Anti-CCR8 antibodies and uses thereof

Abstract

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 producing 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 producing the antibody, and a composition comprising the antibody. Background Art

[0002] CCR8 (CC motif chemokine receptor 8) is mainly expressed in T reg Cells and Th 2 cells, but not Th 1 It has been shown that CD4 + Foxp3 + T reg Cells (CCR8 + T reg T cells) subsets are the main drivers of immune suppression and are important for reg In addition, CCR8 is a molecule that is expressed by tumor-resident T cells in several tumor types. reg Many reports have shown that CCR8 + T reg In clinical practice, T cells are found in the tumor microenvironment of breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer, and many other cancer types. reg Increased T cells are associated with poor prognosis. reg The cells not only inhibit broad 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 for CCR8, which in turn activates CCR8 + Treg cells are recruited into the tumor microenvironment. reg CCR8 inhibitors have been shown to reduce tumor-infiltrating T cells. 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 to meet the needs in 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, comprising: a heavy chain variable region (VH), wherein the heavy chain variable region 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), wherein the light chain variable region 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.

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

[0007] In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises CDR-H1, CDR-H2 and CDR-H3 from the VH set forth in SEQ ID NO: 10, and CDR-L1, CDR-L2 and CDR-L3 from the VL set forth in SEQ ID NO: 11, e.g., 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, and the heavy chain constant region 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, and the light chain constant region 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 selected from Fv, scFv, Fab, Fab', F(ab') 2In 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 antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:

[0011] (1) Ability to specifically bind to huCCR8 protein;

[0012] (2) Ability to specifically bind to cells expressing huCCR8 protein, such as 293T cells;

[0013] (3) capable of specifically binding to 293T cells expressing huCCR8 protein with an EC50 value of 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) able to specifically bind to cynoCCR8 protein;

[0015] (5) can specifically bind to cells expressing cynoCCR8 protein, such as 293T cells;

[0016] (6) capable of specifically binding to 293T cells expressing cynoCCR8 protein with an EC50 value of 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) cannot cross-react with huCCR1 protein;

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

[0019] (9) cannot cross-react with huCCR4 protein;

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

[0021] (11) Ability to achieve ADCC;

[0022] (12) Ability to achieve NK cell-mediated ADCC;

[0023] (13) capable of achieving NK cell-mediated ADCC with an EC50 value of 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) Capable of achieving NK cell-mediated ADCC at an Emax value greater than 30, 35, 40, 45, 50, 55 or 60%.

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

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

[0027] In one aspect, the present invention provides a vector comprising a polynucleotide of the present invention. In one embodiment, the vector is a plasmid, a cosmid, a phage, a phagemid or a virus.

[0028] In one aspect, the present invention provides a host cell comprising a 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 an Escherichia coli.

[0029] In one aspect, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, comprising: (a) culturing a 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] In one aspect, the present invention provides a conjugate comprising an antibody or antigen-binding fragment thereof of the present invention. In one embodiment, the conjugate further comprises an effector molecule. In another embodiment, the conjugate further comprises a linker connecting the antibody or antigen-binding fragment to the effector molecule.

[0031] In one aspect, the present invention provides a composition comprising the antibody or antigen-binding fragment thereof of the present invention, 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] In one aspect, the present invention provides an antibody or 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 for use as a medicament.

[0033] In one aspect, the present invention provides an antibody or 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 for use in treating a CCR8-related disease.

[0034] In one aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof, the polynucleotide, the vector, the host cell, the conjugate, or the composition of the present invention for preparing a medicament for treating a CCR8-related disease.

[0035] In one aspect, the present invention provides a method for treating a CCR8-related disease in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or 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 a 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 carcinoma cell lung cancer. In another embodiment, CCR8 antibody or its antigen binding fragment is combined with other therapeutic agents, such as 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, comprising contacting the biological sample with the antibody or antigen-binding fragment thereof of the present invention or the conjugate of the present invention.

[0038] In one aspect, the present invention provides a method of diagnosing a CCR8-related disease in an individual, comprising contacting a biological sample from the individual with an antibody or 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 comprising a container containing the antibody or antigen-binding fragment thereof of the present invention or the conjugate of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The NK cell-mediated cytotoxicity by the anti-human CCR8 chimeric antibody, Ch403, was shown using primary human NK cells as effector cells and CHO-K1-NanoLuc-CCR8 as target cells to measure NK cell-mediated cytotoxicity. DETAILED DESCRIPTION

[0041] the term

[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 the invention belongs. Otherwise, certain terms used herein have the meaning as set forth in the specification.

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

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

[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 ascertain many equivalents of the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the present invention.

[0046] As used herein, the terms "comprises," "including," "having," or "containing," or any other variation thereof, will be understood to imply inclusion of the stated integer or group of integers, but not exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus comprising 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 apparatus. In addition, unless expressly stated to the contrary, "or" refers to an inclusive or rather than an exclusive or. For example, a condition A or B satisfies any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0047] As used herein, the connection term "and / or" between multiple listed elements is understood to cover both individual and combined options. For example, where two elements are connected 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 of these options are understood to fall within the meaning, thus satisfying the requirements of the term "and / or" as used herein. More than one of the options that are applicable at the same time are also understood to fall within the meaning, thus satisfying the requirements of the term "and / or".

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

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

[0050] As used herein, "subject" or "individual" means any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" 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., more preferably humans.

[0051] It should also be understood that the terms "about," "approximately," "substantially," "substantially," and similar terms used herein when referring to dimensions or features of components of the preferred invention indicate that the described dimensions / features are not strict boundaries or parameters and do not exclude minor variations that are functionally the same or similar, as will be understood by one of ordinary skill in the art. At a minimum, such references including 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] The terms "same" or "percent identity" 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) mean that two or more sequences or subsequences are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0053] For sequence comparison, usually one sequence is used as a reference sequence, and the test sequence is compared with it. 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 sequence identity percentage of one or more test sequences relative to the reference sequence based on the specified program parameters.

[0054] Optimal alignment of sequences for comparison can be performed, for example, 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, Wisconsin), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, Greene Publishing Group, 1996). A joint venture between Wiley Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (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 analysis 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 match or meet a certain positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighboring word score threshold (Altschul et al., supra). These initial neighboring word hits are used as seeds to start searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far 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 mismatching residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score drops by the amount X from its maximum achieved value; when the cumulative score becomes zero or below due to the accumulation of one or more negative scoring 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 (E) of 10, M=5, N=-4, and a comparison of both chains as default settings. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSEIM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. ETSA 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. ETSA 90:5873-5787 (1993)). One similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to 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.

[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 one polypeptide and a second polypeptide differ only in conservative substitutions, the two peptides are typically 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, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides as used herein are interchangeable. It is common knowledge to those skilled in the art that nucleic acids are polynucleotides and that polynucleotides 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 common cloning techniques and PCR TMetc.) and by synthetic means.

[0060] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting 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 constitute a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains of many types, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active 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] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy chain and / or light chain that binds to an antigen of interest, in a particularly preferred embodiment described herein, the antigen is the CC motif chemokine receptor 8 (CCR8). In this regard, the antigen-binding fragment of an antibody described herein may comprise a V described herein from an antibody that binds to CCR8. H and / or V L One, two, three, four, five or all six CDRs of the sequence. The antigen binding fragments of the CCR8-specific antibodies described herein are 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 modulates the biological activity of CCR8.

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

[0063] The term "epitope" includes any determinant, preferably a polypeptide determinant, that is capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, epitope determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics 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, the equilibrium dissociation constant for 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 to 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 suitable for transforming a host cell and containing 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] CC 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 shown in SEQ ID NO: 1. CCL1 is known to be the endogenous ligand of CCR8. Human CCR8 cDNA consists of the nucleotide sequence represented by GenBank ACC No. M_005201.3, and mouse CCR8 cDNA consists of the nucleotide sequence represented by GenBank ACC No. 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 the antibodies and expression vectors, recombinant cells containing the vectors, and compositions containing the antibodies. Methods for preparing the antibodies and methods for using the antibodies to treat diseases including cancer are also provided. 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 subjects and animal models in need when administered alone or in combination with other anti-cancer therapies.

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

[0071] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules, including monoclonal or polyclonal human antibodies, humanized antibodies, composite antibodies and chimeric antibodies and antibody fragments. Generally speaking, antibodies are proteins or peptide chains that show binding specificity to specific antigens. Antibody structure is well known. According to the heavy chain constant domain amino acid sequence, immunoglobulins can be classified into five main categories (i.e., IgA, IgD, IgE, IgG and IgM). IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Therefore, the antibodies of the present invention can belong to any of the five main categories 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 chain of vertebrate species can be classified into one of two significantly different types (i.e., κ and λ). Therefore, the antibodies of the present invention can contain κ or λ light chain constant domains. According to specific embodiments, the antibody of the present invention includes heavy chain and / or light chain constant regions from rat or human antibodies. In addition to heavy chain and light chain constant domains, the antibody also contains an antigen binding region consisting of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementary determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domain may alternatively be referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domain may alternatively be referred to as HCDR1, HCDR2, and HCDR3.

[0072] In addition, the scope of the antibodies disclosed herein also includes various forms, such as those selected from Fv, scFv, Fab, Fab', F(ab') 2 The scope of the antibodies disclosed herein also includes derivative forms thereof, such as multispecific antibodies, or antibody derivatives in which the antibodies are further linked to other agents, such as antibody-drug conjugates (ADCs), and the like.

[0073] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to CCR8 is substantially free of antibodies that do not bind to CCR8). In addition, the 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 antibody population, i.e., the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in small amounts. The monoclonal antibodies of the present invention can be prepared by hybridoma methods, phage display technology, single lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic non-human animals (such as transgenic mice or rats) 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, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv1), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab), a scFv dimer (bivalent diabody), a multispecific antibody formed by an antibody portion comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen binding fragment is capable of binding to the same antigen to which a parent antibody or parent antibody fragment binds. According to an embodiment, the antigen binding fragment comprises a light chain variable region, a light chain constant region, and an 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, which comprises a heavy chain variable region and a light chain variable region connected 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, which comprises a heavy chain variable region and a heavy chain constant region or 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 prepared using any technique known in the art. This definition of a human antibody includes complete or full-length 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 sequence homology with a human antibody such that the antigen-binding properties of the antibody are retained but its antigenicity in the human body 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 usually correspond to the variable regions of antibodies with the desired specificity, affinity and capacity derived from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant region corresponds to the sequence of an antibody derived from another mammalian species (e.g., human) to avoid eliciting an immune response in that species.

[0080] As used herein, the term "multi-specific antibody" refers to an antibody comprising multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence in the multiple has binding specificity to the first epitope, and the second immunoglobulin variable domain sequence in the multiple has binding specificity to the 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 multi-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 multi-protein)). In one embodiment, the multi-specific antibody comprises the third, fourth or fifth immunoglobulin variable domain. In one embodiment, the multi-specific antibody is a bispecific antibody molecule, a tri-specific antibody molecule or a tetra-specific 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. The feature of a bispecific antibody is that the first immunoglobulin variable domain sequence has binding specificity to the first epitope and the second immunoglobulin variable domain sequence has binding specificity to the 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 are on different antigens (e.g., different proteins (or different subunits of a multimeric protein)). In one embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the second epitope. In one embodiment, a bispecific antibody comprises a half antibody or a fragment thereof having binding specificity to the first epitope and a half antibody or a fragment thereof having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a scFv or fragment thereof having binding specificity to a first epitope and a scFv or fragment thereof having binding specificity to 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, TIP-1, CD3 and / or other tumor-associated immunosuppressive factors or surface antigens.

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

[0083] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include heavy chain and light chain CDR groups inserted between heavy chain and light chain framework region (FR) groups, respectively, and the framework region provides support for CDR and defines the spatial relationship of CDR relative to each other. As used herein, the term "CDR group" refers to three hypervariable regions in heavy chain or light chain V region. Starting from the N-terminus of the heavy chain or light chain, these regions are respectively represented as "CDR1", "CDR2" and "CDR3". Therefore, the antigen binding site includes six CDRs, including CDR sets from each of the heavy chain and light chain V regions. A polypeptide comprising 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 shown that the amino acid residues of CDR form extensive contacts with the bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Therefore, the molecular recognition unit is primarily responsible for the specificity of the antigen binding site.

[0084] As used herein, the term "FR group" refers to the four flanking amino acid sequences that are the framework of the CDR of the CDR group of the heavy or light chain V region. Some FR residues can contact the bound antigen; however, FR is primarily responsible for folding the V region into an antigen binding site, particularly the FR residues directly adjacent to the CDR. Within the FR, certain amino acid residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V region is folded into a binding site, the CDR is displayed as a protruding loop motif that forms an antigen binding surface. It is generally recognized that there are conserved structural regions of FR that affect the folding shape of the CDR loop into certain "canonical" structures, regardless of the precise CDR amino acid sequence. In addition, it is known that certain FR residues are involved in non-covalent domain contacts that stabilize the interaction of antibody heavy and light chains.

[0085] The structure and location of immunoglobulin variable regions can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, 1987 and updated versions 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 antigen binding sites is also described in the following literature: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, 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. JE (eds.), 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 about 26-35 (HCDR1), 51-57 (HCDR2) and 93-102 (HCDR3), and the CDR amino acid residues in VL are numbered about 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 all antibody molecules in their naturally occurring conformations. The heavy chains from any vertebrate species can be classified 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 differences in sequence and function, the IgG and IgA classes are further divided into subclasses. People 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 conformations in all antibody molecules. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

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

[0089] The antibody of the present invention may be fused to another protein at its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). The protein 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, 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, comprising a heavy chain variable region having a polypeptide sequence that is 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%) identical to SEQ ID NO: 10, or a light chain variable region having a polypeptide sequence that is 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%) identical 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 antibodies 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 with similar or similar properties to form polypeptides. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0093] Table A

[0094]

[0095]

[0096] The present invention relates to isolated nucleic acids encoding monoclonal antibodies or antigen-binding fragments thereof of the present invention. It will be appreciated by those skilled in the art that the coding sequence of a protein may be altered (e.g., replaced, deleted, inserted, etc.) without altering the amino acid sequence of the protein. Thus, it will be appreciated by those skilled in the art that the nucleic acid sequence encoding the monoclonal antibodies or antigen-binding fragments thereof of the present invention may be altered without altering the amino acid sequence of the protein.

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

[0098] The present invention also provides polynucleotides encoding any one of the antibodies disclosed herein. In some embodiments, disclosed herein are isolated polynucleotides encoding antibodies or antibody fragments that bind to CCR8, wherein the antibodies or antibody fragments comprise 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, disclosed herein are isolated polynucleotides encoding antibodies or antibody fragments that bind to CCR8, wherein the antibodies or antibody fragments comprise 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, disclosed herein are isolated polynucleotides encoding antibodies or antibody fragments that bind to CCR8, wherein the antibodies or antibody fragments comprise a heavy chain variable region and a light chain variable region having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 10 and 11.

[0099] The present invention also provides a vector comprising an isolated nucleic acid molecule encoding a monoclonal antibody or its antigen-binding fragment of the present invention. Any vector known to those skilled in the art in view of the present disclosure, such as a plasmid, a cosmid, a phage vector or a viral vector, can be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element of the conventional function of establishing an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selective marker and an origin of replication. The promoter may be a constitutive, inducible or repressible promoter. Many expression vectors capable of delivering nucleic acids to cells are known in the art and may be used herein to produce antibodies or their antigen-binding fragments in cells. Conventional cloning techniques or artificial gene synthesis may be used to produce recombinant expression vectors according to 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 a host cell, the host cell comprises the nucleic acid molecule encoding the monoclonal antibody of the present invention or its Fab separation. 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 of the present invention or its Fab. 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 an embodiment, the recombinant expression vector is transformed into a 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 a monoclonal antibody or an antigen-binding fragment thereof of the present invention, the method comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or an antigen-binding fragment thereof under conditions that produce the monoclonal antibody or an antigen-binding fragment thereof of the present invention, and recovering the antibody or an antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibody or an 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 appreciated by those skilled in the art, polynucleotides may include genomic sequences, extragenomic and plasmid-encoded sequences, and smaller engineered gene segments that express or may be adapted to express proteins, polypeptides, peptides, etc. Such segments may be isolated from nature or synthetically modified by a skilled artisan.

[0103] As the skilled person will also recognize, 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 that contain introns and correspond to DNA molecules in a one-to-one manner and mRNA molecules that do not contain introns. Additional coding or non-coding sequences can but need not be present in polynucleotides according to the present disclosure, and polynucleotides can but need not be connected to other molecules and / or supporting materials. Polynucleotides can comprise native sequences or can comprise 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 sequences specifically set forth herein.

[0105] The polynucleotides described herein or fragments thereof (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, multiple cloning sites, other coding segments, etc.) so that their total lengths may vary greatly. Thus, it is contemplated that nucleic acid fragments of almost any length may be employed, the total length preferably being limited by the ease of preparation and use in the expected recombinant DNA protocol. For example, illustrative polynucleotide segments of expected total lengths 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 useful.

[0106] Site-specific mutagenesis allows the generation of mutants by using a specific oligonucleotide sequence encoding the desired mutant DNA sequence and a sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being crossed. Mutations can be employed in selected polynucleotide sequences to improve, alter, reduce, modify or otherwise alter the properties of the polynucleotide itself, and / or to alter 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 nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause target cell lysis. In a preferred embodiment, such cells are human cells. Although it is not desirable to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs). Primary cell NK cells used to mediate ADCC express FcγRIII, while monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9: 457-92 (1991). In order to assess the ADCC activity of a molecule, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo (e.g., in an animal model, such as Clynes et al., PNAS (USA), 95: 652-656 (1998) disclosed in the animal model).

[0109] "Effector cells" are leukocytes 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 leukocytes 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. The preferred FcR is a native sequence human FcR. In addition, the preferred FcR is an FcR that binds to IgG antibodies (gamma receptors), and includes receptors of the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily 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 inhibition 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 deHaas 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 responsible for the transfer of 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 lyse a target in the presence of complement. The complement activation pathway is initiated by binding of 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 may be performed, for example as described in Gazzano-Santaro et al., J. Immunol. Methods, 202: 163 (1996).

[0113] Antibody-drug conjugates (ADCs)

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

[0115] Typically, 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. In addition, 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 effector molecule according to the present invention can be coupled by a coupling agent. Examples of coupling agents can be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. A non-selective coupling agent refers to a compound that connects the effector molecule to the antibody via a covalent bond, such as glutaraldehyde. The coupling agent utilizing a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0117] Certain residues on antibodies (such as Cys or Lys, etc.) are used to attach 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. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection agents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly or indirectly linked to antibodies via linkers.

[0118] Antibodies can be conjugated with drugs to form antibody-drug conjugates (ADCs). Typically, ADCs include a joint between a drug and an antibody. The joint can be a degradable or non-degradable joint. Typically, a degradable joint is easily degraded in the intracellular environment, for example, the joint is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable joints include, for example, enzyme-degradable joints, including peptidyl joints that can be degraded by proteases (e.g., lysosomal proteases or endosomal proteases) in cells; or sugar joints, such as glucuronidase-containing joints. Peptidyl joints can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable joints include, for example, pH-sensitive joints (e.g., joints that are hydrolyzed at a pH lower than 5.5, such as hydrazone joints) and joints that degrade under reducing conditions (e.g., disulfide joints). Non-degradable joints typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0119] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred, and 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-(mercurymethyl) dioxane, wherein the counter ion is CH 3 COO - , Cl - or NO 3 - ) and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via thiosuccinimide.

[0120] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In one embodiment, the antibody is connected 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, carboxyl, thiol, hydroxyl or keto group that can form a bond with the linker. When the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0121] Useful drugs include, for example, anti-tubulin drugs, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. 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 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, drug-linkers can be used to form ADCs in a simple step process. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, in a first step, a cysteine ​​residue is reacted with a reactive part of a linker, and then in a subsequent step, a functional group on the linker is reacted with a drug to form an ADC.

[0123] Generally speaking, the functional group on the joint is selected so that it can react specifically with the appropriate reactive group on the drug part. As a non-limiting example, the azide-based part can be used to react specifically with the reactive alkynyl on the drug part. The drug is covalently bound to the joint by 1,3-dipolar cycloaddition between the azide and the alkynyl. 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 connection strategies (for example, those described in Bioconjugation Technology (2nd Edition (Elsevier)) are well known to those skilled in the art. It will be appreciated by those skilled in the art that when selecting the reactive functional group of the complementary pair for the selective reaction between the drug part and the joint, each member of the complementary pair can be used for the joint, and can also be used for the drug.

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

[0125] In certain embodiments, the method according to the invention comprises: combining the antibody with the bifunctional linker compound under conditions sufficient to form the antibody-linker conjugate. In these embodiments, the method according to the invention further comprises: combining the antibody-linker conjugate with the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

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

[0127] in:

[0128] Ab is antibody;

[0129] L is the connector;

[0130] D is a drug; and

[0131] p is a load, a value selected from 1 to 8 (eg, an integer).

[0132] Pharmaceutical 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 specifically bind to CCR8 with unexpectedly high affinity. + T reg Cells are beneficial for tumor escape mechanisms. In some aspects, the present invention provides a method for inhibiting the expression of CCR8 + T reg The invention discloses a method for reducing the number or activity of tumor-infiltrating regulatory T cells (TITR) in a tumor present in a subject by immunosuppression mediated by cells, and provides a pharmaceutical composition for treating cancer via this 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, the invention provides a method for increasing the amount of T effector cells in a subject's tumor by administering an anti-CCR8 antibody to the subject. Cytotoxicity may be antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The agent may be an antibody (e.g., peptide) small molecule, a protein drug conjugate or an interfering nucleic acid. In addition, the present invention may also regulate the CCL1 / CCR8 axis, thereby possibly treating diseases such as diabetic neuropathy, spinal cord injury and IgG4-related diseases (such as sclerosing cholangitis (ISC)). The amino acid sequence of an illustrative antibody or its antigen-binding fragment or their complementary determining region (CDR).

[0134] The present invention also provides a pharmaceutical composition comprising the isolated monoclonal antibody or its antigen-binding fragment 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 its antigen-binding fragment in the first aspect, the recombinant protein in the second aspect, the isolated nucleic acid (especially DNA or RNA) in the third aspect, the carrier in the fourth aspect, the antibody conjugate in the sixth aspect, the immune cell in the seventh aspect, or a combination thereof. The active ingredient of the present invention and the composition comprising them can also be used to manufacture medicines for the therapeutic applications mentioned herein.

[0135] As used herein, the term "treating" a disease refers to 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 such variables as the type and extent of the disease or indication being treated, the patient's general health, the in vivo efficacy of the antibody, the pharmaceutical formulation, the serum half-life of the antibody, and the route of administration.

[0137] The frequency of administration may vary depending on factors such as route of administration, dose, 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 levels 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 antibodies or ADCs thereof according to the present invention can be used in detection applications, for example for use in detecting samples 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. Therefore, the biopsy specimens used in the present invention can include, for example, excision samples of tumors and tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

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

[0143] The present invention further provides a kit, which only comprises the antibody (or fragment thereof) according to the present invention; in a preferred example of the present invention, the kit further comprises a container, instructions, 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, the kit comprising an anti-CCR8 antibody. The kit of the present invention may further comprise tools and / or reagents for ELISA known in the art. If necessary, the kit of the present invention may further comprise tubes, well plates, instruction manuals describing how to use, etc. for mixing various components.

[0146] Example

[0147] The present invention is further described by reference to the following examples. It should be understood that the following examples are only used to describe the present invention, rather than to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are generally 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 antibodies

[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 immunogen combinations (including: human CCR8 expression plasmid, huCCR8 DNA; L929 cells overexpressing human CCR8, L929-huCCR8; and L929 cells overexpressing cynomolgus monkey CCR8, L929-cynoCCR8). Human, cynomolgus monkey and mouse CCR8 protein sequences are shown in SEQ ID NOs: 1 to 3 in Table 1. As previously described (Jiang et al., J. Virol. 2017 Apr 13; 91(9): e02052-16), human CCR8 expression plasmid (huCCR8 DNA) (50 μg per mouse) was delivered intramuscularly using an EPT-I delivery device (TERESA, Shanghai, China) by in vivo electroporation. L929-huCCR8 and L929-cynoCCR8 cells (5 × 10 per mouse 6 Cells) are injected intraperitoneally. Typically, 5-7 immunizations are performed at intervals of three weeks to induce a powerful anti-CCR8 humoral immune response. On the 14th day after each immunization, blood is collected, and the serum titer of 293T cells (293T-huCCR8) for overexpressing human CCR8 and 293T cells (293T-cynoCCR8) for overexpressing cynomolgus monkey CCR8 is determined by FACS. Animals with sufficient levels of anti-CCR8 serum antibodies are selected for final reinforcement.

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

[0152] 3-5 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. 6 / ml of enriched plasma cells were introduced into the channel and placed into the NanoPen chamber of the OptoSelect 14K chip (BerkeleyLights). In order to screen huCCR8-specific plasma cells, a density of 1×10 8293T-huCCR9 cells of 5 μg / ml and AlexaFluor 647 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) of concentration are introduced into the channel.After importing, the freezing valve is opened, and the exposure time of the CY5 channel of the Alexa Fluor 647 fluorophore is set to 3000ms.The pattern positive signal is captured by time-lapse imaging set to 6 minutes and 10 cycles.After completing huCCR8 specific plasma cell screening with 293T-huCCR8 cells, cynoCCR8 specific plasma cells are screened with 293T-cynoCCR8 cells.Plasma cells showing positive signals for both 293T-huCCR8 and 293T-cyoCCR8 cells are respectively output to 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 Opto Plasma B Discovery cDNA synthesis kit (Berkeley Lights) was used to synthesize the first strand of cDNA and amplify the total cDNA. According to the manufacturer's instructions, the Opto Plasma B Discovery Sanger Prep Kit (Berkeley Lights) was used to amplify the antibody VH and VL genes. The amplified VH and VL genes were cloned into mammalian expression vectors containing human IgG1 and kappa constant region genes, respectively. The three HCDRs, three LCDRs, amino acid sequences of VH and VL of the chimeric antibody Ch403, and the DNA sequences of VH and VL are shown in SEQ ID NOs: 4-13 in Table 1. The Expi293 TM The cells expressed the chimeric antibody and were purified by affinity chromatography.

[0155] Table 1: Sequences

[0156]

[0157]

[0158] Example 3: Determination of binding affinity and specificity of anti-CCR8 antibodies

[0159] To determine the binding affinity, the purified anti-CCR8 chimeric antibody was assayed on EasySep TMBuffer (purchased from STEMCELL Technologies) was serially diluted, with the initial antibody concentration of 200nM, and the concentrations were 200nM, 50nM, 12.5nM, 3.125nM, 0.781nM, 0.195nM, 0.0488nM, and 0.0122nM after 4-fold gradient dilution. The above anti-CCR chimeric antibody was incubated with 293T-huCCR8 or 293T-cynoCCR8 cells at 4°C for 30 minutes, and 50μL / well of the above gradient diluted antibody was added to 1E5 / well of the above cells. 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 incubated at 4°C in the dark for 30 minutes. After washing twice with FACS buffer, the cells were resuspended with FACS buffer (cell density was 1E6 / mL) and stained with LSR Fortessa TM Data were collected on a cell analyzer (Beckton Dickinson). Titration curves were generated using a sigmoidal dose-response with nonlinear fitting from GraphPad (1995-2022 GraphPad Software, LLC, Version: Prism 9). The EC50 of the chimeric antibody Ch403 is shown in Table 2.

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

[0161] clone EC50 against 293T-huCCR8 EC50 for 293T-cynoCCR8 Ch403 0.88 0.78

[0162] To determine nonspecific binding, purified anti-CCR8 chimeric antibodies were assayed on EasySep TM Buffer was diluted to 50 nM, and 50 μl / well of the diluted antibody was incubated with 1E5 / well of 293T-huCCR1, 293T-huCCR4 and 293T parental cells at 4°C for 30 minutes. After washing twice with FACS buffer, Alexa Fluor 647 diluted 1:1000 was added to each well. TM 50 μl of goat anti-human IgG secondary antibody was added and incubated at 4°C in the dark for 30 minutes. After washing twice with FACS buffer, the cells were resuspended with FACS buffer (cell density was 1E6 / mL) and then stained with LSR Fortessa TMData were collected on a cell analyzer. Chimeric antibody Ch403 showed no non-specific binding to 293T-huCCR1, 293T-huCCR4 or 293T parental cells.

[0163] Example 4: Anti-CCR8 antibodies enhance 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] In brief, primary NK cells were isolated from PBMC as effector cells. CHO-K1-NanoLuc-CCR8 cell lines were generated as target cells by co-transduction of CCR8 and NanoLuc luciferase genes into CHO-K1 cell lines (ATCC). 100 microliters / 200,000 effector cells were pre-incubated at 37°C for 2 hours with 11.1 microliters of a 10-fold dilution series of anti-CCR8 antibodies (highest final concentration 100 nM, 4-fold dilution, 7 concentration points in total) or 11.1 microliters of 10-fold control human IgG (highest final concentration 100 nM, 4-fold dilution, 7 concentration points in total). Then, 100 microliters / 4000 target cells were added and mixed thoroughly (effector cells: target cells = 50: 1). Set up target cell wells alone (4000 target cells, 222.2 μl), effector cell wells alone (200,000 effector cells, 222.2 μl), and medium wells alone (222.2 μl) as controls. After incubation for 5 hours, 24.6 μl of 10X lysis buffer (Promega, Cat. No. G1821) was added to the target cell control wells (i.e., target cell maximum lysis wells) and the medium control wells (i.e., medium lysis wells), and incubated at 37°C for 45 minutes. The cell mixture was centrifuged at 2000 rpm for 5 minutes at 4°C and lysed using Cytotoxicity was assessed using the luciferase assay system (Promega).

[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 are calculated using the following formula:

[0168]

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

[0170] Antibody EC50(nM) Emax(%) Ch403 0.109 102.1 hIgG ＞100 -

[0171] It should be understood that after reading the teachings of the present invention, those skilled in the art may 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), 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), 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.

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

11.

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

11.

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

15. 5 . The antibody or antigen-binding fragment thereof according to claim 1 , comprising a heavy chain constant region, wherein the heavy chain constant region is from IgG1, IgG2, IgG3 or IgG4 subclass.

6. The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain constant region, wherein the light chain constant region is derived from a λ light chain or a κ light chain. 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 the group consisting of 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. The antibody or antigen-binding fragment thereof according to claim 1 , which is a multispecific antibody.

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

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

13. A vector comprising the polynucleotide according to claim 11 or 12. The vector according to claim 13 , which is a plasmid, a cosmid, or a virus.

15. A host cell comprising the polynucleotide according to claim 11 or 12 or the vector according to claim 13 or 14. The host cell according to claim 15 , which is a eukaryotic cell. The host cell according to claim 16 , which is a CHO cell. The host cell according to claim 15 , which is a prokaryotic cell. The host cell according to claim 18 , which is Escherichia coli.

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

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

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

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

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

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