Anti-CCR8 Antibodies and Their Uses

Abstract

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human CCR8. The present invention also provides a polynucleotide encoding the antibody or antigen-binding fragment thereof, a vector comprising the polynucleotide, a host cell comprising the vector, a method for generating the antibody, and a composition comprising the antibody.

Description

Technical Field

[0001] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human CCR8. The present invention also provides a polynucleotide encoding the antibody or the antigen-binding fragment thereof, a vector comprising the polynucleotide, a host cell comprising the vector, a method for generating the antibody, and a composition comprising the antibody. Background Art

[0002] CCR8 (C-C motif chemokine receptor 8) is mainly expressed on T reg cells and Th2 cells, but not on Th1 cells. It has been demonstrated that CD4 + Foxp3 + T reg cells (CCR8 + T reg cells) subset is a major driver of immunosuppression and is crucial for T reg function and suppression. In addition, CCR8 is a specific marker selectively upregulated on tumor-resident T reg cells in several tumor types. Many reports have shown that the increase in CCR8 + T reg cells is beneficial for tumor escape mechanisms. Clinically, the increase in T reg cells in the tumor microenvironment of breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer, and many other cancer types is associated with poor prognosis. Mechanistically, T reg cells not only inhibit a broad range of anti-tumor immune responses but also promote the regeneration of blood vessels in the tumor microenvironment. Cancer cells and immune cells in the tumor microenvironment secrete CCL1 (a specific ligand of CCR8), thereby recruiting CCR8 + Treg cells into the tumor microenvironment. CCR8 also plays a role in the proliferation and expansion of T reg cells in the tumor microenvironment. It has been shown that CCR8 inhibitors can reduce tumor-infiltrating T reg cells, thereby preventing tumor growth. Therefore, CCR8 is considered a potential therapeutic target for cancer.

[0003] In summary, there is a need in the art for therapeutic agents comprising anti-CCR8 antibodies. Summary of the Invention

[0004] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human CCR8, meeting the needs of the art.

[0005] On the one hand, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human CCR8, which comprises: a heavy chain variable region (VH), the heavy chain variable region comprising CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6; and a light chain variable region (VL), the light chain variable region comprising CDR-L1 as shown in SEQ ID NO: 7, CDR-L2 as shown in SEQ ID NO: 8, and CDR-L3 as shown in SEQ ID NO: 9.

[0006] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: VH as shown in SEQ ID NO: 10 and VL as shown in SEQ ID NO: 11; or a VH having at least 95%, 96%, 97%, 98%, 99% sequence identity to the VH shown in SEQ ID NO: 10 and a VL having at least 95%, 96%, 97%, 98%, 99% sequence identity to the VL shown in SEQ ID NO: 11.

[0007] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises CDR-H1, CDR-H2, and CDR-H3 from the VH shown in SEQ ID NO: 10, and CDR-L1, CDR-L2, and CDR-L3 from the VL shown in SEQ ID NO: 11, for example, according to the Kabat definition, the Chothia definition, or the IMGT definition.

[0008] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain constant region as shown in SEQ ID NO: 14 and a light chain constant region as shown in SEQ ID NO: 15.

[0009] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises an alpha heavy chain, a delta heavy chain, an epsilon heavy chain, a gamma heavy chain, or a mu heavy chain. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region that is from an IgG1, IgG2, IgG3, or IgG4 subclass. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a light chain constant region that is from a lambda light chain or a kappa light chain. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a full-length antibody. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, and xFab. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a chimeric antibody or a humanized antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a multispecific antibody, such as a bispecific antibody.

[0010] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention has one or more of the following characteristics:

[0011] (1) It is capable of specifically binding to the huCCR8 protein;

[0012] (2) It is capable of specifically binding to cells expressing the huCCR8 protein, such as 293T cells;

[0013] (3) It is capable of specifically binding to 293T cells expressing the huCCR8 protein with an EC50 value less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6 or 1.55 nM;

[0014] (4) It is capable of specifically binding to the cynoCCR8 protein;

[0015] (5) It is capable of specifically binding to cells expressing the cynoCCR8 protein, such as 293T cells;

[0016] (6) It is capable of specifically binding to 293T cells expressing the cynoCCR8 protein with an EC50 value less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6 or 1.55 nM;

[0017] (7) It does not cross-react with the huCCR1 protein;

[0018] (8) It does not cross-react with cells expressing the huCCR1 protein, such as 293T cells;

[0019] (9) It does not cross-react with the huCCR4 protein;

[0020] (10) It does not cross-react with cells expressing the huCCR4 protein, such as 293T cells;

[0021] (11) It is capable of effecting ADCC;

[0022] (12) It is capable of effecting NK cell-mediated ADCC;

[0023] (13) It is capable of effecting NK cell-mediated ADCC with an EC50 value less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or 0.05 nM; and / or

[0024] (14) It is capable of effecting NK cell-mediated ADCC with an Emax value greater than 30, 35, 40, 45, 50, 55 or 60%.

[0025] On the one hand, the present invention provides a polynucleotide that encodes an antibody of the present invention or an antigen-binding fragment thereof. In one embodiment, the polynucleotide is codon-optimized for expression in mammals.

[0026] In one embodiment, the polynucleotide of the present invention comprises: SEQ ID NO: 12 and / or 13.

[0027] On the one hand, the present invention provides a vector that contains the polynucleotide of the present invention. In one embodiment, the vector is a plasmid, cosmid, phage, phagemid, or virus.

[0028] On the one hand, the present invention provides a host cell that contains the polynucleotide or vector of the present invention. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a CHO cell. In one embodiment, the host cell is a prokaryotic cell. In one embodiment, the host cell is Escherichia coli.

[0029] On the one hand, the present invention provides a method for generating an antibody or an antigen-binding fragment thereof, which includes: (a) culturing the host cell of the present invention under conditions suitable for expressing the antibody or the antigen-binding fragment thereof, and (b) optionally, recovering the antibody or the antigen-binding fragment thereof.

[0030] On the one hand, the present invention provides a conjugate that contains the antibody of the present invention or an antigen-binding fragment thereof. In one embodiment, the conjugate further contains an effector molecule. In another embodiment, the conjugate further contains a linker that connects the antibody or antigen-binding fragment to the effector molecule.

[0031] On the one hand, the present invention provides a composition that contains the antibody of the present invention or an antigen-binding fragment thereof, the polynucleotide of the present invention, the vector of the present invention, the host cell of the present invention, or the conjugate of the present invention.

[0032] On the one hand, the present invention provides the antibody of the present invention or an antigen-binding fragment thereof, the polynucleotide of the present invention, the vector of the present invention, the host cell of the present invention, the conjugate of the present invention, or the composition of the present invention for use as a medicament.

[0033] On the one hand, the present invention provides the antibody of the present invention or an antigen-binding fragment thereof, the polynucleotide of the present invention, the vector of the present invention, the host cell of the present invention, the conjugate of the present invention, or the composition of the present invention for the treatment of CCR8-related diseases.

[0034] On the one hand, the present invention provides the use of the antibody of the present invention or an antigen-binding fragment thereof, the polynucleotide of the present invention, the vector of the present invention, the host cell of the present invention, the conjugate of the present invention, or the composition of the present invention in the preparation of a medicament for the treatment of CCR8-related diseases.

[0035] In one aspect, the present invention provides a method for treating CCR8-related diseases in an individual, which comprises administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof of the present invention, a polynucleotide of the present invention, a vector of the present invention, a host cell of the present invention, a conjugate of the present invention, or a composition of the present invention.

[0036] In one embodiment, the CCR8-related disease is cancer, such as cancer expressing CCR8. In one embodiment, the cancer is head and neck cancer, nasopharyngeal cancer, colon cancer, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, colorectal cancer, esophageal cancer, ovarian cancer, liver cancer, non-small cell lung cancer, or small cell lung cancer. In another embodiment, a CCR8 antibody or an antigen-binding fragment thereof is administered in combination with other therapeutic agents, such as a PD-1 antibody (e.g., Tislelizumab), to treat CCR8-related diseases.

[0037] In one aspect, the present invention provides a method for detecting CCR8 in a biological sample, which comprises contacting the biological sample with an antibody or an antigen-binding fragment thereof of the present invention or a conjugate of the present invention.

[0038] In one aspect, the present invention provides a method for diagnosing CCR8-related diseases in an individual, which comprises contacting a biological sample from the individual with an antibody or an antigen-binding fragment thereof of the present invention or a conjugate of the present invention.

[0039] In one aspect, the present invention provides a kit, which comprises a container containing an antibody or an antigen-binding fragment thereof of the present invention or a conjugate of the present invention. Brief Description of the Drawings

[0040] Figure 1 Shows NK cell-mediated cytotoxicity by the anti-human CCR8 chimeric antibody, Ch401. Among them, primary human NK cells were used as effector cells, and CHO-K1-NanoLuc-CCR8 was used as target cells to measure NK cell-mediated cytotoxicity. Detailed Description

[0041] Terms

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meanings set forth in the specification.

[0043] It must be noted that, unless the context clearly dictates otherwise, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents.

[0044] Unless otherwise stated, any numerical values (such as concentrations or concentration ranges described herein) should be understood to be modified by the term "about" in all cases. Thus, numerical values generally include ±10% of the recited value. For example, a concentration of 1 mg / mL includes from 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes from 0.9% (w / v) to 11% (w / v). Unless the context clearly dictates otherwise, as used herein, the use of a numerical range expressly includes all possible sub-ranges, all individual numerical values within the range, including integers and fractions of such values within the range.

[0045] Unless otherwise indicated, the term "at least" before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be covered by the present invention.

[0046] As used herein, the terms "comprising", "including", "having", or "containing" or any other variation thereof will be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers, and is intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that comprises a series of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" means an inclusive or rather than an exclusive or. For example, condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0047] As used herein, the conjunctive term "and / or" between a plurality of recited elements is understood to cover both the separate and the combined options. For example, in the case where two elements are joined by "and / or", the first option means that the first element applies and the second element does not apply. The second option means that the second element applies and the first element does not apply. The third option means that the first element and the second element apply together. Any one of these options is understood to fall within the meaning, and thus satisfies the requirements of the term "and / or" as used herein. More than one of the said options applying simultaneously is also understood to fall within the meaning, and thus satisfies the requirements of the term "and / or".

[0048] As used herein, the term "consisting of" or variations thereof as used throughout the specification and claims indicates the inclusion of any recited integer or group of integers, but no additional integer or group of integers may be added to the specified method, structure, or composition.

[0049] As used herein, the term "consisting essentially of" or variations thereof, as used throughout the specification and claims, indicates the inclusion of any recited integer or group of integers, and optionally any recited integer or group of integers that do not materially alter the basic or novel characteristics of the specified method, structure, or composition.

[0050] As used herein, "subject" or "individual" means any animal, preferably a mammal, and most preferably a human. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., and more preferably humans.

[0051] It should also be understood that the terms "about," "approximately," "substantially," "essentially," and similar terms, as used herein in reference to dimensions or characteristics of components of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references to numerical parameters will include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0052] In the context of two or more nucleic acid or polypeptide sequences (e.g., anti-CCR8 antibodies and polynucleotides encoding them, CCR8 polypeptides and CCR8 polynucleotides encoding them), the term "identical" or "identity" percentage refers to the percentage of amino acid residues or nucleotides that are the same or have a specified percentage of identity when two or more sequences or subsequences are compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0053] For sequence comparison, typically one sequence is used as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage of sequence identity of one or more test sequences relative to the reference sequence based on the specified program parameters.

[0054] Optimal alignments of sequences can be performed for comparison, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package of the Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Current Protocols in Molecular Biology, F. M. Ausubel et al. eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (suppl. 1995) (Ausubel)).

[0055] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. Then the word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased.

[0056] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatch residue; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of a word hit in each direction is stopped when the cumulative alignment score drops by the quantity X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negatively scored residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands as the default settings. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix as the default settings (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0057] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences occurred by chance. For example, if the minimum sum probability in a comparison of a test nucleic acid with a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0058] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, where a polypeptide and a second polypeptide differ only by conservative substitutions, the two peptides are generally substantially identical. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0059] The term "polynucleotide" as used herein is defined as a chain of nucleotides. In addition, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the common knowledge that a nucleic acid is a polynucleotide and that a polynucleotide can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means (i.e., using conventional cloning techniques and PCR) TMetc., cloning nucleic acid sequences from recombinant libraries or cellular genomes) and by synthetic means.

[0060] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains that are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, as well as long chains of many types that are commonly referred to in the art as proteins. "Polypeptide" includes, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0061] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy chain and / or light chain that binds to a target antigen. In a particularly preferred embodiment described herein, the antigen is C-C motif chemokine receptor 8 (CCR8). In this regard, the antigen-binding fragment of the antibody described herein may contain one, two, three, four, five, or all six CDRs of the V H and / or V L sequences from an antibody that binds CCR8. The antigen-binding fragment of the CCR8-specific antibody described herein is capable of binding to CCR8. In other embodiments, the binding of the antigen-binding fragment prevents or inhibits the binding of one or more CCR8 ligands to the CCR8 receptor, thereby interrupting the biological response that would otherwise be caused by the binding of the ligand to the receptor. In certain embodiments, the antigen-binding fragment specifically binds to CCR8 and / or inhibits or regulates the biological activity of CCR8.

[0062] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binding agent, such as an antibody, and can additionally be used in an animal to generate an antibody capable of binding to an epitope of the antigen. An antigen can have one or more epitopes.

[0063] The term "epitope" includes any determinant capable of specific binding to an immunoglobulin or a T cell receptor, preferably a polypeptide determinant. An epitope is the antigenic region that is bound by an antibody. In certain embodiments, the epitope determinant includes the chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural features and / or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. According to certain embodiments, when the equilibrium dissociation constant of antibody-antigen binding is less than or equal to 10 -6 M or less than or equal to 10 -7 M or less than or equal to 10 -8 M, it can be said that the antibody specifically binds the antigen. In some embodiments, the equilibrium dissociation constant can be less than or equal to 10 - 9 M or less than or equal to 10 -10 M.

[0064] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid or virus) used to transfer coding information into a host cell. The term "expression vector" refers to a vector that is suitable for transforming a host cell and contains a nucleic acid sequence that directs and / or controls the expression of an inserted heterologous nucleic acid sequence. Expression includes but is not limited to processes such as transcription, translation and RNA splicing (if introns are present).

[0065] C-C motif chemokine receptor 8 (CCR8)

[0066] CCR8 (formerly also known as Cy6, CKR-L1 or TER1) is a G protein-coupled 7-transmembrane CC chemokine receptor protein expressed in the thymus, spleen, etc. The gene encoding this protein is located on human chromosome 3p21. "Human CCR8" consists of 355 amino acids, and its amino acid sequence is as shown in SEQ ID NO:1. CCL1 is known to be an endogenous ligand of CCR8. Human CCR8 cDNA consists of the nucleotide sequence represented by GenBank ACC number M_005201.3, and mouse CCR8 cDNA consists of the nucleotide sequence represented by GenBank ACC number NM_007720.2.

[0067] The CCR8 of the present invention includes those derived from mice, rats, hamsters, guinea pigs, dogs, pigs and primate mammals (including monkeys and humans). Human CCR8 is preferred.

[0068] Antibody

[0069] The present invention generally relates to isolated anti-CCR8 antibodies, nucleic acids encoding said antibodies and expression vectors, recombinant cells containing said vectors, and compositions comprising said antibodies. Also provided are methods for preparing said antibodies and methods for using said antibodies to treat diseases including cancer. The antibodies of the present invention have one or more desired functional properties, including but not limited to high affinity binding to CCR8, high specificity for CCR8, and the ability to inhibit tumor growth in a subject in need and in animal models when administered alone or in combination with other anti-cancer therapies.

[0070] In one general aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds CCR8.

[0071] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules, including monoclonal or polyclonal human antibodies, humanized antibodies, bispecific antibodies, and chimeric antibodies, as well as antibody fragments. Generally speaking, an antibody is a protein or polypeptide chain that exhibits binding specificity for a specific antigen. Antibody structures are well-known. Immunoglobulins can be classified into five main classes (i.e., IgA, IgD, IgE, IgG, and IgM) according to the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, the antibodies of the present invention can belong to any of the five main classes or corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of the constant domain, the antibody light chains of vertebrate species can be classified into one of two distinct types (i.e., κ and λ). Thus, the antibodies of the present invention can contain κ or λ light chain constant domains. According to certain embodiments, the antibodies of the present invention include heavy and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light chain constant domains, an antibody also contains an antigen-binding region composed of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains can alternatively be referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains can alternatively be referred to as HCDR1, HCDR2, and HCDR3.

[0072] In addition, the scope of the antibodies of the present disclosure also includes various forms, such as antibody fragments selected from Fv, scFv, Fab, Fab', F(ab')2, and xFab, etc. The scope of the antibodies of the present disclosure also includes their derivative forms, such as bispecific antibodies, or antibody derivatives in which the antibody is further linked to other reagents, such as antibody-drug conjugates (ADCs), etc.

[0073] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CCR8 is substantially free of antibodies that do not bind to CCR8). Additionally, an isolated antibody is substantially free of other cellular materials and / or chemicals.

[0074] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody comprising the population is identical, except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the invention can be prepared by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal (such as a transgenic mouse or rat) having a genome comprising a human heavy chain transgene and a light chain transgene.

[0075] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv1), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from antibody moieties comprising one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parental antibody or parental antibody fragment binds. According to embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and the Fd segment of a heavy chain. According to other embodiments, the antigen-binding fragment comprises Fab and F(ab').

[0076] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that comprises a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region or comprises only a heavy chain variable region.

[0077] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human using any technique known in the art. This definition of a human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide.

[0078] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase its sequence homology with a human antibody such that the antigen-binding properties of the antibody are retained, but its antigenicity in humans is reduced.

[0079] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable regions of the light and heavy chains generally correspond to the variable regions of an antibody having the desired specificity, affinity, and capacity from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions correspond to the sequences of an antibody from another mammalian species (e.g., human) to avoid eliciting an immune response in that species.

[0080] As used herein, the term "multispecific antibody" refers to an antibody that contains multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the multiple has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the multiple has binding specificity for a second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen (e.g., the same protein (or subunit of a multimeric protein)). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope do not overlap or substantially do not overlap. In one embodiment, the first epitope and the second epitope are on different antigens (e.g., different proteins (or different subunits of a multimeric protein)). In one embodiment, the multispecific antibody contains a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetravalent antibody molecule.

[0081] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or two antigens. A bispecific antibody is characterized in that a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen (e.g., the same protein (or subunit of a polyprotein)). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope are on different antigens (e.g., different proteins (or different subunits of a polyprotein)). In one embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a half antibody or a fragment thereof that has binding specificity for a first epitope and a half antibody or a fragment thereof that has binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a scFv or a fragment thereof that has binding specificity for a first epitope and a scFv or a fragment thereof that has binding specificity for a second epitope. In one embodiment, the first epitope is located on CCR8, and the second epitope is located on PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD47, CD73, apelin, DLL3, Claudin 18.2, TIPE-1, CD3, and / or other tumor-associated immunosuppressive factors or surface antigens.

[0082] As used herein with respect to antibodies, the term "specifically binds" means an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species may also bind that antigen from one or more species. However, such cross-species reactivity per se does not alter the specific classification of the antibody. In another example, an antibody that specifically binds an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity per se does not alter the specific classification of the antibody. In some cases, the term "specifically binds" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; e.g., an antibody recognizes and binds to a specific protein structure, rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0083] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include sets of heavy-chain and light-chain CDRs inserted respectively between groups of heavy-chain and light-chain framework regions (FRs) that provide support for the CDRs and define the spatial relationship of the CDRs relative to one another. As used herein, the term "set of CDRs" refers to the three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen-binding site includes six CDRs, including the set of CDRs from each of the heavy-chain and light-chain V regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analysis of many antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs make extensive contacts with the bound antigen, with the most extensive antigen contacts being with the heavy-chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0084] As used herein, the term "set of FRs" refers to the four flanking amino acid sequences that are the framework for the set of CDRs of the heavy or light chain V region. Some FR residues can contact the bound antigen; however, the FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues immediately adjacent to the CDRs. Within the FRs, certain amino residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are shown to form protruding loop motifs that make up the antigen-binding surface. It is generally recognized that there are conserved structural regions of the FRs that influence the folding of the CDR loops into certain "canonical" structures, regardless of the precise CDR amino acid sequence. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction between the heavy and light chains of the antibody.

[0085] The structure and location of the immunoglobulin variable regions can be determined by reference to: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, 1987 and updates thereof (now available on the Internet (immuno.bme.nwu.edu)), Chothia, AbM, and IMGT (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme). The definition of the antigen-binding site is also described in: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).For example, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31 - 35 (HCDR1), 50 - 65 (HCDR2), and 95 - 102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24 - 34 (LCDR1), 50 - 56 (LCDR2), and 89 - 97 (LCDR3). According to IMGT, the CDR amino acid residues in VH are numbered approximately 26 - 35 (HCDR1), 51 - 57 (HCDR2), and 93 - 102 (HCDR3), and the CDR amino acid residues in VL are numbered approximately 27 - 32 (LCDR1), 50 - 52 (LCDR2), and 89 - 97 (LCDR3) (according to Kabat numbering). According to IMGT, the program IMGT / DomainGap Align can be used to determine the CDR regions of an antibody. Unless otherwise specified, the positions of the CDRs and framework regions disclosed herein are determined according to the IMGT numbering scheme.

[0086] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in their naturally occurring conformation in all antibody molecules. Heavy chains from any vertebrate species can be grouped into one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated as α, δ, ε, γ, and μ, respectively. Based on sequence and functional differences, the IgG and IgA classes are further divided into subclasses. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0087] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains that exist in their naturally occurring conformation in all antibody molecules. κ and λ light chains refer to the two major antibody light chain isotypes.

[0088] As used herein, the term "synthetic antibody" means an antibody produced using recombinant DNA techniques, such as an antibody expressed by a phage as described herein. The term should also be interpreted to mean an antibody produced by synthesizing a DNA molecule encoding the antibody (and the DNA molecule expresses the antibody protein) or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0089] The antibodies of the present invention can be fused to additional proteins at their N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274 - 1281). The proteins to be fused can be appropriately selected by those skilled in the art.

[0090] In a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. The polypeptide sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 4, 5, 6, 7, 8, and 9, respectively, wherein the antibody or antigen-binding fragment thereof specifically binds to CCR8, preferably human CCR8, and wherein the positions of the CDRs are determined according to the Kabat numbering scheme.

[0091] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having a polypeptide sequence with at least about 85%, preferably at least about 90%, more preferably at least about 95% or more (such as 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 10, or a light chain variable region having a polypeptide sequence with at least about 85%, preferably at least about 90%, more preferably at least about 95% or more (such as 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 11.

[0092] In the present invention, the antibodies of the present invention also include conservative variants thereof, which means that compared with the amino acid sequence of the antibody of the present invention, up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids having similar or similar properties to form a polypeptide. Preferably, these conservative variant polypeptides are produced by amino acid substitutions according to Table A.

[0093] Table A

[0094]

[0095]

[0096] The present invention relates to an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0097] Polynucleotides, vectors, host cells, and preparation methods

[0098] The present invention also provides polynucleotides encoding any of the antibodies disclosed herein. In some embodiments, isolated polynucleotides encoding an antibody or antibody fragment that binds to CCR8 are disclosed herein, wherein the antibody or antibody fragment comprises three light chain CDRs having the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9; and / or three heavy chain CDRs having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6. In some embodiments, isolated polynucleotides encoding an antibody or antibody fragment that binds to CCR8 are disclosed herein, wherein the antibody or antibody fragment comprises three light chain CDRs having the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9; and three heavy chain CDRs having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6. In some embodiments, isolated polynucleotides encoding an antibody or antibody fragment that binds to CCR8 are disclosed herein, wherein the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region having a polypeptide sequence selected from: SEQ ID NOs: 10 and 11.

[0099] The present invention also provides vectors comprising an isolated nucleic acid molecule encoding a monoclonal antibody of the invention or an antigen-binding fragment thereof. Any vector known to those skilled in the art in view of the present disclosure can be used, such as plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any element that performs the conventional functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Many expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce an antibody or an antigen-binding fragment thereof in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate the recombinant expression vectors according to the embodiments of the present invention. Such techniques are well known to those skilled in the art in view of the present disclosure.

[0100] The present invention also provides host cells, which contain an isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. Any host cell known to those skilled in the art in view of the present disclosure can be used for the recombinant expression of the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is Escherichia coli (E. coli) TG1 or BL21 cells (for expressing, for example, scFv or Fab antibodies), CHO-DG44 cells, 293F cells, CHO-K1 cells or HEK293 cells (for expressing, for example, full-length IgG antibodies). According to the embodiment, the recombinant expression vector is transformed into the host cell by conventional methods (such as chemical transfection, heat shock or electroporation), in which it is stably integrated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0101] The present invention also provides a method for producing the monoclonal antibody or antigen-binding fragment thereof of the present invention, the method comprising culturing a cell containing a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions for producing the monoclonal antibody or antigen-binding fragment thereof of the present invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (for example, from the supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cell and purified according to conventional techniques known in the art and as described herein.

[0102] As will be understood by those skilled in the art, polynucleotides can include genomic sequences, episomal and plasmid-encoded sequences, and smaller engineered gene segments that express or are adapted to express proteins, polypeptides, peptides, etc. Such segments can be naturally isolated or synthetically modified by those skilled in the art.

[0103] As will also be recognized by those skilled in the art, polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules can include hnRNA molecules containing introns and corresponding to DNA molecules in a one-to-one manner and mRNA molecules not containing introns. Additional coding or non-coding sequences can, but need not, be present within the polynucleotides according to the present disclosure, and the polynucleotides can, but need not, be linked to other molecules and / or support materials. Polynucleotides can contain native sequences or can contain sequences encoding variants or derivatives of such sequences.

[0104] Generally, polynucleotide variants will contain one or more substitutions, additions, deletions and / or insertions, preferably such that the binding affinity of the antibody encoded by the variant polynucleotide is not substantially diminished relative to the antibody encoded by the polynucleotide sequence specifically set forth herein.

[0105] The polynucleotides or fragments thereof described herein (regardless of the length of the coding sequence itself) can be combined with other DNA sequences such as promoters, polyadenylation signals, additional restriction enzyme sites, polylinker sites, other coding regions, etc., such that their total lengths can vary widely. Thus, nucleic acid fragments of almost any length are contemplated, with the total length preferably being limited by ease of preparation and use in the intended recombinant DNA protocols. For example, illustrative polynucleotide segments of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, etc. (including all intermediate lengths) are contemplated to be useful.

[0106] Site-directed mutagenesis allows the generation of mutants by using a specific oligonucleotide sequence encoding the desired mutation and a sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence complexity to form stable duplexes on both sides of the deletion junction being traversed. Mutations can be introduced into the selected polynucleotide sequence to improve, alter, reduce, modify, or otherwise change the properties of the polynucleotide itself, and / or the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0107] Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0108] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. In preferred embodiments, such cells are human cells. While not wishing to be limited to any particular mechanism of action, these cytotoxic cells mediating ADCC generally express Fc receptors (FcR). The primary cell NK cells that mediate ADCC express FcγRIII, while monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. The expression of FcR on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To assess the ADCC activity of a molecule, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively or additionally, the ADCC activity of a target molecule can be evaluated in vivo (e.g., in an animal model such as the animal model disclosed in Clynes et al., PNAS (USA), 95:652-656 (1998)).

[0109] "Effector cells" are white blood cells that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRI, FcγRII, FcγRIII, and / or FcγRIV and perform ADCC effector functions. Examples of human white blood cells that mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells, and neutrophils; of which PBMCs and NK cells are preferred. In a preferred embodiment, the effector cells are human cells.

[0110] The terms "Fc receptor" or "FcR" are used to describe receptors that bind to the Fc region of an antibody. Preferred FcRs are natural sequence human FcRs. In addition, preferred FcRs are FcRs (γ receptors) that bind IgG antibodies and include receptors of the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See Annu.Rev.Immunol., 15:203-234 (1997)). FcRs are reviewed in Ravetech and Kinet, Annu.Rev.Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J.Lab.Clin.Med., 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn that is responsible for transferring maternal IgG to the fetus (Guyer et al., Immunol., 117:587 (1976) and Kim et al., J.Immunol., 24:249 (1994)).

[0111] Complement-dependent cytotoxicity (CDC)

[0112] Complement-dependent cytotoxicity (CDC) refers to the ability of a molecule to initiate complement activation and lysis of a target in the presence of complement. Complement activation pathways are initiated by binding the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed, such as that described by Gazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996).

[0113] Antibody-drug conjugate (ADC)

[0114] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody according to the present invention.

[0115] Generally, an antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, and chemical conjugation is preferred. Preferably, the effector molecule is a therapeutically active drug. Additionally, the effector molecule can be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0116] The antibody and the effector molecule according to the present invention can be conjugated by a coupling agent. Examples of coupling agents can be any one or more of a non-selective coupling agent, a coupling agent utilizing carboxyl groups, a peptide chain, and a coupling agent utilizing disulfide bonds. A non-selective coupling agent is a compound that causes a covalent bond connection between the effector molecule and the antibody, such as glutaraldehyde, etc. The coupling agent utilizing carboxyl groups can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and a hydrazone coupling agent (the coupling site is a hydrazone).

[0117] Certain residues on the antibody (such as Cys or Lys, etc.) are used to link a variety of functional groups, including imaging agents (such as chromophores and fluorophores), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as poly(ethylene glycol)), and therapeutic agents. The antibody can be conjugated with a functional agent to form an antibody-functional agent conjugate. The functional agent (e.g., a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly linked to the antibody via a linker.

[0118] Antibodies can be conjugated with drugs to form antibody-drug conjugates (ADCs). Generally, an ADC contains a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Generally, a degradable linker is prone to degradation in the intracellular environment. For example, the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-based linkers that can be degraded by proteases in cells (e.g., lysosomal proteases or endosomal proteases); or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptide-based linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers generally release the drug under conditions where the antibody is hydrolyzed by proteases.

[0119] Before being linked to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the linkage is achieved through the reactive group. Thiol-specific reactive groups are preferred, which include, for example, maleimide compounds, halogenated (e.g., iodine-, bromine-, or chlorine-substituted) amides, halogenated (e.g., iodine-, bromine-, or chlorine-substituted) esters, halogenated (e.g., iodine-, bromine-, or chlorine-substituted) methyl ketones, benzyl halides (e.g., iodide, bromide, or chloride), vinyl sulfones, pyridyl disulfides, mercury derivatives (such as 3,6-di-(mercurimethyl) dioxane, where the counterion is CH3COO - 、Cl - or NO3 - ) and polymethylene dimethyl sulfide thiosulfonate. The linker can include, for example, a maleimide linked to the antibody via a thiosuccinimide.

[0120] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In one embodiment, the antibody is linked to the drug via a linker, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group that can form a bond with the linker. When the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0121] Useful drugs include, for example, tubulin-binding drugs, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors; typical cytotoxic drugs include, for example, auristatin, camptothecin, docamycin / duocarmycin, etoposide, maytansine and maytansine-like compounds (e.g., DM1 and DM4), taxanes, benzodiazepines or drugs containing benzodiazepines (e.g., pyrrolo[1,4]benzodiazepine (PBD), indolinobenzodiazepine, and oxazolidinobenzodiazepine), and vinca alkaloids.

[0122] In the present invention, the drug-linker can be used to form an ADC in a simple step process. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step process. For example, in a first step, a cysteine residue is reacted with a reactive moiety of the linker, and then in a subsequent step, a functional group on the linker is reacted with the drug to form an ADC.

[0123] Generally, the functional groups on the linker are selected such that they can specifically react with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with a reactive alkyne group on the drug moiety. The drug is covalently linked to the linker through a 1,3-dipolar cycloaddition between the azide and the alkyne. Other useful functional groups include, for example, ketones and aldehydes (suitable for reacting with hydrazides and alkoxyamines); phosphine compounds (suitable for reacting with azides); isocyanates and isothiocyanates (suitable for reacting with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reacting with amines and alcohols). These and other ligation strategies (e.g., those described in Bioconjugation Technology (2nd Edition (Elsevier))) are well known to those skilled in the art. Those skilled in the art can understand that when selecting a complementary pair of reactive functional groups for the selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for the linker and can also be used for the drug.

[0124] The present invention further provides a method for preparing an ADC, which may further include: binding an antibody to a drug-linker compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0125] In certain embodiments, the methods according to the invention include: binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods according to the invention further include: binding the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0126] In some embodiments, an antibody-drug conjugate (ADC) has the following formula: Ab-(L-D) p ,

[0127] Where:

[0128] Ab is an antibody;

[0129] L is a linker;

[0130] D is a drug; and

[0131] p is a payload, selected from values of 1 to 8 (e.g., integers).

[0132] Drug Compositions and Other Uses

[0133] CCL1 / CCR8 signaling is an important pathway in the pathogenesis of several diseases including cancer, inflammatory diseases, and diabetic neuropathy. In particular, the antibodies described herein bind specifically to CCR8 with unexpectedly high affinity. CCR8 is known + T reg cells are beneficial for tumor escape mechanisms. In some aspects, provided herein are methods for reducing the number or activity of tumor-infiltrating regulatory T cells (TITRs) in a tumor present in a subject by inhibiting immunosuppression mediated by CCR8 + T reg cells and the like, and drug compositions for treating cancer via such a mechanism. Such cancers include, but are not limited to, breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer, pancreatic cancer, and many other cancer types associated with poor prognosis. In some aspects, provided herein are methods for increasing the amount of T effector cells in a subject's tumor by administering an anti-CCR8 antibody to the subject. The cytotoxicity can be antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The agent can be an antibody (e.g., a peptide) small molecule, protein-drug conjugate, or interfering nucleic acid. Additionally, the present invention can also modulate the CCL1 / CCR8 axis, thereby potentially treating diseases such as diabetic neuropathy, spinal cord injury, and IgG4-related diseases (such as sclerosing cholangitis (ISC)). Illustrative amino acid sequences of antibodies or antigen-binding fragments thereof or their complementarity-determining regions (CDRs).

[0134] The present invention also provides a pharmaceutical composition, which comprises the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" means a product comprising the active ingredient of the present invention and a pharmaceutically acceptable carrier, wherein the active ingredient is selected from: the isolated monoclonal antibody or antigen-binding fragment thereof in the first aspect, the recombinant protein in the second aspect, the isolated nucleic acid (especially DNA or RNA) in the third aspect, the vector in the fourth aspect, the antibody conjugate in the sixth aspect, the immune cell in the seventh aspect, or a combination thereof. The active ingredients of the present invention and the compositions containing them can also be used for manufacturing drugs for the therapeutic applications mentioned herein.

[0135] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0136] The amount administered will depend on variables such as the type and extent of the disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, the serum half-life of the antibody, and the route of administration.

[0137] The frequency of administration can vary depending on factors such as the route of administration, the dose, the serum half-life of the antibody or fusion protein, and the disease being treated.

[0138] In some embodiments, the antibodies of the present invention are used for non-therapeutic purposes, such as diagnostic tests and assays. For example, the antibodies can be used to determine the CCR8 level in a sample from a subject. A method is provided by contacting a sample with a CCR8-specific antibody of the present invention and detecting the immunoreactivity between the antibody and CCR8 in the sample.

[0139] Detection applications and kits

[0140] The antibody or its ADC according to the present invention can be used in detection applications, for example, for use in a test sample to provide diagnostic information.

[0141] In the present invention, the specimens (samples) used include cells, tissue samples, and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Thus, the biopsy specimens used in the present invention can include, for example, excised samples of tumors and tissue samples prepared by endoscopic methods or puncture or needle biopsies of organs.

[0142] The samples used in the present invention include fixed or preserved cell or tissue samples.

[0143] The present invention further provides a kit, which only contains the antibody (or its fragment) according to the present invention; in a preferred example of the present invention, the kit further includes a container, an instruction manual, a buffer, etc. In a preferred example, the antibody according to the present invention can be immobilized on a test plate.

[0144] According to another aspect of the present invention, a CCR8-mediated disease in a subject is diagnosed by detecting the presence or amount of CCR8 protein in a sample.

[0145] The present invention provides a kit for predicting or diagnosing cancer prognosis, which contains an anti-CCR8 antibody. The kit of the present invention can further contain tools and / or reagents known in the art for ELISA. If necessary, the kit of the present invention can further contain tubes, well plates for mixing various components, an instruction manual describing how to use, etc.

[0146] Examples

[0147] The present invention is further described by referring to the following examples. It should be understood that the following examples are only used to describe the present invention, rather than limiting the scope of the present invention. The experimental methods without indicating specific conditions in the following examples are usually carried out according to conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer.

[0148] Example 1: Generation of mouse anti-CCR8 antibody

[0149] Mouse immunization

[0150] To generate antibodies against CCR8, a total of 50 adult female mice from different strains (BALB / c, C57BL / 6, SJL, and MRL / lpr) were immunized with different combinations of immunogens, including: plasmid expressing human CCR8, huCCR8 DNA; L929 cells overexpressing human CCR8, L929-huCCR8; and L929 cells overexpressing cynomolgus monkey CCR8, L929-cynoCCR8. The human, cynomolgus monkey, and mouse CCR8 protein sequences are shown as SEQ ID NO:1 to 3 in Table 1. As previously described (Jiang et al., J. Virol. 2017 Apr 13;91(9):e02052-16), the human CCR8 expression plasmid (huCCR8 DNA) (50 μg per mouse) was delivered intramuscularly by in vivo electroporation using an EPT-I delivery device (TERESA, Shanghai, China). L929-huCCR8 and L929-cynoCCR8 cells (5×10 6 cells per mouse) were injected intraperitoneally. Typically, 5 - 7 immunizations at three-week intervals were performed to induce a robust anti-CCR8 humoral immune response. Fourteen days after each immunization, blood was collected, and serum titers against 293T cells overexpressing human CCR8 (293T-huCCR8) and 293T cells overexpressing cynomolgus monkey CCR8 (293T-cynoCCR8) were determined by FACS. Animals with sufficient levels of anti-CCR8 serum antibodies were selected for final boost.

[0151] Screening of plasma cells with the Beacon Optofluidic system

[0152] Three to five days after the final boost, spleens were collected and mashed into single-cell suspensions. Plasma cells were isolated using a mouse CD138 positive selection kit (STEMCELL) according to the manufacturer's instructions. Enriched plasma cells at a density of 6.25×10 6 / ml were introduced into the channels and placed in the NanoPen chambers of an OptoSelect 14K chip (BerkeleyLights) according to the manufacturer's instructions. To screen for huCCR8-specific plasma cells, at a density of 1×10 8293T-huCCR9 cells at / ml and Alexa Fluor 647 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) at a concentration of 5 μg / ml were introduced into the channel. After introduction, the cryo-valve was opened, and the exposure time of the CY5 channel for the Alexa Fluor 647 fluorophore was set to 3000 ms. The pattern positive signal was captured by time-lapse imaging set for a 6-minute period and 10 cycles. After screening for huCCR8-specific plasma cells using 293T-huCCR8 cells, cynoCCR8-specific plasma cells were screened using 293T-cynoCCR8 cells. Plasma cells showing positive signals for both 293T-huCCR8 and 293T-cyoCCR8 cells were separately output into 96-well plates filled with lysis buffer.

[0153] Example 2: Antibody VH and VL Gene Cloning, Sequencing, and Chimeric Antibody Expression

[0154] According to the manufacturer's instructions, the first strand of cDNA was synthesized and total cDNA was amplified using the Opto Plasma B Discovery cDNA Synthesis Kit (Berkeley Lights). According to the manufacturer's instructions, the antibody VH and VL genes were amplified using the OptoPlasma B Discovery Sanger Prep Kit (Berkeley Lights). The amplified VH and VL genes were separately cloned into mammalian expression vectors containing the human IgG1 and κ constant region genes. The amino acid sequences of the 3 HCDRs, 3 LCDRs, VH, and VL of the chimeric antibody Ch401, as well as the DNA sequences of VH and VL, are shown as SEQ ID NO:4-13 in Table 1. The chimeric antibody was expressed using Expi293 TM cells and purified by affinity chromatography.

[0155] Table 1: Sequences

[0156]

[0157]

[0158] Example 3: Determination of the Binding Affinity and Specificity of Anti-CCR8 Antibodies

[0159] To determine the binding affinity, the purified anti-CCR8 chimeric antibody was used in EasySep TMThe Buffer (purchased from STEMCELL Technologies) was serially diluted, and the initial antibody concentration was 200 nM. It was diluted 4-fold successively, with concentrations of 200 nM, 50 nM, 12.5 nM, 3.125 nM, 0.781 nM, 0.195 nM, 0.0488 nM, and 0.0122 nM respectively. The above anti-CCR chimeric antibody was incubated with 293T-huCCR8 or 293T-cynoCCR8 cells at 4 °C for 30 minutes. The above serially diluted antibody was added to the above cells at 1E5 / well at 50 μL / well. After washing twice with FACS buffer, Alexa Fluor647 diluted 1:1000 was added to each well TM 50 μl of goat anti-human IgG secondary antibody (purchased from Jackson ImmunoResearch, Cat. Log. No. 109-605-098) was added, and the mixture was incubated at 4 °C for 30 minutes in the dark. After washing twice with FACS buffer, the cells were resuspended with FACS buffer (cell density was 1E6 / mL), and data was collected on an LSR Fortessa TM cell analyzer (Beckton Dickinson). A titration curve was generated using a sigmoidal dose-response of non-linear fitting from GraphPad (1995 - 2022 GraphPad Software, LLC, version number: Prism 9). The EC50 of the chimeric antibody Ch401 is shown in Table 2

[0160] Table 2: EC50 (nM) of anti-CCR8 antibody against 293T-huCCR8 and 293T-cynoCCR8 cells

[0161] Clone EC50 for 293T-huCCR8 EC50 for 293T-cynoCCR8 Ch401 1.47 1.35

[0162] To determine non-specific binding, the purified anti-CCR8 chimeric antibody was diluted to 50 nM in EasySep TM Buffer, and 50 μl / well of the above diluted antibody was incubated with 293T-huCCR1, 293T-huCCR4, and 293T parental cells at 1E5 / well at 4 °C for 30 minutes. After washing twice with FACS buffer, Alexa Fluor647 diluted 1:1000 was added to each well TM 50 μl of goat anti-human IgG secondary antibody was added, and the mixture was incubated at 4 °C for 30 minutes in the dark. After washing twice with FACS buffer, the cells were resuspended with FACS buffer (cell density was 1E6 / mL), and data was collected on an LSR Fortessa TMCollect data on a cell analyzer. The chimeric antibody Ch401 did not show non-specific binding to 293T-huCCR1, 293T-huCCR4, or 293T parental cells.

[0163] Example 4: Anti-CCR8 antibody enhances NK cell-mediated cytotoxicity

[0164] An ADCC assay based on the Nano-Glo luciferase assay was established to explore whether anti-CCR8 antibodies could kill target cells expressing CCR8.

[0165] Briefly, primary NK cells were isolated from PBMCs as effector cells. The CHO-K1-NanoLuc-CCR8 cell line was generated as a target cell by co-transducing the CCR8 and NanoLuc luciferase genes into the CHO-K1 cell line (ATCC). 100 μL / 200,000 effector cells were pre-incubated with 11.1 μL of a 10-fold dilution series of anti-CCR8 antibody (highest final concentration 100 nM, 4-fold dilution, 7 concentration points in total) or 11.1 μL of 10-fold control human IgG (highest final concentration 100 nM, 4-fold dilution, 7 concentration points in total) at 37 °C for 2 hours. Then, 100 μL / 4000 target cells were added and mixed well (effector cell: target cell = 50:1). Separate target cell wells (4000 target cells, 222.2 μL), separate effector cell wells (200,000 effector cells, 222.2 μL), and separate medium wells (222.2 μL) were set as controls. After incubation for 5 hours, 24.6 μL of 10X lysis buffer (Promega, catalog number G1821) was added to the target cell control well (i.e., the target cell maximum lysis well) and the medium control well (i.e., the medium lysis well), and incubated at 37 °C for 45 minutes. The cell mixture was centrifuged at 2000 rpm for 5 minutes at 4 °C and the luciferase assay system (Promega) was used to evaluate cytotoxicity.

[0166] As shown in Table 3, the tested anti-CCR8 chimeric antibodies had potent ADCC activity with an EC50 of less than 1 nM.

[0167] The results were calculated using the following formula:

[0168]

[0169] Table 3: Comparison of cytotoxicity of anti-CCR8 antibodies

[0170] Antibody EC50 (nM) Emax (%) Ch401 0.042 118.5 hIgG ＞100 -

[0171] It should be understood that after reading the teachings of the present invention, those skilled in the art can make various modifications and changes to the present invention, and these equivalent solutions also fall within the scope defined by the claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human CCR8, comprising: A heavy chain variable region (VH) that comprises CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6; And A light chain variable region (VL) that comprises CDR-L1 as shown in SEQ ID NO: 7, CDR-L2 as shown in SEQ ID NO: 8, and CDR-L3 as shown in SEQ ID NO:

9.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: a VH having at least 95% sequence identity with SEQ ID NO: 10 and a VL having at least 95% sequence identity with SEQ ID NO:

11.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising: a VH as shown in SEQ ID NO: 10 and a VL as shown in SEQ ID NO:

11.

4. The antibody or antigen-binding fragment thereof according to claim 1, comprising: A heavy chain constant region as shown in SEQ ID NO: 14 and a light chain constant region as shown in SEQ ID NO:

15.

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain constant region that is from an IgG1, IgG2, IgG3, or IgG4 subclass.

6. The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain constant region that is from a lambda light chain or a kappa light chain.

7. The antibody or antigen-binding fragment thereof according to claim 1, which is a full-length antibody.

8. The antibody or antigen-binding fragment thereof according to claim 1, which is an antibody fragment selected from Fv, scFv, Fab, Fab', and F(ab′)2.

9. The antibody or antigen-binding fragment thereof according to claim 1, which is a chimeric antibody or a humanized antibody.

10. The antibody or antigen-binding fragment thereof according to claim 1, which is a multispecific antibody.

11. A polynucleotide that encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-10.

12. The polynucleotide according to claim 11, comprising: SEQ ID NO: 12 and / or 13.

13. A vector that comprises the polynucleotide according to claim 11 or 12.

14. The vector according to claim 13, which is a plasmid or a virus.

15. A host cell that comprises the polynucleotide according to claim 11 or 12 or the vector according to claim 13 or 14.

16. The host cell according to claim 15, which is a eukaryotic cell.

17. The host cell according to claim 16, which is a CHO cell.

18. The host cell according to claim 15, which is a prokaryotic cell.

19. The host cell according to claim 18, which is Escherichia coli.

20. A method for generating an antibody or an antigen-binding fragment thereof, comprising: (a) culturing a host cell according to any one of claims 15-19 under conditions suitable for expressing the antibody or the antigen-binding fragment thereof, and (b) optionally, recovering the antibody or the antigen-binding fragment thereof.

21. A conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-10.

22. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-10, a polynucleotide according to claim 11 or 12, a vector according to claim 13 or 14, a host cell according to any one of claims 15-19, or a conjugate according to claim 21.

23. A non-diagnostic method for detecting CCR8 in a biological sample, comprising contacting the biological sample with an antibody or an antigen-binding fragment thereof according to any one of claims 1-10 or a conjugate according to claim 21.

24. A kit comprising a container containing an antibody or an antigen-binding fragment thereof according to any one of claims 1-10 or a conjugate according to claim 21.

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