WO2023125793A1

WO2023125793A1 - Anti-ccr6 antibodies and uses thereof

Info

Publication number: WO2023125793A1
Application number: PCT/CN2022/143352
Authority: WO
Inventors: Juan Du; Na Wang; Guohuang FAN; Jianfei Wang
Original assignee: Nanjing Immunophage Biotech Co., Ltd
Priority date: 2021-12-29
Filing date: 2022-12-29
Publication date: 2023-07-06

Abstract

Provided are anti-CCR6 antibodies, or an antigen-binding fragment thereof, a nucleic acid encoding the antibody or the antigen-binding fragment thereof, a vector and a host cell including the nucleic acid, a method for producing the antibody or the antigen-binding fragment thereof, a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof as an active ingredient, and the use of the antibody in treating the diseases mediated by CCR6.

Description

ANTI-CCR6 ANTIBODIES AND USES THEREOF

Technical Field

The present invention relates to novel anti-CCR6 antibodies, or an antigen-binding fragment thereof, a nucleic acid encoding the antibody or the antigen-binding fragment thereof, a vector and a host cell including the nucleic acid, a method for producing the antibody or the antigen-binding fragment thereof, a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof as an active ingredient, and the use of the antibody in treating the diseases mediated by CCR6.

Background of the Invention

Chemokines are a family of low molecular weight chemotactic cytokines involved in cell recruitment and activation in inflammation. Chemokines regulate a broad spectrum of cellular functions and exert their actions by binding to chemokine receptors which are G protein-coupled receptors, causing chemotaxis and activation of various subpopulations of cells in the immune system. Chemokines are divided into different classes, including CC, CXC, CX3C, and XC, based on the positions of the N-terminal cysteine residues within the protein. The CC class of chemokines contains the CC motif in which the first two cysteines are not separated by any amino acids, whereas the CXC class of chemokines contains the CXC motif in which the first two cysteines are separated by a random amino acid. The activity of chemokines is mediated primarily through tight binding to their receptors on the surface of leukocytes.

In normal physiological conditions, the expression of chemokines and chemokine receptors are delicately and tightly regulated, dysregulated expression and activation of either chemokines and chemokine receptors often result in maladies such as autoimmune diseases or cancers. Among the chemokine repertoire, the CC chemokine receptor-ligand pair, CC chemokine receptor 6 (CCR6) , and CC chemokine ligand 20 (CCL20) are becoming recognized for their invaluable therapeutic potential in immunological 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, subsets of CD4 ⁺ and CD8 ⁺ T cells, and NKT cells. Additionally, CCR6 is expressed by both central memory and effector memory T cells. Recent studies have also shown that CCR6 is a specific marker for Th17 cells and regulatory T cells. Interestingly, CCR6 is also expressed on some cancer cells. CCR6 expression has been reported on multiple DC subsets including CD11b ⁺CD8α-myeloid DCs, Langerhans cells (LC) , CD34 ⁺ cell-derived immature myeloid DCs, and immature monocyte-derived DCs. As DCs represent the most potent class of APCs in the immune system with the unique ability to induce primary immune responses against invading pathogens, therefore, CCR6 may regulate the activity of the DCs population involved in driving the innate immune response.

CCL20, which is known by several names, including macrophage inflammatory protein (MIP) -3α, Exodus-1, and liver and activation-regulated chemokine (LARC) , is expressed in various human tissues and immune cells and has been observed mainly in the lymph node, lung, and liver. At the cellular level, endothelial cells, neutrophils, B cells, natural killer cells, dendritic cells (DC) , and macrophages have been reported to secrete CCL20. CCL20 is expressed only by T helper lymphocyte 17 (Th17) cells and not by regulatory T cells or other T helper subsets. The importance of the CCR6–CCL20 axis was emphasized by the discovery of the CCR6-expressing CD4+ T helper cell subpopulations, Th17, and regulatory Treg cells, which form a platform that stringently regulates immune tolerance in healthy individuals. Disruption of this delicate alliance will tip the balance in favour of either the pro-inflammatory signature cell population, Th17, or its immune regulatory partner, the regulatory Treg cells, which are avidly recruited to the sites of infection or injury.

CCR6 and CCL20 have exhibited characteristics in tune with both immune homeostasis and immune activation. It is already established that the immunological impact of this chemokine receptor-ligand partnership has far-reaching consequences in health and disease that affect multiple organs of the human body. A plethora of research studies has demonstrated that the CCR6 and CCL20 axis directly influences the nervous, respiratory, gastrointestinal, excretory, skeletal, and reproductive systems via pleiotropic immune mechanisms, manifesting as diseases with high mortality rates.

The CCL20-CCR6 axis not only has long been known to be involved in inflammatory and infectious diseases, such as rheumatoid arthritis and human immunodeficiency virus infections but also is associated with several cancers, including hepatocellular carcinoma, colorectal cancer, breast cancer, pancreatic cancer, cervical cancer, and kidney cancer. The CCL20-CCR6 axis promotes cancer progression directly by enhancing the migration and proliferation of cancer cells and indirectly by re-modeling the tumor microenvironment through immune cell control.

In light of the role that CCR6-CCL20 plays in the pathogenesis of various diseases, it is desirable to prepare antibodies that inhibit CCR6 activity, which is useful in the treatment of diseases mediated by CCR6, such as autoimmune diseases and cancers.

Therefore, there is a need in the art for more effective therapeutics comprising anti-CCR6 antibodies that effectively inhibit the CCR6 signaling while causing minimal adverse side effects in humans.

Summary of the Invention

Accordingly, as a result of intensive efforts to develop a novel antibody against chemokine receptor CCR6, in particular human CCR6, the present inventors have invented novel anti-CCR6 antibodies against chemokine receptor CCR6 by blocking CCR6-CCL20 signaling, thus completing the present invention.

In the first aspect, disclosed herein is a novel anti-CCR6 antibody or an antigen-binding fragment thereof.

In the second aspect, disclosed herein is a method for treating a disease mediated by CCR6.

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

In the fourth aspect, disclosed herein is a nucleic acid encoding the anti-CCR6 antibody or the antigen-binding fragment thereof, a vector and a host cell containing the nucleic acid.

Brief Description of the Drawings

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

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

Figure 1B shows the representative antibodies disclosed herein do not have the binding ability to the 293T parent cells in the cell-based binding assay.

Figure 2 shows the inhibitory effect of the antibodies disclosed herein on the cell migration.

Figure 3A shows the binding specificity of Mab7 (V _H2-NG/NA+V _L3) , which bound 293T cells expressing human CCR6.

Figure 3B shows the binding specificity of Mab7 (V _H2-NG/NA+V _L3) , which did not bind 293T cells expressing rat CCR6.

Figure 3C shows the binding specificity of Mab7 (V _H2-NG/NA+V _L3) , which did not bind 293T cells expressing dog CCR6.

Figure 3D shows the binding specificity of Mab7 (V _H2-NG/NA+V _L3) , which did not bind 293T cells expressing mouse CCR6.

Figure 3E shows the binding specificity of Mab7 (V _H2-NG/NA+V _L3) , which bound 293T cells expressing cynomolgus CCR6.

Figure 4 shows the binding specificity of humanized Mab7 (V _H2-NG/NA+V _L3) antibody, which bound 293T cells expressing human CCR6, but did not bind 293T cells expressing human CXCR2.

Definitions

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

As used herein, including the appended claims, the singular forms of words such as “a, ” “an, ” and “the, ” include their corresponding plural references unless the context clearly dictates otherwise.

The term “or” is used to mean, and is used interchangeably with, the term “and/or” unless the context clearly dictates otherwise.

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

The terms “administration, ” “administering, ” “treating, ” and “treatment” as used herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as the contact of a reagent to a fluid, where the fluid is in contact with the cell. The term “administration” and “treatment” also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. As used herein, treating any disease or disorder refer to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof) ; or alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient; or modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom) , physiologically, (e.g., stabilization of a physical parameter) , or both; or preventing or delaying the onset or development or progression of the disease or disorder.

The term “affinity” as used herein refers to the strength of interaction between antibody and antigen. Within the antigen, the variable regions of the antibody interact through non-covalent forces with the antigen at numerous sites. In general, the more interactions, the stronger the affinity.

The term “antibody” as used herein refers to a polypeptide of the immunoglobulin family that can bind a corresponding antigen non-covalently, reversibly, and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH and VL are composed of three CDRs and four framework regions (FRs) arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The variable regions of each light/heavy chain (VL/VH) pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same in primary sequence. The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term “antibody” includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. The antibodies 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 antibodies comprise at least one antigen-binding site, at least a variable region. In some embodiments, the anti-CCR6 antibodies comprise an antigen-binding fragment from a CCR6 antibody disclosed herein.

The term “monoclonal antibody” or “mAb” or “Mab” as used herein means a population of substantially homogeneous antibodies, i.e., the antibody molecules comprised in the population are identical in amino acid sequence except for possible naturally occurring mutations that can be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies comprising different amino acid sequences in their variable domains, particularly their complementarity determining regions (CDRs) , which are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies 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. A hybridoma producing a monoclonal antibody can be cultivated in vitro or in vivo. High titers of monoclonal antibodies can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired antibodies. Monoclonal antibodies of isotype IgM or IgG can be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair comprising one “light chain” (about 25 kDa) and one “heavy chain” (about 50-70 kDa) . The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain can define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as α, δ, ε, γ, or μ, and define the antibody's isotypes as IgA, IgD, IgE, IgG, and IgM, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids.

The positions of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, 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) ) . Definitions of antigen combining sites are also described in the following: Ruiz et al., Nucleic Acids Res., 28: 219-221 (2000) ; and Lefranc, M. P., Nucleic Acids Res., 29: 207-209 (2001) ; MacCallum et al., J. Mol. Biol., 262: 732-745 (1996) ; and Martin et al., Proc. Natl. Acad. Sci. USA, 86: 9268-9272 (1989) ; Martin et al., Methods Enzymol., 203: 121-153 (1991) ; and Rees et al., In Sternberg M. J. E. (ed. ) , Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996) . For example, under 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) . Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1) , 52-56 (HCDR2) , and 95-102 (HCDR3) ; and the amino acid residues in VL are numbered 26-32 (LCDR1) , 50-52 (LCDR2) , and 91-96 (LCDR3) . By combining the CDR definitions of both Kabat and Chothia, the CDRs are numbered 26-35 (HCDR1) , 50-65 (HCDR2) , and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1) , 50-56 (LCDR2) , and 89-97 (LCDR3) in human VL. Under IMGT the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1) , 51-57 (HCDR2) , and 93-102 (HCDR3) , and the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1) , 50-52 (LCDR2) , and 89-97 (LCDR3) (numbering according to Kabat) . Under IMGT, the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.

The term “hypervariable region” means the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “CDR” (e.g., LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain) . See, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence) ; see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an antibody by structure) . The term “framework” or “FR” residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

Unless otherwise indicated, an “antigen-binding fragment” means antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g., fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but not limited to, Fab, Fab', F (ab') 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., single-chain Fv (ScFv) ; nanobodies and multispecific antibodies formed from antibody fragments.

As used herein, an antibody “specifically binds” to a target protein, meaning the antibody exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity. An antibody “specifically binds” or “selectively binds, ” is used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, or antigen binding antibody fragment, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologics, for example, in a biological sample, blood, serum, plasma or tissue sample. Thus, under certain designated immunoassay conditions, the antibodies or antigen-binding fragments thereof specifically bind to a particular antigen at least two times greater when compared to the background level and do not specifically bind in a significant amount to other antigens present in the sample. In one aspect, under designated immunoassay conditions, the antibody or antigen-binding fragment thereof, specifically bind to a particular antigen at least ten (10) times greater when compared to the background level of binding and does not specifically bind in a significant amount to other antigens present in the sample.

The term “human antibody” herein means an antibody that comprises human immunoglobulin protein sequences only. A human antibody can contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” means an antibody that comprises only mouse or rat immunoglobulin protein sequences, respectively.

The term “humanized” or “humanized antibody” means forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. The prefix “hum, ” “hu, ” “Hu, ” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions can be included to increase affinity, increase the stability of the humanized antibody, remove a post-translational modification or for other reasons.

The term “corresponding human germline sequence” refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. The corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementary determining regions, a variable segment (as defined above) , or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods disclosed herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm 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 contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296: 57-86, 2000.

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms, which are described in Altschul et al, Nuc. Acids Res. 25: 3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215: 403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as values for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, 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 the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; 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) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLAST program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787, 1993) . One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N) ) , which provides an indication of the probability by which 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 the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also be determined using the algorithm which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 (E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, (1988) ) . In addition, the percent identity between two amino acid sequences can be determined using the algorithm which has been incorporated into the GAP program in the GCG software package using either a BLOSUM62 matrix or a PAM250 matrix, and 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 substituting one or more amino acids by one or more amino acids having similar biological or biochemical properties that do not cause loss of the biological or biochemical functions of the polypeptide. The term “conservative amino acid substitution” refers to a substitution to replace an amino acid residue by an amino acid residue having a similar side chain. Classes of the amino acid residue having a similar side chain are defined and well-known in the art. Such classes include amino acids with basic side chains (e.g., lysine, arginine, histidine) , amino acids with acidic side chains (e.g., aspartic acid, glutamic acid) , amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . It is considered that the antibody according to the present invention has a conservative amino acid substitution and still retains activity.

The term “anti-CCR6 antibody” , “antibody against chemokine receptor CCR6” , or “antibody that binds CCR6” , as used herein equivalently and include an antibody that is capable of inhibiting and/or neutralizing the biological signaling activity of CCR6, for example by blocking binding or substantially reducing binding of CCR6 to its ligand CCL20 and thus inhibiting or reducing the signaling pathway triggered by CCR6 and/or inhibiting or reducing a CCR6-mediated cell response.

The term “anti-CCR6 antibody” as used herein includes both a polyclonal antibody and a monoclonal antibody, is preferably a monoclonal antibody and may have a whole antibody. The whole antibody is a structure having two full-length light chains and two full-length heavy chains, including a constant region, wherein each light chain is linked to the corresponding heavy chain by a disulfide bond.

The whole antibody of the anti-CCR6 antibody disclosed herein includes 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 human antibody libraries, but the present invention is not limited thereto.

As used herein, the term “antigen binding fragment” of the anti-CCR6 antibody refers to a fragment having a function capable of binding to an antigen of the anti-CCR6 antibody, that is, CCR6 and encompasses Fab, Fab', F (ab') ₂, scFv, (scFv) ₂, scFv-Fc, Fv, Fab, Fab', F (ab') ₂, minibody, and diabody and the like, which is used interchangeably with “antibody fragment” .

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

An Fv (variable fragment) including a variable region of each of the heavy chain and the light chain is the minimal antibody fragment having the original specificity of parent immunoglobulin. Double chain Fv (dsFv, disulfide-stabilized Fv) is formed by binding the variable region of the light chain to the variable region of the heavy chain via a disulfide bond. Single chain Fv (scFv) is an Fv wherein the respective variable regions of the heavy chain and the light chain are covalently linked via a peptide linker. These antibody fragments can be obtained by treating the whole antibody with a protease (for example, Fab can be obtained by restriction-cleaving the whole antibody with papain, and the F (ab’ ) ₂ fragment can be obtained by restriction-cleaving the whole antibody with pepsin) and are preferably constructed by genetic recombination technology (for example, by amplifying a DNA encoding the heavy chain of the antibody or a variable region thereof or a DNA encoding the light chain or a variable region thereof as a template by PCR (polymerase chain reaction) using a pair of primers, and amplifying using a combination of a pair of primers to link DNA encoding a peptide linker and each of both ends thereof to the heavy chain or a variable region thereof and the light chain or a variable region thereof) .

The term “nucleic acid” as used herein is interchangeable with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs) . The nucleic acid may be present in a cell or a cell lysate, or in a partially purified form or in a substantially pure form. The nucleic acid may be “isolated” or “substantially pure” , when purified from other cellular components or other contaminants, for example, nucleic acids or proteins of other cells, by standard techniques including, for example, alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well-known in the art. The nucleic acid of the present invention may, for example, be DNA or RNA, and may or may not include an intron sequence.

For expression of the antibody or the antigen-binding fragment thereof disclosed herein, DNA encoding partial-or full-length light and heavy chains are obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the target antibody) , and the DNA may be “operably bound” to transcription and translation control sequences to be inserted into the expression vector.

The term “operably bound” as used herein may indicate that the gene encoding the antibody is ligated into the vector so that the transcription and translation control sequences can serve the intended function of regulating the transcription and translation of the antibody genes.

The expression vector and expression control sequences which are compatible with the host cell used for expression are selected. The light chain genes of the antibody and the heavy chain genes of the antibody are inserted into separate vectors, or both the genes are inserted into the same expression vector. Antibodies are inserted into expression vectors by standard methods (e.g., ligation of an antibody gene fragment and complementary restriction enzyme sites on vectors, or blunt end ligation when there is no restriction enzyme site) . In some cases, the recombinant expression vectors may encode signal peptides that facilitate the secretion of the antibody chains from host cells. The antibody chain genes may be cloned into vectors such that signal peptides are attached to the amino terminus of the antibody chain genes in accordance with the frame. The signal peptides may be immunoglobulin signal peptides or heterologous signal peptides (i.e., signal peptides derived from proteins excluding immunoglobulin) . In addition, the recombinant expression vectors have regulatory sequences that control the expression of the antibody chain genes in the host cells. “Regulatory sequences” may include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control transcription or translation of the antibody chain genes. It will be appreciated by those skilled in the art that the design of expression vectors can be varied by selecting different regulatory sequences depending on factors such as the choice of host cells to be transformed and the levels of protein expression.

The term “host cell” refers to any cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. The host cell disclosed herein is preferably selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli and insect cells, but the present invention is not limited thereto. More specifically, the host cell disclosed herein may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp. In addition, the host cell may be selected from fungi such as Aspergillus sp., yeast such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. or Neurospora crassa, and other eukaryotic cells including lower eukaryotic cells, and higher eukaryotic cells derived from insects. The host cell may also be derived from plants or mammals. Preferably, the host cell is 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. Particularly preferably, CHO cells are used. Mammalian host cells are used to express and produce the polypeptides disclosed herein. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line harboring an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B-cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89: 49-68, 1986) , and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter) , the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

The term “vector” as used here refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid” , which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) . Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors” ) . In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. As disclosed herein, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors are also encompassed by the disclosure herein, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.

The nucleic acid or the vector is transformed or transfected into a host cell. Various techniques commonly used to introduce foreign nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for “transformation” or “transfection” include electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, lipofection or the like. Various expression host/vector combinations may be used to express the antibody according to the invention.

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

In some cases, the expressed antibody may be separated from the host cells and purified to homogeneity. The separation or purification of the antibody can be carried out by separation and purification methods commonly used for proteins, for example, chromatography. The chromatography may, for example, include affinity chromatography including a protein A column and a protein G column, ion-exchange chromatography or hydrophobic chromatography. In addition to the chromatography, the antibody can be separated and purified by a combination of filtration, ultrafiltration, salting out, dialysis or the like.

The term “pharmaceutically acceptable carrier” refers to a substance that can be added to the active ingredient to help formulate or stabilize the formulation and does not cause significantly harmful toxic effects on patients.

The term “therapeutically effective amount” as used herein refers to an amount of a combination of an antibody or an antigen-binding fragment thereof required to cause measurable benefits in vivo in a patient in need of treatment. The exact amount will depend on a number of factors including, but not limited to, the ingredients and physical properties of the therapeutic composition, the population of intended patients and considerations of respective patients and can be readily determined by those skilled in the art. When fully taking these factors into consideration, it is important to administer a minimum amount sufficient to achieve maximum effects without causing adverse effects, and this dose can be easily determined by an expert in the field.

The terms “cancer” or “tumor” as used herein has the broadest meaning as understood in the art and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. In the context disclosed herein, the cancer is not limited to a certain type or location.

Anti-CCR6 antibodies

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

In some embodiments, the antibody or an antigen-binding fragment thereof binds human chemokine receptor CCR6, and optionally (1) binds cynomolgus chemokine receptor CCR6, (2) does not bind mouse chemokine receptor CCR6, (3) does not bind rat chemokine receptor CCR6, and/or (4) does not bind dog chemokine receptor CCR6.

In some embodiments, the anti-CCR6 antibody or an antigen-binding fragment thereof disclosed herein is humanized or is a fully human antibody.

In some embodiments, the anti-CCR6 antibody disclosed herein is an antigen-binding fragment of the full-size monoclonal antibody.

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

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

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

In some embodiments, the anti-CCR6 antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 in a row of Table 1 and/or a light chain variable region comprising LCDR1, LCDR2, and LCDR3 in a row of Table 1. In some embodiments, the anti-CCR6 antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 in a row of Table 1 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 in the same row of Table 1. The amino acid sequence ID numbers of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (based on Kabat numbering scheme) is presented in Table 1 (see Table 3 and 6 for sequence) :

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

HCDR1	HCDR2	HCDR3	LCDR1	LCDR2	LCDR3
SEQ ID NO: 46	SEQ ID NO: 47	SEQ ID NO: 48	SEQ ID NO: 49	SEQ ID NO: 7	SEQ ID NO: 50
SEQ ID NO: 77	SEQ ID NO: 78	SEQ ID NO: 79	SEQ ID NO: 80	SEQ ID NO: 7	SEQ ID NO: 81
SEQ ID NO: 3	SEQ ID NO: 264	SEQ ID NO: 92	SEQ ID NO: 61	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 3	SEQ ID NO: 4	SEQ ID NO: 5	SEQ ID NO: 6	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 11	SEQ ID NO: 12	SEQ ID NO: 13	SEQ ID NO: 14	SEQ ID NO: 15	SEQ ID NO: 16
SEQ ID NO: 19	SEQ ID NO: 20	SEQ ID NO: 21	SEQ ID NO: 22	SEQ ID NO: 7	SEQ ID NO: 23
SEQ ID NO: 26	SEQ ID NO: 27	SEQ ID NO: 28	SEQ ID NO: 29	SEQ ID NO: 7	SEQ ID NO: 30
SEQ ID NO: 33	SEQ ID NO: 34	SEQ ID NO: 35	SEQ ID NO: 36	SEQ ID NO: 7	SEQ ID NO: 37
SEQ ID NO: 3	SEQ ID NO: 40	SEQ ID NO: 41	SEQ ID NO: 42	SEQ ID NO: 43	SEQ ID NO: 8
SEQ ID NO: 53	SEQ ID NO: 54	SEQ ID NO: 55	SEQ ID NO: 56	SEQ ID NO: 57	SEQ ID NO: 30
SEQ ID NO: 3	SEQ ID NO: 60	SEQ ID NO: 41	SEQ ID NO: 61	SEQ ID NO: 43	SEQ ID NO: 62
SEQ ID NO: 3	SEQ ID NO: 65	SEQ ID NO: 66	SEQ ID NO: 67	SEQ ID NO: 68	SEQ ID NO: 8
SEQ ID NO: 26	SEQ ID NO: 71	SEQ ID NO: 72	SEQ ID NO: 22	SEQ ID NO: 7	SEQ ID NO: 30
SEQ ID NO: 3	SEQ ID NO: 60	SEQ ID NO: 41	SEQ ID NO: 61	SEQ ID NO: 43	SEQ ID NO: 74
SEQ ID NO: 84	SEQ ID NO: 85	SEQ ID NO: 86	SEQ ID NO: 87	SEQ ID NO: 88	SEQ ID NO: 8
SEQ ID NO: 3	SEQ ID NO: 91	SEQ ID NO: 92	SEQ ID NO: 6	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 3	SEQ ID NO: 95	SEQ ID NO: 92	SEQ ID NO: 61	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 98	SEQ ID NO: 99	SEQ ID NO: 100	SEQ ID NO: 101	SEQ ID NO: 102	SEQ ID NO: 30
SEQ ID NO: 105	SEQ ID NO: 106	SEQ ID NO: 79	SEQ ID NO: 107	SEQ ID NO: 7	SEQ ID NO: 81
SEQ ID NO: 110	SEQ ID NO: 111	SEQ ID NO: 112	SEQ ID NO: 113	SEQ ID NO: 7	SEQ ID NO: 114
SEQ ID NO: 117	SEQ ID NO: 118	SEQ ID NO: 119	SEQ ID NO: 113	SEQ ID NO: 7	SEQ ID NO: 120
SEQ ID NO: 26	SEQ ID NO: 123	SEQ ID NO: 124	SEQ ID NO: 125	SEQ ID NO: 57	SEQ ID NO: 126

SEQ ID NO: 129	SEQ ID NO: 130	SEQ ID NO: 131	SEQ ID NO: 125	SEQ ID NO: 7	SEQ ID NO: 120
SEQ ID NO: 134	SEQ ID NO: 135	SEQ ID NO: 136	SEQ ID NO: 137	SEQ ID NO: 138	SEQ ID NO: 139
SEQ ID NO: 142	SEQ ID NO: 130	SEQ ID NO: 131	SEQ ID NO: 125	SEQ ID NO: 7	SEQ ID NO: 120
SEQ ID NO: 145	SEQ ID NO: 146	SEQ ID NO: 147	SEQ ID NO: 148	SEQ ID NO: 149	SEQ ID NO: 150
SEQ ID NO: 19	SEQ ID NO: 153	SEQ ID NO: 154	SEQ ID NO: 22	SEQ ID NO: 7	SEQ ID NO: 23
SEQ ID NO: 33	SEQ ID NO: 34	SEQ ID NO: 157	SEQ ID NO: 36	SEQ ID NO: 7	SEQ ID NO: 37
SEQ ID NO: 160	SEQ ID NO: 161	SEQ ID NO: 162	SEQ ID NO: 163	SEQ ID NO: 7	SEQ ID NO: 164
SEQ ID NO: 26	SEQ ID NO: 167	SEQ ID NO: 168	SEQ ID NO: 29	SEQ ID NO: 7	SEQ ID NO: 37
SEQ ID NO: 3	SEQ ID NO: 171	SEQ ID NO: 172	SEQ ID NO: 87	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 3	SEQ ID NO: 175	SEQ ID NO: 41	SEQ ID NO: 61	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 178	SEQ ID NO: 179	SEQ ID NO: 180	SEQ ID NO: 101	SEQ ID NO: 7	SEQ ID NO: 181
SEQ ID NO: 3	SEQ ID NO: 65	SEQ ID NO: 66	SEQ ID NO: 61	SEQ ID NO: 183	SEQ ID NO: 8
SEQ ID NO: 26	SEQ ID NO: 186	SEQ ID NO: 187	SEQ ID NO: 188	SEQ ID NO: 88	SEQ ID NO: 189
SEQ ID NO: 3	SEQ ID NO: 192	SEQ ID NO: 92	SEQ ID NO: 61	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 195	SEQ ID NO: 196	SEQ ID NO: 197	SEQ ID NO: 198	SEQ ID NO: 199	SEQ ID NO: 200
SEQ ID NO: 203	SEQ ID NO: 204	SEQ ID NO: 205	SEQ ID NO: 206	SEQ ID NO: 7	SEQ ID NO: 207
SEQ ID NO: 145	SEQ ID NO: 210	SEQ ID NO: 147	SEQ ID NO: 211	SEQ ID NO: 149	SEQ ID NO: 150
SEQ ID NO: 214	SEQ ID NO: 215	SEQ ID NO: 216	SEQ ID NO: 113	SEQ ID NO: 7	SEQ ID NO: 120
SEQ ID NO: 3	SEQ ID NO: 219	SEQ ID NO: 220	SEQ ID NO: 221	SEQ ID NO: 43	SEQ ID NO: 8
SEQ ID NO: 11	SEQ ID NO: 224	SEQ ID NO: 225	SEQ ID NO: 226	SEQ ID NO: 7	SEQ ID NO: 16
SEQ ID NO: 19	SEQ ID NO: 20	SEQ ID NO: 229	SEQ ID NO: 22	SEQ ID NO: 7	SEQ ID NO: 23
SEQ ID NO: 234	SEQ ID NO: 235	SEQ ID NO: 236	SEQ ID NO: 237	SEQ ID NO: 7	SEQ ID NO: 30
SEQ ID NO: 11	SEQ ID NO: 240	SEQ ID NO: 241	SEQ ID NO: 14	SEQ ID NO: 242	SEQ ID NO: 243
SEQ ID NO: 247	SEQ ID NO: 248	SEQ ID NO: 249	SEQ ID NO: 250	SEQ ID NO: 251	SEQ ID NO: 252
SEQ ID NO: 255	SEQ ID NO: 256	SEQ ID NO: 257	SEQ ID NO: 258	SEQ ID NO: 259	SEQ ID NO: 260
SEQ ID NO: 267	SEQ ID NO: 268	SEQ ID NO: 269	SEQ ID NO: 270	SEQ ID NO: 7	SEQ ID NO: 271
SEQ ID NO: 160	SEQ ID NO: 274	SEQ ID NO: 275	SEQ ID NO: 276	SEQ ID NO: 7	SEQ ID NO: 277
SEQ ID NO: 279	SEQ ID NO: 175	SEQ ID NO: 280	SEQ ID NO: 61	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 46	SEQ ID NO: 47	SEQ ID NO: 283	SEQ ID NO: 284	SEQ ID NO: 7	SEQ ID NO: 50
SEQ ID NO: 287	SEQ ID NO: 288	SEQ ID NO: 289	SEQ ID NO: 113	SEQ ID NO: 7	SEQ ID NO: 139
SEQ ID NO: 287	SEQ ID NO: 292	SEQ ID NO: 293	SEQ ID NO: 294	SEQ ID NO: 7	SEQ ID NO: 207
SEQ ID NO: 84	SEQ ID NO: 65	SEQ ID NO: 297	SEQ ID NO: 87	SEQ ID NO: 298	SEQ ID NO: 8
SEQ ID NO: 3	SEQ ID NO: 40	SEQ ID NO: 303	SEQ ID NO: 61	SEQ ID NO: 7	SEQ ID NO: 8
SEQ ID NO: 306	SEQ ID NO: 307	SEQ ID NO: 308	SEQ ID NO: 309	SEQ ID NO: 7	SEQ ID NO: 310

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, particularly 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, particularly NA.

In some embodiments, the anti-CCR6 antibody or an antigen-binding fragment thereof disclosed herein comprises

(i) a heavy chain variable region (VH) that comprises a HCDR1 (Heavy Chain Complementarity Determining Region 1) of SEQ ID NO: 46, a HCDR2 of SEQ ID NO: 47 and a HCDR3 of SEQ ID NO: 48; and a light chain variable region (VL) that comprises a LCDR1 (Light Chain Complementarity Determining Region 1) of SEQ ID NO: 49, a LCDR2 of SEQ ID NO: 7, and (f) a LCDR3 of SEQ ID NO: 50; or

(ii) a heavy chain variable region (VH) that comprises a HCDR1 (Heavy Chain Complementarity Determining Region 1) of SEQ ID NO: 77, a HCDR2 of SEQ ID NO: 78 and a HCDR3 of SEQ ID NO: 79; and a light chain variable region (VL) that comprises a LCDR1 (Light Chain Complementarity Determining Region 1) of SEQ ID NO: 80, a LCDR2 of SEQ ID NO: 7, and (f) a LCDR3 of SEQ ID NO: 81; or

(iii) a heavy chain variable region (VH) that comprises a HCDR1 (Heavy Chain Complementarity Determining Region 1) of SEQ ID NO: 3, a HCDR2 of SEQ ID NO: 264 and a HCDR3 of SEQ ID NO: 92; and a light chain variable region (VL) that comprises a LCDR1 (Light Chain Complementarity Determining Region 1) of SEQ ID NO: 61, a LCDR2 of SEQ ID NO: 7, and (f) a LCDR3 of SEQ ID NO: 8;

wherein optionally, NG in SEQ ID NO: 47 is mutated to NA or QG; and 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, particularly GE, and/or NG in SEQ ID NO: 80 is mutated to QG or NA; and optionally, NG in SEQ ID NO: 61 is mutated to QG or NA, particularly NA.

(i) a heavy chain variable region (VH) that comprises a HCDR1 (Heavy Chain Complementarity Determining Region 1) of SEQ ID NO: 46, a HCDR2 of SEQ ID NO: 47 and a HCDR3 of SEQ ID NO: 48; and a light chain variable region (VL) that comprises a LCDR1 (Light Chain Complementarity Determining Region 1) of SEQ ID NO: 49, a LCDR2 of SEQ ID NO: 7, and (f) a LCDR3 of SEQ ID NO: 50;

wherein optionally, NG in SEQ ID NO: 47 is mutated to NA or QG.

(ii) a heavy chain variable region (VH) that comprises a HCDR1 (Heavy Chain Complementarity Determining Region 1) of SEQ ID NO: 77, a HCDR2 of SEQ ID NO: 78 and a HCDR3 of SEQ ID NO: 79; and a light chain variable region (VL) that comprises a LCDR1 (Light Chain Complementarity Determining Region 1) of SEQ ID NO: 80, a LCDR2 of SEQ ID NO: 7, and (f) a 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, particularly GE, and/or NG in SEQ ID NO: 80 is mutated to QG or NA.

wherein optionally, NG in SEQ ID NO: 61 is mutated to QG or NA, particularly NA.

In some embodiments, the anti-CCR6 antibody or an antigen-binding fragment thereof disclosed herein comprises (a) a heavy chain variable region comprising an 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 at least 95%, 96%, 97%, 98%or 99%identical to any one 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; and/or (b) a light chain variable region comprising an 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 at least 95%, 96%, 97%, 98%or 99%identical to any one 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. In some embodiments, NG in SEQ ID NO: 47, if present, is mutated to NA or QG. In some embodiments, DG in SEQ ID NO: 78, if present, is mutated to EG or DA, and/or GD in SEQ ID NO: 79, if present, is mutated to AD, SD, GE, or GN, particularly GE, and/or Rat N-terminal to SEQ ID NO: 79, if present, is mutated to K or Q, and/or NG in SEQ ID NO: 80, if present, is mutated to QG or NA. In some embodiments, NG in SEQ ID NO: 61, if present, is mutated to QG or NA, particularly NA.

In some embodiments, the anti-CCR6 antibody or an antigen-binding fragment thereof disclosed herein comprises (a) a heavy chain variable region comprising an 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 with one, two, or three amino acid substitutions in the amino acid sequence of any one 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; and/or (b) a light chain variable region comprising an 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 with one, two, or three amino acid substitutions in the amino acid sequence of any one 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. In some other embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, NG in SEQ ID NO: 47, if present, is mutated to NA or QG. In some embodiments, DG in SEQ ID NO: 78, if present, is mutated to EG or DA, and/or GD in SEQ ID NO: 79, if present, is mutated to AD, SD, GE, or GN, particularly GE, and/or Rat N-terminal to SEQ ID NO: 79, if present, is mutated to K or Q, and/or NG in SEQ ID NO: 80, if present, is mutated to QG or NA. In some embodiments, NG in SEQ ID NO: 61, if present, is mutated to QG or NA, particularly NA.

In some embodiments, the antibody or an antigen-binding fragment thereof disclosed herein comprises 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 antibody or an antigen-binding fragment thereof disclosed herein comprises 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. The amino acid sequence ID numbers of VH and VL is presented in Table 2 (see Table 3 and 6 for sequence) :

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

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

In some embodiments, the antibody or an antigen-binding fragment thereof disclosed herein comprises 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 some embodiments, the antibody or an antigen-binding fragment thereof disclosed herein comprises 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) of selected from SEQ ID NO: 76, 334 or 335.

In some embodiments, the antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) of SEQ ID NO: 262 and a light chain variable region (VL) of selected from SEQ ID NO: 263, 341 or 342.

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

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

(b) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 331, or 332 and a light chain variable region comprising an 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, particularly GE, and/or Rat N-terminal to HCDR3 is mutated to K or Q, and/or NG in CDR-L1 in CDR-L1 is mutated to GQ or NA; or

(c) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 337, 338, 339, or 340 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 343, 344, 345, or 346, optionally NG in CDR-L1 is mutated to QG or NA, particularly NA.

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

(b) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 318 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 321;

(c) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 318 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 320;

(d) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 330 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 336;

(e) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 332 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 333;

(f) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 337 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 347;

(g) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 337 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 348; or

(h) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 337 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 349.

In some embodiments, the antibody of the present disclosure is of IgG1, IgG2, IgG3, or IgG4 isotype. In a more specific embodiment, the antibody of the present disclosure comprises the Fc domain of wild-type human IgG1 (also referred to as human IgG1wt or huIgG1) or IgG2.

The amino acid sequences of the heavy chain (VH) and light chain (VL) and the variable region sequences of the heavy chain CDRs and light chain CDRs of the anti-CCR6 antibody or the antigen-binding fragment thereof disclosed herein are as shown in Table 3 and 6.

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

The disease mediated by CCR6

The disease mediated by CCR6 disclosed herein refers to any disease in which the mediation of CCR6 is involved. In some embodiments, the disease is caused by ligand-mediated overactivity of CCR6. In some embodiments, the disease mediated by CCR6 is selected from autoimmune diseases or inflammatory diseases, infectious diseases and cancers. 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, COPD, optic neuritis, age-related macular degeneration, SLE, Sjogen's syndrome, Scleroderma, systemic sclerosis, chronic kidney disease, liver fibrosis, tuberculosis, idiopathic pulmonary fibrosis, tuberculosis induced lung fibrosis, retroperitoneal fibrosis, pulmonary fibrosis, cystic fibrosis, endomyocardial fibrosis, atrial fibrosis, mediastinal fibrosis, myelofibrosis (bone marrow) , retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, arthrofibrosis, inflammatory bowel diseases, ulcerative colitis, Crohn's disease, transplant rejection, central nervous system injury, atherosclerosis, or infectious diseases.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, it will be obvious to those skilled in the art that these examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.

Cell lines:

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

Example 1. Generation 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 by polybrene (8 μg/mL) , selected in media containing puromycin (2 μg/mL) , and tested for the expression of hCCR6 by fluorescence-activated cell sorting (FACS) . Individual clones with the greatest Mean Fluorescence Intensity to hCCR6 were selected for subsequent studies.

Spleen and lymph node cells from these mice were fused with myeloma cells (Sp2/0) by standard methods to generate hybridomas producing unique antibodies. Supernatants containing antibodies produced by pools of these cells were tested by cell-based ELISA for reactivity with hCCR6 overexpressing cells. Cell-based ELISA was generally carried out as follows. Approximately 3x10 ⁴ 293T-hCCR6 cells per well were seeded in a 96-well plate, and after overnight culture, the cells were washed with 1x PBS with 0.5%BSA followed by the addition of 100 μL of 4%paraformaldehyde solution to fix and cross-link the cells to the microplate. The cells were washed with 1x PBS-T twice. Then the cells were incubated with neat supernatants taken from hybridoma cultures including blank and positive control at 37℃ for 60 minutes. Subsequently, the cells were washed with 1x PBS-T four times. The cells were then incubated with goat anti-mouse IgG second antibody at 1: 10000 dilution in 100 μL PBS at 37℃ for 60 minutes, followed by four times washing with 1x PBS-T. 100 μL TMB was added to the 96-well plate. After 10-12 minutes of incubation, the plate was scanned with detection at 450 nm wavelength.

Supernatants of these positive clones were further confirmed by FACS. FACS analyses were generally carried out 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 distributed into 96-well round-bottom polystyrene plates and incubated with neat supernatants taken from hybridoma cultures on ice for 20-30 minutes. Next, the cells were washed with PBS/0.5%BSA and centrifuged. The pelleted cell samples were re-suspended in 100 μL PBS/0.5%BSA containing anti-mouse IgG-FITC antibody at 1: 300 dilution on ice for 30 minutes, and then washed with PBS/0.5%BSA, followed by pelleting the cells. The cell pellets were re-suspended in PBS/0.5%BSA and analyzed on a CYTOFLEX (Beckman) . Generally, the same supernatants were tested on non-transfected parental cells to confirm that the reactive antibody recognized hCCR6 specifically.

Positive pools were identified and subcloned by limiting dilution. After three fusions, clones producing unique antibodies which recognized human CCR6 were obtained, they were: 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, Mab23, 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 hybridomas were referred to the same designation as the antibodies produced where were from (e.g., hybridoma Mab59 produces antibody Mab59) . All the anti-CCR6 antibody clones have high-affinity antibodies whose amino acid sequences of VH and VL were shown in Table 2.

Example 2. The binding affinity of anti-CCR6 antibodies against hCCR6 over-expressing cells

The affinity of anti-CCR6 antibodies was evaluated by FACS. 293T-hCCR6, which had a high level of human CCR6 expressed on the cell surface, was used in the binding test. Affinity analyses were generally carried out as follows. Approximately 5x10 ⁵ 293T-hCCR6 cells per sample were prepared and blocked with Human TruStain FcX ^TM (Fc Receptor Blocking Solution, Biolegend, 422302) . The test antibodies were diluted with PBS/0.5%BSA (1: 3 series dilution from 225 μg/mL to 0.00381 μg/mL) . The 293T-hCCR6 cells along with parent non-transfected cells were distributed into 96-well round-bottom polystyrene plates and incubated with the diluted antibodies on ice for 20-30 minutes. Next, the cells were washed with PBS/0.5%BSA and centrifuged. The pelleted cell samples were re-suspended in 100 μL PBS/0.5%BSA containing anti-mouse IgG-FITC antibody at 1: 300 dilution on ice for 30 minutes, and then washed with PBS/0.5%BSA, followed by pelleting the cells. The cell pellets were re-suspended in PBS/0.5%BSA and analyzed on a CYTOFLEX (Beckman) .

The results showed that these antibodies bound to the human CCR6 overexpressing 293T cells, but didn’ t bind to cells without CCR6 overexpression, as shown in Figure 1. Figure 1A demonstrates that these antibodies have binding ability to CCR6 overexpressing 293T cells (293T-CCR6) , but not the 293T parent cells (Figure 1B) .

Example 3. The Tango Assay of anti-CCR6 Antibodies

The Tango system is a cell-based assay to measure the GPCR activation and arrestin recruitment. The change in the luminescence intensity is directly correlated to the amount of arrestin recruitment in response to ligand activation of the receptor of interest. The assay is used to determine which of the antibodies described above can block the CCL20-CCR6 signal.

The Tango-CCR6-Gal4-CHO-K1 cells (constructed by Genomeditech) passed in complete medium (F12K medium, 10%FBS, 1%penicillin-streptomycin, 4 μg/mL puromycin, 4 μg/mL blasticidin, 100 μg/mL hygromycin) in an incubator (37℃, 5%CO ₂) were used in the Tango assay.

The Tango-CCR6-Gal4-CHO-K1 cells were pelleted and re-suspended at 1x10 ⁴ cells/70 μL/well, the cells were distributed into a 96-well plate and incubated in a starving medium (F12K, 1%FBS, 1%penicillin-streptomycin) in 5%CO ₂ at 37℃ for 6 hours. Then the test antibodies at desired concentration (3×) in a 50 μL starving medium were added into each well and incubated with the cells in 5%CO ₂ at 37℃ for 1 hour. After that, a 30 μL starving medium with or without CCL20 was added into the desired wells and incubated with the cells in 5%CO ₂ at 37℃ for 24 hours. After overnight culture, the cells were incubated with ONE-Glo working reagent at room temperature in dark for 10 minutes, and then the relative luminescence units (RLU) of each sample were measured using a microplate luminescence reader at 560 nm and recorded. The RLU value was plotted against the antibody concentration and analyzed in GraphPad Prism for concentration curve generation.

The antibodies from the clones such as Mab50 showed a strong CCL20-CCR6 signaling block. The signal blocking ability of the representative antibodies disclosed herein is shown in Table 4.

Table 4. Tango assay result of the anti-CCR6 antibodies.

Clone

Tango IC ₅₀ (nM)

Mab7	8.04
Mab13	1.712
Mab50	0.88
Mab20	5.22
Mab28	3.49
Mab60	168.07
Mab58	22.5
Mab59	9.07

Example 4. The Calcium Mobilization Assay of anti-CCR6 Antibodies

Calcium mobilization assay is a cell-based second messenger assay to measure the calcium flux associated with G-protein coupled receptor activation or inhibition. The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of the receptor of interest. The assay is used to determine which of the antibodies described above can block the CCL20-CCR6 signal.

The CCR6-Gα15-CHO K1 cells (constructed by Genomeditech) passed in a complete medium (F12K medium, 10%FBS, 1%penicillin-streptomycin, 4 μg/mL puromycin, 4 μg/mL blasticidin) in an incubator (37℃, 5%CO2) were used in the calcium mobilization assay.

The fluorescent membrane-permeable calcium-binding dye (the FLIPR Calcium 6 Assay Kit) was dissolved in assay buffer (20 mM HEPES buffer with 1x Hank’s Balanced Salt Solution (HBSS) , pH 7.4) . The loading buffer was prepared with the dye solution containing 5 mM probenecid. The probenecid was prepared into 500 mM stock solution in 1 N NaOH, and then diluted to 250 mM in HBSS buffer before use.

Approximately 1.5x10 ⁴ hCCR6-Gα15-CHO K1 cells were seeded into a 384-well plate and incubated in 25 μL starving medium (F12K, 1%FBS, 1%penicillin-streptomycin) in 5%CO ₂ at 37℃ for 16 hours. Then, the starving medium was completely changed with 25 μL assay buffer, and 25 μL loading buffer was added to the desired wells. After adding dye, the cell plate was incubated for 2 hours at 37℃ with 5%CO ₂ and then kept at room temperature until used. The compounds in 12.5 μL assay buffer at desired concentration (5×) were added into each well and incubated with cells for 30 minutes at room temperature. After incubation, the microplate was transferred to the FLIPR instrument and the calcium assay was started as described in the user guide for the instrument. 12.5 μL assay buffer with or without CCL20 was added during the assay. The MAX ratio value was plotted against the antibody concentration and analyzed in GraphPad Prism for concentration curve generation.

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

Table 5. Calcium mobilization assay result of the anti-CCR6 antibodies.

Clone	Calcium mobilization 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. The Chemotaxis Biological Function Assay of anti-CCR6 Antibodies

A chemotaxis assay is conducted to analyze whether or not a cell type directly orients and migrates toward a defined chemoattractant. Since the CCL20-CCR6 axis plays an important role in mediating immune cell migration, the function of anti-CCR6 antibodies was evaluated using a cell-based chemotaxis assay in vitro.

The BaF3 mouse pro-B cells transfected with human CCR6 (as constructed as stably transfected cell lines) were used to monitor the inhibitory effect of antibodies on CCL20-mediated chemotaxis. All the antibodies are investigated based on the following assay methods.

The BaF3 mouse pro-B cells transfected with human CCR6 were passed in a complete medium (RPMI1640, 10%FBS, 1%penicillin-streptomycin, 10 ng/mL IL-3) in an incubator (37 ℃, 5%CO ₂) .

Prior to the migration assay, the cells were kept in a starving medium for 3 hours (RPMI 1640, 1%FBS, 1%penicillin-streptomycin) .

Chemotaxis was performed using HTS Transwell-96 plates with 5.0 μm pore polycarbonate membranes according to the manufacturer’s protocol. In brief, lower chambers were filled with 100 μL RPMI1640 media containing 0.5%BSA and 100 ng/mL CCL20. After insertion of the filters, 1x 10 ⁵ cells which were pre-treated with antibodies at 10 μg/mL in 75 μL RPMI1640 media containing 0.5%BSA were added to the upper chamber. After 3 hours of incubation at 37 ℃, cell numbers in the lower chamber were analyzed by flow cytometry. Chemotaxis is expressed as the total number of cells in the lower chambers.

The antibodies from the clones such as Mab45, Mab35, Mab7, Mab20, Mab50, Mab14, Mab13, and Mab47 showed a strong inhibitory effect on the cell migration.

Example 6. The binding ability of the anti-CCR6 antibody to cynomolgus CCR6 and mouse CCR6

Through the Tango, calcium mobilization assay and chemotaxis biological function assay, the antibodies from the clones such as Mab7, Mab13, and Mab50 showed better functional activity. The binding ability of these antibodies to cynomolgus CCR6 and mouse CCR6 was tested by FACS.

293T cells were transfected with lentiviral vectors encoding cynomolgus CCR6 and mouse CCR6 by polybrene (8 μg/mL) , selected in media containing puromycin (2 μg/mL) , and tested for the expression of cynomolgus CCR6 and mouse CCR6 by FACS.

Antibodies were studied for specific binding to cynomolgus and mouse CCR6 by flow cytometry using cynomolgus and mouse CCR6-overexpressing cell line. The results showed that the antibodies bound 293T cells expressing cynomolgus CCR6, but did not bind 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 lead antibodies into selected human IgG germline frameworks. Human germline IGHV1-2*02, and IGKV2-30*02 were selected based on sequence similarity within both frameworks (FR) for Mab7. Human germline IGHV1-46*02, and IGKV2-30*02 were selected based on sequence similarity within both frameworks (FR) for Mab13. Human germline IGHV3-48*03, and IGKV2-29*02 were selected based on sequence similarity within both frameworks (FR) for Mab50. To maintain canonical loop structure and chain interface, certain residues in human germline frameworks were back mutated to corresponding mouse residues (Table 6) .

In silico prediction implied high risk sequence liabilities in CDRs of Mab7, Mab13 and Mab50. For Mab7, there is an NG motif in the CDR-H2 region. One liability mutation at the N54 position in the heavy chain was evaluated to see if the potential deamidation site in the VH could be removed without affecting activity. For Mab13, there is a DG motif in the CDR-H2 region, an NG motif in CDR-L1 and an RGD motif in CDR-H3. All of the motifs in the heavy chain and light chain were evaluated to see if the DG, NG and RGD sites in the VH and VL could be removed without affecting activity. For Mab50, there is an NG motif in the CDR-L1 region. One liability mutation at this position in the light chain was evaluated to see if the potential deamidation site in VL could be removed without affecting activity.

Humanization of Mab7, Mab13 and Mab50 resulted in monoclonal antibodies that were confirmed to bind to human CCR6-expressing 293T. For Mab7, the PTM site NG mutant on the CDR-H2 into QG or NA has no effect on binding affinity. The three antibodies of Mab7 (V _H2+V _L2, V _H2+V _L3 and V _H3+V _L2) with selected PTM removal design (NG/NA) were constructed and transfected in Expi293F cell culture. For Mab13, the PTM site DG mutant on the CDR-H2 into EG or DA has no effect on binding affinity. The PTM site NG mutant on the CDR-L1 into QG or NA has no effect on binding affinity. Changing D to E in RGD did not affect the affinity of the antibody, which changes the EC50 from 0.39 nM to 0.24 nM. The antibodies of Mab13 (V _H6+V _L5 and V _H9+V _L1) were constructed and transfected in ExpiCHO cell culture. For Mab50, the PTM site NG mutant on CDR-L1 into NA has no effect on binding affinity. The three antibodies of Mab50 (V _H1+V _L1, V _H1+V _L2 and V _H1+V _L3) with selected PTM removal design (NG/NA) were constructed and transfected in Expi293F cell culture. ALL affinity constants for the humanized version of antibodies Mab7, Mab13 and Mab50 are shown in Table 7.

Table 6: The VHs and VLs of humanization of Mab7, Mab13 and Mab50

Table7 Anti-CCR6 humanized antibody affinity

Example 8. The Calcium Mobilization Assay of Humanized Antibodies

The second round of humanized antibodies was tested by the calcium mobilization assay to determine which of the humanized antibodies described above can block the CCL20-CCR6 signal better.

The humanized antibodies showed a CCL20-CCR8 signaling block and Mab7 (VH2-NG/NA+VL3) showed the strongest CCL20-CCR8 signaling block. The signal blocking ability of the representative antibodies disclosed herein is shown in Table 8.

Table 8: Calcium mobilization assay result of humanized anti-CCR6 antibodies.

Clone	Calcium mobilization IC ₅₀ (nM)
Mab7 (V _H2-NG/NA+V _L2)	27.2
Mab7 (V _H3-NG/NA+V _L2)	54.1
Mab7 (V _H2-NG/NA+V _L3)	6.86

Example 9. Mab7 (V _H2-NG/NA+V _L3) cross-reactivity with cynomolgus CCR6, but not with rat, dog, mouse CCR6

293T cells were transfected with lentiviral vectors encoding mouse, rat and dog CCR6 by polybrene (8 μg/mL) , selected in media containing puromycin (2 μg/mL) , and tested for the expression of mouse, rat and dog CCR6 by FACS.

Humanized antibodies were studied for specific binding to human, rat, dog, mouse and cynomolgus CCR6 by flow cytometry using human, rat, dog, mouse and cynomolgus CCR6-overexpressing and parental 293T cell line.

The results are shown in Figure 3A-3E. According to binding affinity, Mab7 (V _H2-NG/NA+V _L3) bound 293T cells expressing human (3A) , rat (3B) , dog (3C) , mouse (3D) and cynomolgus CCR6 (3E) , but not parental cells.

Example 10. Study of Mab7 (V _H2-NG/NA+V _L3) humanized antibody for binding to human CXCR2

Mab7 (V _H2-NG/NA+V _L3) humanized antibody was tested for binding to CXCR2 overexpressing cells by flow cytometry. Briefly, 293T cells were transfected with lentiviral vectors encoding human CXCR2 by polybrene (8 μg/mL) , selected in media containing puromycin (2 μg/mL) , and tested for the expression of human CXCR2 by FACS using CXCR2 (Biolegend, 320705) . The binding evaluation was performed by expressing human CXCR2 in 293T cells, and reacting the cells with an antibody solution of a humanized Mab7 (V _H2-NG/NA+V _L3) antibody prepared by 14 serially diluting by 3-fold from 25 μg/mL. After reacting at 4℃ for 1 hour, it was reacted with Alexa Fluor 488 affinipure goat anti-human Ig (H+L) (Jackson, 109-545-003) and flow cytometry analysis was performed.

As shown in Figure 4, humanized Mab7 (V _H2-NG/NA+V _L3) antibody bound 293T cells expressing human CCR6, but did not bind 293T cells expressing human CXCR2.

Example 11. Humanized antibody stability validation

Monoclonal antibodies are proteinaceous in nature and are subject to instability issues. The stability testing of monoclonal antibodies is a critical regulatory requirement in their development and commercialization as therapeutic biological molecules. The stability and activity of humanized antibodies Mab7 (V _H2-NG/NA+V _L3) -hIgG1 and Mab7 (V _H2-NG/NA+V _L3) -hIgG4 (S228P) were tested under the stress condition shown in Table 9.

Table 9. Stability test item

^***5FT refers to 5 cycles of Freeze/Thaw

In a similar manner to Examples 2, the binding affinity of humanized antibodies at different time points were performed. The results are shown in Table 10.

Table 9: The affinity EC50 of humanized antibody in stress condition

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

Claims

An isolated antibody or an antigen-binding fragment thereof which specifically binds chemokine receptor CCR6.
The antibody or antigen-binding fragment of claim 1, which binds human chemokine receptor CCR6, and optionally (1) binds cynomolgus chemokine receptor CCR6, (2) does not bind mouse chemokine receptor CCR6, (3) does not bind rat chemokine receptor CCR6, and/or (4) does not bind dog chemokine receptor CCR6.

The antibody or antigen-binding fragment of claim 1 or 2, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and/or a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with the amino acid sequences set forth below (based on Kabat numbering scheme) :

HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 7 SEQ ID NO: 50 SEQ ID NO: 77 SEQ ID NO: 78 SEQ ID NO: 79 SEQ ID NO: 80 SEQ ID NO: 7 SEQ ID NO: 81 SEQ ID NO: 3 SEQ ID NO: 264 SEQ ID NO: 92 SEQ ID NO: 61 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 7 SEQ ID NO: 23 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 7 SEQ ID NO: 30 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 7 SEQ ID NO: 37 SEQ ID NO: 3 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 8 SEQ ID NO: 53 SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID NO: 56 SEQ ID NO: 57 SEQ ID NO: 30 SEQ ID NO: 3 SEQ ID NO: 60 SEQ ID NO: 41 SEQ ID NO: 61 SEQ ID NO: 43 SEQ ID NO: 62 SEQ ID NO: 3 SEQ ID NO: 65 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 68 SEQ ID NO: 8 SEQ ID NO: 26 SEQ ID NO: 71 SEQ ID NO: 72 SEQ ID NO: 22 SEQ ID NO: 7 SEQ ID NO: 30 SEQ ID NO: 3 SEQ ID NO: 60 SEQ ID NO: 41 SEQ ID NO: 61 SEQ ID NO: 43 SEQ ID NO: 74 SEQ ID NO: 84 SEQ ID NO: 85 SEQ ID NO: 86 SEQ ID NO: 87 SEQ ID NO: 88 SEQ ID NO: 8 SEQ ID NO: 3 SEQ ID NO: 91 SEQ ID NO: 92 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 3 SEQ ID NO: 95 SEQ ID NO: 92 SEQ ID NO: 61 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 98 SEQ ID NO: 99 SEQ ID NO: 100 SEQ ID NO: 101 SEQ ID NO: 102 SEQ ID NO: 30 SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 79 SEQ ID NO: 107 SEQ ID NO: 7 SEQ ID NO: 81 SEQ ID NO: 110 SEQ ID NO: 111 SEQ ID NO: 112 SEQ ID NO: 113 SEQ ID NO: 7 SEQ ID NO: 114 SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 113 SEQ ID NO: 7 SEQ ID NO: 120 SEQ ID NO: 26 SEQ ID NO: 123 SEQ ID NO: 124 SEQ ID NO: 125 SEQ ID NO: 57 SEQ ID NO: 126 SEQ ID NO: 129 SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 125 SEQ ID NO: 7 SEQ ID NO: 120 SEQ ID NO: 134 SEQ ID NO: 135 SEQ ID NO: 136 SEQ ID NO: 137 SEQ ID NO: 138 SEQ ID NO: 139 SEQ ID NO: 142 SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 125 SEQ ID NO: 7 SEQ ID NO: 120 SEQ ID NO: 145 SEQ ID NO: 146 SEQ ID NO: 147 SEQ ID NO: 148 SEQ ID NO: 149 SEQ ID NO: 150 SEQ ID NO: 19 SEQ ID NO: 153 SEQ ID NO: 154 SEQ ID NO: 22 SEQ ID NO: 7 SEQ ID NO: 23 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 157 SEQ ID NO: 36 SEQ ID NO: 7 SEQ ID NO: 37 SEQ ID NO: 160 SEQ ID NO: 161 SEQ ID NO: 162 SEQ ID NO: 163 SEQ ID NO: 7 SEQ ID NO: 164

SEQ ID NO: 26 SEQ ID NO: 167 SEQ ID NO: 168 SEQ ID NO: 29 SEQ ID NO: 7 SEQ ID NO: 37 SEQ ID NO: 3 SEQ ID NO: 171 SEQ ID NO: 172 SEQ ID NO: 87 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 3 SEQ ID NO: 175 SEQ ID NO: 41 SEQ ID NO: 61 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 178 SEQ ID NO: 179 SEQ ID NO: 180 SEQ ID NO: 101 SEQ ID NO: 7 SEQ ID NO: 181 SEQ ID NO: 3 SEQ ID NO: 65 SEQ ID NO: 66 SEQ ID NO: 61 SEQ ID NO: 183 SEQ ID NO: 8 SEQ ID NO: 26 SEQ ID NO: 186 SEQ ID NO: 187 SEQ ID NO: 188 SEQ ID NO: 88 SEQ ID NO: 189 SEQ ID NO: 3 SEQ ID NO: 192 SEQ ID NO: 92 SEQ ID NO: 61 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 195 SEQ ID NO: 196 SEQ ID NO: 197 SEQ ID NO: 198 SEQ ID NO: 199 SEQ ID NO: 200 SEQ ID NO: 203 SEQ ID NO: 204 SEQ ID NO: 205 SEQ ID NO: 206 SEQ ID NO: 7 SEQ ID NO: 207 SEQ ID NO: 145 SEQ ID NO: 210 SEQ ID NO: 147 SEQ ID NO: 211 SEQ ID NO: 149 SEQ ID NO: 150 SEQ ID NO: 214 SEQ ID NO: 215 SEQ ID NO: 216 SEQ ID NO: 113 SEQ ID NO: 7 SEQ ID NO: 120 SEQ ID NO: 3 SEQ ID NO: 219 SEQ ID NO: 220 SEQ ID NO: 221 SEQ ID NO: 43 SEQ ID NO: 8 SEQ ID NO: 11 SEQ ID NO: 224 SEQ ID NO: 225 SEQ ID NO: 226 SEQ ID NO: 7 SEQ ID NO: 16 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 229 SEQ ID NO: 22 SEQ ID NO: 7 SEQ ID NO: 23 SEQ ID NO: 234 SEQ ID NO: 235 SEQ ID NO: 236 SEQ ID NO: 237 SEQ ID NO: 7 SEQ ID NO: 30 SEQ ID NO: 11 SEQ ID NO: 240 SEQ ID NO: 241 SEQ ID NO: 14 SEQ ID NO: 242 SEQ ID NO: 243 SEQ ID NO: 247 SEQ ID NO: 248 SEQ ID NO: 249 SEQ ID NO: 250 SEQ ID NO: 251 SEQ ID NO: 252 SEQ ID NO: 255 SEQ ID NO: 256 SEQ ID NO: 257 SEQ ID NO: 258 SEQ ID NO: 259 SEQ ID NO: 260 SEQ ID NO: 267 SEQ ID NO: 268 SEQ ID NO: 269 SEQ ID NO: 270 SEQ ID NO: 7 SEQ ID NO: 271 SEQ ID NO: 160 SEQ ID NO: 274 SEQ ID NO: 275 SEQ ID NO: 276 SEQ ID NO: 7 SEQ ID NO: 277 SEQ ID NO: 279 SEQ ID NO: 175 SEQ ID NO: 280 SEQ ID NO: 61 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 283 SEQ ID NO: 284 SEQ ID NO: 7 SEQ ID NO: 50 SEQ ID NO: 287 SEQ ID NO: 288 SEQ ID NO: 289 SEQ ID NO: 113 SEQ ID NO: 7 SEQ ID NO: 139 SEQ ID NO: 287 SEQ ID NO: 292 SEQ ID NO: 293 SEQ ID NO: 294 SEQ ID NO: 7 SEQ ID NO: 207 SEQ ID NO: 84 SEQ ID NO: 65 SEQ ID NO: 297 SEQ ID NO: 87 SEQ ID NO: 298 SEQ ID NO: 8 SEQ ID NO: 3 SEQ ID NO: 40 SEQ ID NO: 303 SEQ ID NO: 61 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 306 SEQ ID NO: 307 SEQ ID NO: 308 SEQ ID NO: 309 SEQ ID NO: 7 SEQ ID NO: 310

optionally

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

(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, particularly GE, and/or NG in SEQ ID NO: 80 is mutated to QG or NA; or

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

The antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising (a) a heavy chain variable region comprising an 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 at least 95%, 96%, 97%, 98%or 99%identical to any one 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; and/or (b) a light chain variable region comprising an 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 at least 95%, 96%, 97%, 98%or 99%identical to any one 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,

optionally

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

(2) DG in SEQ ID NO: 78, if present, is mutated to EG or DA, and/or GD in SEQ ID NO: 79, if present, is mutated to AD, SD, GE, or GN, particularly GE, and/or R at N-terminal to SEQ ID NO: 79, if present, is mutated to K or Q, and/or NG in SEQ ID NO: 80, if present, is mutated to QG or NA; or

(3) NG in SEQ ID NO: 61, if present, is mutated to QG or NA, particularly NA.
The antibody or antigen-binding fragment thereof of any one of claim 1-3, comprising (a) a heavy chain variable region comprising an 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 with one, two, or three amino acid substitutions in the amino acid sequence of any one 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; and/or (b) a light chain variable region comprising an 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 with one, two, or three amino acid substitutions in the amino acid sequence of any one 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,

optionally, the amino acid substitutions are conservative amino acid substitutions,

optionally

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

(2) DG in SEQ ID NO: 78, if present, is mutated to EG or DA, and/or GD in SEQ ID NO: 79, if present, is mutated to AD, SD, GE, or GN, particularly GE, and/or R at N-terminal to SEQ ID NO: 79, if present, is mutated to K or Q, and/or NG in SEQ ID NO: 80, if present, is mutated to QG or NA; or

(3) NG in SEQ ID NO: 61, if present, is mutated to QG or NA, particularly NA.
The antibody or antigen-binding fragment thereof of claim 5, wherein the amino acid substitutions are conservative amino acid substitutions.
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 of which comprises an amino acid sequence as set forth below:

Optionally

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

(2) DG in SEQ ID NO: 78, if present, is mutated to EG or DA, and/or GD in SEQ ID NO: 79, if present, is mutated to AD, SD, GE, or GN, particularly GE, and/or R at N-terminal to SEQ ID NO: 79, if present, is mutated to K or Q, and/or NG in SEQ ID NO: 80, if present, is mutated to QG or NA; or

(3) NG in SEQ ID NO: 61, if present, is mutated to QG or NA, particularly NA.
The antibody or antigen-binding fragment thereof of any one of claims 1-7, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human-engineered antibody.
The antibody or antigen-binding fragment thereof of claim 8, wherein the humanized antibody comprising

(a) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 311, 312, 313, or 314 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 319, 320, 321, or 322, optionally, NG in HCDR2 is mutated to NA or QG;

(b) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 331, or 332 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 333 or 336, optionally DG in HCDR2 is mutated to EG or DA, and/or GD in HCDR3 is mutated to AD, SD, GE, or GN, particularly GE, and/or R at N-terminal to HCDR3 is mutated to K or Q, and/or NG in CDR-L1 in LCDR1 is mutated to GQ or NA; or

(c) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 337, 338, 339, or 340 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 343, 344, 345, or 346, optionally NG in LCDR1 is mutated to QG or NA, particularly NA.
The antibody or antigen-binding fragment thereof of claim 9, wherein the humanized antibody comprising

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

(b) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 318 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 321;

(c) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 318 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 320;

(d) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 330 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 336;

(e) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 332 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 333;

(f) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 337 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 347;

(g) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 337 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 348; or

(h) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 337 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 349.
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.
An isolated nucleic acid that encodes a heavy chain variable region and/or light chain variable region of the antibody or antigen-binding fragment of any one of claims 1 to 11.
A vector comprising the nucleic acid of claim 12.
A host cell comprising the nucleic acid of claim 12 or the vector of claim 13.
A process for producing an antibody or antigen-binding fragment thereof according to any one of claims 1-11, comprising cultivating the host cell of claim 14 and recovering the antibody or antigen-binding fragment from the culture.
A pharmaceutical composition comprising 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 as an active ingredient and a pharmaceutically acceptable carrier.
A method of treating a disease mediated by CCR6 comprising administering to a patient in need an effective amount of the antibody or antigen-binding fragment of any one of claims 1-11.
The method of claim 17, wherein the cancer is hepatocellular carcinoma, colorectal cancer, breast cancer, pancreatic cancer, cervical cancer, kidney cancer, lung cancer, leukemia or lymphoma, or chronic lymphocytic leukemia; rheumatoid arthritis, multiple sclerosis (MS) , psoriasis, graft versus host disease, lupus, COPD, optic neuritis, age-related macular degeneration, SLE, Sjogren's syndrome, Scleroderma, systemic sclerosis, chronic kidney disease, liver fibrosis, tuberculosis, idiopathic pulmonary fibrosis, tuberculosis induced lung fibrosis, retroperitoneal fibrosis, pulmonary fibrosis, cystic fibrosis, endomyocardial fibrosis, atrial Fibrosis, mediastinal fibrosis, myelofibrosis (bone marrow) , retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, arthrofibrosis, inflammatory bowel diseases, ulcerative colitis, Crohn's disease, atherosclerosis, transplant rejection, central nervous system injury, or infectious diseases.