CN118786143A - Anti-CCR6 antibodies and uses thereof - Google Patents

Abstract

The present invention relates to novel anti-CCR 6 antibodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies or antigen-binding fragments thereof, vectors and host cells comprising the nucleic acids, methods of producing the antibodies or antigen-binding fragments thereof, pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof as active ingredient, and the use of the antibodies in the treatment of CCR6 mediated diseases.

Description

Anti-CCR6 antibodies and uses thereof

Technical Field

The present invention relates to novel anti-CCR6 antibodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies or antigen-binding fragments thereof, vectors and host cells comprising the nucleic acids, methods for producing the antibodies or antigen-binding fragments thereof, drugs containing the antibodies or antigen-binding fragments thereof as active ingredients, and use of the antibodies in treating diseases mediated by CCR6.

Background of the Invention

Chemokines are small secreted proteins that participate in the recruitment and activation of cells during inflammation. Chemokines regulate a variety of cellular functions and exert their effects by binding to chemokine receptors (G protein-coupled receptors), thereby causing chemotaxis and activation of various subsets of cells in the immune system. Chemokines are divided into different classes, including CC, CXC, CX3C, and XC, based on the location of the N-terminal cysteine residues of the protein. CC-class chemokines contain a CC sequence in which the first two cysteines are not separated by any amino acids, while CXC-class chemokines contain a CXC sequence in which the first two cysteines are separated by random amino acids. The activity of chemokines is mainly mediated by tight binding to receptors on the surface of leukocytes.

Under normal physiological conditions, the expression of chemokines and chemokine receptors is delicately and strictly regulated, and dysregulated expression and activation of chemokines and chemokine receptors often lead to autoimmune diseases or malignant diseases such as cancer. Among the chemokine series, CC chemokine receptor 6 (CCR6) and CC chemokine ligand 20 (CCL20) are gradually being recognized for their therapeutic potential in immunology research.

The human CCR6 gene is located on chromosome 6q27, and the mouse CCR6 gene is located on chromosome 17. CCR6 is expressed on immature DCs, most B cells, CD4+ and CD8 ⁺ T cell subsets, and NKT cells. In addition, both central memory and effector memory T cells express CCR6. Recent studies have also shown that CCR6 is a specific marker for Th17 cells and regulatory T cells. Interestingly, some cancer cells also express CCR6. CCR6 has been reported to be expressed on a variety of DC subsets, including CD11b ⁺ CD8α ^- myeloid DCs, Langerhans cells (LCs), CD34 ⁺ cell-derived immature myeloid DCs, and immature monocyte-derived DCs. Since DCs are the most potent APCs in the immune system and have the unique ability to induce primary immune responses against invading pathogens, CCR6 may regulate the activity of DC cell populations that participate in and drive innate immune responses.

CCL20, which has various names including macrophage inflammatory protein (MIP)-3α, Exodus-1, and liver and activation-regulated chemokine (LARC), is expressed in a variety of tissues and immune cells in the human body and is mainly observed in lymph nodes, lungs, and liver. At the cellular level, endothelial cells, neutrophils, B cells, natural killer cells, dendritic cells (DCs), and macrophages are reported to secrete CCL20. CCL20 is only expressed by T helper lymphocytes 17 (Th17), not regulatory T cells or other T helper subsets. The CCR6-CCL20 axis is found in CD4+ T helper cell subsets expressing CCR6, Th17, and regulatory Treg cells, which strictly regulate the immune balance of a healthy person. Disruption of this delicate balance will tip the scales toward the pro-inflammatory signature cell Th17 population or the regulatory Treg cell population, the latter of which will be actively recruited to sites of infection or injury.

CCR6 and CCL20 exhibit properties that are compatible with immune balance and immune activation. It has been confirmed that the immunological effects of this chemokine receptor-ligand have a profound impact on the health and disease of multiple organs in the human body. A large number of studies have shown that the CCR6 and CCL20 axis directly affects the nervous, respiratory, gastrointestinal, excretory, skeletal and reproductive systems through multi-effect immune mechanisms, and plays an important role in some diseases with high mortality.

It is well known that the CCL20-CCR6 axis is not only associated with inflammatory and infectious diseases such as rheumatoid arthritis and human immunodeficiency virus infection, but also with a variety of cancers such as hepatocellular carcinoma, colorectal cancer, breast cancer, pancreatic cancer, cervical cancer, and renal cancer. The CCL20-CCR6 axis directly promotes cancer progression by enhancing the migration and proliferation of cancer cells and indirectly promotes cancer progression by reshaping the tumor microenvironment through immune cell control.

In view of the role played by CCR6-CCL20 in the pathogenesis of various diseases, it is desirable to prepare antibodies that inhibit CCR6 activity, which would be helpful for the treatment of CCR6-mediated diseases such as autoimmune diseases and cancer.

Therefore, there is a need in the art for more effective therapies consisting of anti-CCR6 antibodies that can effectively inhibit CCR6 signaling while minimizing adverse side effects to the human body.

SUMMARY OF THE INVENTION

Through unremitting efforts, the present inventors have developed a novel antibody against chemokine receptor CCR6, especially human CCR6, and invented a novel anti-CCR6 antibody by blocking CCR6-CCL20 signal transduction, thereby completing the present invention.

First, disclosed herein is a novel anti-CCR6 antibody or an antigen-binding fragment thereof.

Second, disclosed herein is a method for treating a disease mediated by CCR6.

Third, disclosed herein is a pharmaceutical composition comprising the anti-CCR6 antibody or antigen-binding fragment thereof disclosed herein as an active ingredient.

Fourth, the present invention discloses a nucleic acid encoding an anti-CCR6 antibody or an antigen-binding fragment thereof, a vector and a host cell containing the nucleic acid.

Legend

The above and other objects, features and other advantages of the present invention will be more clearly understood through and in conjunction with the detailed description of the following drawings:

FIG. 1A shows the ability of representative antibodies disclosed herein to bind to 293T cells overexpressing CCR6 (293T-CCR6) in a cell-based binding assay.

Figure 1B shows that representative antibodies disclosed herein are incapable of binding to 293T cells in a cell-based binding assay.

FIG2 shows the inhibitory effect of the antibodies disclosed herein on cell migration.

FIG. 3A shows the binding specificity of Mab7 (VH2-NG/NA+VL3) binding to 293T cells expressing human CCR6.

Figure 3B shows the binding specificity of Mab7 (VH2-NG/NA+VL3), which did not bind to 293T cells expressing rat CCR6.

Figure 3C shows the binding specificity of Mab7 (VH2-NG/NA+VL3), which did not bind to 293T cells expressing dog CCR6.

Figure 3D shows the binding specificity of Mab7 (VH2-NG/NA+VL3), which did not bind to 293T cells expressing mouse CCR6.

Figure 3E shows the specificity of Mab7 (VH2-NG/NA+VL3) binding to 293T cells expressing canine CCR6.

Figure 4 shows the binding specificity of the humanized Mab7 (VH2-NG/NA+VL3) antibody, which binds to 293T cells expressing human CCR6 but not to 293T cells expressing human CXCR2.

definition

Unless specifically defined otherwise in this document, all other technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

The term "or" means "and/or" and is used interchangeably with the term "and/or" unless the context clearly requires otherwise.

The term "subject" as used herein includes any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog, cat, rabbit), and most preferably a human. The term "patient" as used herein refers to a human patient.

As used herein, the terms "administration," "administration," "treatment," and "treating" as applied to an animal, human, subject, cell, tissue, organ, or biological fluid refer to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid (fluid-to-cell contact). The terms "administration" and "treatment" also refer to in vitro and in vivo treatments, e.g., treatment of a cell with an agent, a diagnostic agent, a binding compound, or another cell. As used herein, treatment of any disease or disorder refers to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof); or alleviating or improving at least one physical parameter, including those that may not be discernible by the patient; or modulating the disease or disorder physically (e.g., stabilizing), physiologically (e.g., stabilizing a physical parameter), or both; or preventing or delaying the onset, development, or exacerbation of the disease or disorder.

The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. The variable region of an antibody interacts with many sites of an antigen through non-covalent forces. Generally speaking, the more interactions there are, the stronger the affinity.

The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that can non-covalently and reversibly bind to the corresponding antigen in a specific manner. For example, a naturally occurring IgG antibody is a tetramer composed of at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a domain CL. The VH and VL regions can be further subdivided into variable regions, called complementarity determining regions (CDRs), and more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four framework regions (FRs), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The variable regions of each pair of light chain/heavy chain (VL/VH) constitute the antibody binding site. Therefore, in general, a complete antibody has two binding sites. Except for bifunctional or bispecific antibodies, the primary sequences of these two binding sites are generally the same. The CDRs are usually aligned by the framework region so that they bind to specific epitopes. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the classical complement system Clq.

The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-allogeneic (anti-Id) antibodies. The antibody can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

In some embodiments, the anti-CCR6 antibody comprises at least one antigen binding site, at least one variable region. In certain embodiments, the anti-CCR6 antibody comprises an antigen binding fragment of a CCR6 antibody disclosed herein.

The terms "monoclonal antibody" or "mAb" or "Mab" as used herein refer to a substantially homogeneous antibody population, i.e., the antibody molecules in the antibody population are identical in amino acid sequence except for a small amount of natural mutations that may be present. In contrast, traditional (polyclonal) antibody preparations typically include a variety of different antibodies whose variable regions, particularly the amino acid sequences in the complementary determining regions (CDRs), are different and typically specific for different epitopes. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population and is not to be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497; U.S. Pat. No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo. High titers of monoclonal antibodies can be obtained in vivo production, in which cells of a single hybridoma are intraperitoneally injected into mice, such as Balb/c mice, to produce ascites containing high concentrations of the desired antibody. Isotype IgM or IgG monoclonal antibodies can be purified from ascites fluid or culture supernatant using column chromatography methods well known to those skilled in the art.

In general, the basic antibody structural unit consists of a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair of polypeptide chains includes a "light chain" (about 25kDa) and a "heavy chain" (about 50-70kDa). The amino-terminal portion of each chain includes a variable region consisting of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain can determine a constant region that is primarily responsible for effector function. Human light chains are generally divided into kappa and lambda light chains. In addition, human heavy chains are generally divided into α, δ, ε, γ or μ, and define the isotype of the antibody as IgA, IgD, IgE, IgG and IgM, respectively. In the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 more amino acids.

The positions of CDRs and framework regions can be determined using various definitions well-known in the art, such as Kabat, Chothia, AbM, and IMGT (see 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., 196:901-917 (1987). Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 196:901-917 (1987). 7-883 (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. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme). The definition of antigen binding sites is also described in: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Molecular Biology, 262:7-89 (2001). Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, according to the Kabat method, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable region (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Under Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). Combining the CDR definitions of Kabat and Chothia, the CDRs in human VH are numbered 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3), and the amino acid residues of CDRs in human VL are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Under 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). Under IMGT, the IMGT/DomainGap Align program can be used to determine the CDR regions of an antibody.

The term "variable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. The variable region includes amino acid residues from "CDR" (e.g., LCDR1, LCDR2 and LCDR3 in the light chain variable region and HCDR1, HCDR2 and HCDR3 in the heavy chain variable region). See Kabat et al. (1991), "Protein Sequences with Immunological Significance", 5th edition. Public Health Service, National Institutes of Health, Bethesda, Maryland (CDR regions of antibodies are defined by sequence); see also Chothia and Lesk (1987) J.Mol.196:901-917 (CDR regions of antibodies are defined by structure). The term "framework" or "FR" residues refers to residues other than the variable region residues defined herein as CDR residues.

Unless otherwise indicated, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the antigen-specific binding ability to bind to a full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as single-chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments.

As used herein, an antibody "specifically binds" to a target protein means that the antibody preferentially binds to the target protein compared to other proteins, but such specificity does not require absolute binding specificity. Antibody "specific binding" or "selective binding" is used to describe the interaction between an antigen (e.g., protein) and an antibody or antigen-binding antibody fragment, and refers to a binding reaction that determines whether the antigen is present in a heterogeneous population of proteins and other biological products, such as a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, the antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least twice the background level without significantly binding to other antigens present in the sample. In one aspect, under specified immunoassay conditions, the antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least ten (10) times the background level without significantly binding to other antigens in the sample.

Here, "human antibodies" refer to antibodies that contain only human immunoglobulin sequences. If the antibody is produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell, the human antibody may contain mouse carbohydrate chains. Similarly, "mouse antibodies" or "rat antibodies" refer to antibodies that contain only mouse or rat immunoglobulin sequences, respectively.

The term "humanized" or "humanized antibody" refers to an antibody form that contains non-human (e.g., murine) antibody sequences and human antibody sequences. Such antibodies contain minimal sequence from a non-human immunoglobulin. In general, a humanized antibody will contain at least one, usually two, variable domains in which all or substantially all of the variable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. The humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin. Where necessary, the prefix "hum", "hu", "Hu" or "h" is added to the antibody clone name to distinguish the humanized antibody from the parent murine antibody. Humanized forms of rodent antibodies usually contain the same CDR sequences as the parent rodent antibody, but certain amino acid substitutions may be made to increase affinity, improve the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that has the highest determined amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to variable region amino acid sequences or subsequences encoded by all other known human germline immunoglobulin variable region sequences. A corresponding human germline sequence may also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other variable region amino acid sequences that have been evaluated. A corresponding human germline sequence may be a framework region only, a complementarity determining region only, a framework and complementarity determining region, a variable segment (as defined above), or other combinations of sequences or subsequences that make up a variable region. Sequence identity can be determined using the methods disclosed herein, for example, by aligning two sequences using BLAST, ALIGN, or other alignment algorithms known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence. In addition, if the antibody comprises FR regions, the FR regions are also from such human sequences, e.g., human germline sequences, or mutant versions of human germline sequences, or antibodies comprising consensus framework sequences from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol 296:57-86, 2000. Biol. 296:57-86, 2000.

An algorithm suitable for determining percentages of sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., Nuc. 25: 3389-3402, 1977; and Altschul et al. Biol. 215: 403-410, 1990. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm first determines high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy a positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold. These initial neighborhood word hits can be used to initiate searches to find longer HSPs containing these words. Word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (a reward score for a pair of matching residues; always > 0) and N (a penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction ceases when: the cumulative match score falls by X from the maximum; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue matches; or 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) defaults to a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both chains. For amino acid sequences, the BLAST program defaults to a word length of 3, an expectation (E) of 10, and uses the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Sci. USA 89:10915) with a permutation (B) of 50, an expectation (E) of 10, M=5, N=4, and a comparison of both chains.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see Karlin and Altschul, Proc. Natl. Sci. USA 90:5873-5787, 1993). One similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, if the smallest sum probability of a test nucleic acid compared to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

The percent identity between two amino acid sequences can also be determined using the algorithm incorporated into the ALIGN program (version 2.0), which uses the PAM120 weight residue table, a gap penalty of 12, and a gap penalty of 4 (E. Meyers and W. Miller, Comput. Appl. 4: 11-17, (1988)). In addition, the percent identity between two amino acid sequences can be determined by the algorithm of the GAP program in the GCG software package, which uses the BLOSUM62 matrix or the PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6 (Needleman and Wunsch, J. Mol. Biol. 48: 444-453, (1970)).

As used herein, the term "conservative substitution" refers to a modification of a polypeptide, including the substitution of one or more amino acids with one or more amino acids having similar biological or biochemical properties, but does not cause the polypeptide to lose biological or biochemical function. The term "conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. The categories of amino acid residues having similar side chains have been defined and are well known in the art. These categories include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (such as alanine, valine, leucine), alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (such as threonine, valine, isoleucine) and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). We believe that antibodies according to the present invention have conservative amino acid substitutions and still retain activity.

The terms "anti-CCR6 antibody", "antibody against chemokine receptor CCR6" or "antibody that binds to CCR6" as used herein are equivalent to and include antibodies that can inhibit and/or neutralize the biological signaling activity of CCR6, for example, by blocking the binding of CCR6 to its ligand CCL20 or greatly reducing the binding of CCR6 to its ligand CCL20, thereby inhibiting or reducing the signaling pathway triggered by CCR6 and/or inhibiting or reducing CCR6-mediated cellular responses.

The term "anti-CCR6 antibody" as used herein includes polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies, which may be whole antibodies. Whole antibodies have a structure of two full-length light chains and two full-length heavy chains (including a constant region), wherein each light chain is connected to the corresponding heavy chain by a disulfide bond.

The whole antibodies of the anti-CCR6 antibodies disclosed herein include IgA, IgD, IgE, IgM and IgG forms, and IgG includes subtypes IgG1, IgG2, IgG3 and IgG4.

The anti-CCR6 antibody disclosed herein is preferably a fully human antibody screened from a human antibody library, but the present invention is not limited thereto.

The "antigen-binding fragment" of an anti-CCR6 antibody used herein refers to a fragment that has the function of binding to the antigen of the anti-CCR6 antibody (i.e., CCR6), including Fab, Fab', F(ab') ₂ , scFv, (scFv) ₂ , scFv-Fc, Fv, Fab, Fab', F(ab') ₂ , minibody and diabody, etc., which can be used interchangeably with "antibody fragment".

Fab includes a variable region of each of the heavy and light chains, a constant region of the light chain, and the first constant region of the heavy chain (CH1 domain), each of which has an antigen binding site. Fab' differs from Fab in that it also has a hinge region, including at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. F(ab') ₂ is formed by disulfide bonds between the cysteine residues in the hinge region of Fab'.

Fv (variable fragment) includes the variable regions of each of the heavy chain and the light chain, and is the smallest antibody fragment with the original specificity of the parent immunoglobulin. Double-chain Fv (dsFv, disulfide-stabilized Fv) is formed by combining the variable region of the light chain with the variable region of the heavy chain through a disulfide bond. Single-chain Fv (scFv) is a type of Fv in which the variable regions of each of the heavy chain and the light chain are covalently linked by a peptide bond. These antibody fragments can be obtained by treating the whole antibody with a protease (for example, Fab can be obtained by restrictive cleavage of the whole antibody with papain, and F(ab') ₂ fragments can be obtained by restrictive cleavage of the whole antibody with pepsin), preferably constructed by genetic recombination technology (for example, constructing Fv fragments by amplifying DNA encoding Fv, using a pair of primers, amplifying the DNA encoding the heavy chain or its variable region or the DNA encoding the light chain or its variable region as a template by PCR (polymerase chain reaction), and then amplifying using a combination of a pair of primers, connecting the DNA encoding the peptide linker and its two ends to the heavy chain or its variable region and the light chain or its variable region, respectively).

As used herein, the term "nucleic acid" is interchangeable with the term "polynucleotide" and refers to deoxynucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs). Nucleic acids may be present in cells or cell lysates, or in partially purified form, or in substantially purified form. When nucleic acids are purified from other cellular components or other impurities (e.g., nucleic acids or proteins of other cells), the nucleic acids may be "isolated" or "substantially purified", and standard techniques for purification include alkali/SDS treatment, cesium chloride stripping, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. For example, the nucleic acids of the present invention may be DNA or RNA, and may or may not include intron sequences.

To express the antibodies or antigen-binding fragments thereof disclosed herein, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the target antibody), and the DNA can be "operably associated" with transcription and translation control sequences and inserted into an expression vector.

The term "operably associated" as used herein may mean that the gene encoding the antibody is linked to the vector so that the transcription and translation control sequences can play the intended function of regulating the transcription and translation of the antibody gene.

Select an expression vector and expression control sequence that are compatible with the host cell for expression. The light chain gene of the antibody and the heavy chain gene of the antibody are inserted into different vectors, respectively, or the two genes are inserted into the same expression vector. The antibody can be inserted into the expression vector by standard methods (such as connecting the antibody gene fragment to the complementary restriction enzyme site on the vector, or blunt-end connection when there is no restriction enzyme site). In some cases, the recombinant expression vector can encode a signal peptide to promote the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector so that the signal peptide is connected to the amino terminus of the antibody chain gene according to the framework. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a protein other than an immunoglobulin). In addition, the recombinant expression vector also has a regulatory sequence that controls the expression of the antibody chain gene in the host cell. "Regulatory sequence" may include promoters, enhancers, and other expression control elements (such as polyadenylation signals) that control the transcription or translation of the antibody chain gene. It will be appreciated by those skilled in the art that the design of the expression vector can be achieved by selecting different regulatory sequences based on factors such as the selection of the host cell to be transformed and the expression level of the protein.

The term "host cell" refers to any cell into which a recombinant expression vector is introduced. It should be understood that these terms refer not only to a specific subject cell, but also to the offspring of such a cell. Due to mutation or environmental influences, some changes may occur in offspring cells, so these offspring cells may not actually be completely identical to the parent cell, but are still included in the scope of the term "host cell" used herein. The host cells disclosed herein are preferably selected from animal cells, plant cells, yeast, Escherichia coli and insect cells, but the present invention is not limited thereto. More specifically, the host cells disclosed herein can be prokaryotic cells, such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus or Staphylococcus, In addition, the host cell can also be selected from fungi, such as Aspergillus; yeast, such as Faun Saccharomyces, Saccharomyces cerevisiae, Schizosaccharomyces or cricket neurospores; and other eukaryotic cells, including lower eukaryotic cells and higher eukaryotic cells derived from insects. The host cell can also be from a plant or a mammal. The host cell is preferably selected from the group consisting of monkey kidney cells (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells, but the present invention is not limited thereto. CHO cells are particularly preferred. Mammalian host cells can be used to express and produce the polypeptides disclosed in the present invention. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines carrying exogenous expression vectors. These cells include any normal mortal cells or normal or abnormal immortal animals or human cells. For example, several suitable host cell lines that can secrete complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B cells and hybridomas. For a general discussion of using mammalian tissue cell culture to express polypeptides, see Winnacker, From Genes to Clones, VCH Publishers, NY, N.Y., 1987. Expression vectors for mammalian host cells may include expression control sequences, such as replication origins, promoters and enhancers (see Queen et al., Immunol. Rev. 89: 49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific and/or adjustable or regulatable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (such as human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

The term "vector" as used herein refers to a nucleic acid molecule that is capable of transporting another nucleic acid connected to it. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which other DNA fragments can be connected. Another type of vector is a viral vector, which can connect additional DNA fragments to the viral genome. Certain vectors are capable of autonomous replication in the introduced host cell (e.g., bacterial vectors with bacterial replication origins and exonic mammalian vectors). Other vectors (such as non-exonic mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome. In addition, some vectors can also guide the expression of genes operatively connected to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in DNA recombinant technology are usually in the form of plasmids. As described herein, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the content disclosed herein also includes other forms of expression vectors, such as viral vectors (such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have the same functions.

The nucleic acid or vector is transformed or transfected into a host cell. Various common techniques for introducing foreign nucleic acid (DNA or RNA) into prokaryotic or eukaryotic host cells for "transformation" or "transfection" include electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, etc. Various expression host/vector combinations can be used to express the antibodies of the present invention.

When a recombinant expression vector capable of expressing an antibody or an antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody can be produced by culturing the cell for a sufficient period of time to allow the antibody to be expressed in the host cell, more preferably, by culturing the cell for a sufficient period of time to allow the antibody to be secreted into the culture medium.

In some cases, the expressed antibodies can be separated from the host cells and purified to homogeneity. The separation or purification of antibodies can be carried out by separation and purification methods commonly used for proteins, such as chromatography. For example, chromatography can include affinity chromatography (including protein A column and protein G column), ion exchange chromatography or hydrophobic chromatography. In addition to chromatography, antibodies can also be separated and purified by combined methods such as filtration, ultrafiltration, salting out, dialysis, etc.

The term "pharmaceutically acceptable formulation" refers to a substance that can be added to an active ingredient to aid in formulating or stabilizing the formulation without causing significant adverse toxic effects to the patient.

As used herein, the term "therapeutically effective amount" refers to the amount of the composition of the antibody or antigen-binding fragment thereof required to produce a measurable benefit in a patient in need of treatment. The exact amount depends on a variety of factors, including but not limited to the ingredients and physical properties of the therapeutic composition, the target patient population, and individual patient considerations. With full consideration of these factors, it is important to administer a sufficient minimum dose to achieve the maximum effect without causing adverse reactions, and this dose can be determined by experts in the field.

The term "cancer" or "tumor" as used herein has the broadest meaning as understood in the art and refers to a physiological condition in mammals that is typically characterized by irregular cell growth. In the context of this disclosure, cancer is not limited to a certain type or location.

CCR6 Antibody

Disclosed herein is an antibody or antigen-binding fragment thereof, which can specifically bind to the chemokine receptor CCR6. The antibody or antigen-binding fragment thereof disclosed herein includes but is not limited to the antibody or antigen-binding fragment thereof generated as described below.

In certain embodiments, the antibody or antigen-binding fragment thereof binds to human chemokine receptor CCR6 and may selectively (1) bind to monkey chemokine receptor CCR6, (2) not bind to mouse chemokine receptor CCR6, (3) not bind to rat chemokine receptor CCR6, and/or (4) not bind to dog chemokine receptor CCR6.

In certain embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein are humanized or fully human antibodies.

In certain embodiments, the anti-CCR6 antibodies disclosed herein are antigen-binding fragments of full-length monoclonal antibodies.

In certain embodiments, the anti-CCR6 antibody is isolated or recombinant.

In some embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein comprise a HCDR (heavy chain complementarity determining region) comprising the amino acid sequence of any one of the HCDRs listed in Table 1 (based on the Kabat numbering scheme). In other embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein comprise (or alternatively comprise) one, two, three or more HCDRs comprising the amino acid sequence of any one of the HCDRs listed in Table 1 (based on the Kabat numbering scheme).

In some embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include a LCDR (light chain complementarity determining region) comprising the amino acid sequence of any one of the LCDRs listed in Table 1 (based on the Kabat numbering scheme). In other embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include (or alternatively include) one, two, three or more LCDRs comprising the amino acid sequence of any one of the LCDRs listed in Table 1 (based on the Kabat numbering scheme).

In some embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include a heavy chain variable region consisting of HCDR1, HCDR2 and HCDR3 in a row of Table 1 and/or a light chain variable region consisting of LCDR1, LCDR2 and LCDR3 in a row of Table 1. In some embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include a heavy chain variable region consisting of HCDR1, HCDR2 and HCDR3 in a row of Table 1 and a light chain variable region consisting of LCDR1, LCDR2 and LCDR3 in the same row of Table 1. Table 1 lists the amino acid sequence ID numbers (based on the Kabat numbering scheme) of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (sequences are shown in Tables 3 and 6):

Table 1: Amino acid sequence ID numbers of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3:

Alternatively, NG in SEQ ID NO: 47 is mutated to NA or QG. Alternatively, DG in SEQ ID NO: 78 is mutated to EG or DA, and/or GD in SEQ ID NO: 79 is mutated to AD, SD, GE or GN, especially GE, and/or NG in SEQ ID NO: 80 is mutated to QG or NA. Alternatively, NG in SEQ ID NO: 61 is mutated to QG or NA, especially NA.

In certain embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include

(i) a heavy chain variable region (VH) comprising HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 46, HCDR2 of SEQ ID NO: 47, and HCDR3 of SEQ ID NO: 48; and a light chain variable region (VL) comprising LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 49, LCDR2 of SEQ ID NO: 7, and (f) LCDR3 of SEQ ID NO: 50; or

(ii) a heavy chain variable region (VH) comprising HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 77, HCDR2 of SEQ ID NO: 78, and HCDR3 of SEQ ID NO: 79; and a light chain variable region (VL) comprising LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 80, LCDR2 of SEQ ID NO: 7, and (f) LCDR3 of SEQ ID NO: 81; or

(iii) a heavy chain variable region (VH) comprising HCDR1 (heavy chain complementary determining region 1) of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 264, and HCDR3 of SEQ ID NO: 92; and a light chain variable region (VL) comprising LCDR1 (light chain complementary determining region 1) of SEQ ID NO: 61, LCDR2 of SEQ ID NO: 7, and (f) LCDR3 of SEQ ID NO: 8;

wherein optionally, NG in SEQ ID NO:47 is mutated to NA or QG; optionally, DG in SEQ ID NO:78 is mutated to EG or DA, and/or GD in SEQ ID NO:79 is mutated to AD, SD, GE or GN, in particular GE, and/or NG in SEQ ID NO:80 is mutated to QG or NA; optionally, NG in SEQ ID NO:61 is mutated to QG or NA, in particular NA.

In certain embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include

(i) a heavy chain variable region (VH) comprising HCDR1 (heavy chain complementary determining region 1) of SEQ ID NO:46, HCDR2 of SEQ ID NO:47, and HCDR3 of SEQ ID NO:48; and a light chain variable region (VL) comprising LCDR1 (light chain complementary determining region 1) of SEQ ID NO:49, LCDR2 of SEQ ID NO:7, and (f) LCDR3 of SEQ ID NO:50;

Among them, NG in SEQ ID NO:47 can be selected to be mutated to NA or QG.

In certain embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include

(ii) a heavy chain variable region (VH) comprising HCDR1 (heavy chain complementary determining region 1) of SEQ ID NO: 77, HCDR2 of SEQ ID NO: 78, and HCDR3 of SEQ ID NO: 79; and a light chain variable region (VL) comprising LCDR1 (light chain complementary determining region 1) of SEQ ID NO: 80, LCDR2 of SEQ ID NO: 7, and (f) LCDR3 of SEQ ID NO: 81;

wherein, optionally, DG in SEQ ID NO:78 is mutated to EG or DA, and/or GD in SEQ ID NO:79 is mutated to AD, SD, GE or GN, especially GE, and/or NG in SEQ ID NO:80 is mutated to QG or

NA.

In certain embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include

(iii) a heavy chain variable region (VH) comprising HCDR1 (heavy chain complementary determining region 1) of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 264, and HCDR3 of SEQ ID NO: 92; and a light chain variable region (VL) comprising LCDR1 (light chain complementary determining region 1) of SEQ ID NO: 61, LCDR2 of SEQ ID NO: 7, and (f) LCDR3 of SEQ ID NO: 8;

Among them, NG in SEQ ID NO:61 can be selected to be mutated to QG or NA, especially NA.

In some embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO; (c) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, 9, 17, 24, 31, 38, 44, 315, 316, 51, 58, 63, 69, 58, 75, 323, 324, 325, 326, 327, 328, 329, 330, 82, 89, 93, 96, 103, 108, 115, 121, 127, 132, 140, 143, 144, 145 3,151,155,158,165,169,173,176,184,190,193,201,208,212,217,222,227,230,232,238,244,245,253,261,262,265,272,278,281,285,290,295,299,301, or 304 (Table 2, 3 or 6), or with SEQ ID Any of the following amino acid sequences that are at least 95%, 96%, 97%, 98% or 99% identical to NO: 1,9,17,24,31,38,44,315,316,51,58,63,69,58,75,323,324,325,326,327,328,329,330,82,89,93,96,103,108,115,121,127, 132,140,143,151,155,158,165,169,173,176,184,190,193,201,208,212,217,222,227,230,232,238,244,245,253,261,262,265,272,278,281,285,290,295,299,301, or 304; and/or (b) a polypeptide consisting of SEQ ID The amino acid sequence of NO in the light chain variable region is: 2,10,18,25,32,39,45,52,59,64,70,73,76,334,335,83,90,94,97,104,109,116,122,128,133,141,144,152,156,159,166,170,17 4,177,182,185,191,194,202,209,213,218,223,228,231,233,239,246,254,263,341,342,266,273,282,286,291,296,300,302,or305 (Table 2, 3 or 6), or with SEQ ID Any amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any of NO: 2, 10, 18, 25, 32, 39, 45, 52, 59, 64, 70, 73, 76, 334, 335, 83, 90, 94, 97, 104, 109, 116, 122, 128, 133, 141, 144, 152 , 156,159,166,170,174,177,182,185,191,194,202,209,213,218,223,228,231,233,239,246,254,263,341,342,266,273,282,286,291,296,300,302, or 305. In some embodiments, NG in SEQ ID NO:47 (if present) is mutated to NA or QG. In some embodiments, DG (if present) in SEQ ID NO: 78 is mutated to EG or DA, and/or GD (if present) in SEQ ID NO: 79 is mutated to AD, SD, GE or GN, especially GE, and/or the Rat N-terminus of SEQ ID NO: 79 (if present) is mutated to K or Q, and/or NG (if present) in SEQ ID NO: 80 is mutated to QG or NA. In certain embodiments, NG (if present) in SEQ ID NO: 61 is mutated to QG or NA, especially NA.

In some embodiments, the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein include (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO; (c) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, 9, 17, 24, 31, 38, 44, 315, 316, 51, 58, 63, 69, 58, 75, 323, 324, 325, 326, 327, 328, 329, 330, 82, 89, 93, 96, 103, 108, 115, 121, 127, 132, 140, 143, 151, 1 55,158,165,169,173,176,184,190,193,201,208,212,217,222,227,230,232,238,244,245,253,261,262,265,272,278,281,285,290,295,299,301, or (Table 2, 3 or 6), or in SEQ ID An amino acid sequence having one, two or three amino acid substitutions in the amino acid sequence of any of NO: 1,9,17,24,31,38,44,315,316,51,58,63,69,58,75,323,324,325,326,327,328,329,330,82,89,93,96,103,108,115,121,127,1 32,140,143,151,155,158,165,169,173,176,184,190,193,201,208,212,217,222,227,230,232,238,244,245,253,261,262,265,272,278,281,285,290,295,299,301, or 304; and/or (b) a polypeptide consisting of SEQ ID The amino acid sequence of NO in the light chain variable region is: 2,10,18,25,32,39,45,52,59,64,70,73,76,334,335,83,90,94,97,104,109,116,122,128,133,141,144,152,156,159,166,170,17 4,177,182,185,191,194,202,209,213,218,223,228,231,233,239,246,254,263,341,342,266,273,282,286,291,296,300,302,or305 (Table 2, 3, or 6), or in SEQ. an amino acid sequence having one, two or three amino acid substitutions in the amino acid sequence of any one of ID NOs: 2, 10, 18, 25, 32, 39, 45, 52, 59, 64, 70, 73, 76, 334, 335, 83, 90, 94, 97, 104, 109, 116, 122, 128, 133, 141, 144, 152, 156, 159, 166, 170, 174, 177, 182, 185, 191, 194, 202, 209, 213, 218, 223, 228, 231, 233, 239, 246, 254, 263, 341, 342, 266, 273, 282, 286, 291, 296, 300, 302, or 305. In some other embodiments, the amino acid substitution is a conservative amino acid substitution. In some embodiments, NG (if present) in SEQ ID NO: 47 is mutated to NA or QG. In some embodiments, DG (if present) in SEQ ID NO: 78 is mutated to EG or DA, and/or GD (if present) in SEQ ID NO: 79 is mutated to AD, SD, GE or GN, especially GE, and/or the Rat N-terminus of SEQ ID NO: 79 (if present) is mutated to K or Q, and/or NG (if present) in SEQ ID NO: 80 is mutated to QG or NA. In certain embodiments, NG (if present) in SEQ ID NO: 61 is mutated to QG or NA, especially NA.

In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include a heavy chain variable region (VH) in any row of Table 2 and/or a light chain variable region (VL) in any row of Table 2. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include a heavy chain variable region (VH) in any row of Table 2 and a light chain variable region (VL) in the same row of Table 2. Table 2 lists the amino acid sequence ID numbers of VH and VL (sequences are shown in Tables 3 and 6):

Table 2: Amino acid sequence ID numbers of VH and VL

In some embodiments, NG (if present) in SEQ ID NO: 47 is mutated to NA or QG. In some embodiments, DG (if present) in SEQ ID NO: 78 is mutated to EG or DA, and/or GD (if present) in SEQ ID NO: 79 is mutated to AD, SD, GE or GN, especially GE, and/or the Rat N-terminus of SEQ ID NO: 79 (if present) is mutated to K or Q, and/or NG (if present) in SEQ ID NO: 80 is mutated to QG or NA. In certain embodiments, NG (if present) in SEQ ID NO: 61 is mutated to QG or NA, especially NA.

In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include a heavy chain variable region (VH) selected from SEQ ID NO:44, 315 or 316 and a light chain variable region (VL) of SEQ ID NO:45.

In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include a heavy chain variable region (VH) selected from SEQ ID NO: 75, 323, 324, 325, 326, 327, 328, 329 or 330 and a light chain variable region (VL) selected from SEQ ID NO: 76, 334 or 335.

In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include a heavy chain variable region (VH) of SEQ ID NO: 262 and a light chain variable region (VL) of SEQ ID NO: 263, 341, or 342.

In certain embodiments of the humanized antibody, the antibody or antigen-binding fragment thereof comprises

(a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 311, 312, 313 or 314, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 319, 320, 321 or 322, and optionally, the NG in CDR-H2 is mutated to NA or QG;

(b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 331 or 332 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 333 or 336, optionally, DG in CDR-H2 is mutated to EG or DA, and/or GD in CDR-H3 is mutated to AD, SD, GE or GN, in particular GE, and/or the Rat N-terminus of HCDR3 is mutated to K or Q, and/or NG in CDR-L1 is mutated to GQ or NA; or

(c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 337, 338, 339 or 340, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 343, 344, 345 or 346, and the NG in CDR-L1 can be selectively mutated to QG or NA, especially NA.

In certain embodiments of the humanized antibody, the antibody or antigen-binding fragment thereof comprises

(a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 317, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 320;

(b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 318, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 321;

(c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 318, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 320;

(d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 330, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 336;

(e) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 332, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 333;

(f) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 337, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 347;

(g) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 337, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 348; or

(h) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 337, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 349.

In certain embodiments, the antibodies of the present disclosure are of the IgG1, IgG2, IgG3 or IgG4 isotype. In a more specific embodiment, the antibodies of the present disclosure comprise the Fc domain of wild-type human IgG1 (also referred to as human IgG1 wt or huIgG1) or IgG2.

The amino acid sequences of the heavy chain (VH) and light chain (VL) of the anti-CCR6 antibodies or antigen-binding fragments thereof disclosed herein and the variable region sequences of the heavy chain CDR and light chain CDR are shown in Tables 3 and 6.

Table 3: Amino acid sequences of the heavy chain (VH) and light chain (VL) of the anti-CCR6 antibodies disclosed herein and the amino acid sequences of the heavy chain CDRs and light chain CDRs, wherein the CDRs are based on the Kabat numbering scheme:

CCR6-mediated diseases

The diseases mediated by CCR6 disclosed herein refer to any diseases mediated by CCR6. In certain embodiments, the disease is caused by ligand-mediated CCR6 overactivity. In some embodiments, the disease mediated by CCR6 is selected from autoimmune diseases or inflammatory diseases, infectious diseases and cancer. In some further embodiments, the disease is hepatocellular carcinoma, colorectal cancer, breast cancer, pancreatic cancer, cervical cancer, kidney cancer, lung cancer, leukemia or lymphoma or chronic lymphocytic leukemia (CLL). In some further embodiments, the disease is rheumatoid arthritis, multiple sclerosis (MS), psoriasis, graft-versus-host disease (GVHD), lupus, chronic obstructive pulmonary disease, optic neuritis, age-related macular degeneration, systemic lupus erythematosus, Sjogen's syndrome, scleroderma, systemic sclerosis, chronic kidney disease, liver fibrosis, tuberculosis, idiopathic pulmonary fibrosis, tuberculosis-induced pulmonary fibrosis, scleroderma, systemic sclerosis, chronic kidney disease, liver fibrosis, tuberculosis, idiopathic pulmonary fibrosis, tuberculosis-induced pulmonary fibrosis, retroperitoneal fibrosis, pulmonary fibrosis, cystic fibrosis, endocardial fibrosis, atrial fibrosis, mediastinal fibrosis, myelofibrosis (bone marrow), retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, arthrofibrosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, transplant rejection, central nervous system injury, atherosclerosis, or an infectious disease.

The present invention will be described in more detail below with reference to the following examples. However, it is obvious to those skilled in the art that these examples are only used to illustrate the present invention and should not be understood as limiting the scope of the present invention.

Cell lines:

293T cell line was obtained from ATCC (CRL-3216) and cultured in DMEM + 10% FBS + 1 mM sodium pyruvate.

Example 1. Development of anti-CCR6 monoclonal antibodies

Anti-CCR6 monoclonal antibodies were developed by immunizing SJL/JOrlIcoCrl mice (6-8 weeks, female, vitalriver) with 293T cells overexpressing human CCR6 (hCCR6). Briefly, 293T cells were transfected with lentiviral vectors encoding hCCR6 with polybrene (8 μg/mL), selected in medium containing puromycin (2 μg/mL), and hCCR6 expression was detected by fluorescence activated cell sorting (FACS). Single clones with the highest mean fluorescence intensity of hCCR6 were selected for subsequent studies.

The spleen and lymph node cells of these mice were fused with myeloma cells (Sp2/0) using standard methods to produce hybridoma cells that secrete unique antibodies. The supernatant of the hybridoma cells was screened by cell ELISA and FACS of hCCR6 overexpressing cells. The cell ELISA was generally performed as follows. Approximately 3x10 ⁴ 293T-hCCR6 cells were inoculated per well in a 96-well plate. After overnight culture, the cells were rinsed with 1x PBS containing 0.5% BSA, and then 100 μL of 4% paraformaldehyde solution was added to fix and crosslink the cells to the microwell plate. The cells were rinsed twice with 1x PBS-T. The cells were then incubated with hybridoma culture supernatant (including blank control and positive control) at 37°C for 60 minutes. Then, the cells were rinsed four times with 1x PBS-T. Then incubate the cells with 100 μL of goat anti-mouse IgG secondary antibody at a dilution of 1:10,000 in PBS at 37°C for 60 minutes and then rinse four times with 1x PBS-T. Add 100 μL of TMB to the 96-well plate. After incubation for 10-12 minutes, scan and detect at a wavelength of 450 nm.

The supernatants of these positive clones were further confirmed by FACS. FACS analysis was generally performed as follows. Approximately 5x10 ⁵ CHOK1-hCCR6 cells per sample were prepared and blocked with TruStain FcX ^TM (anti-mouse CD16/32) antibody (Biolegend, 101320). The cells were spread into 96-well round-bottom polystyrene plates and incubated with the supernatant of the hybridoma cells on ice for 20-30 minutes. Then, the cells were washed with PBS/0.5% BSA and centrifuged. The cells were then washed with PBS/0.5% BSA and the cell pellet was resuspended in 100 μL PBS/0.5% BSA containing anti-mouse IgG-FITC antibody at a dilution of 1:300 and incubated on ice for 30 minutes. The cell pellet was resuspended in PBS/0.5% BSA and analyzed on CYTOFLEX (Beckman). Generally, the same supernatant was tested on untransfected parental cells to confirm that the reactive antibody specifically recognized hCCR6.

After the positive clones were identified, they were subcloned by limiting dilution. After three fusions, unique antibody clones that can recognize human CCR6 were obtained, which are: Mab6, Mab12, Mab50, Mab3, Mab30, Mab22, Mab59, Mab1, Mab7, Mab8, Mab17, Mab5, Mab10, Mab27, Mab49, Mab35, Mab26, Mab2, Mab24, Mab18, Mab13, Mab33, Mab4, Mab40, Mab54, Mab 23, Mab21, Mab58, Mab44, Mab16, Mab38, Mab19, Mab42, Mab31, Mab43, Mab34, Mab9, Mab15, Mab11, Mab46, Mab14, Mab29, Mab53, Mab45, Mab41, Mab20, Mab25, Mab28, Mab57, Mab56, Mab39, Mab36, Mab37, Mab48, Mab60 and Mab47. The name of the hybridoma is the same as the name of the antibody produced (for example, hybridoma Mab59 produces antibody Mab59). All anti-CCR6 antibody clones have high affinity antibodies, and the amino acid sequences of their VH and VL are shown in Table 2.

Example 2. Binding affinity of anti-CCR6 antibodies to hCCR6-overexpressing cells

The affinity of anti-CCR6 antibodies was evaluated by FACS. 293T-hCCR6 expresses high levels of human CCR6 on the cell surface and is used for binding tests. Affinity analysis is generally performed as follows. Approximately 5x10 ⁵ 293T-hCCR6 cells are prepared for each sample and blocked with Human TruStain FcX ^TM (Fc receptor blocking solution, Biolegend, 422302). The test antibody is diluted with PBS/0.5% BSA (1:3 serial dilutions from 225μg/mL to 0.00381μg/mL). 293T-hCCR6 cells and untransfected parent cells are placed in a 96-well round-bottom polystyrene plate and incubated on ice with the diluted antibody for 20-30 minutes. Then, the cells are rinsed with PBS/0.5% BSA and centrifuged. The cells were then washed with PBS/0.5% BSA and the cell pellet was resuspended in 100 μL PBS/0.5% BSA containing anti-mouse IgG-FITC antibody at a dilution of 1:300 and incubated on ice for 30 minutes. The cell pellet was resuspended in PBS/0.5% BSA and analyzed on CYTOFLEX (Beckman).

The results showed that these antibodies could bind to 293T cells overexpressing human CCR6, but could not bind to parent cells without CCR6 overexpression, as shown in Figure 1. Figure 1A shows that these antibodies have the ability to bind to 293T cells overexpressing CCR6 (293T-CCR6), but have no ability to bind to 293T parent cells (Figure 1B).

Example 3. Tango assay of anti-CCR6 antibodies

The Tango system is a cell-based assay that measures GPCR activation and recruitment of β-arrestin. The change in luminescence intensity directly correlates to the amount of β-arrestin recruited when the ligand activates the associated receptor. This assay can be used to determine which of the above antibodies blocks CCL20-CCR6 signaling.

Tango-CCR6-Gal4-CHO-K1 cells (constructed by Genomeditech) were cultured in complete medium (F12K medium, 10% FBS, 1% penicillin-streptomycin, 4 μg/mL puromycin, 4 μg/mL blasticidin, 100 μg/mL hygromycin) and the Tango assay was performed in an incubator (37°C, 5% CO2).

Tango-CCR6-Gal4-CHO-K1 cells were centrifuged and resuspended at a ratio of 1x10 ⁴ cells/70 μL/well, and the cells were dispersed into 96-well plates and incubated at 37°C under 5% CO2 for 6 hours in starvation medium (F12K, 1% FBS, 1% penicillin-streptomycin). Then, the detection antibody at the required concentration (3×) was added to 50 μL of starvation medium and incubated with the cells at 37°C under 5% CO2 for 1 hour. Then, 30 μL of starvation medium with or without CCL20 was added to the required wells and cultured with the cells at 5% CO2 and 37°C for 24 hours. After overnight incubation, the cells were incubated in the dark at room temperature with ONE-Glo working reagent for 10 minutes, and then the relative luminescence units (RLU) of each sample were measured and recorded at a wavelength of 560 nm using a microplate luminescence reader. The RLU values were plotted against the antibody concentrations, and concentration curve analysis was performed in GraphPad Prism.

Clonal antibodies such as Mab50 showed strong CCL20-CCR6 signal blocking ability. The signal blocking ability of representative antibodies disclosed herein is shown in Table 4.

Table 4. Tango detection results of anti-CCR6 antibodies

Example 4. Calcium flux assay of anti-CCR6 antibodies

The calcium flux assay is a cell-based second messenger assay used to measure calcium flux associated with G protein-coupled receptor activation or inhibition. The change in fluorescence intensity directly correlates to the amount of intracellular calcium released into the cytoplasm upon ligand activation of the associated receptor. This assay was used to determine which of the above antibodies could block CCL20-CCR6 signaling.

CCR6-Gα15-CHO K1 cells (produced by Genomeditech) were passaged in complete medium (F12K medium, 10% FBS, 1% penicillin-streptomycin, 4 μg/mL puromycin, 4 μg/mL beauvericin) and used for calcium flux assay in an incubator (37°C, 5% CO2).

Calcium binding dye (FLIPR Calcium 6 Assay Kit) was dissolved in assay buffer (20 mM HEPES buffer and 1x Hank's Balanced Salt Solution (HBSS), pH 7.4). The loading buffer was prepared with a dye solution containing 5 mM probenecid. Probenecid was prepared as a 500 mM stock solution in 1 N NaOH and diluted to 250 mM in HBSS buffer before use.

Approximately 1.5x10 ⁴ hCCR6-Gα15-CHO K1 cells were seeded into 384-well plates and incubated in 25 μL of starvation medium (F12K, 1% FBS, 1% penicillin-streptomycin) at 5% CO2, 37°C for 16 hours. Then, the starvation medium was completely replaced with 25 μL of assay buffer, and 25 μL of loading buffer was added to the desired wells. After dye addition, the cell plates were incubated at 37°C, 5% CO2 for 2 hours and then stored at room temperature until use. 12.5 μL of compound in assay buffer at the desired concentration (5×) was added to each well and incubated with the cells at room temperature for 30 minutes. At the end of the incubation, the microplate was transferred to the FLIPR instrument and the calcium assay was started as described in the instrument user guide. 12.5 μL of assay buffer with or without CCL20 was added during the assay. MAX ratios were plotted against antibody concentration and analyzed in GraphPad Prism to generate concentration curves.

Antibodies from clones such as Mab44 showed strong CCL20-CCR6 signal blocking ability. The signal blocking ability of representative antibodies disclosed herein is shown in Table 5.

Table 5. Results of calcium mobilization assay of anti-CCR6 antibodies

Clone number Calcium flux IC ₅₀ (nM) Mab7 3.96 Mab13 5.88 Mab39 32.67 Mab14 13.74 Mab44 1.7 Mab46 6.4 Mab27 12.99 Mab49 56.8 Mab50 6.66

Example 5. Migration experiment of anti-CCR6 antibody

Migration assays are designed to analyze whether a cell is directed toward and migrates toward a defined chemotactic agent. Because the CCL20-CCR6 axis plays an important role in mediating immune cell migration, the functionality of anti-CCR6 antibodies was evaluated using a cell-based in vitro chemotaxis assay.

BaF3 mouse proB cells transfected with human CCR6 were used as a constructed stably transfected cell line to monitor the inhibitory effect of antibodies on CCL20-mediated migration. All antibodies were studied based on the following assays.

BaF3 mouse proB cells transfected with human CCR6 were cultured in complete medium (RPMI1640, 10% FBS, 1% penicillin-streptomycin, 10 ng/mL IL-3) (37°C, 5% CO2)

Cells were kept in starvation medium (RPMI 1640, 1% FBS, 1% penicillin-streptomycin) for 3 hours before migration assays.

Chemotaxis assays were performed using HTS Transwell-96 plates with 5.0 μm pore size polycarbonate membranes according to the manufacturer's protocol. Briefly, the lower chamber was filled with 100 μL RPMI1640 medium containing 0.5% BSA and 100 ng/mL CCL20. After inserting the filter, 1x 105 cells pre-treated with 10 μg/mL antibody in 75 μL RPMI1640 medium containing 0.5% BSA were added to the upper chamber. After incubation at 37°C for 3 hours, the number of cells in the lower chamber was analyzed by flow cytometry. Migration was expressed as the total number of cells in the lower chamber.

Cloned antibodies such as Mab45, Mab35, Mab7, Mab20, Mab50, Mab14, Mab13 and Mab47 have a strong inhibitory effect on cell migration.

Example 6. Binding ability of anti-CCR6 antibodies to canine CCR6 and mouse CCR6

The cloned antibodies Mab7, Mab13 and Mab50 showed better functional activity by Tango, calcium flux and migration bioassays. The binding ability of these antibodies to canine CCR6 and mouse CCR6 was detected by FACS.

293T cells were transfected with lentiviral vectors encoding canine CCR6 and mouse CCR6, polybrene (8 μg/mL) was added, and selection was performed in medium containing puromycin (2 μg/mL), and the expression of canine CCR6 and mouse CCR6 in overexpressing cells was detected by FACS.

The researchers used FACS to study the specific binding of the antibody to canine and mouse CCR6 using canine and mouse CCR6 overexpressing cell lines. The results showed that the antibody could bind to 293T cells expressing canine CCR6, but not to 293T cells expressing mouse CCR6.

Example 7. Humanization of antibodies Mab7, Mab13 and Mab50

Antibodies Mab7, Mab13, and Mab50 were humanized by grafting the CDRs of the lead antibodies into selected human IgG frameworks. Human IGHV1-2*02 and IGKV2-30*02 were selected based on sequence similarity within both frameworks (FR) of Mab7. Human IGHV1-46*02 and IGKV2-30*02 were selected based on sequence similarity to Mab13 within both frameworks (FR). Human IGHV3-48*03 and IGKV2-29*02 were selected based on sequence similarity to Mab50 within both frameworks (FR). To maintain typical loop structures and chain interfaces, certain residues in the human frameworks were back-mutated to the corresponding mouse residues (Table 6)

PTM prediction indicated that the CDRs of Mab7, Mab13, and Mab50 had high-risk sequence defects. The CDR-H2 region of Mab7 has an NG motif. We evaluated possible mutations at position N54 in the heavy chain to determine whether the potential deamination site in VH could be removed without affecting activity. The CDR-H2 region of Mab13 has a DG group, the CDR-L1 region has an NG group, and the CDR-H3 region has an RGD group. We evaluated all sequences in the heavy and light chains to determine whether the DG, NG, and RGD sites in VH and VL could be removed without affecting activity. For Mab50, the CDR-L1 region has an NG motif. We evaluated possible mutations at this position in the light chain to determine whether the potential deamination site in VL could be removed without affecting activity.

Humanized monoclonal antibodies from Mab7, Mab13, and Mab50 were shown to bind to 293T expressing human CCR6. For Mab7, mutation of the PTM site NG on CDR-H2 to QG or NA had no effect on binding affinity. Three antibodies of Mab7 (VH2+VL2, VH2+VL3, and VH3+VL2) were constructed and transfected into Expi293F cells. For Mab13, mutation of the PTM site DG on CDR-H2 to EG or DA had no effect on binding affinity. Mutation of the PTM site NG on CDR-L1 to QG or NA had no effect on binding affinity. Changing D to E in RGD did not affect antibody affinity, but changed the EC50 from 0.39 nM to 0.24 nM. Antibodies of Mab13 (VH6+VL5 and VH9+VL1) were constructed and transfected in ExpiCHO cell culture. The mutation of the PTM site NG to NA on the CDR-L1 of Mab50 had no effect on binding affinity. Three Mab50 antibodies (VH1+VL1, VH1+VL2 and VH1+VL3) with selective removal of PTM sites (NG/NA) were constructed and transfected in Expi293F cell culture. Table 7 lists the ALL affinity constants of humanized antibodies Mab7, Mab13 and Mab50.

Table 6: Humanized VHs and VLs of Mab7, Mab13 and Mab50

Table 7 Anti-CCR6 humanized antibody affinity

Example 8. Calcium flux assay of humanized antibodies

The second round of humanized antibodies was tested by calcium flux assay to determine which of the above humanized antibodies could better block CCL20-CCR6 signaling.

Humanized antibodies showed CCL20-CCR6 signal blocking, and Mab7 (VH2-NG/NA+VL3) showed the strongest CCL20-CCR6 signal blocking. Table 8 lists the signal blocking capabilities of representative antibodies disclosed herein.

Table 8: Results of calcium mobilization assay of humanized anti-CCR6 antibodies

Clone number Calcium flux IC ₅₀ (nM) Mab7( _VH2-NG/NA + _VL2 ) 27.2 Mab7( _VH3-NG/NA + _VL2 ) 54.1 Mab7( _VH2-NG/NA + _VL3 ) 6.86

Example 9. Mab7 (VH2-NG/NA+VL3) cross-reacts with monkey CCR6, but not with rat, dog, or mouse CCR6

Lentiviral vectors encoding mouse, rat and canine CCR6 were transfected into 293T cells with polybrene (8 μg/mL), selected in medium containing puromycin (2 μg/mL), and the expression of mouse, rat and canine CCR6 was detected by FACS.

Human, rat, dog, mouse and monkey CCR6 high-expressing cell lines and parental 293T cell lines were used to study the specific binding of humanized antibodies to human, rat, dog, mouse and monkey CCR6 by flow cytometry.

The results are shown in Figures 3A-3E. Based on the binding affinity, Mab7 (VH2-NG/NA+VL3) binds to 293T cells expressing human (3A), rat (3B), canine (3C), mouse (3D) and monkey CCR6 (3E), but not to the parental cells.

Example 10. Study on the binding of Mab7 (VH2-NG/NA+VL3) humanized antibody to human CXCR2

The binding of Mab7 (VH2-NG/NA+VL3) humanized antibody to cells overexpressing CXCR2 was detected by FACS. Briefly, lentiviral vectors encoding human CXCR2 were transfected into 293T cells with polybrene (8 μg/mL), selected in medium containing puromycin (2 μg/mL), and the expression of human CXCR2 was detected by FACS using CXCR2 antibody (Biolegend, 320705). Binding assessment was performed by reacting 293T cells overexpressing human CXCR2 with humanized Mab7 (VH2-NG/NA+VL3) antibody prepared by 3-fold dilution at 25 μg/mL. After 1 hour of reaction at 4°C, it was reacted with Alexa Fluor 488 affinity pure goat anti-human Ig (H+L) (Jackson, 109-545-003) and analyzed by flow cytometry.

As shown in Figure 4, the humanized Mab7 (VH2-NG/NA+VL3) antibody can bind to 293T cells expressing human CCR6, but cannot bind to 293T cells expressing human CXCR2.

Example 11. Humanized Antibody Stability Verification

Monoclonal antibodies are essentially proteins and have instability issues. Stability testing of monoclonal antibodies is a key requirement for their development and commercialization as therapeutic biomolecules. The stability and activity of humanized antibodies Mab7(VH2-NG/NA+VL3)-hIgG1 and Mab7(VH2-NG/NA+VL3)-hIgG4(S228P) were tested under the stress conditions shown in Table 9.

Table 9. Stability test

^*** 5FT means 5 freeze-thaw cycles

The binding affinity of the humanized antibody at different time points was measured in a manner similar to Example 2. The results are shown in Table 10.

Table 9: Affinity EC50 of humanized antibodies under stress conditions

It should be understood that if any prior art publication is cited herein, such citation does not constitute an admission that the publication forms part of the technical general knowledge in any country. The disclosures of all publications, patents, patent applications and published patent applications referred to herein by identifying citations are incorporated herein by reference in their entirety.

Claims (18)

1. An isolated antibody or antigen binding fragment thereof that specifically binds the chemokine receptor CCR 6.

2. The antibody or antigen binding fragment of claim 1 that binds to human chemokine receptor CCR6 and optionally (1) binds to monkey chemokine receptor CCR6, (2) does not bind to mouse chemokine receptor CCR6, (3) does not bind to rat chemokine receptor CCR6, and/or (4) does not bind to dog chemokine receptor CCR6.

3. The antibody or antigen binding fragment of claim 1 or 2, comprising a heavy chain variable region consisting of HCDR1, HCDR2 and HCDR3 and/or a light chain variable region consisting of LCDR1, LCDR2 and LCDR3, having the amino acid sequence as follows (based on the Kabat numbering scheme):

selectable (optional)

(1) NG in SEQ ID NO. 47 is mutated to NA or;

(2) DG in SEQ ID NO. 78 is mutated to EG or DA and/or GD in SEQ ID NO. 79 is mutated to AD, SD, GE or GN, in particular GE, and/or NG in SEQ ID NO. 80 is mutated to QG or NA; or (b)

(3) NG in SEQ ID NO. 61 is mutated to QG or NA, especially NA.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO; (b) Antigen binding fragments consisting of the amino acid sequence of SEQ ID NO :1,9,17,24,31,38,44,315,316,51,58,63,69,58,75,323,324,325,326,327,328,329,330,82,89,93,96,103,108,115,121,127,132,140,143,151,155,158,165,169,173,176,184,190,193,201,208,212,217,222,227,230,

232,238,244,245,253,261,262,265,272,278,281,285,290,295,299,301,Or 304 (tables 2,3 or 6),

Or an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to any of the SEQ ID NOs 1,9,17,24,

31,38,44,315,316,51,58,63,69,58,75,323,324,325,326,327,328,329,330,82,89,93,96,103,

108,115,121,127,132,140,143,151,155,158,165,169,173,176,184,190,193,201,208,212,217,

222,227,230,232,238,244,245,253,261,262,265,272,278,281,285,290,295,299,301,Or 304; and/or (b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO :2,10,18,25,32,39,45,52,59,64,70,73,76,334,335,83,90,94,97,104,109,116,122,128,133,141,144,152,156,159,166,170,174,177,182,

185,191,194,202,209,213,218,223,228,231,233,239,246,254,263,341,342,266,273,282,286,291,296,300,302,or 305( Table 2, 3 or 6), or an amino acid sequence :2,10,18,25,32,39,45,52,59,64,70,73,76,334,335,83,90,94,97,104,109,116,122,128,133,141,144,152,156,159,166,170,174,177,182,185,191,194,202,209,213,218,223,228,231,233,239,246,254,263,341,342,266,273,282,286,291,296,300,302,or 305, that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs

Selectable (optional)

(1) NG (if present) in SEQ ID NO. 47 is mutated to NA or QG;

(2) DG (if present) in SEQ ID NO. 78 to EG or DA, and/or GD (if present) in SEQ ID NO. 79 to AD, SD, GE or GN, especially GE, and/or R (if present) at the N-terminus of SEQ ID NO. 79 to K or Q, and/or NG (if present) in SEQ ID NO. 80 to QG or NA; or (b)

(3) NG in SEQ ID NO. 61 (if present) is mutated to QG or NA, especially NA.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO; (b) An antigen binding fragment consisting of the amino acid sequence of SEQ ID NO:

1,9,17,24,31,38,44,315,316,51,58,63,69,58,75,323,324,325,326,327,328,329,330,82,89,93,96,103,108,115,121,127,132,140,143,151,155,158,165,169,173,176,184,190,193,201,208,212,217,222,227,230,232,238,244,245,253,261,262,265,272,278,281,285,290,295,299,301,or 304( Table 2, 3 or 6), or an amino acid sequence :1,9,17,24,31,38,44,315,316,51,58,63,69,58,75,323,324,325,326,327,328,329,330,82,89,93,96,103,108,115,121,127,132,140,143,151,155,158,165,169,173,176,184,190,193,201,208,212,217,222,227,230,232,238,244,245,253,261,262,265,272,278,281,285,290,295,299,301,or 304; having one, two or three amino acid substitutions in the amino acid sequence of any of SEQ ID NOs and/or (b) a light chain variable region :2,10,18,25,32,39,45,52,59,64,70,73,76,334,335,83,90,94,97,104,109,116,122,128,133,141,144,152,156,159,166,170,174,177,182,185,191,194,202,209,213,218,223,228,231,233,239,246,254,263,341,342,266,273,282,286,291,296,300,302,or 305( table 2, 3 or 6) consisting of the amino acid sequence of SEQ ID NOs, or an amino acid sequence :2,10,18,25,32,39,45,52,59,64,70,73,76,334,335,83,90,94,97,104,109,116,122,128,133,141,144,152,156,159,166,170,174,177,182,185,191,194,202,209,213,218,223,228,231,233,239,246,254,263,341,342,266,273,282,286,291,296,300,302,or 305, having one, two or three amino acid substitutions in the amino acid sequence of any of SEQ ID NOs

Alternatively, the amino acid substitutions are conservative amino acid substitutions,

Selectable (optional)

(1) NG (if present) in SEQ ID NO. 47 is mutated to NA or QG;

(2) DG (if present) in SEQ ID NO. 78 to EG or DA, and/or GD (if present) in SEQ ID NO. 79 to AD, SD, GE or GN, especially GE, and/or R (if present) at the N-terminus of SEQ ID NO. 79 to K or Q, and/or NG (if present) in SEQ ID NO. 80 to QG or NA; or (b)

(3) NG in SEQ ID NO. 61 (if present) is mutated to QG or NA, especially NA.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the amino acid substitution is a conservative amino acid substitution.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising a heavy chain variable region and a light chain variable region, each variable region comprising the amino acid sequence of seq id no:

Selectable item

(1) NG (if present) in SEQ ID NO. 47 is mutated to NA or QG;

(2) DG (if present) in SEQ ID NO. 78 to EG or DA, and/or GD (if present) in SEQ ID NO. 79 to AD, SD, GE or GN, especially GE, and/or R (if present) at the N-terminus of SEQ ID NO. 79 to K or Q, and/or NG (if present) in SEQ ID NO. 80 to QG or NA; or (b)

(3) NG in SEQ ID NO. 61 (if present) is mutated to QG or NA, especially NA.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, which is a monoclonal antibody, chimeric antibody, humanized antibody, or humanized engineered antibody.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the humanized antibody comprises

(A) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 311, 312, 313 or 314 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 319, 320, 321 or 322, optionally with NG mutation in HCDR2 to NA or QG;

(b) A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO. 331 or 332 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO. 333 or 336, optionally mutating DG in HCDR2 to EG or DA, and/or mutating GD in HCDR3 to AD, SD, GE or GN, especially GE, and/or mutating R at the N-terminus of HCDR3 to K or Q, and/or mutating NG of CDR-L1 in LCDR1 to GQ or NA; or (b)

(C) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 337, 338, 339 or 340 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 343, 344, 345 or 346, NG in LCDR1 being optionally mutated to QG or NA, in particular NA.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the humanized antibody comprises

(A) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 317 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 320;

(b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 318, and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 321;

(c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 318 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 320;

(d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 330 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 336;

(e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 332 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 333;

(f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 337 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 347;

(g) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 337 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 348; or (b)

(H) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 337 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 349.

11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv) ₂、scFv-Fc、Fv、Fab、Fab'、F(ab')₂, minibody, and diabody.

12. An isolated nucleic acid encoding the heavy chain variable region and/or the light chain variable region of the antibody or antigen-binding fragment of any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13.

15. A process for the production of an antibody or antigen-binding fragment thereof according to any one of claims 1-11, comprising culturing the host cell of claim 14 and recovering the antibody or antigen-binding fragment from the culture.

16. A pharmaceutical composition comprising as active ingredient the antibody or antigen-binding fragment thereof of any one of claims 1-11, the nucleic acid of claim 12, the vector of claim 13 or the host cell of claim 14, and a pharmaceutically acceptable formulation.

17. A method of treating a CCR6 mediated disease comprising administering to a patient in need thereof an effective amount of the antibody or antigen binding fragment of any one of claims 1-11.

18. The method of claim 17, wherein the cancer is hepatocellular carcinoma, colorectal cancer, breast cancer, pancreatic cancer, cervical cancer, renal cancer, lung cancer, leukemia or lymphoma, or chronic lymphocytic leukemia; rheumatoid arthritis, multiple Sclerosis (MS), psoriasis, graft versus host disease, lupus, chronic obstructive pulmonary disease, optic neuritis, age-related macular degeneration, systemic lupus erythematosus, sjogren's syndrome, scleroderma, systemic sclerosis, chronic kidney disease, liver fibrosis, tuberculosis, idiopathic pulmonary fibrosis, tuberculosis-induced pulmonary fibrosis, retroperitoneal fibrosis, pulmonary fibrosis, cystic fibrosis, endocardial fibrosis, atrial fibrosis, mediastinal fibrosis, bone marrow fibrosis (bone marrow), retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, joint fibrosis, inflammatory bowel disease, ulcerative colitis, crohn's disease, atherosclerosis, graft rejection, central nervous system injury, or infectious disease.