA.

[0236] In some embodiments, the antibody or its antigen-binding fragment, nucleic acid, expression vector, pharmaceutical composition, or drug according to the invention is used in combination with a second antibody, which induces cell death in cells expressing the antigen bound by the second antibody via ADCC. In one embodiment, the second antibody (e.g., IgG1 or IgG3 isotype) induces ADCC against cells bound by the antibody. NK cells play an important role in inducing ADCC, and the increased reactivity of NK cells can be directed to target cells by using such a second antibody. In one embodiment, the second antibody is specific to cell surface antigens (e.g., membrane antigens). In some embodiments, the second antibody is specific to tumor antigens (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2 / Neu), CD33, CD22, MUC-1, CEA, KDR, αVβ3, etc., particularly lymphoma antigens (e.g., CD20). Therefore, the present invention also provides a method for enhancing the antitumor activity of monoclonal antibodies against tumor antigens.

[0237] The present invention also relates to isolated antibodies or antigen-binding fragments thereof, nucleic acids, carriers or pharmaceutical compositions according to the present invention, which are used as pharmaceuticals.

[0238] The present invention also relates to isolated antibodies or antigen-binding fragments thereof, nucleic acids, carriers, pharmaceutical compositions or drugs according to the present invention for the treatment and / or prevention of disease in subjects in need, preferably CCR8-related diseases.

[0239] In some implementations, the disease (e.g., CCR8-related disease) is cancer.

[0240] In some embodiments, the disease is a cancer selected from the group consisting of or including: colon cancer, melanoma, breast cancer, urothelial carcinoma, adenoid cystic carcinoma, adrenal carcinoma, ataxia-telangiectasia, atypical nevus syndrome, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, carcinoid tumor, cervical cancer, colorectal cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, islet cell tumor, kidney cancer, laryngeal cancer, leukemia, liver cancer, lobular carcinoma, hepatocellular carcinoma, lung cancer, glioma, meningioma, nasopharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, peritoneal cancer, pituitary adenoma, polycythemia vera, prostate cancer, renal cell carcinoma, retinoblastoma, salivary gland cancer, sarcoma, small intestine cancer, gastric cancer, testicular cancer, thyroid cancer, uterine (endometrial) cancer, and vaginal cancer.

[0241] In some implementations, the disease is a cancer selected from the group consisting of colorectal cancer, breast cancer, kidney cancer, and sarcoma.

[0242] In some implementations, the cancer is selected from the group consisting of colon cancer, melanoma, breast cancer, and urothelial carcinoma.

[0243] In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is urothelial carcinoma.

[0244] In some implementations, the disease (e.g., CCR8-related disease) is a disease associated with or related to impaired immune response.

[0245] In some embodiments, the disease associated with or related to impaired immune response is an inflammatory disease or a hypersensitivity disease. Non-limiting examples of inflammatory diseases according to the invention include cholangitis and arthritis.

[0246] The present invention also relates to a method for treating and / or preventing diseases (preferably CCR8-related diseases, more preferably cancer), comprising administering to a subject in need a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof, nucleic acid, carrier, pharmaceutical composition or drug according to the present invention.

[0247] The present invention also relates to the use of isolated antibodies or antigen-binding fragments thereof, nucleic acids, carriers or pharmaceutical compositions according to the present invention in the preparation of medicaments, preferably for the treatment and / or prevention of CCR8-related diseases, more preferably cancer.

[0248] The present invention also relates to isolated antibodies or antigen-binding fragments thereof, nucleic acids, vectors or pharmaceutical compositions according to the present invention for depleting CCR8-expressing tumor T cells. reg Uses of the cells. In some embodiments, the T... regOverexpression of CCR8.

[0249] Another object of the present invention relates to the use of an antibody or antigen-binding fragment thereof as described above in combination with another therapeutic agent in treating a disease in a subject in need, wherein the antibody or antigen-binding fragment thereof serves as an adjuvant to the therapeutic agent.

[0250] Therefore, this invention relates to antibodies or antigen-binding fragments thereof (preferably in pharmaceutical compositions or medicaments) as described herein, used as adjuvants in cancer therapy. Therefore, this invention relates to antibodies or antigen-binding fragments thereof (preferably in pharmaceutical compositions or medicaments) as described herein, used as adjuvants in the therapy of infectious diseases.

[0251] In one embodiment, the present invention relates to the use of antibodies or fragments thereof, as described herein, for enhancing immune responses induced by cancer therapies in patients in need.

[0252] In one embodiment, the antibody or fragment thereof according to the invention can be used as an immunotherapeutic agent, particularly for the treatment of a wide variety of cancers (e.g., cancers associated with immunosuppression and / or immune depletion).

[0253] In one embodiment, the antibody or fragment thereof according to the invention can enhance an immune response induced by cancer therapy in a patient, including administering an effective amount of the antibody or fragment thereof to a subject to enhance the immune response induced by cancer therapy in the patient.

[0254] In another embodiment, an antibody or fragment thereof, as described herein, can increase or improve the immune response of a subject.

[0255] Like other known immunotherapeutic agents, the ability of antibodies or fragments thereof according to the present invention to enhance immune responses in patients may have broader therapeutic implications beyond the field of cancer. For example, immune enhancers have been proposed for the treatment of a wide variety of infectious diseases, particularly pathogens that promote immunosuppression and / or immune depletion. Additionally, such immune enhancers can be used to enhance the efficacy of vaccines, such as vaccines for infectious diseases and cancer.

[0256] Another object of the present invention relates to an antibody or antigen-binding fragment thereof according to the invention for depleting CCR8-expressing T cells in subjects in need. reg The use of cells in which a therapeutically effective amount of the antibody or a fragment thereof of the present invention will be administered to the subject.

[0257] Therefore, the present invention also relates to a method for depleting CCR8-expressing T in subjects in need. reg A cellular method comprising administering to the subject an antibody or antigen-binding fragment thereof as described herein, a pharmaceutical composition or a drug.

[0258] In one embodiment, the antibody or its antigen-binding fragment as described above (preferably in the composition, pharmaceutical composition or medicament described above) is used to deplete CCR8-expressing T cells. reg cell.

[0259] In one implementation, the CCR8 expresses T reg The cells are tumor-infiltrating T cells reg .

[0260] Another object of the present invention relates to a method for desensitizing cells to CCL1 stimulation in a subject in need, comprising administering to the subject an antibody or antigen-binding fragment thereof according to the invention, a pharmaceutical composition or a drug.

[0261] Therefore, the present invention also relates to the use of antibodies or antigen-binding fragments or immunotoxin fusion proteins according to the present invention for killing CCR8-expressing cells (e.g., Treg cells or cancer cells).

[0262] Another object of the present invention is the use of antibodies or antigen-binding fragments thereof targeting both human and rabbit CCR8 according to the present invention in rabbit models for preclinical studies. Attached Figure Description

[0263] Figure 1 This is a chromatogram showing the purity of the mAb2 antibody and subsequent quantification. Electrophoresis was performed under denaturing conditions (SDS-PAGE) on a Bio-Rad 15% gel (Bio-Rad 4568084), and mAb2 was labeled.

[0264] Figures 2A-2B This is a composite diagram showing the flow cytometry analysis used to detect CCL1. The diagram shown here is the positive control CCL1 (…). Figure 2A ) and negative control C3a ( Figure 2B ).

[0265] Figure 3 This is a histogram representing the percentage of CCL1 binding on HuT-78 in the flow cytometry assay illustrated in Figure 2 using mAb1, mAb2, mAb3, mAb4, and mAb5.

[0266] Figure 4 This indicates that the human T was detected using mAb as described above. reg Flow cytometry image of cells (cells expressing FoxP3).

[0267] Figure 5 This is a flow cytometry plot showing the detection of rabbit CD4-positive cells using the mAb described above. 1.5% corresponds to peripheral T cells. reg The expected proportion of cells.

[0268] Figure 6. Humanized Fab expression and ligand binding. The three figures A, B and C list the Fab for each test on the x-axis, respectively involving the original expression fluorescence of fluorescein (FITC) (A), the relative binding fluorescence / expression fluorescence ×100 of the KLH peptide (B), and the relative binding fluorescence / expression fluorescence ×100 of the sulfated peptide (C).

[0269] Example

[0270] The present invention is further illustrated by the following embodiments.

[0271] Example 1: Antibody Production

[0272] Materials and methods

[0273] Mouse immunization and fusion: Anti-human CCR8 monoclonal antibody (mAb) was obtained by immunizing BalB-C mice with a stable hCCR8 syngeneic cell line, following the previously described method (WO 02 / 081523). Following immunization, spleen cells (50 × 10⁶ cells / year) were harvested. 6 (cells) and fused with mouse cell line SP2O (20 × 10⁻⁶ cells) via electrofusion. 6 Hybridomas producing mAbs were obtained by fusing 10 cells to 20 cells. The fusion product was diluted in 400 ml of HAT-containing medium (DMEM 10% FCS) and aliquoted into 20 96-well plates. One week after fusion, the supernatant of the growing hybridomas was screened.

[0274] Hybridoma screening: Hybridoma supernatant was screened by flow cytometry on the human HuT-78 cell line (ATCC TIB 161) that naturally expresses CCR8. 50 µl of the supernatant was added at a 10⁻¹⁰ ratio. 6 HuT-78 cells diluted 2.5 µl / ml in PBS were allocated to 96-well plates. 50 µl of hybridoma supernatant was added. 50 µl of culture medium was added as a negative control. 50 µl of mouse anti-hCCR8 (diluted 2.5 µl / ml) (Biolegend L263G8) was added as a positive control. After incubation at 4°C for 10 min, the plates were centrifuged (1200 g, 10 min) and the supernatant was discarded. For color development, 50 µl of goat anti-mouse IgG Alexa Fluor 488 (Biolegend L263G8) diluted 100-fold in PBS was added. After incubation at 4°C for 5 min, the plates were centrifuged and the supernatant was discarded. The cell pellet was resuspended in 100 µl of PBS and analyzed by flow cytometry on BD Accuri. Fluorescence (expressed as mean fluorescence) was calculated using BDCSampler software.

[0275] Antibody isotype identification: The mAbs were identified by flow cytometry using the above protocol, with the difference being that the chromogenic antibody was either rat anti-mouse IgG1 FITC (LO-MG1.2 Synabs), rat anti-mouse IgG2a FITC (LO-MG2a7 Synabs), rat anti-mouse IgG2b FITC (LO-MG2b2 Synabs), rat anti-mouse IgG3 FITC (LO-MG3-7 Synabs), or rat anti-mouse IgM FITC (LO-MM9 Synabs).

[0276] result

[0277] Forty-one positive hybridomas were obtained and classified according to their fluorescence intensity (Table 1).

[0278]

[0279] Table 1: Fluorescence obtained from antibodies produced by hybridomas

[0280] A significant intensity variation was observed between the first 5 hybridomas and the last 37 hybridomas. It is speculated that the first 5 mAbs identified a particularly exposed epitope.

[0281] These 5 mAbs were isotyped by flow cytometry (Table 2).

[0282]

[0283] Table 2: Results of Homotype Identification

[0284] Five hybridomas were cultured in culture flasks (200 ml of medium). mAbs were purified from the culture supernatant on a rat anti-mouse column. The supernatants of hybridomas 1, 2, 3, and 5, representing IgG2a, were purified on a LO-MG2a column (from Synabs). The supernatant of hybridoma 4, representing IgG1, was purified on a LO-MG1 column (from Synabs). The purified mAbs were resuspended in PBS. The purity of the mAbs was then controlled by electrophoresis on a polyacrylamide gel under both denaturing and non-denaturing conditions. The purity of each mAb was greater than 90% (see [link to relevant documentation]). Figure 1 (Example of the purity of mAb2).

[0285] Example 2: Epitope Determination

[0286] CCR8 exhibits four extracellular regions: an N-terminal tail, a C-terminal tail, an extracellular loop (ECL), and two intracellular loops (ICL).

[0287] The corresponding sequence is highlighted in bold in the full-length hCCR8 sequence (genebank P51685): MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGK LLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTY YLLDQWVFGTVM CKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVF YQVASEDGVLQCYSFYNQQTLKWKIFTNFKM NILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSL HSMHILDGCSISQQLTY ATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL (SEQ ID NO: 3) Materials and methods To determine the epitopes recognized by the selected antibody, a peptide scan was performed. This peptide scan first involves coating an ELISA plate with several peptides corresponding to the entire extracellular region of CCR8, and then adding the antibody.

[0288] The peptides used (purchased from Eurogentec) are: - Peptide 1: MDYTLDLSVTTV (SEQ ID NO: 4) -Peptide 2: VTTVTDYYYPDIFSSPC (SEQ ID NO: 1) - Peptide 3: FSSPCDAELIQTNGK (SEQ ID NO: 5) -Peptide 4: YLLDQWVFGTVMC (SEQ ID NO: 6) - Peptide 5: YQVASEDGVLQC (SEQ ID NO: 7) -Peptide 6: VLQCYSFYNQQTLK (SEQ ID NO: 8) - Peptide 7: QTLKWKIFTNFKM (SEQ ID NO: 9) The peptides were coated overnight at 4°C with 5 µl / ml of borate buffer at pH 9. After washing with PBS buffer, different mAbs diluted in PBS at 5 µl / ml were added to the plate and incubated at 37°C for 1 hour. After washing with PBS buffer, 1000-fold diluted goat anti-mouse IgG Hrp (Icl GGHL-90P) was added to the plate and incubated at 37°C for 30 minutes. After washing with PBS, TMB solution was added to the plate and incubated at room temperature for 10 minutes. The colorimetric reaction was terminated by adding sulfuric acid solution at pH 2. The yellow color of the ELISA plate was read at 450 nm.

[0289] result

[0290] All five mAbs recognize only peptide 2. Peptide 2 corresponds to the AA 9-25 region of the full-length human CCR8 sequence.

[0291] The region corresponding to this epitope has previously been described as the CCL1 binding region. Sulfonation (i.e., posttranscriptional addition of SO3H groups) at tyrosine residues Y15 and Y17 has also been described (Yu, Hoffhiness et al., 2017. Nature Method.4: 583).

[0292] To verify whether these 5 mAbs can recognize both sulfonated and non-sulfonated forms of CCR8, a comparative ELISA was performed with coatings of non-sulfonated peptide 2 (VTTVTDYYYPDIFSSPC) and Y15, Y17 sulfonated peptide 2 (VTTVTDY(SO3H)YY(SO3H)PDIFSSPC).

[0293] Sulfonated peptide 2 was also purchased from Eurogentec. The ELISA protocol is similar to that detailed for peptide scanning (see Example 1).

[0294] ELISA (Table 3) showed that sulfonation only slightly reduced mAb binding to 5 mAbs.

[0295]

[0296] Table 3: Absorbance at 450 nm for peptide 2 or sulfonated peptide 2 using mAb

[0297] These five mAbs recognized the same epitope, and four of them showed the same IgG2a isotype. This result may mean that some mAbs are identical and originated from primitive activated B lymphocytes that have already divided extensively in the spleen. To verify this, the corresponding heavy and light chain variable region mRNAs of each mAb were sequenced.

[0298] Example 3: Inhibition of CCL1 binding

[0299] Since the epitopes identified by the five mAbs are described as corresponding to CCR8 ligand binding, it was explored whether some of these mAbs could block CCL1 binding.

[0300] As previously mentioned (Liu, Doijen et al., 2021. Biochemical Pharmacology. 188:114565), the ability of mAbs to block CCL1 binding can be evaluated using Alexa fluor 647-labeled CCL1 (Almac UK cat: CAF07).

[0301] Materials and methods

[0302] Positive control: 50 µl of solution was suspended in PBS (10... 6 HUT-78 ( / ml) was incubated at 4°C with 50µl PBS for 10 minutes, then 50µl of 10-fold diluted CCL1 Alexa Fluor647 (Almac cat: CAFO7) was added and incubated for another 10 minutes at 4°C.

[0303] Negative control: 50 µl of solution was suspended in PBS (10... 6 HUT-78 in 50 µl PBS was incubated at 4°C for 10 minutes with 50 µl of 10-fold diluted C3a Alexa Fluor647 (Almac cat CAF91A1) and incubated at 4°C for another 10 minutes.

[0304] Assay: 50 µl of the solution was suspended in PBS (10) 6 HUT-78 in 50 µl of each mAb diluted 5 µg / ml in PBS was incubated at 4°C for 10 min, followed by incubation at 4°C for another 10 min with a 10-fold diluted CCL1 Alexa Fluor647 (Almac cat: CAFO7). After incubation, the plates were washed three times with PBS and analyzed by flow cytometry.

[0305] An example of flow cytometry analysis of mAb 2 is shown in Figures 2A-2B .

[0306] result

[0307] The results are shown in Table 4.

[0308]

[0309] Table 4: Average fluorescence (CCL1 binding)

[0310] Results expressed as CCL1 binding percentage (mean fluorescence assay - mean fluorescence negative control / mean fluorescence positive control - mean fluorescence negative control) are shown in Figure 3 .

[0311] The results showed that all mAbs fully or partially competed with CCL1 (mAb2).

[0312] Example 4: Antibody internalization

[0313] When the ligand CCL1 binds to a GPCR, it first induces an intracellular activation cascade, then initiates the internalization of the ligand-GPCR complex. This phenomenon, known as desensitization, temporarily renders the cell insensitive to any further stimulation of the same ligand. Here we demonstrate that antibodies also induce the internalization of the antibody-GPCR complex.

[0314] Materials and methods

[0315] Suspend 200 µl in the culture medium (10) 6 HUT-78 (at a concentration of 5 µg / ml) was dispensed into 96-well plates and incubated at 37°C. mAb 1 was added at different time points (0 min, 1 h, 2 h, 3 h, 3 h 30 min, and 3 h 55 min) to a final concentration of 5 µg / ml. A negative control without mAb 1 was also tested.

[0316] After 4 hours of incubation, the plates were centrifuged and the supernatant was discarded. For color development, 50 µl of goat anti-mouse IgG Alexa Fluor 488 (Biolegend L263G8) diluted 100-fold with PBS was added. After incubation at 4°C for 5 minutes, the plates were centrifuged and the supernatant was discarded. The cell pellet was resuspended in 100 µl of PBS and analyzed by flow cytometry on BD Accuri. Fluorescence (expressed as mean fluorescence) was calculated using BD CSampler software.

[0317] result

[0318] The results are shown in Table 5.

[0319]

[0320] Table 5: Average Fluorescence

[0321] The results showed that mAb1, through ligand-mimicking binding, also induced the internalization of the mAb-CCR8 complex, leading to the downregulation of CCR8 expression.

[0322] Example 5: Human and Rabbit T reg Identification

[0323] T regCharacterized by expression of the transcription factor FoxP3. The study investigated whether these five mAbs also recognize T. reg .

[0324] Materials and methods

[0325] Flow cytometry (human): Human PBMCs (peripheral blood mononuclear cells) isolated from fresh blood by Ficoll centrifugation were tested by flow cytometry. After permeabilization (FIX & PERM® Cell Permeabilization Kit Thermofisher GAS003), the isolated PBMCs were double-labeled with FOXP3 Alexa 488 (BD Biosciences) and each mAb Alexa Fluor647.

[0326] Flow cytometry (rabbit): Rabbit PBMCs (separated from fresh blood by Ficoll centrifugation) were double-labeled with mouse anti-rabbit CD4 FITC mAb (Mybiosource cat: MBS 216606) and one of the five mAbs, Alexa Fluor 647.

[0327] Note: Anti-FOXP3 double labeling was not performed because there is currently no anti-FOXP3 available for flow cytometry.

[0328] ELISA: See Example 2.

[0329] result

[0330] All five mAbs tested identified FOXP3-positive cells. As an example, cell surface analysis of FOXP3-mAb1 dual-labeled PBMCs performed on a FaCScan flow cytometer (Becton Dickinson) is shown in [the image / data]. Figure 4 .

[0331] The human CCR8 epitope recognized by five monoclonal antibodies (mAbs) is similar to the corresponding rabbit CCR8 (genebank G1T2B2), except for one AA: - Human epitope: VTTVTDYYYPDIFSSPC (SEQ ID NO: 1) - Rabbit epitope: VTAVTDYYYPDIFSSPC (SEQ ID NO: 2) To verify whether these five mAbs also recognize rabbit CCR8, a comparative ELISA was performed on human peptide 2 (VTTVTDYYYPDIFSSPC) and its corresponding rabbit peptide (VTAVTDYYYPDIFSSPC). The results are shown in Table 6.

[0332]

[0333] Table 6: Absorbance at 450 nm for human or rabbit peptide 2 using mAb

[0334] Clones 1, 3, and 5 recognize rabbit peptides.

[0335] Flow cytometry was also performed to verify whether these five mAbs could bind to a subset of peripheral rabbit CD4+ lymphocytes. Clones 1, 3, and 5 identified 1.5% of CD4-positive cells, corresponding to peripheral T cells. reg proportion ( Figure 5 This result confirms the findings obtained by ELISA.

[0336] Example 6: Validation of yeast-based screening for Fab antibodies targeting sulfonated and non-sulfonated CCR8 fragments

[0337] Materials and methods

[0338] Affinity was determined using a yeast-based assay.

[0339] Yeast expressing Fab was induced to express the antigen. In short, the yeast was washed for 10 minutes with PBSF buffer (PBS, BSA 0.1%). 5 Two yeast cells each.

[0340] Yeast cells were incubated with KLH peptide (or KLH alone) at concentrations ranging from 1000 ng / mL to 0.32 nM (10000 to 3.2 ng / mL for peptides) for 1 hour, or with sulfated peptides ranging from 100 ng / mL to 0.032 ng / mL. Yeast cells were washed twice with PBSF buffer (PBS, BSA 0.1%).

[0341] Yeast cells were then incubated on ice for 15 minutes with a fluorescent reporter, using streptavidin-PE (for peptide binding) and a combination of anti-V5 and anti-mouse-FITC (for Fab expression). Cells were washed once and resuspended in PBSF buffer.

[0342] The yeast cells were then analyzed using a Cytoflex flow cytometer (Beckman Coulter Life Sciences).

[0343] Thermal stability determination method.

[0344] Yeast expressing WT SynCCR8 (SEQ ID NO: 1) and selected Fab candidates (including Fab11 (including the heavy chain variable region of SEQ ID NO: 22 and the light chain variable region of SEQ ID NO: 29), or Fab14 (including the heavy chain variable region of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 29), or Fab15 (including the heavy chain variable region of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 30), or Fab17 (including the heavy chain variable region of SEQ ID NO: 24 and the light chain variable region of SEQ ID NO: 29))) were subjected to Fab expression. 5 × 10⁶ cells expressing WT SynCCR8 were used for Fab expression. 5 One yeast cell and 5 × 10⁸ cells expressing humanized Fab. 5 Mix the yeast cells and wash twice with PBSF buffer (PBS, BSA 0.1%).

[0345] Incubate the yeast at 20 or 60°C for 10 minutes and place it directly on ice. Then incubate the yeast cells with 30 ng / ml of biotinylated sulfated peptide for 2 hours.

[0346] Yeast cells were then incubated on ice for 15 minutes with a fluorescent reporter, using a combination of streptavidin PE (for peptide binding) and anti-V5 and anti-mouse-FITC (for Fab expression). Cells were washed once and resuspended in PBSF buffer.

[0347] Yeast cells were then analyzed on a Cytoflex flow cytometer (Beckman Coulter Life Sciences). The percentage of remaining bound fluorescence was calculated as ((PE / FITC at 60°C) / (PE / FITC at 20°C) × 100).

[0348] result

[0349] Both light and heavy chains were expressed and displayed on the yeast surface. Strong binding signals were further detected on KLH peptides (affinity) and sulfonated peptides (affinity), while no binding was observed on KLH alone (data not shown).

[0350] Therefore, the yeast-based assay protocol, which includes all humanized conjugates tested, is validated.

[0351] Example X: Humanized Fab retains functional binding properties against sulfated CCR8-derived peptides

[0352] The same yeast-based protocol was followed for each humanized conjugate. In short, five humanized heavy chain variable chains were tested in combination with four humanized light chain variable chains. Expression of each test conjugate candidate and binding fluorescence intensity for assessing ligand binding were measured in the presence of sulfated peptides.

[0353] The humanized clones tested are presented below.

[0354]

[0355] Table 7: A comprehensive chart including WT and variant Fab (i.e., from Fab1 to Fab21) and their corresponding variable regions, and the estimated percentage of humanization relative to the reference hominid.

[0356] The variable regions reported in this article correspond to SEQ ID Nos. 21 to 31.

[0357] Overall, the humanized Fab variants showed variable expression levels in yeast-based assays, including three sets of experiments, as reported in Figure 6.

[0358] In summary, the proposed construct was effectively expressed on the yeast surface, although the expression of the conjugate including the light chain LB (SEQ ID NO28) was relatively low.

[0359] Based on the results of the conjugate including the heavy chain HF (i.e., Fab1), a slight effect of deamination on binding affinity was found. The heavy chain HF (SEQ ID NO 26) is characterized by the N-to-D mutation in the H-CDR2 region.

[0360] Based on the results of the conjugate including the light chain LF (i.e., Fab2), no effect of glycosylation on antigen binding was found. The light chain LF (SEQ ID NO 31) is characterized by an N-to-D mutation in the FR3 frame region.

[0361] Overall, a group of humanized Fab conjugates were found to exhibit similar antigen affinity when compared with the original (WT) mouse conjugates.

[0362] Interestingly, it was further discovered that some of these conjugates exhibited improved thermal stability compared to the original (WT) mouse conjugates. This was particularly true for the Fab15 and Fab17 conjugates.

[0363] In conclusion, the humanization steps are applicable to the disclosed antigen-binding fragments, specifically the CCR8-derived peptide.

[0364] sequence list

[0365] The sequences reported in this specification are summarized below according to their respective numbers SEQ ID NO X.

[0366] 。

Claims

1. An isolated antibody or antigen-binding fragment thereof, said isolated antibody or antigen-binding fragment thereof binding to chemokine (CC motif) receptor 8 (CCR8), wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), said heavy chain variable region comprising three complementarity-determining regions (CDRs): - VH-CDR1: SEQ ID NO: 10, - VH-CDR2: GINPKX1GVTRYNQKFKG, where X1 is any amino acid (SEQ ID NO: 38), and - VH-CDR3: SEQ ID NO: 12, The heavy chain variable region (VH) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 21 to 26; Furthermore, the antibody or its antigen-binding fragment further includes a light chain variable region (VL), which comprises the following three complementarity-determining regions (CDRs): - VL-CDR1: SEQ ID NO: 13, - VL-CDR2: SEQ ID NO: 14, and - VL-CDR3: SEQ ID NO: 15 The light chain variable region (VL) has at least 80% sequence identity with the polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

2. The isolated antibody or its antigen-binding fragment according to claim 1, wherein VH-CDR2 is SEQ ID NO:

11.

3. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein: The VH is characterized in that the VH comprises a polypeptide sequence selected from the group consisting of SEQ ID NO 21 to 26; and / or The VL is characterized in that it comprises a polypeptide sequence selected from the group consisting of SEQ ID NO 27 to 31.

4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the isolated antibody or antigen-binding fragment thereof comprises: - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 21 (H-WT) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 25 (HE) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 27 (L-WT); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 28 (LB); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 26 (HF) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 31 (LF).

5. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the isolated antibody or antigen-binding fragment thereof comprises: - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 22 (HA) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD); or - The heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 23 (HC) and the light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 30 (LE).

6. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having at least 80% sequence identity with SEQ ID NO 24 (HD) and a light chain variable region (VL) having at least 80% sequence identity with SEQ ID NO 29 (LD).

7. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the isolated antibody or antigen-binding fragment thereof is monoclonal.

8. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the isolated antibody or antigen-binding fragment thereof recognizes an epitope consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a post-transcriptionally modified form thereof; particularly its sulfated or sulfonated form.

9. A nucleic acid encoding an isolated antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid according to claim 9.

11. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, a nucleic acid according to claim 9 or a carrier according to claim 10, and at least one pharmaceutically acceptable excipient.

12. The pharmaceutical composition of claim 11 further comprises at least one additional therapeutic agent, preferably a programmed cell death protein 1 (PD-1) inhibitor.

13. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the nucleic acid according to claim 9, the carrier according to claim 10, or the pharmaceutical composition according to any one of claims 11 or 12, for use as a pharmaceutical remedy.

14. Use of the isolated antibody or its antigen-binding fragment, nucleic acid, carrier or pharmaceutical composition according to claim 13 in methods for treating and / or preventing and / or reducing the likelihood of cancer.

15. The isolated antibody or antigen-binding fragment thereof, nucleic acid, carrier or pharmaceutical composition for use according to claim 14, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, kidney cancer and sarcoma.

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