Citrullinated antigen-specific chimeric antigen receptors for targeting regulatory T cells to treat hidradenitis suppurativa

Abstract

Disclosed herein are chimeric antigen receptors (CARs) containing an antigen-binding site that recognizes citrullinated polypeptides. Citrullinated polypeptides, such as citrullinated vimentin, are expressed in skin lesions of subjects with hidradenitis suppurativa. Also disclosed are T cells, particularly Treg cells, that express these CARs. Administration of these CAR-T cells is useful for treating hidradenitis suppurativa. The present disclosure relates to chimeric antigen receptors that react with citrullinated antigens and regulatory T cells that express the receptors for treating hidradenitis suppurativa.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 519,184, filed August 11, 2023, and U.S. Provisional Application No. 63 / 408,046, filed September 19, 2022, each of which is incorporated by reference in its entirety.

[0002] Reference to the electronic sequence listing The contents of the electronic sequence listing (237752000740seqlist.xml, size: 66,037 bytes, and created on September 13, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to chimeric antigen receptors that react with citrullinated antigens and regulatory T cells that express the receptors for the treatment of hidradenitis suppurativa. [Background technology]

[0004] Hidradenitis suppurativa (HS) is a chronic inflammatory skin condition manifested by the development of painful skin lesions formed by follicular obstruction caused by hyperkeratosis and hyperplasia of the infundibulum epithelium. Disease progression ultimately leads to the "blockage" and rupture of follicular units, which initiates a chronic cutaneous inflammatory response. HS has a significant negative impact on patients due to the disease-related pain and limited movement, the foul odor and discharge from affected skin, and the disfigurement associated with HS symptoms. Current treatment options are inadequate to resolve symptoms, and there remains an unmet need for effective treatments.

[0005] Regulatory T (Treg) cells have been found to be enriched in HS skin lesions, yet an imbalance exists between the ratio of Tregs to CD4+ helper T17 (Th17) cells, favoring Th17. Normalizing this ratio may improve immune dysregulation in HS. Preclinical studies have demonstrated the potential of antigen-specific and autologous Treg cells to treat systemic inflammation and organ damage, supporting the hypothesis that Treg cells can provide transformative and long-lasting treatment. Recent studies have demonstrated the therapeutic potential of Treg-related immunotherapies, including low-dose interleukin-2 (IL-2) and IL-2 mutant proteins, in various autoimmune conditions. Clinical trials are also evaluating the potential of polyclonal Treg cells as a treatment option for organ transplant recipients and patients suffering from autoimmune conditions. Treg cell infusions have been well tolerated but have shown limited efficacy.

[0006] Currently, there is no cure for HS, and many other chronic inflammatory diseases. This is due, in part, to the fact that the cause of HS is unknown. In fact, HS is frequently treated with corticosteroids or other anti-inflammatory agents, which may predispose recipients to infections. Therefore, what is needed in the art is a treatment that targets antigens preferentially found in HS lesions. Summary of the Invention

[0007] The present disclosure relates to chimeric antigen receptors (CARs) that react with citrullinated antigens and regulatory T cells expressing the same for treating hidradenitis suppurativa. In particular, the CARs expressed by Treg cells of the present disclosure specifically bind to citrullinated polypeptides, including vimentin, citrullinated filaggrin, and citrullinated fibrinogen, as well as citrullinated fragments thereof. In some embodiments, the single-chain fragment variable (scFv) portion of the CAR is derived from an antibody highly specific for one or more citrullinated proteins. In one embodiment, a specific SCFv chain is inserted into a second-generation CAR construct. In some embodiments, the SCFv chain is inserted into a CAR construct cloned into a lentiviral vector. In the detailed description, reference to an antibody is applicable to the antigen-binding domain of the CAR of the present disclosure, unless the context indicates otherwise.

[0008] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate exemplary embodiments and, together with the description, further serve to enable one of ordinary skill in the art to make and use these and other embodiments apparent to those skilled in the relevant arts. The present invention is described in more detail in conjunction with the following drawings: [Brief explanation of the drawings]

[0009] [Figure 1] A: Citrullinated protein (CitP) is frequently present in samples of hidradenitis suppurativa (HS) lesions but absent in samples of normal skin (non-HS). B: CitP is also frequently present in synovial tissue samples from rheumatoid arthritis (RA) patients but absent in synovial tissue samples from healthy subjects (non-RA). CitP reactivity was measured by detecting binding of anti-citrullinated vimentin (CV) antibodies (e.g., anti-CV Ab referred to herein as S01-mIgG2a).

[0010] [Figure 2A]HS skin lesions have higher levels of inflammation and CitP target antigens compared to normal skin, and HS skin lesions have a higher percentage of inflammatory cells than normal skin. [Figure 2B] HS skin lesions have higher levels of inflammation and CitP target antigens compared to normal skin. HS skin lesions have higher numbers of CitP+ inflammatory cells than normal skin. [Figure 2C] HS skin lesions have higher levels of inflammation and CitP target antigens compared to normal skin. HS skin lesions have higher numbers of CitP+ non-inflammatory cells than normal skin.

[0011] [Figure 3] An exemplary CV-reactive chimeric antigen receptor (CAR) expressed on the surface of a reporter cell line is activated by citrullinated vimentin, fibrinogen, and two different filaggrin proteins, but not by non-citrullinated vimentin, fibrinogen, and filaggrin proteins.

[0012] [Figure 4A] Higher levels of CitP are present in sera from patients with HS and RA than in sera from normal control subjects, as determined by measuring binding of anti-CV Ab (S01-mIgG2a) in a cell-based assay and ELISA, respectively. [Figure 4B] Higher levels of CitP are present in sera from patients with HS and RA than in sera from normal control subjects, as determined by measuring binding of anti-CV Ab (S01-mIgG2a) in a cell-based assay and ELISA, respectively. [Figure 4C] Higher levels of citrullinated and MMP-degraded vimentin (VICM) are present in the serum of HS and RA patients than in the serum of normal control subjects.

[0013] [Figure 5]A-B: Serum samples from HS patients have elevated levels of C-reactive protein (CRP) and anti-citrullinated protein antibodies (ACPA) compared to normal serum samples.

[0014] [Figure 6] A: Schematic of the cell-based assay for detecting CitP. B: Serum samples from HS patients have elevated levels of citrullinated antigen (CitP) compared to normal serum samples.

[0015] [Figure 7A] HS patients have Treg frequencies and phenotypes comparable to healthy donors (HD). [Figure 7B] HS patients have Treg frequencies and phenotypes comparable to healthy donors (HD). [Figure 7C] HS patients have Treg frequencies and phenotypes comparable to healthy donors (HD). [Figure 7D] HS patients have Treg frequencies and phenotypes comparable to healthy donors (HD).

[0016] [Figure 8] A: The frequency and recovery of Tregs during the generation of CV-CAR Treg cells are comparable between HS patients (HS, Hurley2, and Hurley3) and HD. B: The frequency and recovery of Tregs during the generation of CV-CAR Treg cells are comparable between HS patients (HS, Hurley2, and Hurley3) and HD. C: CV-CAR Treg cells derived from PBMCs of HD and HS patients (HS, Hurley2, and Hurley3) express comparable levels of FOXP3. Hurley2 and Hurley3 represent patients with moderate and severe HS, respectively.

[0017] [Figure 9A] CV-CAR Treg cells derived from PBMCs of HD or HS patients expanded and were similarly activated by CAR stimulation (pCV beads). [Figure 9B]CV-CAR Treg cells derived from PBMCs of HD or HS patients expanded and were similarly activated by CAR stimulation (pCV beads).

[0018] [Figure 10A] Treg cells derived from PBMCs of HD or HS patients exhibit similar suppressive activity. [Figure 10B] Treg cells derived from PBMCs of HD or HS patients exhibit similar suppressive activity. DETAILED DESCRIPTION OF THE INVENTION

[0019] Protein citrullination is an irreversible post-translational modification that affects protein structure and is involved in regulating the function of histones, cytoskeleton, and secreted proteins. Furthermore, citrullinated proteins accumulate at sites of chronic inflammation. Citrulline is generated through the post-translational conversion of arginine to citrulline by the peptidylarginine deiminase (PAD) enzyme. PAD enzymes have been shown to be highly expressed in activated myeloid cells, including macrophages and neutrophils. PAD enzymes play an important role in the formation of neutrophil extracellular traps (NETs), a phenomenon that externalizes autoantigens and immune stimulatory molecules. During NETosis, neutrophils externalize citrullinated autoantigens and release DAMPs as innate immune activators involved in promoting the pathogenesis of HS. While expressed intracellularly, aberrant PAD activity can lead to the deposition of citrullinated proteins in many tissues, including joints, lungs, lymph nodes, and periodontal tissues, in patients with inflammatory diseases. PAD enzyme activity is increased in the inflamed skin of HS patients, particularly in neutrophils, which release citrullinated proteins and other autoantigens during the progression of NETosis. Citrullinated vimentin (CV) and fibrinogen are found in the extracellular matrix of inflamed skin in patients with hidradenitis suppurativa (HS). Citrullination also leads to the generation of autoantigens during inflammatory responses. Citrullinated autoantigens are highly immunogenic and can be presented on MHC molecules, inducing T cell-mediated B cell activation. These autoantigens lead to the development of pathogenic anti-citrullinated protein antibodies (ACPAs) in the serum of HS patients and at the site of skin lesions. ACPAs can react with several citrullinated proteins, including citrullinated fibrinogen, myeloperoxidase, nucleosomes, histone H4, and dsDNA. Taken together, the presence of NETs, ​​citrullinated proteins, and ACPAs in HS skin lesions indicates a pathogenic role of citrullination in the pathogenesis of HS disease.

[0020] This disclosure describes a targeted antigen-specific approach to increase the function and quantity of Treg cells that can be activated at disease sites, such as HS lesions. As described herein, targeting citrullinated proteins with CAR-Treg cells is believed to be a novel approach for the treatment of HS.

[0021] I. Definition Unless otherwise specified, terms and symbols in biochemistry, nucleic acid chemistry, molecular biology, developmental biology, and molecular genetics follow those of standard treatises and textbooks in the field, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, 1989); Alberts and Singer, Developmental Biology, Eighth Edition (Sinauer Associates Inc., Sunderland, MA, 2006); Kornberg and Baker, DNA Replication, Second Edition (WH Freeman, New York, 1992); Gaits, ed., Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991), etc.

[0022] As used herein, the terms "antigen," "immunogen," and "antibody target" refer to a molecule, compound, or complex that is recognized by an antibody, i.e., that can be bound by an antibody. The terms can refer to any molecule that can be recognized by an antibody, such as a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.). Those skilled in the art will understand that the terms do not indicate that a molecule is immunogenic in all contexts, but simply that the molecule can be targeted by an antibody.

[0023] As used herein, the term "epitope" refers to a localized site on an antigen that is recognized and bound by an antibody. An epitope can include a few amino acids, or a portion of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or a portion thereof. In some cases, an epitope includes non-protein components derived, for example, from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is three-dimensional. Thus, for example, if the target is a protein, an epitope can consist of contiguous amino acids or amino acids from different parts of the protein that are brought into close proximity by protein folding (e.g., a discontinuous epitope).

[0024] As used herein, the term "antibody" refers to a polypeptide comprising a framework region derived from an immunoglobulin gene that specifically binds and recognizes an antigen. Typically, the "variable region" comprises the antigen-binding region of an antibody (or its functional equivalent) and is most important in determining the specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD).

[0025] Antibodies can be of (i) any of the five major classes of immunoglobulins, namely, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), and mu (IgM), based on the identity of the heavy chain constant domain, or (ii) any subclass (isotype) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Light chains can be either lambda or kappa.

[0026] Below is a non-exhaustive list of different antibody forms, all of which retain antigen-binding activity. (1) Whole immunoglobulins (also called "intact" antibodies) (two light chains and two heavy chains, e.g., tetramers), (2) immunoglobulin polypeptides (light or heavy chains); (3) antibody fragments, such as Fv (monovalent or bivalent variable region fragments, including the variable region (e.g., V L and / or V H ), Fab(V L C L V H C H ), F(ab')2, Fv(V L V H ), scFv (single-chain Fv) (V linked by a linker, e.g., a peptide linker L and V H (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(Fv)2 fused to a CH3 domain), diabody (non-covalent dimer of single chain Fv (scFv) fragments consisting of heavy chain variable (VH) and light chain variable (VL) regions linked by a small peptide linker), triabody is a trivalent sc(Fv)3 or trispecific sc(Fv)3 (4) multivalent antibodies (antibodies that contain binding regions that bind to two different epitopes or proteins, e.g., "scorpion" antibodies, (5) fusion proteins containing the binding portion of an immunoglobulin fused to another amino acid sequence (such as a fluorescent protein); and (6) A heavy-chain-only antibody or antibody fragment, which has only two heavy chains and lacks the two light chains normally found in antibodies.

[0027] The production and properties of tandem scFvs and diabodies are described, for example, in Asano et al. (2011) J Biol. Chem. 286:1812; Kenanova et al. (2010) Prot Eng Design Sel 23:789; Asano et al. (2008) Prot Eng Design Sel 21:597.

[0028] As used herein, the phrase "set of CDR sequences" refers to the three heavy chain and / or three light chain CDRs of a particular antibody described herein. A "light chain" CDR sequence set refers to the light chain CDR sequences. A "heavy chain" CDR sequence set refers to the heavy chain CDR sequences. A "complete" CDR sequence set refers to both the heavy and light chain CDR sequences. CDRs are predicted based on IMGT sequence alignment.

[0029] As used herein, the term "chimeric antibody" refers to an antibody having amino acid sequences derived from more than one species. In one embodiment, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and function, and the constant regions are homologous to sequences from another species (usually in the subject to be treated, e.g., human) to avoid eliciting an immune response.

[0030] As used herein, the term "humanized antibody" refers to a chimeric antibody in which CDRs from the VH and VL regions of a non-human antibody with desired specificity, affinity, and capacity are grafted onto human framework sequences. In one embodiment, the framework residues of the humanized antibody are modified to improve and optimize the specificity, affinity, and capacity of the antibody. Humanization, i.e., replacing non-human CDR sequences with the corresponding sequences of a human antibody, is carried out according to the methods described, for example, in U.S. Pat. Nos. 5,545,806; 5,569,825; 5,633,425; 5,661,016; Riechmann et al., Nature 332:323-327 (1988); Marks et al., Bio / Technology 10:779-783 (1992); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996).

[0031] As used herein, "human antibody" means an antibody produced by a human or an antibody having an amino acid sequence corresponding thereto and made using any technique known in the art.

[0032] Specificity of binding can generally be defined by the comparative dissociation constant (Kd) of the antibody (or other targeting moiety) for the target compared to the dissociation constants for the antibody and other substances or unrelated molecules in the environment. A larger (higher) Kd is a Kd that represents a lower affinity interaction. Conversely, a smaller (lower) Kd is a Kd that represents a higher affinity interaction or tighter binding. By way of example only, the Kd of an antibody that specifically binds to a target may be femtomolar, picomolar, nanomolar, or micromolar, while the Kd of an antibody that binds to unrelated material may be millimolar or greater. Binding affinity can be in the micromolar range (kD = 10 -4 ~10 -6 ), nanomolar range (kD = 10 -7 M~10 -9 M), picomolar range (kD = 10 -10 M~10-12 M), or femtomolar range (kD = 10 -13 M~10 -15 M).

[0033] As used herein, an antibody has a Kd of 10 -4 An agent "binds" or "recognizes" an antigen or epitope if it binds to the antigen or epitope at less than M (i.e., in the micromolar range). The term "binds" with respect to a cell type (e.g., an antibody binding to cells expressing the antigen) typically indicates that the agent binds to the majority of cells in a pure population of those cells. For example, an antibody that binds to a given cell type will usually bind to at least two-thirds of the cells in the population of cells shown (e.g., 67, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some cases, binding to a polypeptide can be assayed by comparing antibody binding to cells displaying the polypeptide with antibody binding (or lack thereof) to cells that do not express the polypeptide. Those skilled in the art will recognize that some variability will occur depending on the method and / or threshold used to measure binding. The affinity of an antibody for a target can be determined according to methods known in the art, for example, as reviewed in Ernst et al. Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009).

[0034] As used herein, the term "higher affinity" refers to the relative degree of antibody binding where antibody X binds to target Y more strongly (K) and / or with a smaller dissociation constant (K) than target Z, in this context, antibody X has a higher affinity for target Y than Z. Similarly, the term "lower affinity" refers to the degree of antibody binding where antibody X binds to target Y more weakly and / or with a larger dissociation constant than target Z, in this context, antibody X has a lower affinity for target Y than Z. The binding affinity between an antibody and its target antigen can be expressed as K = 1 / K, where K is equal to k / k. The values ​​of k and k can be measured using surface plasmon resonance technology, for example, using the Molecular Affinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany). Antagonists or blocking antibodies are antibodies that partially or completely block, inhibit, or neutralize the biological activity associated with a target antigen compared to the activity under similar physiological conditions in the absence of the antibody. Antagonists can be competitive, non-competitive, or irreversible. Competitive antagonists bind to the natural ligand or receptor at the same site as the natural ligand-receptor interaction, or bind allosterically in a manner that induces changes that prevent normal binding. Non-competitive antagonists bind at a site different from the natural ligand-receptor interaction, but the resulting KD or signal is low. Irreversible inhibitors cause covalent modifications to the receptor, preventing subsequent binding.

[0035] As used herein, the term "avidity" refers to the overall stability of the binding complex between an antibody and a target antigen. It is governed by three factors: (i) the intrinsic affinity of the antibody for the antigen, (2) the valency of the antibody, and (3) the geometric arrangement of the interacting components. Affinity is the strength of the interaction between an antibody and a single target, while avidity is the cumulative strength of multiple affinities. In one embodiment, the antibodies provided herein are bivalent.

[0036] As used herein, an antibody "preferentially binds" a first antigen compared to a second antigen if the antibody binds to the first antigen with higher affinity than the second antigen. Preferential binding can be at least 2-fold, 5-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold.

[0037] As used herein, an antibody is defined as a molecule containing at least 1 x 10 antibodies to a target antigen or each member of a target group of antigens. -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 M, and, for example, binds to the target antigen or each member of the target antigen group with an affinity that is at least 2-fold higher than its affinity for a non-target antigen to which it is being compared. Typically, specific binding is characterized by the antibody binding to an antigen with sufficient affinity that it is useful as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent in targeting the antigen or epitope.

[0038] As used herein, the term "polypeptide" refers to a molecule having a sequence of natural and / or unnatural amino acids linked by peptide bonds. The term "peptide" refers to a short polypeptide, typically 30 amino acids or less in length. The amino acid sequence of a polypeptide is referred to as its "primary structure." The term "protein" refers to a polypeptide having secondary, tertiary, and / or quaternary structure, e.g., a structure stabilized by hydrogen bonds, a relationship between the secondary structure and a structure formed from multiple proteins. Proteins may be further modified by other attached moieties, such as carbohydrates (glycoproteins), lipids (lipoproteins), phosphate groups (phosphoproteins), etc.

[0039] As used herein, an amino acid sequence "consists" only of the amino acids in that sequence.

[0040] As used herein, a first amino acid sequence "consists essentially of" a second amino acid sequence if the first amino acid sequence (1) comprises the second amino acid sequence and (2) is no more than 1 amino acid, no more than 2 amino acids, or no more than 3 amino acids longer than the second amino acid sequence.

[0041] As used herein, a first amino acid sequence is a "fragment" of a second amino acid sequence if the second amino acid sequence comprises the first amino acid sequence. In certain embodiments, a first amino acid sequence that is a fragment of a second amino acid sequence has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fewer amino acids than the second amino acid sequence.

[0042] As used herein, the "functional equivalent" of a reference amino acid sequence is a sequence that is not identical to the reference sequence, but contains minor changes, such as the insertion, deletion, or substitution of one or several amino acids.A functionally equivalent sequence retains the function (e.g., immunogenicity) of the reference sequence to which it is equivalent.When a functionally equivalent amino acid sequence contains one or more amino acid substitutions with respect to the reference sequence, these are generally conservative amino acid substitutions.

[0043] As used herein, a "conservative amino acid substitution" refers to a substitution of one amino acid residue with another without abolishing the desired properties of the protein. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R chain length for each other. See, for example, Watson, et al., "Molecular Biology of the Gene," 4th Edition, 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, p. 224. Examples of conservative amino acid substitutions include the following (note that some categories are not mutually exclusive): [Table 1]

[0044] As used herein, the term "substantially identical" refers to identity between a first amino acid sequence that includes a sufficient or minimum number of amino acid residues that are i) identical or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences share a common structural domain and / or a common functional activity and / or a common immunogenicity. For example, amino acid sequences that share a common structural or antigenic domain that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical are referred to as being sufficiently or substantially identical. In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that includes a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode polypeptides having a common functional activity or encode a common structural polypeptide domain or a common functional polypeptide activity.

[0045] As used herein, a chemical, such as a polypeptide, is "substantially pure" or "isolated" if it is the predominant chemical of that type (e.g., polypeptide) in a composition. This includes chemicals that represent more than 50%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or more than 99.99% of that type of chemical in a composition. A substantially purified fraction is a composition in which the target species constitutes at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition means that about 80%-90% or more of the macromolecular species present in the composition are the purified target species. A target species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules, stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition.

[0046] The phrase "isolated antibody" refers to an antibody produced in vivo or in vitro that has been removed from the source that produces the antibody, e.g., an animal, hybridoma, or other cell line (e.g., a recombinant insect, yeast, or bacterial cell that produces the antibody, etc.).

[0047] The term "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are the same length. Determining the percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using, for example, the NBLAST nucleotide program parameters set to score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using, for example, the XBLAST program parameters set to score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0048] For antibodies, the percentage sequence identity can be determined when the antibody sequences are maximally aligned by IMGT. After alignment, when a region of a subject antibody (e.g., the entire variable region of a heavy or light chain) is compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody regions, divided by the total number of aligned positions in the two regions, and multiplied by 100 to convert to a percentage.

[0049] Percent amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be obtained from the National Institutes of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, unmask=yes, strand=all, expected frequency of occurrence=10, minimum low complexity length=15 / 5, multipass e-value=0.01, constant for multipass=25, dropoff for final gapped alignments=25, and scoring matrix=BLOSUM62.

[0050] In situations where NCBI-BLAST2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y, where X is the number of amino acid residues scored as a perfect match by the sequence alignment program NCBI-BLAST2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. As used herein, the term "nucleic acid sequence" refers to a sequence of nucleosides or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages, including cDNA. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may contain naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine, and uracil, as well as xanthine and hypoxanthine. It is understood that polynucleotides containing non-transcribed nucleotide bases may be useful, for example, as probes in hybridization assays. Nucleic acids may be either double-stranded or single-stranded, representing the sense or antisense strand. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences, as well as codon-optimized or synonymous codon equivalents.

[0051] As used herein, the term "isolated nucleic acid" refers to a nucleic acid that is substantially free of cellular material and culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid from which it is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0052] Hybridization can occur to all or part of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrid, is determined by its Tm, which, in a sodium-containing buffer, is a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C)-600 / L), or a similar formula). Therefore, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. To identify molecules similar to, but not identical to, a known nucleic acid molecule, for example, if a nucleic acid molecule with >95% identity is desired, it can be assumed that a 1% mismatch will result in a decrease in Tm of approximately 1°C, and the final wash temperature will be decreased by approximately 5°C. Based on these considerations, those skilled in the art can easily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. By way of example, the following conditions may be employed to achieve stringent hybridization: hybridization in 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS at Tm-5°C based on the above formula, followed by a wash in 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a wash step in 3x SSC at 42°C. However, it is understood that equivalent stringency may be achieved using alternative buffers, salts, and temperatures. Further guidance regarding hybridization conditions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 2002, and in: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0053] As used herein, the term "expression construct" refers to a polynucleotide comprising an expression control sequence operably linked to a heterologous nucleotide sequence to be expressed (i.e., a sequence to which the expression control sequence is not normally associated in nature). As used herein, the term "expression vector" refers to a polynucleotide comprising an expression construct and sequences sufficient for replication in a host cell or insertion into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term "expression control sequence" refers to a nucleotide sequence that controls the transcription and / or translation of a nucleotide sequence operably linked to it. Expression control sequences include promoters, enhancers, repressors (transcriptional control sequences) and ribosome binding sites (translation control sequences).

[0054] As used herein, a nucleotide sequence is "operably linked" to an expression control sequence when the expression control sequence functions to control the transcription of the nucleotide sequence in a cell, including promoting transcription of the nucleotide sequence through interaction of a polymerase with a promoter.

[0055] The term "vector" as used herein includes any intermediate vehicle for a nucleic acid molecule that allows the molecule to be introduced into, for example, a prokaryotic and / or eukaryotic cell and / or integrated into the genome, including plasmids, phagemids, bacteriophages, or viral vectors such as retrovirus-based vectors, lentivirus vectors, and adeno-associated virus vectors. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, that can replicate independently of chromosomal DNA.

[0056] "Transfection" refers to the introduction of new genetic material into a cell. It includes transformation (the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane), transduction (the introduction of foreign DNA into a host cell by a bacteriophage virus), and conjugation.

[0057] As used herein, "host cell" refers to a recombinant cell that contains an expression construct.

[0058] As used herein, the term "biological sample" refers to a sample that contains cells (e.g., cells) or biomolecules derived from cells.

[0059] As used herein, the terms "therapy," "treatment," "therapeutic intervention," and "amelioration" refer to any activity that results in a reduction in the severity of symptoms. The terms "treating" and "preventing" are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increased survival time or survival rate, etc. Treatment and prevention can be complete or partial. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient at different time points before or during treatment. In some embodiments, the severity of the disease is reduced by at least 10%, for example, compared to the individual before administration or to a control individual not receiving the treatment. In some embodiments, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, becomes undetectable using standard diagnostic techniques. "Treating" and "treatment" can also mean prolonging survival compared to expected survival if not receiving treatment. As used herein, "treating" and "treatment" include prophylactic treatment.

[0060] Compositions or methods "comprising" or "including" one or more recited elements may include other elements not specifically recited (e.g., without limitation, as open-ended terms). For example, a composition "comprises" or "includes" an antibody may contain the antibody alone or in combination with other components. In contrast, the phrase "consisting of" is closed and indicates that such embodiment does not contain additional elements. The term "consisting essentially of" refers to the inclusion of the recited elements and other elements that do not materially affect the basic and novel characteristics of the claimed combination (e.g., a partially closed term). Aspects and embodiments described herein as "comprising" should be understood to include "consisting of" and "consisting essentially of" embodiments.

[0061] As used herein, the following meanings apply unless otherwise specified: The word "may" is used in its permissive sense (i.e., meaning "could") rather than its obligatory sense (i.e., meaning "should"). The singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "an element" includes a combination of two or more elements, notwithstanding the use of other terms and phrases for one or more elements, such as "one or more." The phrase "at least one" includes "one," "one or more," "one or a plurality," and "a plurality." The term "or," unless otherwise indicated, is non-exclusive, i.e., encompasses both "and" and "or." The term "either" between a modifier and a sequence means that the modifier modifies every member of the sequence. Thus, for example, the phrase "any of at least 1, 2, or 3" means "at least 1, at least 2, or at least 3."

[0062] II. Chimeric Antigen Receptors A "chimeric antigen receptor" or "CAR" is an engineered molecule that contains an optional signal peptide, a target binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain, and an optional costimulatory domain. CARs are based on the structure of the T cell receptor, which is expressed in T cells and is involved in the cellular immune response. The "target binding domain" is also referred to herein as the "antigen binding domain," and as such, the term "target" encompasses "antigen."

[0063] The so-called "first generation" CARs contained a targeting domain and a CD3ξ signaling domain. The so-called "second generation" CARs additionally contained costimulatory domains, such as CD28 or 4-1BB domains. The so-called "third generation" CARs contain multiple costimulatory domains. The so-called "fourth generation" CARs, also known as "TRUCKS," have been engineered to release transgenic cytokines upon CAR signaling.

[0064] A chimeric antigen receptor ("CAR") comprises the following elements: (1) an optional signal peptide, (2) a target binding domain, (3) an optional hinge region, (4) a transmembrane region, and (5) an intracellular domain comprising a signaling domain. Optionally, a CAR can comprise a CD3ζ signaling domain, an Fc receptor signaling domain, or a costimulatory (signaling) domain. That is, these optional elements may be included in addition to or in place of other optional elements. The target binding domain is heterologous to at least one of the other domains. That is, the target binding domain does not naturally occur on a T cell receptor or is not within the same protein as at least one of the other domains.

[0065] The "target binding domain" provides binding specificity for the CAR. The "signal peptide" guides the polypeptide through the cell membrane. The target binding domain can be bound to the domain of an antibody that binds to the target antigen for a so-called "universal CAR." The "hinge region" is a flexible connector region, e.g., a natural or synthetic polypeptide, or any other type of molecule, that provides structural flexibility and spacing between adjacent polypeptide regions. The "transmembrane domain" is a transmembrane protein domain and is usually hydrophobic. The "signal transduction domain" or "signaling domain" transmits a signal through a signal transduction pathway within the cell upon binding. Such signal transduction activates cellular activity. The "costimulatory domain" is an auxiliary signaling domain that further transmits signals.

[0066] In some embodiments, the CAR is (i) a target binding domain (also referred to herein as an antigen binding domain) reactive with a citrullinated protein or a citrullinated fragment thereof, e.g., a VH-VL or VL-VH target binding domain, wherein the two variable domains are separated by a flexible linker of 15 to 25 amino acids in length; (ii) hinge domain, (iii) a costimulatory domain, and (iv) an intracellular signaling domain (also referred to herein as an activation domain) That is, in some embodiments, the CAR comprises an antigen-binding domain fused to a CAR platform comprising a hinge domain, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and an activation domain. The CAR may further comprise a signal peptide (also referred to herein as a leader sequence) to direct expression of the CAR to the surface of cells, such as Tregs.

[0067] In some embodiments, the CAR comprises an antigen binding domain fused in-frame to a CAR platform comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 28, and SEQ ID NO: 19. In other embodiments, the CAR comprises an antigen binding domain fused in-frame to a CAR platform comprising the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 28, and SEQ ID NO: 19. In some embodiments, the CAR comprises an antigen binding domain fused in-frame to a CAR platform comprising the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 29, and SEQ ID NO: 19. In other embodiments, the CAR comprises an antigen binding domain fused in-frame to a CAR platform comprising the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 29, and SEQ ID NO: 19.

[0068] A. Signal Peptide The signal peptide can be any peptide whose function is to allow a polypeptide to cross a cell membrane. The signal peptide can be derived from CD4, CD8, CD28, a TLR, or a receptor of the immunoglobulin family.

[0069] For example, the signal peptide may comprise the following sequence: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 18), or MALPVTALLLPLALLLHAAR (SEQ ID NO: 23).

[0070] B. Target Binding Domain 1. Structure The target binding domain can comprise any polypeptide comprising a target binding function, such as an antibody as defined herein. In one embodiment, the target binding domain can comprise an antibody form that retains the antigen binding activity defined herein. In one embodiment, the target binding domain can comprise a single-chain antibody (scFv). The scFv can be connected to a transmembrane domain via a hinge domain, whose length, flexibility, and origin provide variability in CAR design, and together with the transmembrane domain, influence the association of the CAR with additional proteins required for interaction with antigens, the establishment of an immunological synapse, and the transmission of robust activation signals.

[0071] 2. Target / antigen The chimeric antigen receptors disclosed herein comprise a target-binding domain (also referred to herein as an antigen-binding domain) that binds to a citrullinated antigen, such as one found in skin lesions of subjects with hidradenitis suppurativa. In particular, the target-binding domain can bind to one or more of (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) citrullinated peptides thereof. In some embodiments, the target-binding domain can bind to a citrullinated peptide fragment of (i)-(iii), wherein the peptide fragment is at least 10 amino acids long, e.g., at least 12 amino acids long, at least 14 amino acids long, or at least 16 amino acids long. In some embodiments, the target-binding domain further binds to tenascin-C. In some embodiments, the target binding domain is capable of binding to two or more of (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) tenascin-C, or citrullinated peptide fragments thereof.

[0072] In some embodiments, the targeting domain is one or more citrullinated peptides selected from the following sequences: ST(Cit)SVSSSSY(Cit)(Cit)MFGG (SEQ ID NO: 24), VYAT(Cit)SSAV(Cit)L(Cit)SSV(Sequence number 25), (Cit)PAPPPISGGGY(Cit)A(Cit) (SEQ ID NO: 26), SHQEST(Cit)GRSRGRSGRSGS (SEQ ID NO: 27).

[0073] In some embodiments, the antigen binding domain binds to one or more citrullinated peptides but not to non-citrullinated counterparts. In some embodiments, the antigen binding domain binds to the citrullinated vimentin peptide represented by SEQ ID NO:24 but not to STRSVSSSSYRRMFGG (SEQ ID NO:45). In some embodiments, the antigen binding domain binds to the citrullinated vimentin peptide represented by SEQ ID NO:25 but not to VYATRSSAVRLRSSV (SEQ ID NO:46). In some embodiments, the antigen binding domain binds to the citrullinated fibrinogen peptide represented by SEQ ID NO:26 but not to RPAPPPISGGGYRAR (SEQ ID NO:47). In some embodiments, the antigen binding domain binds to the citrullinated filaggrin peptide represented by SEQ ID NO:27 but not to SHQESTRGRSRGRSGRSGS (SEQ ID NO:48).

[0074] The target binding domain may comprise sequences derived from antibody VH and VL domains. In some embodiments, the target binding domain may comprise sequences derived from a heavy chain-only antibody or an antibody fragment having only two VH domains, including a particular set of CDRs from the VH and VL domains. In some embodiments, the target binding domain comprises CDRs derived from the VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the target binding domain comprises CDRs derived from the VL domain of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the target binding domain comprises CDRs derived from the VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 3, respectively. In some embodiments, the target binding domain comprises CDRs derived from the VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 4, respectively. In some embodiments, the target binding domain comprises CDRs derived from the VH and VL domains of SEQ ID NO: 2 and SEQ ID NO: 3, respectively. In some embodiments, the target binding domain comprises CDRs derived from the VH and VL domains of SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0075] In some embodiments, the target binding domain comprises complementarity determining regions (CDRs) from the VH and VL domains of SEQ ID NO: 1 (S01 VH) and SEQ ID NO: 4 (S01 VL). In some embodiments, the CDRs are as defined using an antibody numbering scheme, such as the Chothia, AbM, Kabat, Contact, and IMGT numbering schemes. In some embodiments, the CDRs are as defined by the Kabat numbering system. In some embodiments, the VH domain of the target binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, and the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. [Table 2] [Table 3]

[0076] In some embodiments, the target binding domain comprises complementarity determining regions (CDRs) from the VH and VL domains of SEQ ID NO:49 (U01 VH) and SEQ ID NO:50 (U01 VL). In some embodiments, the CDRs are as defined using an antibody numbering scheme, such as the Chothia, AbM, Kabat, Contact, and IMGT numbering schemes. In some embodiments, the CDRs are as defined by the Kabat numbering system. In some embodiments, the VH domain of the target binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:53, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:57, and the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:60, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:62, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:64. [Table 4] [Table 5]

[0077] In certain embodiments, the target binding domain comprises a VH sequence selected from the following: (1) SBT01 VH(M) HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 1), (2) SBT01 VH(G) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 2), (3)U01 VH EVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSS (SEQ ID NO: 49), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity to the above sequences, wherein the target binding domain binds to a citrullinated antigen as described herein.

[0078] The target binding region may comprise a VL sequence selected from: (1) SBT01 VL(M) SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVL (SEQ ID NO: 3), (2) SBT01 VL(G) SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVL (SEQ ID NO: 4), (3)U01 VL AIQMTQSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIKRAAA (SEQ ID NO: 50), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity to the above sequences, wherein the target binding domain binds to a citrullinated antigen as described herein.

[0079] In another embodiment, the antigen-binding region comprises an scFV comprising one or more VH domains comprising the amino acid sequence of the VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the antigen-binding region comprises an scFV comprising an amino acid sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 1 or SEQ ID NO: 2, provided that the scFV domain binds to a citrullinated antigen described herein.

[0080] In some embodiments, the scFV comprises a VL domain of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the antigen-binding region comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 3 or SEQ ID NO: 4, wherein the scFV domain binds to a citrullinated antigen described herein.

[0081] In some embodiments, the scFV domain comprises the VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 3, respectively. In some embodiments, the antigen-binding region comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22 is disposed between the VH and VL domains.

[0082] In some embodiments, the scFV domain comprises the VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 4, respectively. In some embodiments, the antigen-binding region comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 1 and SEQ ID NO: 4, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22 is disposed between the VH and VL domains.

[0083] In some embodiments, the scFV domain comprises the VH and VL domains of SEQ ID NO: 2 and SEQ ID NO: 3, respectively. In some embodiments, the antigen-binding region comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 2 and SEQ ID NO: 3, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22 is disposed between the VH and VL domains.

[0084] In some embodiments, the scFV domain comprises the VH and VL domains of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. In some embodiments, the antigen-binding region comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 2 and SEQ ID NO: 4, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22 is disposed between the VH and VL domains.

[0085] In some embodiments, the scFV domain comprises the VH and VL domains of SEQ ID NO: 49 and SEQ ID NO: 50, respectively. In some embodiments, the antigen-binding region comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 49 and SEQ ID NO: 50, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22 is disposed between the VH and VL domains.

[0086] In another embodiment, the antigen-binding region comprises an scFV comprising an amino acid sequence selected from the following: SBT01G-VHVL-GGGSx3 linker-pSB_0149 [ka] SBT01G - VHVL - Whitlow218 linker - pSB_0158 [ka] SBT01G-VHVL-AB pur linker-pSB_0159 [ka] U01 - VHVL - Ab pur linker [ka] or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity to the above sequences, wherein the target binding domain binds to a citrullinated antigen as described herein.

[0087] For example, the linker may comprise the following sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 20) GGGSx3 linker, or GSTSGSGKPGSGEGSTKG (SEQ ID NO: 21) Whitlow218 linker, or ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 22) AB pur linker.

[0088] Optionally, any of the foregoing sequences may comprise a set of CDRs from the VH and VL domains described above.

[0089] C. Hinge region In some embodiments, the hinge region of a CAR of the present disclosure can be selected from the CD8, CD4, or CD28 extracellular domains, the Fc region of an IgG1 antibody, or the extracellular domain of any of the TLR receptors, as are well known in the art and can be found in the GenBank database.

[0090] For example, the hinge region may comprise the following sequence: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 30) (CD28), or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 15) (CD8), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the sequence of the hinge region described above.

[0091] D. Transmembrane domain The transmembrane domain can include the transmembrane domain of an immunoglobulin family receptor, such as CD8. The intracellular domain can be selected from any transmembrane molecule on T cells. For example, the transmembrane (TM) domain of the disclosed CAR can include the TM domain of CD2, CD3, CD16, CD32, CD64, CD28, CD247, 4-1BBL, CD4, or CD8.

[0092] For example, the transmembrane domain may comprise the following sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 16), or IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 17), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the sequence of the hinge region described above.

[0093] E. Signaling Domains 1. CD3ζ signaling domain In some embodiments, the signaling domain comprises a CD3ζ signaling domain. The CD3ζ signaling domain of the disclosed CAR molecules can comprise a CD3ζ amino acid sequence, for example, the signaling domain of CD3 zeta.

[0094] For example, the CD3 zeta signaling domain can comprise the following sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 19), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned CD3 zeta signaling domain sequence.

[0095] For example, the CD3 zeta signaling domain can include amino acids 21-163, 31-142, 68-89, and / or 138-158 of the sequence set forth in SEQ ID NO: 19, or a functional variant thereof (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10-20 amino acid substitutions, deletions, or additions).

[0096] 2. Fc receptor signaling domain In some embodiments, the signaling domain comprises an Fc signaling domain. The Fc signaling domain may be any one of Fc-alpha, Fc-gamma, Fc-epsilon, Fc-mu, and Fc-delta receptors. For example, the signaling domain of an Fc receptor may include amino acids involved in interactions with Src (e.g., Fgr, Fyn, Hck, Lyn, Yes, and Src) and ZAP-70 family kinases, e.g., one or more ITAM domains (e.g., Sanchez-Mejorada et al. (1998) J. Leukocyte Biol. 63:531; Garcia-Garcia et al. (2002) J. Leukocyte Biol. 72:1092). In some embodiments, the Fc receptor signaling domain comprises at least one ITAM domain derived from or substantially identical to any one of Fc-alpha, Fc-gamma, Fc-epsilon, Fc-mu, and Fc-delta receptors.

[0097] The sequence can also be found as follows: [Table 6]

[0098] F. Costimulatory Domain CARs of the present disclosure can include one or more costimulatory domains in addition to the signaling domain of a CD3ζ or Fc receptor. The costimulatory domain can be derived from, for example, CD28, 4-1BB, CD2, CD27, CD30, OX40, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3, B7-H4, etc. CAR constructs can include two or more costimulatory signaling domains (e.g., CD28 and 4-1BB).

[0099] The costimulatory domain or domains can be located between the signaling domain and the transmembrane region.

[0100] In certain embodiments, the CD28 costimulatory domain may comprise the following sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 29), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence.

[0101] In certain embodiments, the 41BB costimulatory domain may comprise the following sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 28), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence.

[0102] III. Nucleic acids A. Nucleic Acid Encoding CAR Disclosed herein is a nucleic acid molecule (polynucleotide) comprising a nucleotide sequence encoding the CAR of the present disclosure. The nucleic acid of the disclosed CAR may be in the form of DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, which may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or non-coding (antisense) strand. RNA includes mRNA, siRNA, sRNA, ssRNA, etc.

[0103] For example, the nucleic acid can comprise a nucleotide sequence encoding the polypeptide of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. In certain embodiments, the nucleotide sequence encoding the VH domain comprises: (1) SBT01 VH(M) CACCTGCACTTGCAGGAGTCGGGCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAACACCC ACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCTCGAGC (SEQ ID NO: 8), (2) SBT01 VH(G) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTA CTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCTCGAGC (SEQ ID NO: 9), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence.

[0104] In certain embodiments, the nucleotide sequence encoding the VL domain comprises: (1) SBT01 VL(M) TCCTATGTCCTGACTCAGCCACCCTCAGTGTCGCTGGCCCCGGGAGAGACGGCCACAATTACTTGTGGTGGAGACGACATTGAAAATCAAAATGTCAACTGGTATCAGCAGAAGTCAGGTCAGGCCCCTATGCTGCTCATCTTCTTTGATACCAGACGGCCCTCAG GGATCCCGGAGCGATTCTCTGGCTCCAGGTCTGAGGACACGGCCAACCTGACCATCACCAGGGTCGAGGCCGGGGATGACGCCGACTATTTCTGTCAGGTGTATGATAGGAAGACTGATCACCAAGTCTTCGGACCTGGGACCACGGTCACCGTCCTA (SEQ ID NO: 10), (2) SBT01 VL(G) TCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGG ATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 11), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence.

[0105] In another embodiment, the nucleic acid molecule encoding the VH domain comprises SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the nucleic acid molecule encoding the VH domain comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 8 or SEQ ID NO: 9, with the proviso that the scFV domain binds to a citrullinated antigen as described herein.

[0106] In some embodiments, the nucleic acid molecule encoding the VL domain comprises SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the nucleic acid molecule encoding the VL domain comprises a nucleic acid sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 10 or SEQ ID NO: 11, provided that the scFV domain binds to a citrullinated antigen described herein.

[0107] In some embodiments, the nucleic acid molecule encoding the scFV domain comprises SEQ ID NO: 8 and SEQ ID NO: 10. In some embodiments, the nucleic acid molecule encoding the scFV comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 8 or SEQ ID NO: 10, provided that the scFV domain binds to a citrullinated antigen as described herein.

[0108] In some embodiments, the nucleic acid molecule encoding the scFV domain comprises SEQ ID NO: 8 and SEQ ID NO: 11. In some embodiments, the nucleic acid molecule encoding the scFV comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 8 or SEQ ID NO: 11, provided that the scFV domain binds to a citrullinated antigen as described herein.

[0109] In some embodiments, the nucleic acid molecule encoding the scFV domain comprises SEQ ID NO: 9 and SEQ ID NO: 10. In some embodiments, the nucleic acid molecule encoding the scFV comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 9 or SEQ ID NO: 10, provided that the scFV domain binds to a citrullinated antigen as described herein.

[0110] In some embodiments, the nucleic acid molecule encoding the scFV domain comprises SEQ ID NO: 9 and SEQ ID NO: 11. In some embodiments, the nucleic acid molecule encoding the scFV comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 9 or SEQ ID NO: 11, provided that the scFV domain binds to a citrullinated antigen described herein.

[0111] In another embodiment, the nucleic acid molecule encodes an scFv molecule and has the following nucleotide sequence: SBT01G-VHVL-GGGSx3 linker-pSB_0149 [ka] or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence.

[0112] In another embodiment, the nucleic acid molecule encodes an scFv molecule and has the following nucleotide sequence: SBT01G - VHVL - Whitlow218 linker - pSB_0158 [ka] or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence.

[0113] In another embodiment, the nucleic acid molecule encodes an scFv molecule and has the following nucleotide sequence: SBT01G-VHVL-AB pur linker-pSB_0159 [ka] or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity with the aforementioned sequence.

[0114] Optionally, these may include sequences encoding a set of CDRs from the VH and VL domains described herein.

[0115] Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions but do not alter the properties or activity of the encoded CAR polypeptide. In some embodiments, polynucleotide variants contain alterations that do not result in any changes to the amino acid sequence. In some embodiments, polynucleotide variants contain "silent" substitutions due to the degeneracy of the genetic code. Polynucleotide variants may be produced for a variety of reasons, such as to optimize codon expression for a particular host.

[0116] In some embodiments, the polynucleotides described herein are isolated.

[0117] The polynucleotide encoding the CAR can be an isolated molecule or can be contained in a vector, such as a plasmid, cosmid, artificial chromosome, or virus. Such vectors can be used to transfect target cells.

[0118] B. Expression Constructs and Vectors A polynucleotide encoding a CAR of the present disclosure can comprise a regulatory element operably linked to the nucleotide sequence encoding the CAR. For example, the polynucleotide can comprise one or more transcriptional regulatory elements, such as a promoter or enhancer, that cause the sequence encoding the CAR to be expressed in the cell when the polynucleotide is present in the cell.

[0119] The nucleic acids disclosed herein can be incorporated into vectors capable of transfecting cells. Such vectors include, but are not limited to, viral vector plasmids and endoplasmic reticulum, such as liposomes. Exemplary viral vectors include adenovirus vectors (Ad), AAV, lentivirus, and vesicular stomatitis virus (VSV), as well as retroviruses. Lentivirus is a genus of the Retroviridae family, including HIV, SIV, and FIV. Lentiviruses can deliver large amounts of genetic material into the DNA of host cells. They can infect non-dividing cells.

[0120] IV.Cells In some embodiments, a recombinant (host) cell harboring a nucleic acid molecule encoding a disclosed CAR, the nucleic acid molecule may further comprise an expression control sequence operably linked to the nucleotide sequence encoding the CAR. Once the CAR is assembled (by synthesis, site-directed mutagenesis, or another method, as known to those skilled in the art), the nucleic acid molecule encoding the disclosed CAR can be inserted into an expression vector and operably linked to expression control sequences appropriate for expression of the disclosed CAR in a desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and / or expression of the CAR polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0121] The present disclosure also provides cells (e.g., recombinant cells) comprising a nucleic acid molecule encoding a CAR and / or a nucleic acid molecule that expresses a CAR.

[0122] Nucleic acid molecules encoding the disclosed CARs can be delivered to host cells, including, but not limited to, T cells, B cells, myeloid progenitor cells, macrophages, etc., by plasmid or viral vectors known in the art. The resulting recombinant (host) cells can include, but are not limited to, T cells, CD4 T cells, Treg cells, CD8 alpha T cells, CD8 beta T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages, and dendritic cells, T memory stem cells, and cells expressing MHC class I or class II, as are known to those of skill in the art. In some embodiments, the recombinant (host) cells carrying the nucleic acid molecules encoding the disclosed CARs can be myeloid progenitor cells selected from the group consisting of general myeloid progenitor cells, granulocyte-macrophage progenitor cells, megakaryocyte-erythroid progenitor cells, granulocyte progenitor cells, and monocyte progenitor cells, as are known to those of skill in the art. In some embodiments, the myeloid cells are autologous or allogeneic cells.

[0123] In some embodiments, the cells expressing a CAR of the present disclosure are Treg cells. reg "T cells" are cells belonging to a specialized subpopulation of T cells that act to suppress immune responses, thereby maintaining homeostasis and self-tolerance. reg can inhibit T cell proliferation and cytokine production, playing a crucial role in preventing autoimmunity. reg is characterized by the expression of FoxP3. regSurface markers for Tregs include CD4, CD25high (high molecular density), and CD127low (low molecular density). Mouse and human Tregs express GITR / AITR and CTLA-4. Human CD4+FoxP3+ Treg cells can be divided into three subpopulations: (1) CD45RA+CD25+FoxP3l0w resting Treg cells, (2) CD45RO+CD25highFoxP3high activated Treg cells, and (3) inflammatory cytokine-producing CD45RO+CD25+FoxP3low non-suppressive effector T cells (Teff).

[0124] The cells transformed with the nucleic acids disclosed herein can be cells obtained from the subject to whom the recombinant cells are administered, thus mitigating the problem of allogeneic immune responses.

[0125] The cells may be expanded ex vivo prior to administration to a subject.

[0126] Treg cells incorporating nucleic acids expressing the CARs of the present disclosure express these CARs and can be used in the methods described herein to treat hidradenitis suppurativa.

[0127] Proteins produced by transformed / recombinant hosts can also be purified according to any appropriate method, including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Proteins can be readily purified by attaching affinity tags, such as hexahistidine, maltose-binding domain, influenza coat sequence, and glutathione-S-transferase, to the protein and passing it through an appropriate affinity column. In some embodiments, proteins can also be physically characterized using techniques such as proteolysis, high-performance liquid chromatography (HPLC), nuclear magnetic resonance, and X-ray crystallography.

[0128] V. Composition Also disclosed are pharmaceutical compositions comprising recombinant cells harboring nucleic acid molecules encoding and / or expressing the disclosed CAR polypeptides and a pharmaceutically acceptable carrier, and methods of use in the treatment of hidradenitis suppurativa.

[0129] As used herein, the term "pharmaceutical composition" refers to a composition comprising a pharmaceutical compound (e.g., a drug or recombinant Treg cells described herein) and a pharmaceutically acceptable carrier.

[0130] As used herein, the term "pharmaceutically acceptable" refers to a carrier that is compatible with the other ingredients of a pharmaceutical composition and that can be safely administered to a subject. This term is used synonymously with "physiologically acceptable" and "pharmacologically acceptable." Pharmaceutical compositions and techniques for their preparation and use will be known to those skilled in the art in light of the present disclosure. For a detailed list of suitable pharmacological compositions and techniques for their administration, reference may be made to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), "Remington: The Principles of Pharmacy Practice," Lippincott Williams & Wilkins, 2008.

[0131] Pharmaceutically acceptable carriers are generally sterile, at least for human use. Pharmaceutical compositions will generally include buffers and preservatives during storage, and may also include buffers and carriers for delivery appropriate for the route of administration. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological (0.9%) saline, phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), and multiple electrolyte solutions such as PlasmaLyte ATM (Baxter).

[0132] Pharmaceutical compositions can be formulated for any route of administration, including mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular, or intra-arterial injection, either bolus or infusion), oral, or transdermal.

[0133] Injectable (e.g., intravenous) compositions can include a solution of the composition suspended in an acceptable carrier, such as an aqueous carrier. Any of a variety of aqueous carriers can be used, including water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may contain glycoproteins to enhance stability, such as albumin, lipoproteins, and globulins. Physiologically buffered saline (135-150 mM NaCl) is often used. The compositions can contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. In some embodiments, the compositions can be formulated in kits for intravenous administration.

[0134] Suitable formulations for parenteral administration, such as intra-articular (intra-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Infusion solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In the practice of the present invention, compositions can be administered, for example, by intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally. Compound formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0135] The cells may be cryopreserved. Cryopreservation may include formulating the cells with a cryopreservative, such as DMSO. Commercially available media include, for example, CryoStor® and pZerve®, available from Millipore Sigma.

[0136] The composition may be formulated into a dosage form for administration. The term "dosage form" refers to the specific form of a pharmaceutical product and depends on the route of administration. Examples of dosage forms include, but are not limited to, dispersions, suppositories, ointments, poultices, pastes, powders, dressings, creams, plasters, solutions, patches, aerosols (e.g., nasal sprays or inhalers), gels, liquid dosage forms suitable for oral or mucosal administration to a patient (including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs), liquid dosage forms suitable for parenteral administration to a patient, and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0137] The terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual in each administration. Dosage will vary depending on numerous factors, including frequency of administration, size and tolerance of the individual, severity of the condition, risk of side effects, route of administration, and imaging modality of detectable label (if any). Those skilled in the art will recognize that dosage can be altered depending on the above factors or based on the course of treatment.

[0138] Pharmaceutical preparations can be packaged or prepared in unit dosage form.In this form, the preparation is divided into unit dosages containing appropriate amounts of active ingredients, for example, according to the dosage of therapeutic agent or the concentration of composition.The unit dosage form can be a packaged preparation, and the package contains a discrete amount of preparation.The composition can also contain other compatible therapeutic agents if desired.

[0139] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising complementarity determining regions (CDRs) from the VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VL domain of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 3. In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 4. In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VH and VL domains of SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VH and VL domains of SEQ ID NO: 2 and SEQ ID NO: 4. In some embodiments, the cells comprise T cells, CD4 T cells, Treg cells, CD8 alpha T cells, CD8 beta T cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages, and dendritic cells, or T memory stem cells. In some embodiments, the cells are Treg cells.

[0140] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising complementarity-determining regions (CDRs) from the VH domain of SEQ ID NO: 49. In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VL domain of SEQ ID NO: 50. In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VH domain and VL domain of SEQ ID NO: 49 and SEQ ID NO: 50. In some embodiments, the cell is a T cell, CD4 T cell, Treg cell, CD8 alpha T cell, CD8 beta T cell, T helper cell, granulocyte (neutrophil, basophil, eosinophil), megakaryocyte, monocyte, macrophage, and dendritic cell, or T memory stem cell. In some embodiments, the cell is a Treg cell.

[0141] In some embodiments, a composition of the invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VH domain and VL domain of SEQ ID NO: 1 (SBT01 VH(M)) and SEQ ID NO: 4 (SBT01 VL(G)). In some embodiments, the VH domain of the CAR polypeptide comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, and the VL domain of the CAR polypeptide comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a T helper cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a T memory stem cell. In some embodiments, the cells are Treg cells.

[0142] In another embodiment, a composition of the invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising one or more VH domains comprising the amino acid sequence of the VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, the cell is a T cell, CD4 T cell, Treg cell, CD8 alpha T cell, CD8 beta T cell, T helper cell, granulocyte (neutrophil, basophil, eosinophil), megakaryocyte, monocyte, macrophage, and dendritic cell, or T memory stem cell. In some embodiments, the cell is a Treg cell.

[0143] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising a VL domain of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO:3 or SEQ ID NO:4, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, the cell is a T cell, CD4 T cell, Treg cell, CD8 alpha T cell, CD8 beta T cell, T helper cell, granulocyte (neutrophil, basophil, eosinophil), megakaryocyte, monocyte, macrophage, and dendritic cell, or T memory stem cell. In some embodiments, the cell is a Treg cell.

[0144] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 3. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, the cell is a T cell, CD4 T cell, Treg cell, CD8 alpha T cell, CD8 beta T cell, T helper cell, granulocyte (neutrophil, basophil, eosinophil), megakaryocyte, monocyte, macrophage, and dendritic cell, or T memory stem cell. In some embodiments, the cell is a Treg cell.

[0145] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO:1 and SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO:1 and SEQ ID NO:4, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, the cell is a T cell, CD4 T cell, Treg cell, CD8 alpha T cell, CD8 beta T cell, T helper cell, granulocyte (neutrophil, basophil, eosinophil), megakaryocyte, monocyte, macrophage, and dendritic cell, or a T memory stem cell. In some embodiments, the cell is a Treg cell.

[0146] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO:2 and SEQ ID NO:3. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO:2 and SEQ ID NO:3, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, the cell is a T cell, CD4 T cell, Treg cell, CD8 alpha T cell, CD8 beta T cell, T helper cell, granulocyte (neutrophil, basophil, eosinophil), megakaryocyte, monocyte, macrophage, and dendritic cell, or T memory stem cell. In some embodiments, the cell is a Treg cell.

[0147] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO:2 and SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO:2 and SEQ ID NO:4, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, the cell is a T cell, CD4 T cell, Treg cell, CD8 alpha T cell, CD8 beta T cell, T helper cell, granulocyte (neutrophil, basophil, eosinophil), megakaryocyte, monocyte, macrophage, and dendritic cell, or T memory stem cell. In some embodiments, the cell is a Treg cell.

[0148] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO: 49 and SEQ ID NO: 50. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO: 49 and SEQ ID NO: 50, wherein the scFV domain binds to a citrullinated antigen described herein. In some embodiments, the cell is a T cell, CD4 T cell, Treg cell, CD8 alpha T cell, CD8 beta T cell, T helper cell, granulocyte (neutrophil, basophil, eosinophil), megakaryocyte, monocyte, macrophage, and dendritic cell, or T memory stem cell. In some embodiments, the cell is a Treg cell.

[0149] VI.How to use T cells, particularly Treg cells expressing the CARs disclosed herein, are useful for treating hidradenitis suppurativa. A method of use includes administering an effective amount of the pharmaceutical composition of the present disclosure to a subject in need thereof, for example, a subject suffering from hidradenitis suppurativa.

[0150] As used herein, the term "subject" refers to an individual animal. As used herein, the term "patient" refers to a subject under the care or supervision of a healthcare provider, such as a doctor or nurse. Subjects include humans and non-human primates, e.g., monkeys, as well as mammals such as dogs, cats, horses, cows, rabbits, rats, mice, goats, pigs, and other mammalian species. Subjects may also include birds. A patient may be an individual seeking treatment, monitoring, adjustment, or modification of an existing therapeutic regimen, etc. The term "hidradenitis suppurativa subject" refers to an individual diagnosed with hidradenitis suppurativa. Hidradenitis suppurativa patients may include individuals who are not receiving treatment, individuals currently receiving treatment, individuals who have received treatment, and individuals who have discontinued treatment.

[0151] As used herein, the terms "effective amount," "effective dose," and "therapeutically effective amount" refer to an amount of an agent sufficient to produce a desired response, e.g., reduce or eliminate signs or symptoms of a disease or ameliorate a disorder. In some examples, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or underlying causes of any of the disorders or diseases and / or prevents progression of the disease. For example, a therapeutically effective amount may result in an increase or decrease of a given parameter by at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy may also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount may result in an effect of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more compared to a control.

[0152] The pharmaceutical composition can be administered by any suitable route, including, but not limited to, intravenous, subcutaneous, intramuscular, or intraperitoneal routes. An example of administration of the pharmaceutical composition involves storing the composition at 10 mg / ml in sterile isotonic saline for injection at 4°C and diluting it with 100 ml or 200 ml of 0.9% sodium chloride for injection before administration to the patient. The pharmaceutical composition is administered by intravenous infusion over a period of 1 hour at a dose of 0.2 to 10 mg / kg. In other embodiments, the pharmaceutical composition is administered by intravenous infusion over a period of 15 minutes to 2 hours. In yet other embodiments, the administration procedure is by subcutaneous bolus injection.

[0153] The dose of the composition is selected to provide effective treatment to the patient and ranges from less than 0.1 mg / kg body weight to about 25 mg / kg body weight per patient, or from 1 mg to 2 g per patient. In some cases, the dose ranges from 1 to 100 mg / kg, or from approximately 50 mg to 8000 mg per patient. The dose may be repeated at an appropriate frequency, which may range from once daily to once every three months, depending on the pharmacokinetics (e.g., half-life of the composition in the circulation) and pharmacodynamic response (e.g., duration of therapeutic effect of the composition). In some embodiments, the in vivo half-life is about 7 to about 25 days, and dosing of the composition is repeated between once weekly and once every three months.

[0154] Administration can be regular.Depending on the administration route, the dosage can be administered, for example, once every 1, 3, 5, 7, 10, 14, 21, or 28 days or more (for example, once every 2, 3, 4, or 6 months).In some cases, administration is more frequent, for example, twice or three times a day.As will be recognized by those skilled in the art, patients can be monitored to adjust dosage and administration frequency according to the course of treatment and any adverse side effects.

[0155] Thus, in some embodiments, additional administrations depend on the patient's progress, e.g., the patient is monitored between doses. For example, the patient's rate of symptom relief can be monitored after an initial dose or multiple doses.

[0156] In certain embodiments, the T cells described herein are administered to a skin lesion in a subject with hidradenitis suppurativa.

[0157] An exemplary method of the present disclosure includes isolating T lymphocytes from a biological sample obtained from a subject. Such T cells can be isolated by immunoaffinity, for example, using a derivatized solid support and an anti-CD4 antibody. CD4+ regulatory T cells (Treg) can be separated from non-Treg cells based on their marker profile. Treg cells are CD4+, CD25+, and CD127lo. Non-Treg cells are CD4+, CD25+, and CD127+. The isolated Treg cells are then transfected with an expression vector encoding a chimeric antigen receptor (CAR) of the present disclosure. The transfected cells are expanded. The expanded cells are administered to the subject.

[0158] VII. Kit As used herein, the term "kit" refers to a collection of items intended to be used together. The kit may optionally include reference agents and / or instructions for their use. The kit may further include a shipping container adapted to hold a container, such as a vial, containing a composition disclosed herein. The kit may include a container that contains the collection of items therein.

[0159] The kits of the present disclosure may include a pharmaceutical composition described herein contained in a container such as a bag or bottle for intravenous administration. A fluid conduit, such as a plastic tube with a drip chamber, may also be included in the kit. The drip chamber may be in communication with an intravenous needle through the fluid conduit. The fluid conduit may also include one or more Y-sites and a roller clamp. VIII. List of Embodiments 1. A method of treating a subject suffering from hidradenitis suppurativa, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a plurality of modified regulatory T (Treg) cells and a pharmaceutically acceptable excipient; the engineered Treg cells express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; the antigen-binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof; The method, wherein the modified Treg cells are CD4+, CD25+, and CD127lo. 2. The method of embodiment 1, wherein the antigen-binding domain of the CAR specifically binds to citrullinated vimentin. 3. The method of embodiment 1, wherein the antigen-binding domain of the CAR binds to all three of: (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptide fragments thereof. 4. The antigen-binding domain of the CAR comprises a VH domain and a VL domain; (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 36; and (ii) The method of embodiment 3, wherein the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. 5. The antigen-binding domain of the CAR comprises a VH domain and a VL domain; (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 57; and (ii) The method of embodiment 3, wherein the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 64. 6. The method of any one of embodiments 1 to 5, wherein the intracellular signaling domain of the CAR is derived from CD3-zeta. 7. The method of any one of embodiments 1-6, wherein said at least one costimulatory domain of said CAR comprises a costimulatory domain of a member of the group consisting of FceR1g, Fcg, CD28, CD134(OX40), CD137(4-1BB), CTLA-4, CTLA-4 / CD-28 hybrid, DAP10, CD27, 2B4, and combinations thereof, and optionally said at least one costimulatory domain comprises a CD28 and / or 4-1BB costimulatory domain. 8. The antigen-binding domain of the CAR is an antibody, an antibody fragment, a camelid 8. The method of any one of embodiments 1 to 7, comprising a nanobody, a heavy chain-only antibody, or an aptamer. 9. The method of any one of embodiments 1 to 8, wherein the transmembrane domain of the CAR is a CD8 transmembrane domain or a CD28 transmembrane domain. 10. The method of any one of embodiments 1-9, wherein the hinge domain of the CAR is a CD8 hinge domain or a CD28 hinge domain. 11. The method of any one of embodiments 1 to 10, wherein the CAR further comprises a signal peptide. 12. The method of embodiment 11, wherein the signal peptide of the CAR is a CD8 signal peptide or a GM-CSF signal peptide. 13. The method of any one of embodiments 1 to 12, wherein the antigen-binding domain of the CAR comprises a single-chain fragment. 14. The method of embodiment 13, wherein the single-chain fragment comprises a single-chain variable fragment (scFv). 15. The method of embodiment 14, wherein the scFv fragment comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1; and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:4. 16. The method of embodiment 14, wherein the scFv fragment comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 49; and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 50. 17. The method of embodiment 14, wherein the scFv fragment comprises: (a) a VH selected from: (1) SBT01 VH(M) HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 1), or (2) SBT01 VH(G) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 2), and (b) VL selected from the following: (1) SBT01 VL(M) SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVL (SEQ ID NO: 3) or (2) SBT01 VL(G) SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVL (SEQ ID NO: 4). 18. The method of any one of embodiments 1 to 17, wherein the VH-VL fragments are linked by a linker selected from the group consisting of: (i) GGGSx3 (SEQ ID NO: 20), (ii) Whitlow218 (SEQ ID NO: 21), and (iii) AB Pur (SEQ ID NO: 22). 19. The method of embodiment 18, wherein the scFv comprises the following amino acid sequence: (a) the SBT01G-VHVL-GGGSx3 linker of SEQ ID NO: 5, or (b) the SBT01G-VHVL-Whitlow218 linker of SEQ ID NO: 6, or (c) the SBT01G-VHVL-AB pur linker of SEQ ID NO: 7, or (d) U01-VHVL-AB pur linker of SEQ ID NO: 51. 20. The method of any one of embodiments 1-19, wherein the modified Treg cells are human T cells and the subject is a human patient. 21. The method of any one of embodiments 1 to 20, wherein the modified Treg cells are primary T cells. 22. The method of any one of embodiments 1-21, wherein the pharmaceutical composition is administered intravenously. 23. A method of treating a subject suffering from hidradenitis suppurativa, comprising: (a) isolating T cells from a biological sample obtained from said subject; (b) enriching the T cells for regulatory T cells (Tregs); (c) transfecting the enriched Treg cells with an expression vector encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain to generate modified Treg cells expressing the CAR; the antigen-binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof; the transfecting, wherein the modified Treg cells are CD4+, CD25+, and CD127lo; (d) expanding the modified Treg cells; and (e) administering the modified Treg cells to the subject. 24. The method of embodiment 23, wherein said expanding comprises using anti-CD3 / CD28 coated beads. 25. The method of embodiment 23, wherein said expanding does not involve using anti-CD3 / CD28 coated beads. 26. The method of any one of embodiments 23 to 25, wherein the transfection occurs by use of a viral vector, electroporation, heat shock, bacteriophage, sonication, or calcium phosphate. 27. The method of any one of embodiments 23-26, further comprising administering to the subject one or more anti-inflammatory and / or therapeutic agents. 28. The method of embodiment 27, wherein the one or more anti-inflammatory agents comprise an antibody that inhibits inflammatory cytokines. 29. The method of embodiment 28, wherein the anti-inflammatory agent comprises an anti-TNF antibody, an anti-IL-6 antibody, or a combination thereof. 30. The method of any one of embodiments 23-29, further comprising the limitations of any one of embodiments 2-22. 31. Use of a plurality of modified regulatory T (Treg) cells in the manufacture of a medicament for treating hidradenitis suppurativa in a subject, comprising: the engineered Treg cells express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; the antigen-binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof; The above use, wherein the modified Treg cells are CD4+, CD25+, and CD127lo. 32. The use of embodiment 31, further comprising the limitations of any one of embodiments 2 to 22. 33. A plurality of modified regulatory T (Treg) cells for use in a method of treating hidradenitis suppurativa in a subject, the method comprising administering to the subject the plurality of modified regulatory T (Treg) cells, wherein the engineered Treg cells express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; the antigen-binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof; The plurality of modified Treg cells, wherein the modified Treg cells are CD4+, CD25+, and CD127lo. 34. The plurality of modified Treg cells for use according to embodiment 33, further comprising the limitations of any one of embodiments 2 to 22. [Example]

[0160] Abbreviations: ACPA (anti-citrullinated protein antibody); CAR (chimeric antigen receptor); CF (citrullinated fibrinogen); Cit (citrullinated); CitP (citrullinated protein); CRP (C-reactive protein); CV (citrullinated vimentin); EGFR (epidermal growth factor receptor); HD (healthy donor); HS (hidradenitis suppurativa); IV (intravenous); LPS (lipopolysaccharide); PAD2 (peptidylarginine deiminase 2); PBMC (peripheral blood mononuclear cells); PBS (phosphate-buffered saline); RA (rheumatoid arthritis); scFv (single-chain variable fragment); SF (synovial fluid); Teff (effector T cell); Treg (regulatory T cell); TSDR (Treg-specific demethylated region); UTD (untransduced); and VICM (citrullinated and MMP-degraded vimentin).

[0161] Example 1: Detection of citrullinated antigens in inflammatory sites in patients with hidradenitis suppurativa and rheumatoid arthritis An immunohistochemistry (IHC) assay was developed to assess the presence of citrullinated antigens in biopsy samples from HS patients. The IHC assay used an anti-citrullinated vimentin antibody (anti-CV Ab) containing the VH and VL of the single-chain antigen-binding domain (scFV portion) of the S01 CAR and the Fc portion of mouse IgG2a (S01-mIgG2a). This assay was optimized for use with fixed tissue using formalin-fixed, paraffin-embedded positive and negative control cell lines. The S01-mIgG2a Ab produced positive staining in SKNBE2 cells overexpressing PAD2 and PAD4 (positive control cell line) but did not react with the negative control cell line, SKNBE2 WT. The specific reactivity of the S01-mIgG2a Ab in all staining runs was consistent with the negative control antibody, which demonstrated a lack of specific reactivity. To detect binding in patient biopsy samples, S01-mIgG2a Ab was added at a concentration of 12 μg / mL to sections of HS skin (n=58) and normal skin from adjacent samples (n=10), then visualized using the EnVision FLEX system on a Dako Autostainer Link 48. Additional sections from all specimens tested were stained with hematoxylin and eosin (H&E) to assess tissue quality and determine the extent of inflammatory cell infiltration. Additionally, all samples were stained with a negative control mouse IgG antibody to confirm the specificity of S01-mIgG2a Ab staining.

[0162] result Strong reactivity to S01-mIgG2a Ab staining was observed across multiple cell types, including epithelial cells, synovial cells, and immune cells, in HS skin biopsy sections, as well as extracellular staining, particularly in areas of necrotic tissue. In contrast, little staining was observed in normal skin sections.

[0163] Figures 1A-1B show that citrullinated protein (CitP) is present in samples of hidradenitis suppurativa (HS) lesions and synovial tissue samples from rheumatoid arthritis (RA) patients, but is absent in samples of normal skin (non-HS) and normal synovial tissue (non-RA).

[0164] Figures 2A-2B show that lesional HS skin exhibited elevated levels of inflammation, including a higher number of CitP+ inflammatory cells, compared with adjacent normal skin. Furthermore, as shown in Figure 2C, lesional HS skin contained a higher number of viable CitP+ non-inflammatory cells compared with adjacent normal skin. Thus, immunohistochemical evaluation demonstrated increased inflammation and increased target antigen (CitP) expression in lesional HS skin.

[0165] Example 2: Detection of citrullinated antigens in sera from patients with hidradenitis suppurativa and rheumatoid arthritis To assess the presence of citrullinated proteins (CitPs) in the serum of HS and RA patients, cell-based assays and ELISAs were performed using CV-CAR-transduced cells and anti-CV-Ab, respectively. Furthermore, serum levels of VICM were measured. Levels of c-reactive protein (CRP) and anti-citrullinated protein antibodies (ACPA) were measured in HS and normal serum samples.

[0166] Jurkat-FF-luciferase transduction: approximately 24 x 10 6 Jurkat-FF-luc cells were pelleted and resuspended in 24 ml of RPMI containing protamine sulfate and virus at an MOI of 3 to express CV-CAR. The cells were mixed and 4 ml aliquots were placed in each well of a 6-well plate. The plate was then rotated and placed in an incubator overnight. The cells were pelleted and replated in 25 ml of RPMI in a T75 flask.

[0167] Stimulation. Transduced cells were pelleted, resuspended in RPMI, and placed in the wells of a black / white plate. Serum samples from HS and RA patients were thawed, vortexed, diluted with RPMI, and then added to the plates containing transduced cells. Normal serum samples served as controls. Alternatively, citrullinated or noncitrullinated proteins were added to the plates as stimuli. Cells were cultured at 37°C in the presence of stimuli.

[0168] Luciferase assay. Approximately 24 hours later, 75 μl of BioGlo® reagent was added to each well, and the plate was incubated in the dark for 2–3 minutes. Luminescence was then read on a plate reader.

[0169] result As shown in Figure 3, the CV-CAR reporter cell line was activated by citrullinated vimentin, fibrinogen, and filaggrin proteins, but not by their non-citrullinated counterparts.

[0170] As shown in Figures 4A-4B, CitP levels are higher in serum from HS or RA patients than in serum from normal control subjects. Similarly, as shown in Figure 4C, serum from HS and RA patients has higher levels of citrullinated and MMP-degraded vimentin (VICM) than serum from normal control subjects.

[0171] As shown in Figures 5A-5B, serum samples from HS patients had elevated levels of CRP and ACPA compared to normal serum samples.

[0172] Figure 6A provides a schematic diagram of the cell-based detection assay used to measure the presence of citrullinated antigens in serum. Figure 6B shows that serum samples from HS patients had elevated levels of citrullinated antigens compared to normal serum samples. Surprisingly, 42 of 65 HS serum samples were found to have Cit-P levels above the detection limit, indicated by the dotted line.

[0173] Example 3: Isolation and characterization of Treg cells from hidradenitis suppurativa patients and healthy donors The immunophenotype of Treg cells recovered from whole blood samples obtained from healthy donors and HS patients was evaluated.

[0174] Isolation of Tregs. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Treg cells were then isolated by gating on CD4+CD25+CD127lo cells using FACS.

[0175] Flow cytometry and FACS analysis: Treg cell cultures were collected, centrifuged at 300 × g for 5 minutes, and then resuspended in 1X Flowstain buffer (Invitrogen) containing anti-Ki67, anti-CTLA4, and anti-HLA-DR surface staining antibodies. Treg cells were incubated at 4 °C for 30 minutes, then centrifuged and washed with 1X Flow stain buffer. Stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry.

[0176] result As shown in Figures 7A-7D, HS patients have Treg frequencies and phenotypes comparable to those of healthy donors. The CD4 and CD8 T cell subset profiles of HS patients are similar to those of HD controls. Tregs from HS patients have the same frequency as Tregs from healthy donors, but the absolute numbers of Tregs from HS patients are higher. This difference is driven by the higher frequency and number of pan-CD4 T cells.

[0177] Example 4: Isolation, transduction, expansion, and characterization of Treg cells Treg cells were collected from whole blood samples obtained from healthy donors and HS patients.

[0178] Isolation and tissue culture of Tregs. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. CD25+ cells were enriched by positive selection. Treg cells were isolated by gating on CD4+CD25+CD127lo cells using FACS. After isolation, 140,000 Treg cells were expanded in 48-well tissue culture plates.

[0179] Transduction of primary Tregs: Primary Treg cells were transduced with the CV-CAR construct by spinoculation in the presence of protamine sulfate on day 2 of expansion.

[0180] Flow cytometry and FACS analysis: On day 14 of expansion, CV-CAR-expressing Tregs were stained for the transcription factors FoxP3 and Helios. Cells were fixed and permeabilized using eBiosciences FoxP3 transcription factor buffer set (eBiosciences).

[0181] result As shown in Figures 8A-8B, the frequency and recovery of Tregs during the generation of CV-CAR Treg cells is comparable between HS and HD patients. This observation holds true for both moderate and severe HS patients (Hurley 2 and Hurley 3). CV-CAR Treg cells can be successfully generated from HS patients, as evidenced by the expression of FOXP3 (Figure 8C) and Helios (data not shown).

[0182] Example 5: Evaluation of CV-CAR Treg suppressive activity and activation by citrullinated vimentin in vitro Treg cells were collected from whole blood samples obtained from healthy donors (N=6) or HS patients (N=7).

[0183] Isolation and tissue culture of Tregs. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. CD25+ cells were enriched by positive selection. Treg cells were then isolated by gating on CD4+CD25+CD127lo cells using FACS. After isolation, 140,000 Treg cells were expanded in 48-well tissue culture plates.

[0184] Transduction of primary Tregs: Primary Treg cells were transduced with the CV-CAR construct by spinoculation in the presence of protamine sulfate on day 2 of expansion.

[0185] Flow cytometry and FACS analysis: Treg cell cultures were collected and centrifuged at 300 × g for 5 minutes, then resuspended in 1X Flowstain buffer (Invitrogen) containing a viability dye (Invitrogen), anti-EGFR, and CD71 surface staining antibodies. Treg cells were incubated at 4°C for 30 minutes, then centrifuged and washed with 1X Flow stain buffer. Stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry. Expression of Helios and FoxP3 by Treg cells was also assessed by flow cytometry.

[0186] Treg activation: Untransduced and CV-CAR-expressing Treg cells were cultured in vitro with citrullinated vimentin beads at pCV bead:Treg ratios ranging from 1:243 to 1:1. Treg activation was assessed by measuring the percentage of proliferating cells and CD71 expression.

[0187] Treg suppression assay: Tregs were co-cultured with either CD3 / CD28-activated T cells or allogeneic whole T cells to assess Treg suppression of target cells. Teff cells were co-cultured at the following Treg:Tresp ratios: 1:32, 1:16, 1:4, 1:2, or 1:1.

[0188] result No disease-specific differences in Treg cell isolation, phenotype, or function have been observed to date, and Tregs from healthy donors and HS patients have similar levels of Helios and FoxP3 (data not shown). Figures 9A-9B and 10A-10B show that Tregs from healthy donors and HS patients have similar proliferation and activation capacity upon CAR stimulation (pCV-beads) and similar suppressive activity.

[0189] It should be understood that the specification and drawings are not intended to limit the invention to the particular forms disclosed, but on the contrary, are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this specification. Accordingly, the specification and drawings are to be construed as merely illustrative and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It should be understood that the forms of the invention shown and described herein are to be considered exemplary embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all of which will be apparent to those skilled in the art upon reading this specification of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention, as set forth in the following claims. The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the specification.

[0190] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method of treating a subject suffering from hidradenitis suppurativa, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a plurality of modified regulatory T (Treg) cells and a pharmaceutically acceptable excipient; the modified Treg cells express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; the antigen-binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof; The method, wherein the modified Treg cells are CD4+, CD25+, and CD127lo.

2. 2. The method of claim 1, wherein the antigen-binding domain of the CAR specifically binds to citrullinated vimentin.

3. 2. The method of claim 1, wherein the antigen-binding domain of the CAR binds to all three of (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptide fragments thereof.

4. 4. The method of claim 3, wherein the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein: (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 36; and (ii) the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:

43.

5. 4. The method of claim 3, wherein the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein: (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 57; and (ii) the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:

64.

6. 2. The method of claim 1, wherein the intracellular signaling domain of the CAR is derived from CD3 zeta.

7. 2. The method of claim 1, wherein the at least one costimulatory domain of the CAR comprises a costimulatory domain of a member of the group consisting of FceR1g, Fcg, CD28, CD134 (OX40), CD137 (4-1BB), CTLA-4, CTLA-4 / CD-28 hybrid, DAP10, CD27, 2B4, and combinations thereof.

8. 2. The method of claim 1, wherein the antigen-binding domain of the CAR comprises an antibody, an antibody fragment, a camelid nanobody, a heavy chain-only antibody, or an aptamer.

9. 2. The method of claim 1, wherein the transmembrane domain of the CAR is a CD8 transmembrane domain or a CD28 transmembrane domain.

10. 2. The method of claim 1, wherein the hinge domain of the CAR is a CD8 hinge domain or a CD28 hinge domain.

11. The method of claim 1, wherein the CAR further comprises a signal peptide.

12. The method of claim 11, wherein the signal peptide of the CAR is a CD8 signal peptide or a GM-CSF signal peptide.

13. 2. The method of claim 1, wherein the antigen-binding domain of the CAR comprises a single-chain fragment.

14. 14. The method of claim 13, wherein the single chain fragment comprises a single chain variable fragment (scFv).

15. 15. The method of claim 14, wherein the scFv fragment comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1; and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

4.

16. 15. The method of claim 14, wherein the scFv fragment comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 49; and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

50.

17. 15. The method of claim 14, wherein the scFv fragment comprises: (a) a VH selected from: (1) SBT01 VH (M) HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 1), or (2) SBT01 VH(G) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 2), and (b) a VL selected from: (1) SBT01 VL(M) SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVL (SEQ ID NO: 3) or (2) SBT01 VL(G) SYVLTQPPSSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVL (SEQ ID NO: 4).

18. 18. The method of claim 17, wherein the VH-VL fragments are linked by a linker selected from the group consisting of: (i) GGGSx3 (SEQ ID NO: 20), (ii) Whitlow218 (SEQ ID NO: 21), and (iii) AB Pur (SEQ ID NO: 22).

19. 19. The method of claim 18, wherein the scFv comprises the amino acid sequence: (a) the SBT01G-VHVL-GGGSx3 linker of SEQ ID NO: 5, or (b) the SBT01G-VHVL-Whitlow218 linker of SEQ ID NO: 6, or (c) the SBT01G-VHVL-AB pur linker of SEQ ID NO: 7, or (d) U01-VHVL-AB pur linker of SEQ ID NO:

51.

20. 2. The method of claim 1, wherein the modified Treg cells are human T cells and the subject is a human patient.

21. 21. The method of claim 20, wherein the modified Treg cells are primary T cells.

22. 22. The method of claim 21, wherein the pharmaceutical composition is administered intravenously.

23. 1. A method of treating a subject suffering from hidradenitis suppurativa, comprising: (a) isolating T cells from a biological sample obtained from said subject; (b) enriching the T cells for regulatory T cells (Tregs); (c) transfecting the enriched Treg cells with an expression vector encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain to generate modified Treg cells expressing the CAR; the antigen-binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof; the transfecting, wherein the modified Treg cells are CD4+, CD25+, and CD127lo; (d) expanding the modified Treg cells; and (e) administering the modified Treg cells to the subject.

24. 24. The method of claim 23, wherein said expanding comprises using anti-CD3 / CD28 coated beads.

25. 24. The method of claim 23, wherein said expanding does not involve using anti-CD3 / CD28 coated beads.

26. 24. The method of claim 23, wherein the transfection occurs through the use of a viral vector, electroporation, heat shock, bacteriophage, sonication, or calcium phosphate.

27. 24. The method of claim 23, further comprising administering one or more anti-inflammatory and / or therapeutic agents to the subject.

28. 28. The method of claim 27, wherein the one or more anti-inflammatory agents comprises an antibody that inhibits a pro-inflammatory cytokine.

29. 29. The method of claim 28, wherein the anti-inflammatory agent comprises an anti-TNF antibody, an anti-IL-6 antibody, or a combination thereof.

30. 30. The method of any one of claims 23 to 29, further comprising the limitations of any one of claims 2 to 22.

31. 1. Use of a plurality of modified regulatory T (Treg) cells in the manufacture of a medicament for treating hidradenitis suppurativa in a subject, comprising: the modified Treg cells express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; the antigen-binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof; The aforementioned use, wherein the modified Treg cells are CD4+, CD25+, and CD127lo.

32. The use according to embodiment 31, further comprising the limitations according to any one of claims 2 to 22.

33. A plurality of modified regulatory T (Treg) cells for use in a method of treating hidradenitis suppurativa in a subject, the method comprising administering to the subject the plurality of modified regulatory T (Treg) cells, the modified Treg cells express a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; the antigen-binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof; The plurality of modified Treg cells, wherein the modified Treg cells are CD4+, CD25+, and CD127lo.

34. 34. The plurality of modified Treg cells for use according to embodiment 33, further comprising the limitations of any one of claims 2 to 22.

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