Antibodies targeting XCL1 and methods of use thereof

Abstract

The present invention provides compounds and methods of targeting human lymphocyte chemokines, including therapeutic antibodies, pharmaceutical compositions, and methods of use thereof, for use in the fields of immune-mediated diseases including ulcerative colitis, vitiligo, multiple sclerosis, alopecia areata, psoriasis, type 1 diabetes, and asthma.

Description

[0001] sequence list file

[0002] This application is accompanied by a sequence list in ST.26 XML format. The sequence list is provided as a file named “30574_SequenceListing”, created on May 6, 2024, and is 43 kilobytes in size. The ST.26 XML format sequence list information is incorporated herein by reference in its entirety. Invention Field

[0003] This invention relates to the field of medicine. More specifically, it relates to antibodies against lymphocyte chemokines, particularly human chemokines XCL1 and XCL2; pharmaceutical compositions comprising such antibodies; and methods of using such antibodies. The antibodies disclosed herein are capable of binding to and neutralizing XCL1 and XCL2. The antibodies and methods of this invention are expected to be applied to the treatment of autoimmune and chronic inflammatory diseases (hereinafter collectively referred to as immune-mediated diseases), particularly alopecia areata, vitiligo, ulcerative colitis (UC), rheumatoid arthritis (RA), multiple sclerosis (MS), type 1 diabetes, psoriasis, asthma, and other diseases, including their treatment. Background of the Invention

[0005] XCL1 and XCL2, also known as lymphocyte chemokine-α and lymphocyte chemokine-β, respectively, are the only two members of the C chemokine / γ chemokine family. XCL1 is expressed and secreted by various immune cells, including activated CD8+ T cells, CD4+ T cells, natural killer (NK) cells, and thymic medullary epithelial cells. XCL2 is a paralog of XCL1, believed to be generated by gene duplication, and its amino acid sequence is almost identical to that of XCL1 (differences only in two amino acids in its N-terminal region). Due to this structural similarity, XCL1 and XCL2 are considered to have similar functions.

[0006] Unlike most other chemokines, which have four cysteine ​​residues near their N-terminus and can form two disulfide bonds, XCL1 and XCL2 have only two such cysteine ​​residues and can only form a single disulfide bond. This allows the chemokines to metamorphose, switching between two conformations: the first conformation has a standard monomeric chemokine fold containing an α-helix structure, while the second conformation is primarily a β-sheet. This form-changing typically occurs several times per second. The monomeric α-helix form can bind to and activate the receptor XCR1, thereby inducing cell migration. The α-helix form then undergoes a conformational change, transforming into the β-sheet form. The β-sheet can exist in monomeric form but is more prone to dimerization. When in the dimer or β-sheet conformation, XCL1 is inactive in binding to XCR1, and the dimer can adhere to glycosaminoglycans (GAGs) on the cell surface. The monomeric α-helix form does not bind to glycosaminoglycans (GAGs). See Lei and Takahama, Microbesand Infection 14 (2012) 262-267; Fox et al., ACS Chem. Biol. 10(11) (2015) 2580-2588. The frequent conversions between these forms, particularly considering the inactivity of the dimer and β-sheet forms and their tendency to aggregate on cell surface GAGs, complicate the design of anti-XCL1 and anti-XCL2 antibodies, especially as therapeutic agents, because antibodies capable of binding to the dimer form of chemokines may primarily bind to the inactive form of the chemokine, leading to unknown properties regarding their pharmacokinetics, immunogenicity, and efficacy.

[0007] Autoimmune diseases are a class of immune-mediated disorders caused by an immune response against the body's own tissues. These diseases are usually chronic and can be debilitating or even life-threatening. Currently, FDA-approved treatments for immune-mediated diseases include corticosteroids (commonly used to treat acute inflammation) and biologics targeting TNFα, interleukin-12, -17, and -23. Although these treatments have proven effective in alleviating symptoms in some patients, others do not respond to existing therapies or gradually lose their response. Therefore, there is still a need to develop other therapies targeting different human targets and pathways to treat immune-mediated diseases.

[0008] Some autoimmune diseases are associated with overactivity of CD8+ T cells and tissue damage caused by CD8+ T cells after they migrate to certain parts of the body, including alopecia areata, vitiligo, UC, type 1 diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, and asthma. However, many specific details of the CD8+ T cell activation mechanisms behind these conditions remain unclear. For example, the specific role of dendritic cells (DCs) in activating CD8+ T cells is usually elucidated in the field of oncology rather than immunology (e.g., in studies of immune checkpoint antagonists). This is the case for the DC subset known as conventional dendritic cells type 1 (cDC1); see, for example, Bowman-Kirigin et al., “The Conventional Dendritic Cell 1 Subset Primes CD8+ T Cells and Traffics Tumor Antigen to Drive Antititumor Immunity in the Brain,” Cancer Immunol Res, 11(1) (2023), pp. 20–37.

[0009] Although XCL1 antibodies are currently available as research tools for detecting and visualizing XCL1, no approved or known XCL1-specific antagonist antibody therapies have been developed to date. Therefore, there remains an urgent need for antibodies, pharmaceutical compositions, and methods targeting human XCL1 for the treatment of immune-mediated diseases such as RA, MS, asthma, psoriasis, UC, type 1 diabetes, alopecia areata, and vitiligo. We require XCL1 antibodies with favorable therapeutic properties, including effective neutralizers (i.e., antagonists) of human XCL1. Advantageous properties of such antibodies may include high affinity for human XCL1 and low nonspecific binding, particularly to other chemokines that may have similar sequences and / or structures to XCL1. Such XCL1 antibodies should also have therapeutically acceptable pharmacokinetic (PK) characteristics and exhibit low immunogenicity. Such XCL1 antibodies should also be suitable for commercial production, including high solubility levels and low aggregation levels. This disclosure provides XCL1 antibodies that meet one or more of the above requirements for the treatment of immune-mediated diseases.

[0010] Therefore, in some embodiments, the present invention provides antibodies against human XCL1. The antibodies disclosed herein can also bind to and neutralize human XCL2. Summary of the Invention

[0011] According to some embodiments, the antibody of the present invention has antagonistic activity against human XCL1, and in some embodiments, antagonistic activity against human XCL2. In one aspect, this document describes an antibody that binds to human XCL1, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR). The HCVR includes complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3. The LCVR comprises CDRs LCDR1, LCDR2, and LCDR3. HCDR1 is or includes SEQ ID NO: 10. HCDR2 is or includes SEQ ID NO: 11. HCDR3 is or includes SEQ ID NO: 12, wherein Z is selected from Q and E. LCDR1 is or includes SEQ ID NO: 5, wherein X is selected from S and D. LCDR2 is or includes SEQ ID NO: 8. LCDR3 is or includes SEQ ID NO: 9.

[0012] In one implementation, Z is E. In another implementation, Z is Q.

[0013] In one implementation, X is D. In another implementation, X is S.

[0014] In one embodiment, HCDR1 is or includes SEQ ID NO: 10; HCDR2 is or includes SEQ ID NO: 11; HCDR3 is or includes SEQ ID NO: 13; LCDR1 is or includes SEQ ID NO: 6; LCDR2 is or includes SEQ ID NO: 8; and LCDR3 is or includes SEQ ID NO: 9.

[0015] In one embodiment, LCVR is or includes SEQ ID NO: 17, and HCVR is or includes SEQ ID NO: 15, wherein X a Selected from Q and pyroglutamic acid. In one embodiment, X... a It's Q.

[0016] In one embodiment, the antibody has a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is or includes SEQ ID NO: 19, wherein X1 is selected from Q and pyroglutamic acid; X2 is G or absent; and X3 is K or absent; and the amino acid sequence of LC is or includes SEQ ID NO: 21. In one embodiment, X1 is Q; X2 is G; and X3 is K.

[0017] In one embodiment, HCDR1 is or includes SEQ ID NO: 10; HCDR2 is or includes SEQ ID NO: 11; HCDR3 is or includes SEQ ID NO: 14; LCDR1 is or includes SEQ ID NO: 7; LCDR2 is or includes SEQ ID NO: 8; and LCDR3 is or includes SEQ ID NO: 9.

[0018] In one embodiment, the amino acid sequence of LCVR is or includes SEQ ID NO: 18, and the amino acid sequence of HCVR is or includes SEQ ID NO: 16, wherein X a Selected from Q and pyroglutamic acid. In one embodiment, X... a It's Q.

[0019] In one embodiment, the antibody has a heavy chain (HC) and a light chain (LC). The HC is or includes SEQ ID NO: 20, wherein X1 is selected from Q and pyroglutamic acid; X2 is G or absent; and X3 is K or absent; the LC is or includes SEQ ID NO: 22. In one embodiment, X1 is Q; X2 is G; and X3 is K.

[0020] In any of the above embodiments, the antibody may be an XCL1 antagonist.

[0021] In any of the above embodiments, the antibody can bind to human XCL2.

[0022] In any of the above embodiments, the antibody may be an XCL2 antagonist.

[0023] In any of the above embodiments, the antibody can bind to the dimer XCL1. The dimer XCL1 can be a homodimer XCL1. The antibody can bind to XCL1 with a β-sheet configuration. The homodimer XCL1 can bind to glycosaminoglycans on the cell surface.

[0024] In any of the above embodiments, the antibody may bind to the dimer XCL2. The dimer XCL2 may be a homodimer XCL2. The antibody may bind to XCL2 with a β-sheet configuration. The homodimer XCL2 may bind to glycosaminoglycans on the cell surface.

[0025] In any of the above embodiments, the antibody may bind to a heterodimeric complex comprising an XCL1 monomer and an XCL2 monomer. In this embodiment, the antibody binds to at least one amino acid of the XCL1 monomer or at least one amino acid of the XCL2 monomer in the heterodimeric complex. The heterodimeric complex may bind to glycosaminoglycans on the cell surface.

[0026] In any of the above embodiments, the antibody does not bind to chemokines other than XCL1 and XCL2.

[0027] In one embodiment, the antibody includes a heavy chain variable region (HCVR) having at least 95% sequence identity with SEQ ID NO: 15 and a light chain variable region (LCVR) having at least 95% sequence identity with SEQ ID NO: 17.

[0028] In one embodiment, the antibody comprises a heavy chain (HC) having at least 95% sequence identity with SEQ ID NO: 19 and a light chain (LC) having at least 95% sequence identity with SEQ ID NO: 21.

[0029] In one embodiment, the antibody includes a heavy chain variable region (HCVR) having at least 95% sequence identity with SEQ ID NO: 16 and a light chain variable region (LCVR) having at least 95% sequence identity with SEQ ID NO: 18.

[0030] In one embodiment, the antibody comprises a heavy chain (HC) having at least 95% sequence identity with SEQ ID NO: 20 and a light chain (LC) having at least 95% sequence identity with SEQ ID NO: 22.

[0031] In one embodiment, the antibody binds to an epitope of XCL1, the epitope comprising one or more amino acids of SEQ ID NO: 27. In one embodiment, the antibody binds to two or more amino acids of SEQ ID NO: 27. In another embodiment, the antibody binds to three or more amino acids of SEQ ID NO: 27.

[0032] In one embodiment, the antibody binds to one or more amino acids of SEQ ID NO: 29.

[0033] In one embodiment, the antibody binds to an epitope of XCL1, the epitope comprising one or more amino acids of SEQ ID NO: 28. In another embodiment, the antibody binds to two or more amino acids of SEQ ID NO: 28.

[0034] In one embodiment, the antibody binds to an epitope of XCL1, the epitope comprising one or more amino acids of SEQ ID NO: 27 and one or more amino acids of SEQ ID NO: 28. In one embodiment, the antibody binds to two or more amino acids of SEQ ID NO: 27 and one or more amino acids of SEQ ID NO: 28. In one embodiment, the antibody binds to one or more amino acids of SEQ ID NO: 29.

[0035] In one embodiment, the antibody has a human IgG1 isotype.

[0036] This disclosure also provides a pharmaceutical composition comprising an antibody as described in any of the embodiments above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0037] This disclosure provides a method for treating alopecia areata, vitiligo, asthma, psoriasis, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis in an individual in need, the method comprising administering an effective amount of an antibody or pharmaceutical composition as described herein to said individual.

[0038] This disclosure provides an antibody or pharmaceutical composition for use in therapies, such as for treating alopecia areata, vitiligo, asthma, psoriasis, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis.

[0039] This disclosure provides the use of antibody or pharmaceutical composition in the manufacture of medicaments for the treatment of alopecia areata, vitiligo, asthma, psoriasis, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis.

[0040] In one aspect, a nucleic acid is provided, comprising a sequence encoding SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In another aspect, a vector is described, comprising a first nucleic acid sequence encoding SEQ ID NO: 23 or SEQ ID NO: 25 and a second nucleic acid sequence encoding SEQ ID NO: 24 or SEQ ID NO: 26, and a cell comprising such a vector. In one embodiment, a method for producing an antibody is disclosed, comprising culturing the cells under conditions that cause antibody expression and recovering the expressed antibody from the culture medium. In one embodiment, an antibody is disclosed, which is produced by culturing the cells under conditions that cause antibody expression and recovering the expressed antibody from the culture medium.

[0041] In one aspect, a protein complex is provided comprising a dimer XCL1 and an antibody that binds to the dimer XCL1. The dimer XCL1 may be a homodimer XCL1. The antibody may bind to at least one amino acid residue on each monomeric unit of the dimer XCL1.

[0042] In one aspect, a protein complex is provided comprising a dimer XCL2 and an antibody that binds to the dimer XCL2. The dimer XCL2 may be a homodimer XCL2. The antibody may bind to at least one amino acid residue on each monomeric unit of the dimer XCL2.

[0043] In one aspect, a protein complex is provided, comprising a heterodimer composed of XCL1 monomer units and XCL2 monomer units, and an antibody bound to the heterodimer. In this embodiment, the antibody may bind to at least one amino acid residue of XCL1, or at least one amino acid residue of XCL2, or at least one residue of XCL1 and at least one residue of XCL2. Invention Details

[0045] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody implementations include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugate antibodies. Antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0046] The exemplary antibody disclosed herein is an immunoglobulin G (IgG) antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC), which are cross-linked by interchain disulfide bonds. Each of the four polypeptide chains has an amino-terminal portion comprising a variable region of approximately 100-125 or more amino acids, which is primarily responsible for antigen recognition. Each of the four polypeptide chains also has a carboxyl-terminal portion comprising a constant region, which is primarily responsible for effector function. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further subdivided into subtypes (e.g., IgG1, IgG2, IgG3, and IgG4).

[0047] The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). CDRs are exposed on the protein surface and are crucial regions for antibody antigen-binding specificity. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this paper, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," while the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." CDRs contain most of the residues that specifically interact with the antigen. The allocation of amino acid residues in the CDR can be performed according to well-known schemes, including those by Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)); Chothia (Chothia et al., “Canonical structures for the hypervariable regions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)); and North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256). (2011) or those described in IMGT (the international ImMunoGeneTics database is available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212).

[0048] As used herein, embodiments of this disclosure may also include antibody fragments or antigen-binding fragments that contain at least a portion of an antibody that retains the ability to specifically interact with an antigen or antigenic epitope, such as Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fv(sdFv), and Fd fragments.

[0049] The antibody of this invention is a monoclonal antibody. A monoclonal antibody is an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, rather than its production method. Monoclonal antibodies can be produced by, for example, hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR transplantation), or combinations of such technologies, or other techniques known in the art.

[0050] Methods for the preparation and purification of antibodies are well known in the art; for example, see Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, Chapters 5-8 and 15, ISBN 0-87969-314-2. For instance, mice or rabbits (including transgenic mice or rabbits known in the art) can be immunized with human XCL1 or a portion thereof, and the resulting antibodies can then be recovered, screened, purified, and their amino acid sequences determined using conventional methods well known in the art.

[0051] In a specific embodiment of the invention, the antibody or the nucleic acid encoding the antibody is provided in an isolated form. As used herein, the term "isolated" means a protein, peptide, or nucleic acid that is free from or substantially free from other macromolecules found in the cellular environment.

[0052] The antibodies of the present invention can be prepared and purified using known methods. For example, cDNA sequences encoding HC (e.g., the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20) and LC (e.g., the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22) can be cloned and engineered into a GS (glutamine synthase) expression vector. In one example, the antibody has a heavy chain with the amino acid sequence shown in SEQ ID NO: 19 and a light chain with the amino acid sequence shown in SEQ ID NO: 21; in this example, cDNA sequences that can be used to express these chains are shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. In another example, the antibody has a heavy chain with the amino acid sequence shown in SEQ ID NO: 20 and a light chain with the amino acid sequence shown in SEQ ID NO: 22; in this example, cDNA sequences that can be used to express these chains are shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

[0053] Subsequently, the engineered immunoglobulin expression vector can be stably transfected into CHO cells. As those skilled in the art will understand, mammalian antibody expression leads to glycosylation, typically occurring at highly conserved N-glycosylation sites in the Fc region. It can be verified whether the stable clone expresses an antibody that specifically binds to human XCL1 (e.g., represented by a peptide derived from a recombinant containing SEQ ID NO: 1). Positive clones can be amplified in serum-free medium for antibody production in a bioreactor. The medium in which the antibody has been secreted can be purified using conventional techniques. For example, the medium can be conveniently applied to a Protein A or G Sepharose FF column equilibrated with a compatible buffer (e.g., phosphate-buffered saline). The column is washed to remove non-specifically bound components. The bound antibody can be eluted, for example, by a pH gradient, and the antibody fraction can be detected by methods such as SDS-PAGE, and then pooled. The antibody can be concentrated and / or sterilely filtered using common techniques. Soluble aggregates and polymers can be effectively removed using common methods, including size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, or hydroxyapatite chromatography. The product can be frozen immediately, for example, by freezing at -70°C or by freeze-drying.

[0054] The antibodies of the present invention can be used to treat patients. More specifically, the antibodies of the present invention are expected to be used to treat immune-mediated diseases or conditions, including alopecia areata, vitiligo, asthma, RA, MS, UC, psoriasis, and type 1 diabetes. As used interchangeably herein, “treatment” and its various morphologies are intended to refer to all processes in which the progression of the disease described herein may be slowed, interrupted, prevented, controlled, terminated, or reversed, but do not necessarily mean the complete elimination of all symptoms of the disease. Treatment includes administering the antibodies of the present invention or pharmaceutical compositions thereof to treat diseases or conditions in the human body that may benefit from reduced XCL1 and / or XCL2 activity, including: (a) inhibiting further progression of the disease, i.e., preventing its development; or (b) alleviating the disease, even if the disease or condition subsides, or reducing its symptoms or complications.

[0055] As used interchangeably herein, the terms “patient,” “subject,” and “individual” all refer to humans. In some embodiments, the patient is further described as having a disease, condition, or illness (e.g., an immune-mediated disease) from which reduced XCL1 and / or XCL2 activity would benefit. In other embodiments, the patient is further described as being at risk of developing an immune-mediated disease, condition, or illness from which reduced XCL1 and / or XCL2 activity would benefit.

[0056] In this article, the term "epitope" refers to an amino acid residue on an antigen that can be bound by an antibody. Epitopes can be linear epitopes, conformational epitopes, or mixed epitopes.

[0057] The term "epitope" can be used to refer to a structural epitope. According to some embodiments, a structural epitope can be used to describe a region on an antigen covered by an antibody (e.g., the footprint of antibody binding to an antigen). In some embodiments, a structural epitope can describe amino acid residues on the antigen that are within a specific distance (e.g., within a specific Angstrom range) from the antibody amino acid residues.

[0058] The term "epitope" can also be used to refer to a functional epitope. According to some implementations, a functional epitope can be used to describe amino acid residues of an antigen that interact with amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody.

[0059] Epitopes can be determined using various experimental techniques, also known as "epitope mapping techniques." It is understood that epitope determination can vary depending on the epitope mapping technique used, as well as the experimental conditions employed, such as antigen conformational changes or fragmentation induced by specific experimental conditions. Epitope mapping techniques are well-known in the field (see, for example, Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd edition, 2018), and include, but are not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species exchange mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

[0060] Unless otherwise stated, the term “binding” and its various morphologies as used herein refer to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in the two proteins or molecules coming into close proximity, as determined by commonly used methods known in the art. Specifically, with respect to the binding of an antibody to an antigen, the term “binding” and its various morphologies refer to the interaction of one or more CDRs in the hypervariable region of the antibody with the antigen; that is, the specific binding of the antibody can be conferred by one or more CDRs.

[0061] As used herein, the terms “specific binding” and “specific binding” refer to a situation where one member of a specific binding pair does not bind significantly to any other molecule besides its specific binding partner. For example, some antibodies may be considered to specifically bind XCL1 and XCL2 but not to other similar proteins (e.g., chemokines). If an antibody binds to an antigen at least about 50%, or 2-fold, or 20-fold, or 50-fold, or 100-fold more strongly than it binds to different antigens or non-antigen targets, such binding is achieved by techniques available in the art (e.g., competitive ELISA or KLA-B assays using BIACORE or KINEXA). D If the antibody is specifically bound to the antigen, then it can be considered that the antibody specifically binds to the antigen.

[0062] The antibody of the present invention can be incorporated into a pharmaceutical composition prepared by methods known in the art, comprising the antibody of the present invention and one or more pharmaceutically acceptable carriers and / or diluents (e.g., Remington, The Science and Practice of Pharmacy, 22nd edition, edited by Loyd V., Pharmaceutical Press, 2012, which provides an outline of various formulation techniques generally known to practitioners). Suitable carriers for pharmaceutical compositions include any substance that, when combined with the antibody of the present invention, retains molecular activity and does not react with the patient's immune system.

[0063] A pharmaceutical composition comprising the antibody of the present invention may be administered via a parenteral route (e.g., subcutaneously, intravenously, intraperitoneally, intramuscularly, or transdermally) to a patient who is at risk of or has exhibited the disease or condition described herein. As used interchangeably herein, the pharmaceutical composition of the present invention contains an “effective” or “therapeuticly effective” amount of the antibody of the present invention. An effective amount refers to the amount required (at a specific dose, duration, and route of administration) to achieve the desired therapeutic effect. The effective amount of an antibody may vary depending on factors such as an individual’s disease state, age, sex, weight, and the antibody’s ability to elicit the desired response in the individual. An effective amount also refers to the amount at which the therapeutic benefit of the antibody of the present invention outweighs any toxic or harmful effects.

[0064] As used in this disclosure, in the context of two or more amino acid sequences, the percentage of homology refers to a specific percentage of identical amino acid residues between two or more sequences when compared and aligned to obtain maximum correspondence, such as by using sequence comparison algorithms (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percentage of homology may exist in a region (e.g., a functional domain) of the sequences being compared, or optionally across the entire length of both sequences being compared. For example, the percentage of homology of a sequence can be compared to a reference sequence. For example, when using a sequence comparison algorithm, a test sequence and a reference sequence can be input into a computer (and, if necessary, further specify subsequence coordinates and sequence algorithm program parameters). The sequence comparison algorithm then calculates the percentage of sequence identity or homology of the test sequence relative to the reference sequence based on the specified program parameters. Exemplary sequence alignment and / or homology algorithms are available from Smith & Waterman, Adv. Appl. Math. 2:482 (1981), Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), GAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Package from the Genetics Computing Group, 575 Science Dr., Madison, Wisconsin), or by visual inspection (see discussion below by Ausubel et al.). One example of an algorithm suitable for determining sequence identity and percentage sequence similarity is the BLAST algorithm, detailed in Altschul et al., J. Mol. Biol. 215:403-410 (1990). The software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0065] It is known that antibody recombinant production can lead to antibody modification under certain circumstances. Examples of such modifications include the loss of lysine or glycine at the C-terminus of the heavy chain (HC); the conversion of glutamine or glutamate at the N-terminus of the heavy chain to pyroglutamate; or the conversion of glutamine or glutamate at the N-terminus of the λ light chain to pyroglutamate. This can result in heterogeneous antibody mixtures, including unmodified antibodies, single-modified antibodies, or antibodies with multiple modifications. The antibodies disclosed herein are susceptible to such modifications and are covered within the scope of the claims derived therefrom.

[0066] In one embodiment, the antibody of the present invention binds to and neutralizes XCL1. In one embodiment, the antibody of the present invention binds to and neutralizes XCL2.

[0067] In one embodiment, the antibody of the present invention inhibits or prevents the migration of cells expressing XCR1. In a specific embodiment, the antibody of the present invention neutralizes the migration of dendritic cells. In one embodiment, the dendritic cells are cDC1.

[0068] In one embodiment, the antibody of the present invention targets the K-type of XCL1. D Less than 5.0 x 10 -11 M, in one embodiment, the antibody of the present invention against K of XCL2 D Less than 1.0 x 10 -11 M.

[0069] In one embodiment, the antibody of the present invention binds to the β-sheet monomeric configuration of XCL1. In one embodiment, the antibody of the present invention binds to the dimeric configuration of XCL1.

[0070] In one embodiment, the antibody of the present invention binds to the β-sheet monomeric configuration of XCL2. In one embodiment, the antibody of the present invention binds to the dimeric configuration of XCL2.

[0071] In one embodiment, the antibody disclosed herein binds to the homodimer XCL1. In one embodiment, the antibody disclosed herein binds to the homodimer XCL2. In one embodiment, the antibody disclosed herein binds to a heterodimeric complex comprising a monomer of XCL1 and a monomer of XCL2. In one embodiment, the antibody disclosed herein binds to free monomers XCL1 and / or XCL2, and / or free homodimers XCL1 and / or free homodimers XCL2, and / or heterodimers of free XCL1 and XCL2. In one embodiment, the antibody disclosed herein binds to homodimers XCL1 bound to GAG, and / or homodimers XCL2 bound to GAG, and / or heterodimers of XCL1 and XCL2 bound to GAG.

[0072] In one embodiment, the antibody binds the sequence shown in SEQ ID NO: 29 (i.e., threonine 16, glutamine 17, arginine 18, leucine 19, and proline 20). In one embodiment, the antibody binds the sequence shown in SEQ ID NO: 29, as well as lysine 42 and arginine 43. In one embodiment, the antibody of the present invention binds one or more amino acids of SEQ ID NO: 27; or two or more amino acids of SEQ ID NO: 27; or three or more amino acids of SEQ ID NO: 27. In one embodiment, the antibody of the present invention binds one or more amino acids of SEQ ID NO: 28; or two or more amino acids of SEQ ID NO: 28. In one embodiment, the antibody of the present invention binds one or more amino acids of SEQ ID NO: 29. In one embodiment, the antibody of the present invention binds one or more amino acids of SEQ ID NO: 27 and one or more amino acids of SEQ ID NO: 28; or two or more amino acids of SEQ ID NO: 27 and two or more amino acids of SEQ ID NO: 28.

[0073] In one embodiment, the antibody of the present invention has anti-inflammatory activity and can be used as a therapeutic agent.

[0074] In one aspect, the antibodies of the present invention bind to XCL1 and / or XCL2, but do not specifically bind to other chemokines. Example

[0075] Example 1: Expression of recombinant XCL1 and its variants and XCL2

[0076] Each protein (human XCL1, SEQ ID NO: 1; human XCL2, SEQ ID NO: 2; CC3, SEQ ID NO: 3; and CC5, SEQ ID NO: 4) was expressed as a fusion protein with the following structure: an N-terminal 8xHis tag, a known BRIL fusion protein, an enterokinase (EK) cleavage site, and a C-terminal specific protein (XCL1, etc.). Stable CHO-K1 cell lines for each protein were constructed using fusion protein expression plasmids according to methods known in the art. The fusion proteins were expressed and secreted in chemically defined media. The media were collected, and the fusion proteins were captured on a Ni-IMAC column (e.g., a column provided by Pierce Corporation) and eluted with 375 mM imidazole at pH 7.5. The Ni-IMAC collection was dialyzed into EK digestion buffer (20 mM Tris, 100 mM NaCl, 5 mM CaCl2; pH 8), and the fusion proteins were incubated with EK overnight. Free proteins were captured using Capto-S resin (Cytiva Cat#17544101) and then purified by size exclusion chromatography (Superdex 75 –Cytiva Cat# 28989333). The identity of the proteins was confirmed by mass spectrometry.

[0077] Example 2: Expression of anti-XCL1 antibody

[0078] Exemplary anti-XCL1 antibodies of the present invention are shown in Table 1 below. The antibodies were produced using a stable CHO-K1 cell line in a chemically defined culture medium. The antibodies were captured with PRISMA resin (Cytiva – Cat# 1754981) and eluted with a pH 3 buffer. The pH of the PRISMA collection was adjusted to 5 to precipitate host cell protein (HCP). The protein was purified using an anion exchange chromatography (AEX) process with a POROS 50 HS (Thermo Fisher Cat# 1335906) and then dialyzed into 10 mM histidine, 275 mM sucrose (pH 6). The material was analyzed by analytical size exclusion chromatography (SEC) and confirmed by mass spectrometry.

[0079] Table 1: SEQ ID No of exemplary antibody amino acid sequences

[0080] .

[0081] A control antibody that specifically binds monomer XCL1 (i.e., binds CC3 with high affinity but not CC5; see examples below) has an HC amino acid sequence shown in SEQ ID NO: 27 and an LC amino acid sequence shown in SEQ ID NO: 28. cDNA sequences that can be used to express said HC and LC are shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively. In this document, this antibody is referred to as a monomer-binding control (MBC).

[0082] The relationships between the regions of an exemplary anti-XCL1 antibody are as follows (amino acid numbering is linear; the allocation of amino acids in the variable domains is based on the International Immunogenetics Information System). ® (The website is www.imgt.org). The amino acid assignment in the CDR domain of each exemplary antibody is based on the well-known North numbering rule, except for HCDR2, whose C-terminus is based on the well-known Kabat numbering rule.

[0083] The exemplary antibodies disclosed herein are identified as having high binding affinity and stable chemical and physical properties (including aggregation and solubility), meeting the requirements for therapeutic parenteral administration. The exemplary antibodies disclosed herein are also identified as having low immunogenicity (including low nonspecific binding and / or serum binding) and pharmacokinetic properties consistent with therapeutic parenteral administration for the treatment of immune-mediated diseases. In one aspect, the antibodies disclosed herein are human antibodies of the IgG1 isotype. The antibodies disclosed herein are monoclonal antibodies. Other isotype antibodies (e.g., IgG4, etc.) are also contemplated in this disclosure.

[0084] Example 3: Characteristics of XCL1 variants CC3 and CC5

[0085] XCL1 is a metamorphic protein that undergoes significant conformational rearrangements depending on salt concentration and temperature conditions. High salt and low temperature (10°C) favor the formation of an active monomeric conformation with a standard α-helical motif that forms a standard chemokine fold, which can transmit signals via its homologous receptor XCR1; while low salt and high temperature (40°C) favor the formation of an inactive β-sheet conformation, which can dimerize and bind endogenous glycosaminoglycans (GAGs).

[0086] To investigate the properties of each conformation individually, engineered variant proteins were expressed and purified. CC3 (SEQ ID NO: 3) is an engineered variant of XCL1 that, when residues 21 and 59 are mutated to cysteine ​​(V21C / V59C), stabilizes it in an active monomeric conformation containing an α-helix by forming disulfide bonds. Similarly, mutating residues 36 and 49 to cysteine ​​(A36C / A49C) forms another disulfide bond structure that stabilizes an inactive β-sheet conformation that tends towards a dimer state, referred to as CC5 (SEQ ID NO: 4). These variants are known in the art and were prepared according to methods described in the literature (see Biopolymers 2021 Oct;112(10):e23402 doi: 10.1002 / bip.23402. 2020.9.28 e-release).

[0087] XCR1 is expressed on various types of dendritic cells (DCs), primarily type 1 conventional dendritic cells (cDC1). cDC1 are antigen-presenting cells (APCs) adept at presenting antigens to CD8+ T cells in the presence of major histocompatibility complex I. When CD8+ T cells secrete XCR1, a chemotactic gradient is generated, attracting XCR1-carrying cells to the site of CD8+ T cells (e.g., within tumors or autoimmune sites), thereby enhancing the immune response. See Dorner et al., Immunity 31 (2009) 823-833; Audsley et al., Cells 9 (2020) 565 1-22.

[0088] Recombinant human XCL1, CC3, and CC5 were prepared and evaluated in a migration assay using a mouse Ba / F3 cell line stably overexpressing human XCR1. This cell line migrated to the XCL1 chemokine gradient in vitro on a 5 µM Boyden Chamber plate. The assay setup was as follows: 150,000 cells / well were added to the upper chamber, and different concentrations of XCL1 were added to the lower chamber. Cells were incubated at 37°C for approximately 4 hours, and the number of migrated cells was quantified using a luminescent cell viability assay. The results are shown in Table 2 below.

[0089] Table 2: Migration of XCR1-expressing cells in the presence of XCL1 variants

[0090] .

[0091] For CC3, compared to wild-type XCL1 (858.4 nM), its EC 50Robust migration occurred at approximately 114.7 nM. No migration was observed for CC5. These data confirm the observations in the literature, indicating that recombinant CC3 and CC5 represent two distinct conformations of XCL1.

[0092] Example 4: Combining kinetics and affinity

[0093] The affinity and binding kinetics of the antibody of this invention and the MBCs of human XCL1, human XCL2, mouse XCL1, cynomolgus monkey XCL1, and rat XCL1 were determined by surface plasmon resonance using a Biacore 8K (GE Healthcare) instrument. The experimental procedures were largely in accordance with the instrument manual. First, the antibody was captured on a Biacore Protein A chip, and then a series of antigen concentrations decreasing from 400 nM to 0.039 nM were sequentially passed through it, with the antigen serially diluted 2-fold in PBS-P20-BSA (0.1% surfactant P20, 0.1 mg / mL BSA). All measurements were performed at 25°C.

[0094] A multi-cycle kinetic setup was employed, running each analyte concentration in separate cycles and regenerating the chip surface after each sample injection. The regeneration process was optimized to maintain consistent surface properties across cycles. Each cycle began with antibody injection at a flow rate of 10 µl / min (approximately 150 response units (RU) capture level) for 3 minutes; followed by antigen injection at a flow rate of 50 µl / min for 3 minutes; and a 15-minute dissociation phase in PBS-P20-BSA. Afterward, 10 mM glycine buffer (pH 1.5) was injected at a flow rate of 50 µl / min for 30 seconds, repeated three times to regenerate the chip surface. Data were fitted to a 1:1 binding model to derive k. a and k d And calculate K d .

[0095] Following the general steps described above, the following parameters of the antibody of this invention can be obtained.

[0096] Table 3: Affinity and kinetics of antibodies to various XCL1 antigens

[0097] .

[0098] Notably, antibodies 1 and 2 bind to CC5 with good affinity but not to CC3, whereas the opposite was observed in monomer-specific binding to MBC. This may be because the introduction of cysteine ​​in CC3 could interfere with the epitope binding of antibodies 1 and 2. Crystallographic experiments support the binding of antibodies 1 and 2 to the β-sheet monomeric form of XCL1. On the other hand, antibodies 1 and 2 can bind primarily and preferentially to the dimer form, indicating that antibodies 1 and 2 can bind to the homodimeric XCL1.

[0099] Example 5: Neutralization of XCL1 and XCL2: Human XCR1 Migration Test

[0100] XCL1 and XCL2 are highly conserved chemokines whose chemokine activity is mediated by the XCR1 receptor. Human XCR1 receptor was stably transfected into the mouse cell line Ba / F3 to assess the ability of XCL1 / 2 to induce migration in vitro.

[0101] In this experiment, 150,000 recombinant hXCR1 / Ba / F3 cells were seeded in the upper chamber of a 5 µM Boyden Chamber plate, while the lower chamber contained XCL1 at a concentration empirically determined for each species. Cells migrated to the XCL1 / 2 gradient for approximately 4 hours, during which cell viability readings in the lower chamber were measured. Migration was induced by human XCL1, human XCL2, mouse, rat, and cynomolgus monkey XCL1 in this experiment. After ligand dose-response assays, a single concentration of XCL1 / 2 was added to the lower chamber of the Boyden Chamber plate, and dose-response assays were performed simultaneously for each test mAb. The neutralizing IC50 of each antibody was determined. 50 Values. As shown in Table 4 below, all antibodies were able to neutralize human XCL1 and XCL2, as well as rat, mouse, and cynomolgus monkey XCL1.

[0102] Table 4: Neutralizing effect of the antibodies of this invention on XCL1 and XCL2

[0103] .

[0104] The results in Tables 3 and 4 demonstrate that both exemplary antibodies bind to both XCL1 and XCL2 with high affinity and neutralize both chemokines in vitro. Therefore, it is understood that the antibodies of this disclosure can be used to bind and neutralize XCL1, XCL2, or both, including in a therapeutic context. It is understood that the antibodies disclosed herein bind to the homodimer XCL1, and also to the homodimer XCL2 due to the sequence similarity between XCL1 and XCL2. These antibodies also bind to heterodimeric complexes that may be formed, comprising an XCL1 monomer and an XCL2 monomer. The antibodies of this disclosure can bind to free monomers XCL1 or XCL2, or free homodimers of XCL1, or free homodimers of XCL2, or heterodimers of free XCL1 and XCL2. The antibody disclosed in this invention can also bind to the homodimer XCL1 bound to GAG, the homodimer XCL2 bound to GAG, or the heterodimer of XCL1 and XCL2 bound to GAG.

[0105] Example 6: Neutralizing effect of antibody on XCL1 activity: dendritic cell migration

[0106] XCL1 and XCL2 stimulated the migration of conventional type 1 dendritic cells (cDC1) expressing XCR1. To verify the neutralizing activity of the XCL1 / 2 antibody, the migration of human cDC1 cells differentiated from CD34+ stem cells to XCL1 cells in Boyden Chamber was detected by flow cytometry. In summary, CD34+ cells derived from human umbilical cord blood were induced to differentiate for 3 weeks on feeder cells expressing DLL1 in a cytokine mixture (adapted from Balan et al., doi: 10.1016 / j.celrep.2018.07.033). Myeloid dendritic cell kit (StemCell; lineage negative, HLA-DR) was used. + CD11c + Cell enrichment. Triple doses of 10 ng / mL XCL1+ antibody were titrated and pre-incubated for 1 hour, then added to the lower chamber of a Boyden Chamber. The antibodies used were antibody 1, antibody 2, and the hIgG1-AAS isotype control antibody. 150,000 enriched differentiated cells were added to the upper chamber of the Boyden Chamber and incubated for 2 hours. Human cDC1 cells migrating in the lower chamber were stained (live / dead, CLEC9A). + XCR1 + The XCL1 / 2 antibody in this study completely inhibited the migration of cDC1 to XCL1 in all three replicate experiments, with an IC50 score of [missing value]. 50 The geometric mean of the value (nM) and its error (Δ method) are shown in Table 5.

[0107] Table 5: Inhibitory efficacy of XCL1 / 2 antibodies against XCL1-stimulated primary human cDC1 migration

[0108] .

[0109] Example 7: Anti-inflammatory effect of antibodies in mice

[0110] The immunosuppressive activities of antibodies 1 and 2 were assessed in the ears of mice with an oxazolone-induced delayed-type hypersensitivity model and compared with anti-CD8 depletion antibody (Bio X Cell #BE0117). On day 0, mice were treated with anti-XCL1 / 2 antibodies, and approximately one hour later, sensitization was achieved using a 3% oxazolone solution in acetone on the shaved ventral skin. On day 6, the ears of mice were challenged with a 2% oxazolone solution, and changes in ear swelling were measured on day 7. The results are shown in Table 6, expressed as changes in ear thickness (Δ) between the anti-XCL1 / 2 antibody group and the isotype control group.

[0111] Table 6: Anti-inflammatory effects in mouse models

[0112] .

[0113] Example 8: Epitope Localization and Measurement

[0114] The binding sites of exemplary antibodies on the XCL1 surface were determined by HDX and X-ray crystallography, techniques well known in the art.

[0115] HDX analysis revealed that antibodies 1 and 2 bind to a nonlinear epitope formed by residues 12 to 20 (SEQ ID NO: 27) and residues 35 to 46 (SEQ ID NO: 28). X-ray crystal structure (2.77 Å resolution) of the antigen-binding region (Fab) of the fragment complexed with human XCL1 further indicated that the specific residues involved in binding are those whose sequences are represented by SEQ ID NO: 29 (i.e., threonine 16, glutamine 17, arginine 18, leucine 19, and proline 20), as well as lysine 42 and arginine 43. Therefore, the antibodies of the present invention can bind one or more amino acids of SEQ ID NO: 27; or two or more amino acids of SEQ ID NO: 27; or three or more amino acids of SEQ ID NO: 27. The antibodies of the present invention can bind one or more amino acids of SEQ ID NO: 28; or two or more amino acids of SEQ ID NO: 28. The antibodies of the present invention can bind one or more amino acids of SEQ ID NO: 29. The antibody of the present invention can bind to one or more amino acids of SEQ ID NO: 27 and one or more amino acids of SEQ ID NO: 28; or two or more amino acids of SEQ ID NO: 27 and two or more amino acids of SEQ ID NO: 28.

[0116] This document exemplifies the binding of nonlinear epitopes of XCL1 / XCL2 to antibodies. In one example, the antibody binds to the monomeric β-sheet configuration of XCL1 or XCL2. In another example, the antibody binds to the dimer form of XCL1 or XCL2. In this case, the antibody may bind to one of the two monomeric subunits of the chemokine, which together define the dimer. For example, the antibody may bind to the amino acid residues defined in SEQ ID NO: 29 and lysine 42 and / or arginine 43 of the first monomeric unit of XCL1. This disclosure also contemplates an antibody that binds to the dimer XCL1 or XCL2 by binding to a nonlinear epitope comprising the two monomeric residues of the dimer. Another binding mode contemplated herein is binding to the monomeric β-sheet of XCL1 or XCL2 in a manner that prevents dimerization.

[0117] Example 9: GAG binding test

[0118] As previously mentioned, the β-sheet conformation of XCL1 / 2 can dimerize and cannot bind to the XCR1 receptor. Instead, in this form, XCL1 / 2 binds to cell surface glycosaminoglycans (GAGs), which is thought to facilitate the establishment of chemotactic gradients. Antibodies 1 and 2 bind to the β-sheet conformation, preferably the dimer form, and their ability to bind to the XCL1 / 2 dimer bound to cell surface GAGs was investigated using flow cytometry.

[0119] 293T cells were incubated for 1 hour with various formulations of XCL1 or XCL2 at 1 µg / mL and 100 nM of antibody 1, antibody 2, or hIgG1-AAS isotype control antibody (“Control”). Subsequently, staining with anti-human Fc / PE secondary antibody (LifeTechnologies 12-4998-82) was performed to detect antibody-XCL1 / 2 complexes bound to GAGs on the cell surface. Under an alternative condition, binding of the antibody-XCL1 / 2 complex was performed in the presence of 100 µg / mL heparin to verify the specificity of GAG binding. Mean fluorescence intensity (MFI) was measured by flow cytometry under each condition. The experiment was repeated three times, and the mean MFI + / - standard deviations for each experiment are shown in Table 7 below.

[0120] Table 7: Flow cytometry detection of the binding of XCL1 / 2 antibody to XCL1 / 2-GAG on the surface of 293T cells (MFI)

[0121] .

[0122] The above results indicate that antibodies 1 and 2 bind to inactive or dimeric XCL1 on the cell surface. Combined with the results of Example 6, this shows that antibodies 1 and 2 can bind to inactive XCL1, isolating it and preventing it from activating XCL1.

[0123] Example 10: XCL1 specificity of an exemplary antibody

[0124] Human serum binding assays were used to identify antibodies 1 and 2, as well as the non-specific targets of MBC. Each antibody was coated in microplates and then treated with serum. After overnight incubation, unbound serum proteins were washed away, followed by the elution of antibody-bound proteins. The samples were digested with trypsin, and peptides were identified by mass spectrometry.

[0125] Antibodies 1 and 2 did not bind to other chemokines, but MBC was found to bind to a large number of a second, non-targeting chemokine. Subsequent experiments showed that in the presence of high concentrations of this chemokine (i.e., as expected under physiological conditions), MBC lost its biological activity, while antibodies 1 and 2 remained unaffected.

[0126] sequence list

[0127] SEQ ID NO: 1 person XCL1

[0128] VGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNMIQTKPTGTQQSTNTAVTLTG

[0129] SEQ ID NO: 2 people XCL2

[0130] VGSEVSHRRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNMIQTKPTGTQQSTNTAVTLTG

[0131] SEQ ID NO: 3 CC3

[0132] VGSEVSDKRTCVSLTTQRLPCSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDCVRSMDRKSNTRNMIQTKPTGTQQSTNTAVTLTG

[0133] SEQ ID NO: 4 CC5

[0134] VGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRCVIFITKRGLKVCCDPQATWVRDVVRSMDRKSNTRNMIQTKPTGTQQSTNTAVTLTG

[0135] SEQ ID NO: 5 Universal LCDR1 sequence of antibodies 1 and 2

[0136] QASQXISDYLN, where X is selected from S and D

[0137] SEQ ID NO: 6 Antibody 1 light chain CDR1

[0138] QASQDISDYLN

[0139] SEQ ID NO: 7 Antibody 2 light chain CDR1

[0140] QASQSISDYLN

[0141] SEQ ID NO: 8 Light chain CDR2 of antibodies 1 and 2

[0142] YDASAKQT

[0143] SEQ ID NO: 9 Light chain CDR3 of antibodies 1 and 2

[0144] QHYDNLGYYT

[0145] SEQ ID NO: 10 Heavy chain CDR1 of antibodies 1 and 2

[0146] KASGYTFTSYYMV

[0147] SEQ ID NO: 11 Heavy chain CDR2 of antibodies 1 and 2

[0148] VIDPSKGSTNYAQKFQG

[0149] SEQ ID NO: 12 HCDR3 CDR2 of antibodies 1 and 2

[0150] AREGLZLLAFDI, where Z is selected from Q and E

[0151] SEQ ID NO: 13 Antibody 1 Heavy Chain CDR3

[0152] AREGLELLAFDI

[0153] SEQ ID NO: 14 Antibody 2 heavy chain CDR3

[0154] AREGLQLLAFDI

[0155] SEQ ID NO: 15 Antibody 1 HCVR

[0156] X a VQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMVWVRQAPGQGLEWMGVIDPSKGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGLELLAFDIWGQGTMVTVSS, where X is Q or pyroglutamic acid.

[0157] SEQ ID NO: 16 Antibody 2 HCVR

[0158] X a VQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMVWVRQAPGQGLEWMGVIDPSKGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGLQLLAFDIWGQGTMVTVSS, where X is Q or pyroglutamic acid.

[0159] SEQ ID NO: 17 Antibody 1 LCVR

[0160] DIQMTQSPSSSLSASVGDRVTITCQASQDISDYLNWYQQKPGKAPKLLIYDASAKQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHYDNLGYYTFGQGTKLEIK

[0161] SEQ ID NO: 18 Antibody 2 LCVR

[0162] DIQMTQSPSSLSASVGDRVTITCQASQSISDYLNWYQQKPGKAPKLLIYDASAKQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHYDNLGYYTFGQGTKLEIK

[0163] SEQ ID NO: 19 Antibody 1 HC

[0164] X1VQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMVWVRQAPGQGLEWMGVIDPSKGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGLELLAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPX2X3, where X1 is Q or pyroglutamic acid; X2 is G or absent; and X3 is K or absent

[0165] SEQ ID NO: 20 Antibody 2 HC

[0166] X1VQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMVWVRQAPGQGLEWMGVIDPSKGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGLQLLAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPX2X3, where X1 is Q or pyroglutamic acid; X2 is G or absent; and X3 is K or absent

[0167] SEQ ID NO: 21 Antibody 1 LC

[0168] DIQMTQSPSSLSASVGDRVTITCQASQDISDYLNWYQQKPGKAPKLLIYDASAKQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHYDNLGYYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0169] SEQ ID NO: 22 Antibody 2 LC

[0170] DIQMTQSPSSLSASVGDRVTITCQASQSISDYLNWYQQKPGKAPKLLIYDASAKQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHYDNLGYYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0171] SEQ ID NO: 23 Heavy chain DNA sequence of Antibody 1

[0172]

[0173] SEQ ID NO: 24 DNA sequence of the light chain of Antibody 1

[0174] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATAAGCGACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCTTCCGCTAAACAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACATTATGATAATCTCGGATACTACACTTTTGGCCAAGGGACCAAGCTGGAGATCAAAagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc

[0175] SEQ ID NO: 25 DNA sequence of the heavy chain of Antibody 2

[0176]

[0177] SEQ ID NO: 26 Antibody 2 light chain DNA sequence

[0178] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGAGCATAAGCGACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCTTCCGCTAAACAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACATTATGATAATCTCGGATACTACACTTTTGGCCAAGGGACCAAGCTGGAGATCAAAAGAACTGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0179] SEQ ID NO: 27 XCL1 epitope 1 (antibodies 1 and 2)

[0180] VSLTTQRLP

[0181] SEQ ID NO: 28 XCL1 epitope 2 (antibodies 1 and 2)

[0182] RAVIFITKRGLK

[0183] SEQ ID NO: 29 XCL1 epitope 1 subsequence (antibodies 1 and 2)

[0184] TQRLP

[0185] SEQ ID NO: 30 Heavy chain variable region of Antibody 1, without variation

[0186] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMVWVRQAPGQGLEWMGVIDPSKGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGLELLAFDIWGQGTMVTVSS

[0187] SEQ ID NO: 31 Heavy chain variable region of Antibody 2, without variation

[0188] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMVWVRQAPGQGLEWMGVIDPSKGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGLQLLAFDIWGQGTMVTVSS

[0189] SEQ ID NO: 32 Heavy chain of Antibody 1, without variation

[0190] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMVWVRQAPGQGLEWMGVIDPSKGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGLELLAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0191] SEQ ID NO: 33 Heavy chain of Antibody 2, without variation

[0192] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMVWVRQAPGQGLEWMGVIDPSKGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGLQLLAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. An antibody binding to human XCL1, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprises CDRs LCDR1, LCDR2, and LCDR3, wherein The HCDR1 contains SEQ ID NO: 10, The HCDR2 contains SEQ ID NO: 11, The HCDR3 comprises SEQ ID NO: 12, wherein Z is selected from Q and E; The LCDR1 contains SEQ ID NO: 5, where X is selected from S and D. The LCDR2 contains SEQ ID NO: 8, and The LCDR3 contains SEQ ID NO:

9.

2. The antibody according to claim 1, wherein Z is E.

3. The antibody according to claim 1 or claim 2, wherein X is D.

4. The antibody according to claim 1, wherein Z is Q.

5. The antibody according to claim 1 or claim 4, wherein X is S.

6. The antibody according to any one of claims 1-3, wherein HCDR1 comprises SEQ ID NO: 10; HCDR2 comprises SEQ ID NO: 11; HCDR3 comprises SEQ ID NO: 13; LCDR1 comprises SEQ ID NO: 6; LCDR2 comprises SEQ ID NO: 8; and LCDR3 comprises SEQ ID NO:

9.

7. The antibody according to any one of claims 1-3 or claim 6, wherein the LCVR comprises SEQ ID NO: 17, and the HCVR comprises SEQ ID NO: 15, wherein X a Selected from Q and pyroglutamic acid.

8. The antibody according to claim 7, wherein X a It's Q.

9. The antibody according to any one of claims 1-3, 6 or 7, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 19, wherein X1 is selected from Q and pyroglutamic acid; X2 is G or absent; and X3 is K or absent; and the LC comprises SEQ ID NO:

21.

10. The antibody according to claim 9, wherein X1 is Q; X2 is G; and X3 is K.

11. The antibody according to any one of claims 1, 4 and 5, wherein HCDR1 comprises SEQ ID NO: 10; HCDR2 comprises SEQ ID NO: 11; HCDR3 comprises SEQ ID NO: 14; LCDR1 comprises SEQ ID NO: 7; LCDR2 comprises SEQ ID NO: 8; and LCDR3 comprises SEQ ID NO:

9.

12. The antibody according to any one of claims 1, 4, 5, and 11, wherein the LCVR comprises SEQ ID NO: 18, and the HCVR comprises SEQ ID NO: 16, wherein X a Selected from Q and pyroglutamic acid.

13. The antibody according to claim 12, wherein X a It's Q.

14. The antibody according to any one of claims 1, 4, 5, 11 and 12, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 20, wherein X1 is selected from Q and pyroglutamic acid; X2 is G or absent; and X3 is K or absent; and the LC comprises SEQ ID NO:

22.

15. The antibody according to claim 14, wherein X1 is Q; X2 is G; and X3 is K.

16. The antibody according to any one of claims 1-15, wherein the antibody is an XCL1 antagonist.

17. The antibody according to any one of claims 1-15, wherein the antibody binds to human XCL2.

18. The antibody according to claim 17, wherein the antibody is an XCL2 antagonist.

19. The antibody according to any one of claims 1-18, which binds to the dimer XCL1.

20. The antibody according to any one of claims 1-19, wherein the dimer XCL1 is a homodimer XCL1.

21. The antibody according to any one of claims 1-20, wherein the antibody binds to XCL1 in a β-sheet configuration.

22. The antibody according to any one of claims 1-21, wherein the homodimer XCL1 binds to glycosaminoglycans on the cell surface.

23. The antibody according to any one of claims 1-22, wherein it binds to the dimer XCL2.

24. The antibody according to any one of claims 1-23, wherein the dimer XCL2 is a homodimer XCL2.

25. The antibody according to any one of claims 1-24, wherein the antibody binds to at least one amino acid residue on each monomer unit of the dimer XCL2.

26. The antibody according to any one of claims 1-25, wherein the homodimer XCL2 binds to glycosaminoglycans on the cell surface.

27. The antibody according to any one of claims 1-26, which binds a heterodimeric complex comprising a monomer of XCL1 and a monomer of XCL2.

28. The antibody according to any one of claims 1-27, wherein the antibody binds at least one amino acid of the XCL1 monomer and at least one amino acid of the XCL2 monomer in the heterodimeric complex.

29. The antibody according to any one of claims 1-28, wherein the heterodimeric complex binds to glycosaminoglycans on the cell surface.

30. The antibody according to any one of claims 1-29, which does not bind to chemokines other than XCL1 and XCL2.

31. An antibody that binds to human XCL1, comprising a heavy chain variable region (HCVR) having at least 95% sequence identity with SEQ ID NO: 15 and a light chain variable region (LCVR) having at least 95% sequence identity with SEQ ID NO:

17.

32. The antibody according to claim 31, comprising a heavy chain (HC) having at least 95% sequence identity with SEQ ID NO: 19 and a light chain (LC) having at least 95% sequence identity with SEQ ID NO:

21.

33. An antibody that binds to human XCL1, comprising a heavy chain variable region (HCVR) having at least 95% sequence identity with SEQ ID NO: 16 and a light chain variable region (LCVR) having at least 95% sequence identity with SEQ ID NO:

18.

34. The antibody according to claim 33, comprising a heavy chain (HC) having at least 95% sequence identity with SEQ ID NO: 20 and a light chain (LC) having at least 95% sequence identity with SEQ ID NO:

22.

35. An antibody that binds to an epitope of XCL1, said epitope comprising one or more amino acids of SEQ ID NO:

27.

36. The antibody according to claim 35, wherein it binds two or more amino acids of SEQ ID NO:

27.

37. The antibody according to claim 36, which binds three or more amino acids of SEQ ID NO:

27.

38. The antibody according to claim 37, which binds to one or more amino acids of SEQ ID NO:

29.

39. An antibody that binds to an epitope of XCL1, said epitope comprising one or more amino acids of SEQ ID NO:

28.

40. The antibody according to claim 39, wherein it binds two or more amino acids of SEQ ID NO:

28.

41. An antibody that binds to an epitope of XCL1, said epitope comprising one or more amino acids of SEQ ID NO: 27 and one or more amino acids of SEQ ID NO:

28.

42. The antibody according to claim 41, which binds two or more amino acids of SEQ ID NO: 27 and one or more amino acids of SEQ ID NO:

28.

43. The antibody according to claim 41, which binds to one or more amino acids of SEQ ID NO:

29.

44. The antibody according to any one of claims 1-43, wherein the antibody has a human IgG1 isotype.

45. A pharmaceutical composition comprising the antibody of any one of claims 1-44 and one or more pharmaceutically acceptable carriers, diluents or excipients.

46. ​​A method for treating alopecia areata, vitiligo, asthma, psoriasis, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis in an individual in need, the method comprising administering to said individual an effective amount of the antibody of any one of claims 1-44 or the pharmaceutical composition of claim 45.

47. The antibody according to any one of claims 1-44, or the pharmaceutical composition according to claim 45, for use in therapy.

48. The antibody according to any one of claims 1-44, or the pharmaceutical composition according to claim 45, for the treatment of alopecia areata, vitiligo, asthma, psoriasis, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis.

49. Use of the antibody according to any one of claims 1-44 or the pharmaceutical composition according to claim 45 in the preparation of a medicament for treating alopecia areata, vitiligo, asthma, psoriasis, multiple sclerosis, rheumatoid arthritis, or ulcerative colitis.

50. A nucleic acid comprising a sequence encoding SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO:

26.

51. A vector comprising a first nucleic acid sequence encoding SEQ ID NO: 23 or SEQ ID NO: 25 and a second nucleic acid sequence encoding SEQ ID NO: 24 or SEQ ID NO:

26.

52. A cell comprising the carrier of claim 51.

53. A method for producing an antibody, the method comprising culturing the cells according to claim 52 under conditions that cause antibody expression, and recovering the expressed antibody from the culture medium.

54. An antibody produced by culturing cells according to claim 52 under conditions that cause antibody expression and recovering the expressed antibody from the culture medium.

55. A protein complex comprising a dimer XCL1 and an antibody bound to the dimer XCL1.

56. The protein complex of claim 55, wherein the dimer XCL1 is a homodimer XCL1.

57. The protein complex of claim 55 or claim 56, wherein the antibody binds to XCL1 in a β-sheet configuration.

58. A protein complex comprising a dimer XCL2 and an antibody bound to the dimer XCL2.

59. The protein complex of claim 58, wherein the dimer XCL2 is a homodimer XCL2.

60. The protein complex of claim 58 or claim 59, wherein the antibody binds to the β-sheet configuration of XCL2.

61. A protein complex comprising: Heterodimers containing monomeric units of XCL1 and XCL2, and Antibodies that bind to the heterodimer.

62. The protein complex of claim 61, wherein the antibody binds to at least one amino acid residue of XCL1.

63. The protein complex of claim 61 or claim 62, wherein the antibody binds to at least one amino acid residue of XCL2.

64. The protein complex according to any one of claims 61-63, wherein the antibody binds at least one residue of the β-sheet configuration of XCL1 or XCL2.