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

Abstract

Disclosed herein are chimeric antigen receptors (CARs) comprising an antigen binding site that recognizes a citrullinated polypeptide. A citrullinated polypeptide, such as a citrullinated waveform protein, is expressed in a skin lesion in a subject suffering from hidradenitis suppurativa. Also disclosed are T cells, and in particular Treg cells that express these CARs. Administration of these CAR-T cells can be used to treat hidradenitis suppurativa.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 519,184, filed on August 11, 2023, and U.S. Provisional Application No. 63 / 408,046, filed on September 19, 2022, both of which are hereby incorporated by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (237752000740seqlist.xml; size: 66037 bytes; and creation date: September 13, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to chimeric antigen receptors reactive with citrullinated antigens and regulatory T cells expressing the receptors for treating hidradenitis suppurativa. Background Art

[0006] Hidradenitis suppurativa (HS) is a chronic inflammatory skin condition characterized by the development of painful skin lesions formed by follicular occlusion caused by hyperkeratinization and hyperplasia of the infundibular epithelium. Disease progression ultimately leads to 'plugging' and rupture of the follicular units, which triggers a chronic skin inflammatory response. HS has a severe negative impact on patients due to the pain and limited mobility associated with the disease, the malodor and discharge from the affected skin, and the disfigurement that accompanies HS symptoms. Current treatment options are inadequate to address symptoms, and there remains an unmet need for effective therapies.

[0007] Regulatory T (Treg) cells have been found to be enriched in HS skin lesions, however, the ratio of Treg to CD4+ helper T17 (Th17) cells is unbalanced in favor of Th17. Normalization of this ratio has the potential to improve immune dysregulation in HS. Preclinical studies have shown that antigen-specific and autologous Treg cells have the potential to treat systemic inflammation and organ damage, supporting the hypothesis that Treg cells may lead to transformative and durable treatments. 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 diseases. Clinical studies have also evaluated the potential of polyclonal Treg cells as a therapeutic option for organ transplant recipients and patients with autoimmune conditions. Although Treg cell infusions are well tolerated, only limited efficacy has been observed.

[0008] Currently, there is no cure for HS, as well as many other chronic inflammatory diseases. This is partly because the cause of HS is still unclear. In fact, HS is often treated with corticosteroids or other anti-inflammatory agents, which may make the recipient more susceptible to infectious diseases. Therefore, what is needed in the art is a treatment that targets antigens that are preferentially found in HS lesions. Summary of the invention

[0009] The present disclosure relates to chimeric antigen receptors reacting with citrullinated antigens and regulatory T cells expressing receptors for treating hidradenitis suppurativa. Specifically, the CAR expressed by the Treg cells of the present disclosure specifically binds to citrullinated polypeptides, including vimentin, citrullinated filamentous protein and citrullinated fibrinogen and citrullinated fragments thereof. In some embodiments, the single-chain fragment variable region (scFv) of CAR is partially obtained from antibodies highly specific to 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, it is mentioned that the antibody is applicable to the antigen binding domain of the CAR of the present disclosure, unless otherwise indicated in the context. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable those skilled in the relevant art to make and use these embodiments and other embodiments that are obvious to those skilled in the art. The present invention will be described in more detail with reference to the following drawings, in which:

[0011] Figure 1A : Citrullinated protein (CitP) is frequently found in samples of hidradenitis suppurativa (HS) lesions but not in samples of normal skin (non-HS). Figure 1B :CitP also frequently appears in synovial tissue samples of patients with rheumatoid arthritis (RA), but not in synovial tissue samples of healthy subjects (non-RA). CitP reactivity is measured by detecting the binding of anti-citrullinated vimentin (CV) antibodies (e.g., anti-CV antibodies referred to herein as S01-mIgG2a).

[0012] FIG. 2A to FIG. 2C : HS skin lesions have higher levels of inflammation and CitP target antigens compared with normal skin. Figure 2A : HS skin lesions have a higher percentage of inflammatory cells than normal skin. Figure 2B : HS skin lesions have a greater number of CitP+ inflammatory cells than normal skin. Figure 2C : HS skin lesions also have a greater number of CitP+ non-inflammatory cells than normal skin.

[0013] 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 filaggrins, but not by non-citrullinated vimentin, fibrinogen, and filaggrin.

[0014] FIG. 4A to FIG. 4B : Higher levels of CitP were present in the sera of patients with HS and RA compared with the sera of normal control subjects, as determined by measuring the 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 sera of patients with HS and RA compared with the sera of normal control subjects.

[0015] FIG. 5A to FIG. 5B : Compared with normal serum samples, HS patient serum samples have elevated levels of C-reactive protein (CRP) and anti-citrullinated protein antibodies (ACPA).

[0016] Fig. 6A : Schematic representation of the cell-based assay for detection of CitP. Figure 6B : Serum samples from HS patients have elevated levels of citrullinated antigens (CitP) compared to samples of normal serum.

[0017] FIG. 7A to FIG. 7D : HS patients have Treg frequencies and phenotypes comparable to healthy donors (HD).

[0018] FIG. 8A to FIG. 8B : During the production of CV-CAR Treg cells, Treg frequency and recovery were comparable between HS patients (HS, Hurley 2, and Hurley 3) and HD patients. Figure 8C : CV-CAR Treg cells derived from PBMCs of HD and HS patients (HS, Hurley 2, and Hurley 3) expressed comparable levels of FOXP3. Hurley 2 and Hurley 3 represent patients with moderate and severe HS, respectively.

[0019] FIG. 9A to FIG. 9B : CV-CAR Treg cells derived from PBMCs of HD or HS patients proliferated and were similarly activated by CAR stimulation (pCV beads).

[0020] FIG. 10A to FIG. 10B : Treg cells derived from PBMCs of HD or HS patients showed similar suppressive activity. DETAILED DESCRIPTION

[0021] Citrullination of proteins is an irreversible post-translational modification that affects protein structure and is associated with the regulation of histone, cytoskeleton and secretory protein function. In addition, citrullinated proteins accumulate at sites of chronic inflammation. Citrulline is produced by the post-translational conversion of arginine to citrulline by the enzyme peptidylarginine deiminase (PAD). PAD enzymes have been shown to be strongly expressed in activated bone marrow cells, including macrophages and neutrophils. PAD enzymes play an important role in the formation of neutrophil extracellular traps (NETs), a phenomenon in which autoantigens and immunostimulatory molecules are externalized. During the NET formation process, neutrophils externalize citrullinated autoantigens, releasing DAMPs as innate immune activators, which are associated with driving the pathogenesis of HS. Although expressed intracellularly, abnormal 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. The activity of PAD enzymes is increased in the inflamed skin of HS patients, especially in neutrophils, releasing citrullinated proteins and other autoantigens during the NET formation process. Citrullinated vimentin (CV) and fibrinogen have been found in the extracellular matrix of inflamed skin from patients with hidradenitis suppurativa (HS). Citrullination also leads to the production of autoantigens during the inflammatory response. Citrullinated autoantigens are highly immunogenic, can be presented on MHC molecules, and induce T-cell-mediated B-cell activation. This leads to the production of pathogenic anti-citrullinated protein antibodies (ACPA), which have been observed in the sera of HS patients and at the sites of skin lesions. ACPA can react with several citrullinated proteins, including citrullinated fibrinogen, myeloperoxidase, nucleosomes, histone H4, and dsDNA. Taken together, the presence of NETs, ​​citrullinated proteins, and ACPA in HS skin lesions indicates a pathogenic role for citrullination in the etiology of HS disease.

[0022] The present disclosure describes a targeted antigen-specific approach to increase the functionality and number 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 considered a new approach to treat HS.

[0023] I. Definitions

[0024] Unless otherwise indicated, the terminology and symbols of biochemistry, nucleic acid chemistry, molecular biology, developmental biology, and molecular genetics follow those of standard conventions and texts in the field, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Press, 1989); Alberts and Singer, Developmental Biology, 8th edition (Sinauer Associates Inc., Sunderland, MA, 2006); Kornberg and Baker, DNA Replication, 2nd edition (WH Freeman, New York, 1992); Gaits, ed., Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Lehninger, Biochemistry, 2nd edition (Worth Publishers, New York, 1975); Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); etc.

[0025] 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., can be bound by an antibody. The term can refer to any molecule that can be recognized by an antibody, such as a polypeptide, a polynucleotide, a carbohydrate, a lipid, a chemical moiety or a combination thereof (e.g., a phosphorylated or glycosylated polypeptide, etc.). Those skilled in the art will understand that the term does not mean that the molecule is immunogenic in every case, but only that it can be targeted by an antibody.

[0026] As used herein, the term "epitope" refers to a localized site on an antigen that is recognized and bound by an antibody. An epitope may include several amino acids or portions of several amino acids, such as 5 or 6, or more, such as 20 or more amino acids, or portions of those amino acids. In some cases, an epitope includes non-protein components, such as components from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional portion. Thus, for example, when the target is a protein, an epitope may be composed of continuous amino acids, or of amino acids from different parts of a protein that are approached by protein folding (e.g., discontinuous epitopes).

[0027] As used herein, the term "antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene that specifically binds and recognizes an antigen. Typically, the "variable region" comprises the antigen-binding region of the antibody (or its functional equivalent) and is most critical in terms of specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" (about 25 kD) chain and a "heavy" chain (about 50-70 kD).

[0028] Antibodies can belong to (i) any of the five major classes of immunoglobulins: α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), or (ii) a subclass (isotype) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of the constant domain of their heavy chains. The light chain can be λ or κ.

[0029] The following is a non-exhaustive list of different antibody formats, all of which retain antigen binding activity:

[0030] (1) Whole immunoglobulin (also called “intact” antibody) (two light chains and two heavy chains, e.g., a tetramer);

[0031] (2) immunoglobulin polypeptide (light chain or heavy chain);

[0032] (3) Antibody fragments, such as Fv (monovalent or bivalent variable region fragments, and may contain only 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) (comprising V L and V H polypeptides), (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, miniantibodies (sc(FV)2 fused to a CH3 domain), diabodies (non-covalent dimers of single-chain Fv (scFv) fragments consisting of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a small peptide linker), triabodies (trivalent sc(Fv)3 or trispecific sc(Fv)3);

[0033] (4) multivalent antibodies (antibodies containing binding regions that bind to two different epitopes or proteins, such as “scorpion” antibodies);

[0034] (5) fusion proteins comprising a binding portion of an immunoglobulin fused to another amino acid sequence (such as a fluorescent protein); and

[0035] (6) Heavy chain-only antibodies or antibody fragments, which have only two heavy chains and lack the two light chains normally found in antibodies.

[0036] The generation and characterization of tandem scFvs and diabodies are described, e.g., 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.

[0037] As used herein, the phrase "CDR sequence set" refers to the three heavy chain and / or three light chain CDRs of a specific 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 "full" CDR sequence set refers to the heavy chain and light chain CDR sequences. CDRs are predicted based on IMGT sequence alignments.

[0038] As used herein, the term "chimeric antibody" refers to an antibody having amino acid sequences from two or more species. In one embodiment, the variable regions of the light and heavy chains correspond to the variable regions of an antibody from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences derived from another species (usually in the subject receiving therapy, such as a human) to avoid eliciting an immune response.

[0039] As used herein, the term "humanized antibody" refers to a chimeric antibody in which the CDRs of the VH and VL regions of non-human antibodies with desired specificity, affinity and capacity are transplanted to human framework sequences. In one embodiment, the framework residues of a humanized antibody are modified to refine and optimize antibody specificity, affinity and capacity. Humanization (i.e., replacing the corresponding sequences of a human antibody with non-human CDR sequences) can be performed following the methods described in, for example, 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).

[0040] As used herein, the term "human antibody" refers to an antibody produced by humans or an antibody having an amino acid sequence corresponding thereto prepared by any technique known in the art.

[0041] The specificity of binding can be defined based on the relative dissociation constant (Kd) of the antibody (or other targeting moiety) for the target, compared to the dissociation constants of the antibody and other substances or generally unrelated molecules in the environment. A larger (higher) Kd is a Kd that describes a lower affinity interaction. Conversely, a smaller (lower) Kd is a Kd that describes a higher affinity interaction or a 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 an unrelated substance may be millimolar or higher. Binding affinity may be in the micromolar range (kD = 10 -4 Up to 10 -6 ), nanomolar range (kD = 10 -7 M to 10 -9 M), picomolar range (kD = 10 -10 M to 10 -12 M) or femtomolar range (kD = 10 -13 M to 10 -15 M).

[0042] As used herein, if the antibody is expressed at less than 10 -4 M (i.e., in the micromolar range) to bind to an antigen or epitope, then the antibody "binds" or "recognizes" the antigen or epitope. The term "binding" with respect to a cell type (e.g., an antibody that binds to a cell expressing an antigen) generally means that the agent binds to the majority of cells in a pure population of these cells. For example, an antibody that binds to a given cell type generally binds to at least 2 / 3 of the cells in a given cell population (e.g., 67%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). In some cases, the binding to a polypeptide can be determined by comparing the binding of an antibody to a cell presenting a polypeptide with the binding (or lack of binding) of an antibody to a cell that does not express a polypeptide. Those skilled in the art will recognize that some variability will occur depending on the method and / or threshold for determining binding. The affinity of an antibody for a target can be determined according to methods known in the art, for example, as described in Ernst et al., Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009).

[0043] As used herein, the term "greater affinity" refers to the relative degree of antibody binding, wherein antibody X binds to target Y more strongly (Kon) and / or has a smaller dissociation constant (Koff) than to target Z, and in this case, antibody X has a greater affinity for target Y than for target Z. Similarly, the term "lesser affinity" refers herein to the degree of antibody binding, wherein antibody X binds to target Y less strongly than to target Z and / or has a larger dissociation constant than to target Z, and in this case, antibody X has a lower affinity for target Y than for Z. The binding affinity between an antibody and its target antigen can be expressed as KA equals 1 / KD, where KD equals kon / koff. The kon and koff values ​​can be measured using surface plasmon resonance technology, for example, using a molecular affinity screening system (MASS-1) (SierraSensors GmbH, Hamburg, Germany). An antagonist or blocking antibody is an antibody that partially or completely blocks, inhibits, or neutralizes a biological activity associated with a target antigen relative to its activity in the absence of the antibody under similar physiological conditions. Antagonists can be competitive, noncompetitive, or irreversible. Competitive antagonists are substances that bind to a natural ligand or receptor at the same site of the natural ligand-receptor interaction, or that bind allosterically in a way that induces changes that prevent normal binding. Noncompetitive antagonists bind at a site different from the natural ligand-receptor interaction, but reduce the KD or signal produced by the interaction. Irreversible inhibitors result in covalent modification of the receptor, thereby preventing any subsequent binding.

[0044] As used herein, the term "affinity" refers to the overall stability of the binding complex between an antibody and a target antigen. It is controlled by three factors: (i) the intrinsic affinity of the antibody for the antigen, (2) the valence 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.

[0045] As used herein, an antibody "preferentially binds" a first antigen relative to a second antigen if the antibody binds the first antigen with greater affinity than the second antigen. Preferential binding can be at least any of 2-fold, 5-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold greater affinity.

[0046] As used herein, if the antibody is expressed at 1 × 10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10M, 1×10 -11 M, 1×10 -12 An antibody "specifically binds" or is "specific for" a target antigen or group of target antigens if the antibody binds to each member of the target antigen or group of target antigens with an affinity greater than at least any one of M, and, for example, if the antibody binds to each member of the target antigen or group of target antigens with an affinity at least two times greater than its affinity for binding to non-target antigens. Typically, specific binding is characterized by binding to the antigen with sufficient affinity so that the antibody can be used as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent targeting the antigen or epitope.

[0047] As used herein, the term "polypeptide" refers to a molecule having a sequence of natural and / or non-natural amino acids linked by peptide bonds. The term "peptide" refers to a short polypeptide, usually no more than 30 amino acids long. 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, such as structures stabilized by hydrogen bonds, relationships between secondary structures, and structures formed by more than one protein. Proteins may be further modified by other attached moieties, such as carbohydrates (glycoproteins), lipids (lipoproteins), phosphate groups (phosphoproteins), etc.

[0048] As used herein, an amino acid sequence "consists of" only the amino acids in that sequence.

[0049] 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, no more than 2, or no more than 3 amino acids longer than the second amino acid sequence.

[0050] 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, the first amino acid sequence, which is a fragment of the second amino acid sequence, may be less than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids than the second amino acid sequence.

[0051] As used herein, a "functional equivalent" of a reference amino acid sequence is a sequence that is different from the reference sequence, but contains minor changes, such as, for example, insertions, deletions or substitutions of one or several amino acids. Functionally equivalent sequences retain the function (e.g., immunogenicity) of the reference sequence to which they are equivalent. If a functionally equivalent amino acid sequence contains one or more amino acid substitutions relative to the reference sequence, these are generally conservative amino acid substitutions.

[0052] As used herein, "conservative amino acid substitution" refers to the substitution of one amino acid residue by another amino acid residue without destroying the desired properties of the protein. Suitable conservative amino acid substitutions can be made by replacing amino acids with similar hydrophobicity, polarity, and R chain length. See, for example, Watson et al., "Molecular Biology of the Gene," 4th edition, 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, page 224. Examples of conservative amino acid substitutions include the following (note that some categories are not mutually exclusive):

[0053]

[0054] As used herein, the term "substantially identical" refers to the identity between a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are i) identical to the aligned amino acid residues in the second amino acid sequence, or ii) conservative substitutions of the aligned amino acid residues in the second amino acid sequence, such that the first amino acid sequence and the second amino acid sequence have a common domain and / or a common functional activity and / or a common immunogenicity. For example, amino acid sequences containing a common structure or antigenic domain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 contains a sufficient or minimum number of nucleotides that are identical to the aligned nucleotides in the second nucleic acid sequence, such that the first nucleotide sequence and the second nucleotide sequence encode a polypeptide having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, or encode a polypeptide having the same immunogenic properties.

[0055] As used herein, a chemical entity (such as a polypeptide) is "substantially pure" or "isolated" if the chemical entity is the predominant chemical entity of its kind (e.g., a polypeptide) in the composition. This includes chemical entities that represent more than 50%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, or more than 99.99% of the chemical entities of its kind in the composition. A substantially purified fraction is a composition in which the target species accounts for at least about 50% (on a molar basis) of all macromolecular species present. Typically, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition are the purified species of interest. If the composition consists essentially of a single macromolecular species, the target species is purified to substantial homogeneity (contaminant species cannot be detected in the composition by conventional detection methods). Solvent species, small molecules, stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species within this definition.

[0056] The phrase "isolated antibody" refers to an in vivo or in vitro produced antibody that is removed from a source from which the antibody is produced, eg, an animal, hybridoma, or other cell line such as recombinant insect, yeast, or bacterial cells that produce the antibody.

[0057] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. In order to determine the percentage of identity of two amino acid sequences or two nucleic acid sequences, for the purpose of optimal comparison, the sequences are compared (for example, a gap can be introduced in the first amino acid or nucleic acid sequence so as to be optimally compared with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage of identity between the 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 of the same length. A mathematical algorithm can also be used to determine the percentage of identity between the two sequences. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as adapted in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This 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 with the NBLAST nucleotide program parameter set (e.g., score = 100, wordlength = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameter set (e.g., to score 50, wordlength = 3) to obtain amino acid sequences homologous to the protein molecules described herein. In order to obtain gap alignments for comparison purposes, gap BLAST as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402 can be utilized. Alternatively, PSI-BLAST can be used to perform an iterative search to detect distance relationships (Id.) between molecules. When using BLAST, gap BLAST and PSI-Blast programs, the default parameters of each program (such as the default parameters of XBLAST and NBLAST) can be used (see, for example, the NCBI website). Another preferred non-limiting example of a mathematical algorithm for sequence comparison is Myers and Miller, 1988, CABIOS 4: 11-17 algorithm. This algorithm is incorporated into the ALIGN program (version 2.0), which is a 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 the two sequences can be determined using techniques similar to those described above, with or without gaps. When calculating percent identity, typically only exact matches are calculated.

[0058] For antibodies, when the antibody sequences are maximally aligned by IMGT, the percent sequence identity can be determined. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percent sequence identity between the subject antibody region and the reference antibody region is the number of positions occupied by the same amino acid in both the subject antibody region and the reference antibody region divided by the total number of aligned positions in the two regions multiplied by 100 to convert to a percentage.

[0059] The amino acid sequence identity percentage 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 is available from the National Institute of Health in Bethesda, Maryland. NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, demasking = yes, chain = all, expected occurrences = 10, minimum low complexity length = 15 / 5, multiple passes e value = 0.01, multiple passes constant = 25, final gap alignment decay = 25, scoring matrix = BLOSUM62.

[0060] In the case of using NCBI-BLAST2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a specific % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in the alignment of A and B in that program, and where Y is the total number of amino acid residues in B. It should be understood that when 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 is not equal to the % amino acid sequence identity of B to A. As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of natural bases, sugars, and intersugar (backbone) bonds, and includes cDNA. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid sequence of the present application can be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and can include naturally occurring bases, including adenine, guanine, cytosine, thymidine and uracil. The sequence can also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. It should be understood that polynucleotides containing non-transcribable nucleotide bases can be used as probes, for example, in hybridization assays. Nucleic acids can be double-stranded or single-stranded, and represent sense strands or antisense strands. In addition, the term "nucleic acid" includes complementary nucleic acid sequences and codon optimization or synonymous codon equivalents.

[0061] As used herein, the term "isolated nucleic acid" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of the sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.

[0062] All or part of the nucleic acid sequence molecule can be hybridized. The length of the hybridization portion is usually at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides. Those skilled in the art will recognize that the stability of the nucleic acid duplex or hybrid is determined by Tm, which is a function of sodium ion concentration and temperature in a sodium-containing buffer (Tm = 81.5°C -16.6 (Log10 [Na+]) + 0.41 (% (G+C) -600 / l), or similar equations). Therefore, the parameters that determine the stability of the hybrid in the washing conditions are sodium ion concentration and temperature. In order to identify molecules that are similar but not identical to known nucleic acid molecules, it can be assumed that a 1% mismatch results in a Tm reduction of about 1°C, for example, if a nucleic acid molecule with> 95% identity is sought, the final washing temperature will be reduced by about 5°C. Based on these considerations, those skilled in the art will be able to easily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. Merely by way of example, stringent hybridization can be achieved using the following conditions: hybridization at 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS at Tm-5°C, based on the above equation, followed by washing with 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. However, it will be appreciated that equivalent stringency can be achieved using alternative buffers, salts, and temperatures. Additional guidance on hybridization conditions can be found in: Current Protocols in Molecular Biology, John Wiley & Sons, NY, 2002, and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0063] As used herein, the term "expression construct" refers to a polynucleotide comprising an expression control sequence that is operably linked to a heterologous nucleotide sequence (i.e., a sequence to which the expression control sequence is not normally linked in nature) that is the object of expression. As used herein, the term "expression vector" refers to a polynucleotide comprising an expression construct and a sequence sufficient to replicate in a host cell or insert 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 regulates the transcription and / or translation of a nucleotide sequence to which it is operably linked. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences), and ribosome binding sites (translation regulatory sequences).

[0064] As used herein, a nucleotide sequence is "operably linked" to an expression control sequence when the expression control sequence functions in a cell to regulate transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through interaction between a polymerase and a promoter.

[0065] As used herein, the term "vector" includes any intermediate vehicle of a nucleic acid molecule that enables the nucleic acid molecule, for example, to be introduced into a prokaryotic and / or eukaryotic cell and / or integrated into the genome, and includes plasmids, phagemids, viral phages or viral vectors, such as retroviral-based vectors, lentiviral vectors, adeno-associated viral vectors, etc. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0066] "Transfection" refers to the introduction of new genetic material into cells. It includes transformation (direct uptake and incorporation of foreign genetic material from the surrounding environment through the cell membrane), transduction (introduction of foreign DNA into host cells by viruses, bacteriophage viruses), and conjugation.

[0067] As used herein, "host cell" refers to a recombinant cell that contains an expression construct.

[0068] As used herein, the term "biological sample" refers to a sample containing cells (eg, cells) or biomolecules derived from cells.

[0069] As used herein, the terms "therapy", "treatment", "therapeutic intervention" and "improvement" refer to any activity that leads to a reduction in the severity of symptoms. The terms "treatment" and "prevention" are not absolute terms. Treatment and prevention can refer to any delay in onset, improvement of symptoms, improvement in patient survival, increase in survival time or survival rate, etc. Treatment and prevention can be complete or partial. The therapeutic effect can be compared with an individual or population that has not received treatment, or with the same patient at different times before or during treatment. In some aspects, the severity of the disease is reduced by at least 10% compared to, for example, an individual before administration or a control individual that has not received treatment. In some aspects, the severe reduction of the disease is reduced by at least 25%, 50%, 75%, 80% or 90%, or in some cases, it is no longer detectable using standard diagnostic techniques. "Treatment" or "treatment" can also mean prolonged survival compared to the expected survival when not receiving treatment. "Treatment" used herein also includes preventive treatment.

[0070] A composition or method that "comprising" or "includes" one or more of the listed elements may include other elements not specifically listed (e.g., open-ended terms mean including, but not limited to). For example, a composition that "comprising" or "includes" an antibody may contain the antibody alone or in combination with other ingredients. In contrast, the phrase "consisting of" is closed, indicating that such an embodiment does not include additional elements. The term "consisting essentially of" means including the listed elements and other elements that do not materially affect the basic and novel characteristics of the claimed combination (e.g., partially closed terms). It should be understood that aspects and embodiments described herein as "comprising" include "consisting of" and "consisting essentially of" embodiments.

[0071] As used herein, the following meanings apply unless otherwise specified. The word "may" is used in a permissive sense (i.e., meaning with some possibility), rather than in a mandatory sense (i.e., meaning must). The singular forms "a / an" and "the" include plural referents. Thus, for example, a submission to "an element" includes a combination of two or more elements, although other terms and phrases, such as "one or more" are used for one or more elements. The phrase "at least one" includes "one", "one or more", "one or aplurality" and "multiple". Unless otherwise specified, the term "or" is non-exclusive, i.e., includes "and" and "or" modifiers and the term "any" between the sequence refers to each member of the modifier modifying the sequence. Thus, for example, the phrase "at least any one of 1, 2 or 3" means "at least 1, at least 2 or at least 3".

[0072] II. Chimeric Antigen Receptors

[0073] "Chimeric antigen receptor" or "CAR" is an engineered molecule comprising an optional signal peptide, a target binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain, and an optional co-stimulatory domain. CAR is based on the structure of the T cell receptor, which is expressed on T cells and participates in cell-mediated immune responses. "Target binding domain" is also referred to as "antigen binding domain" herein, and therefore the term "target" includes "antigen".

[0074] The so-called "first generation" CAR has a targeting domain and a CD3ξ signal transduction domain. The so-called "second generation" CAR further includes a costimulatory domain, such as a CD28 or 4-1BB domain. The so-called "third generation" CAR includes multiple costimulatory domains. The so-called "fourth generation" CAR (also referred to as "TRUCKS") is engineered to release transgenic cytokines during CAR signaling.

[0075] A chimeric antigen receptor ("CAR") includes the following elements: (1) an optional signal peptide, (2) a target binding domain, (3) an optional hinge region; (4) a transmembrane region; (5) an intracellular domain comprising a signal transduction domain. Optionally, CAR may include any of the following: a CD3ζ signal transduction domain, an Fc receptor signal transduction domain, a co-stimulatory (signal transduction) 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 other domain. That is, the target binding domain does not naturally occur on a T cell receptor, or is not in the same protein as at least one other domain.

[0076] The "target binding domain" provides binding specificity to the CAR. The "signal peptide" guides the polypeptide through the cell membrane. The target binding domain can be bound to the domain of the antibody, which binds to the target antigen to form a so-called "universal CAR". The "hinge region" is a flexible connection region, such as a natural or synthetic polypeptide, or any other type of molecule, to provide structural flexibility and spacing for the side polypeptide region. A "transmembrane domain" is a transmembrane protein domain, usually hydrophobic. A "signal transduction domain" or "signaling domain" transmits a signal to the cell through a signal transduction pathway when bound. This signal transduction activates the activity of the cell. A "costimulatory domain" is an accessory signaling domain that further transmits a signal.

[0077] In some embodiments, the CAR comprises:

[0078] (i) a target binding domain (also referred to herein as an antigen binding domain) that reacts with a citrullinated protein or a citrullinated fragment thereof (such as VH-VL or VL-VH), wherein the two variable domains are separated by a flexible linker of 15-25 amino acids in length;

[0079] (ii) hinge domain;

[0080] (iii) a co-stimulatory domain; and

[0081] (iv) intracellular signaling domain (also referred to herein as activation domain). That is, in some embodiments, CAR comprises an antigen binding domain fused to a CAR platform, the CAR platform comprising a hinge domain, a transmembrane domain, and an intracellular domain comprising a co-stimulatory domain and an activation domain. CAR may further include a signal peptide (also referred to herein as a leader sequence) to guide the expression of CAR on the cell surface, such as Treg.

[0082] In some embodiments, the CAR comprises an antigen binding domain fused in frame to a CAR platform comprising an amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 28, and SEQ ID: 19. In other embodiments, the CAR comprises an antigen binding domain fused in frame to a CAR platform comprising an amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 28, and SEQ ID: 19. In some embodiments, the CAR comprises an antigen binding domain fused in frame to a CAR platform comprising an amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 29, and SEQ ID: 19. In other embodiments, the CAR comprises an antigen binding domain fused in frame to a CAR platform comprising an amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 29, and SEQ ID: 19.

[0083] A. Signal peptide

[0084] The signal peptide may be any peptide having the function of allowing the polypeptide to pass through the cell membrane. The signal peptide may be derived from CD4, CD8, CD28, TLR or immunoglobulin receptor family.

[0085] For example, a signal peptide may comprise the following sequence:

[0086] MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO:18); or

[0087] MALPVTALLLPLALLLHAAR (SEQ ID NO: 23).

[0088] B. Target Binding Domain

[0089] 1. Structure

[0090] The target binding domain may include any polypeptide comprising a target binding function, such as an antibody defined herein. In one embodiment, the target binding domain may include an antibody form retaining the antigen binding activity defined herein. In one embodiment, the target binding domain may include a single-chain antibody (scFV). ScFv can be connected to a transmembrane domain via a hinge domain, and the length, flexibility and source of the hinge domain provide variability for the design of CAR, and can contribute to the interaction with the antigen, the establishment of an immune synapse, and affect the combination of CAR with the additional proteins required for transmitting a strong activation signal.

[0091] 2. Target / Antigen

[0092] 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 those found in skin lesions of subjects with hidradenitis suppurativa. In particular, the target binding domain may bind to one or more of the following: (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) citrullinated peptides thereof. In some embodiments, the target binding domain may bind to a citrullinated peptide fragment of (i)-(iii), wherein the peptide fragment is at least 10 amino acids in length, such as at least 12 amino acids, at least 14 amino acids, or at least 16 amino acids in length. In some embodiments, the target binding domain further binds to tenascin C. In some embodiments, the target binding domain may bind to two or more of the following: (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated filaggrin, and (iv) tenascin C or a citrullinated peptide fragment thereof.

[0093] In some embodiments, the target domain is one or more citrullinated peptides selected from the following sequences:

[0094] ST(Cit)SVSSSSY(Cit)(Cit)MFGG(SEQ ID NO:24);

[0095] VYAT(Cit)SSAV(Cit)L(Cit)SSV(SEQ ID NO:25);

[0096] (Cit)PAPPPISGGGY(Cit)A(Cit)(SEQ ID NO:26);

[0097] SHQEST(Cit)GRSRGRSGRSGS (SEQ ID NO: 27).

[0098] In some embodiments, the antigen binding domain binds to one or more citrullinated peptides, but not to the non-citrullinated counterparts. In some embodiments, the antigen binding domain binds to the citrullinated vimentin peptide as shown in 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 as shown in 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 as shown in 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 as shown in SEQ ID NO:27, but not to SHQESTRGRSRGRSGRSGS (SEQ ID NO:48).

[0099] The target binding domain may comprise sequences from antibody VH and VL domains. In some embodiments, the target binding domain may comprise sequences from heavy chain antibodies only or antibody fragments having only two VH domains. This includes specific CDR sets from VH and VL domains. In some embodiments, the target binding domain comprises CDRs from the VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the target binding domain comprises CDRs from the VL domain of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the target binding domain comprises CDRs 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 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 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 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 from the VH and VL domains of SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0100] In some embodiments, the target binding domain comprises complementary determining regions (CDRs) of VH and VL domains from SEQ ID NO: 1 (S01 VH) and SEQ ID NO: 4 (S01 VL). In some embodiments, CDRs are defined using antibody numbering schemes, such as Chothia, AbM, Kabat, Contact, and IMGT numbering schemes. In some embodiments, CDRs are defined using the Kabat numbering scheme. In some embodiments, the VH domain of the target binding domain comprises a VH-CDR1 containing an amino acid sequence of SEQ ID NO: 32, a VH-CDR2 containing an amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 containing an amino acid sequence of SEQ ID NO: 36, and the VL domain of the target binding domain comprises a VL-CDR1 containing an amino acid sequence of SEQ ID NO: 39, a VL-CDR2 containing an amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 containing an amino acid sequence of SEQ ID NO: 43.

[0101]

[0102]

[0103] In some embodiments, the target binding domain comprises complementary determining regions (CDRs) of VH and VL domains from SEQ ID NO: 49 (U01 VH) and SEQ ID NO: 50 (U01 VL). In some embodiments, CDRs are defined using antibody numbering schemes, such as Chothia, AbM, Kabat, Contact, and IMGT numbering schemes. In some embodiments, CDRs are defined using the Kabat numbering scheme. In some embodiments, the VH domain of the target binding domain comprises a VH-CDR1 containing an amino acid sequence of SEQ ID NO: 53, a VH-CDR2 containing an amino acid sequence of SEQ ID NO: 55, and a VH-CDR3 containing an amino acid sequence of SEQ ID NO: 57, and the VL domain of the target binding domain comprises a VL-CDR1 containing an amino acid sequence of SEQ ID NO: 60, a VL-CDR2 containing an amino acid sequence of SEQ ID NO: 62, and a VL-CDR3 containing an amino acid sequence of SEQ ID NO: 64.

[0104]

[0105]

[0106] In certain embodiments, the target binding domain comprises a VH sequence selected from:

[0107] (1)SBT01 VH(M)

[0108] HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPP GKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVT AADTAVYYCARLDPFDYWGRGTLVTVSS(SEQ ID NO:1);

[0109] (2) SBT01 VH(G)

[0110] QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPP GKGLEWIGSIYYSGSTYYNPSLKSRVTISSVDTSKNQFSLKLSSVTA ADTAVYYCARLDPFDYWGRGTLVTVSS(SEQ ID NO:2);

[0111] (3)U01 VH

[0112] OR

[0113] A sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity to the aforementioned sequences, provided that the target binding domain binds to a citrullinated antigen as described herein.

[0114] The target binding region may comprise a VL sequence selected from the group consisting of:

[0115] (1)SBT01 VL(M)

[0116] SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQA PMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQV YDRKTDHQVFGPGTTVTVL(SEQ ID NO:3);

[0117] (2)SBT01 VL(G)

[0118] SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQA PVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQV WDSSSDHQVFGTGTKVTVL(SEQ ID NO:4);

[0119] (3)U01 VL

[0120] OR

[0121] A sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity to the aforementioned sequences, provided that the target binding domain binds to a citrullinated antigen as described herein.

[0122] In another embodiment, the antigen binding region comprises a scFV comprising one or more VH domains comprising the amino acid sequence of a VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the antigen binding region comprises a scFV comprising an amino acid sequence having at least any one 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.

[0123] 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 a scFV comprising an amino acid sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 3 or SEQ ID NO: 4, provided that the scFV domain binds to a citrullinated antigen described herein.

[0124] 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 any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 1 or SEQ ID NO: 3, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 is placed between the VH and VL domains.

[0125] 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 any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 1 and SEQ ID NO: 4, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 is placed between the VH and VL domains.

[0126] 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 any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 2 and SEQ ID NO: 3, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 is placed between the VH and VL domains.

[0127] 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 any one of 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, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 is placed between the VH and VL domains.

[0128] 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 any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 49 and SEQ ID NO: 50, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, a linker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 is placed between the VH and VL domains.

[0129] In another embodiment, the antigen binding region comprises a scFV comprising an amino acid sequence selected from the group consisting of:

[0130] SBT01G-VHVL-GGGSx3 connector-pSB_0149

[0131] HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSSGGGGSG GGGSGGGGSSYVLTQPPSVSVVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR(SEQ ID NO:5) (GGGSx3 adapter in bold); SBT01G-VHVL-Whitlow 218 adapter-pSB_0158

[0132] HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSSGSTSGSGKPGSGEGSTKGSYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR(SEQ ID NO:6) (Whitlow 218 linker is in bold);

[0133] SBT01G-VHVL-AB pur linker-pSB_0159

[0134] HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVT AADTAVYYCARLDPFDYWGRGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARSYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR(SEQ ID NO:7) (AB pur linker is in bold);

[0135] U01-VHVL-Ab pur linker

[0136] EVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARAIQMTQSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIKRAAA(SEQ ID NO:51) (AB pur linker is in bold); or

[0137] A sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity to the aforementioned sequences, provided that the target binding domain binds to a citrullinated antigen as described herein.

[0138] For example, the linker may comprise the following sequence:

[0139] GGGGSGGGGSGGGGS (SEQ ID NO: 20) GGGSx3 linker; or

[0140] GSTSGSGKPGSGEGSTKG (SEQ ID NO: 21) Whitlow 218 linker; or

[0141] ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 22) AB pur linker.

[0142] Optionally, any of the foregoing sequences may include sets of CDRs from the above-described VH and VL domains.

[0143] C. Hinge region

[0144] In some embodiments, the hinge region of the disclosed CAR can be selected from the extracellular domains of CD8, CD4 or CD28, the Fc region of an IgG1 antibody, or the extracellular domain of any TLR receptor, which are known to those skilled in the art and can be found in the GenBank database.

[0145] For example, the hinge region may comprise the following sequence:

[0146] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(SEQ ID NO:30)(CD28); or

[0147] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:15)(CD8); or

[0148] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned hinge region sequence.

[0149] D. Transmembrane domain

[0150] The transmembrane domain may include a transmembrane domain of an immunoglobulin family receptor (such as CD8). The intracellular domain may be selected from any transmembrane molecule on a T cell. For example, the transmembrane (TM) domain of the disclosed CAR may include a TM domain of CD2, CD3, CD16, CD32, CD64, CD28, CD247, 4-1BBL, CD4 or CD8.

[0151] For example, a transmembrane domain may comprise the following sequence:

[0152] FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:16); or

[0153] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:17); or

[0154] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned hinge region sequence.

[0155] E. Signal transduction domain

[0156] 1.CD3ζ signal transduction domain

[0157] In some embodiments, the signal transduction domain comprises a CD3 zeta signaling domain. The CD3 zeta signaling domain of the disclosed CAR molecule may comprise a CD3 zeta amino acid sequence, e.g., a signal transduction domain of CD3 zeta.

[0158] For example, a CD3ζ signaling domain may comprise the following sequence:

[0159] or

[0160] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned CD3 signaling domain sequence.

[0161] For example, the CD3 zeta signaling domain may include amino acids 21-163, 31-142, 68-89 and / or 138-158 of the sequence shown 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).

[0162] 2. Fc receptor signal transduction domain

[0163] In some embodiments, the signal transduction domain comprises an Fc signaling domain. The Fc signaling domain can be any of Fc-α, Fc-γ, Fc-ε, Fc-μ, and Fc-δ receptors. For example, the Fc receptor signaling domain can comprise amino acids involved in interacting with Src (e.g., Fgr, Fyn, Hck, Lyn, Yes, and Src) and ZAP-70 family kinases, such as one or more ITAM domains (see, 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, e.g., from any of Fc-α, Fc-γ, Fc-ε, Fc-mu, and Fc-δ receptors, or substantially the same.

[0164] The sequence can also be found as follows:

[0165]

[0166]

[0167] F. Co-stimulatory domain

[0168] In addition to the signal transduction domains of CD3ζ or Fc receptors, CAR of the present disclosure may include one or more costimulatory domains.Costimulatory domains may 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 may contain two or more costimulatory signaling domains (e.g., CD28 and 4-1BB).

[0169] The costimulatory domain can be located between the signal transduction domain and the transmembrane region.

[0170] In certain embodiments, the CD28 costimulatory domain may comprise the following sequence:

[0171] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:29); or

[0172] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence.

[0173] In certain embodiments, the 41BB costimulatory domain may comprise the following sequence:

[0174] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:28); or

[0175] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence.

[0176] III. Nucleic Acids

[0177] A. Nucleic acid encoding CAR

[0178] Disclosed herein are nucleic acid molecules (polynucleotides) comprising nucleotide sequences encoding the disclosed CAR. The nucleic acid of the disclosed CAR may be in the form of DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if a single strand is present, may be a coding strand or a non-coding (antisense) strand. RNA includes mRNA, siRNA, sRNA, ssRNA, etc.

[0179] For example, the nucleic acid may comprise a nucleotide sequence encoding a polypeptide of any one 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:

[0180] (1)SBT01 VH(M)

[0181] CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAACAC CCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCTCGAGC(SEQ ID NO:8);

[0182] (2) SBT01 VH(G)

[0183] CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATC OR

[0184] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence.

[0185] In certain embodiments, the nucleotide sequence encoding the VL domain comprises:

[0186] (1)SBT01 VL(M)

[0187] TCCTATGTCCTGACTCAGCCACCCTCAGTGTCGCTGGCCCCGGGAGAGACGGCCACAATTACTTGTGGTGGAGACGACATTGAAAATCAAAATGTCAACTGGTATCAGCAGAAGTCAGGTCAGGCCCCTATGCTGCTCATCTTCTTTGATACCAGACGGCCCTCAGGGATCCCGGAGCGATTCTCTGGCTCCAGGTCTGAGGACACGGCCAACCTGACCATCACCAGGGTCGAGGCCGGGGATGACGCCGACTATTTCTGTCAGGTGTATGATAGGAAGACTGATCACCAAGTCTTCGGACCTGGGACCACGGTCACCGTCCTA(SEQ ID NO:10);

[0188] (2)SBT01 VL(G)

[0189] TCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA(SEQ ID NO:11); or

[0190] a sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the foregoing sequence.

[0191] 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 any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 8 or SEQ ID NO: 9, provided that the scFV domain binds to a citrullinated antigen described herein.

[0192] 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 one 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.

[0193] 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 any one of 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 described herein.

[0194] 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 any one of 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 described herein.

[0195] 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 any one of 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 described herein.

[0196] 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 any one of 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.

[0197] In another embodiment, the nucleic acid molecule encodes a scFv molecule and has the nucleotide sequence:

[0198] SBT01G-VHVL-GGGSx3 connector-pSB_0149

[0199] CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAAC ACCCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCTCGAGC GGTGGCGGTGGCTCGGGCGGTGGT GGGTCGGGTGGCGGCGGATCTTCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCA GGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC(SEQ ID NO:12) (GGGSx3 connector underlined); or

[0200] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence.

[0201] In another embodiment, the nucleic acid molecule encodes a scFv molecule and has the nucleotide sequence:

[0202] SBT01G-VHVL-Whitlow 218 connector-pSB_0158

[0203] CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAAC ACCCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCTCGAGC GGAAGCACGAGTGGTTCAGGCAAA CCGGGTTCCGGTGAAGGTTCAACAAAAGGTTCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCA GGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC(SEQ ID NO:13) (Whitlow218 connector underlined); or

[0204] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence.

[0205] In another embodiment, the nucleic acid molecule encodes a scFv molecule and has the nucleotide sequence:

[0206] SBT01G-VHVL-AB pur connector-pSB_0159

[0207] CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAAC ACCCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCTCGAGC GCCTCTAGCGGGGGGAGCACATCA GGAAGCGGCAAGCCCGGTAGCGGCGAAGGCTCCTCTGGCAGCGCCCGCTCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAG GGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC(SEQID NO:14) (AB pur connector is underlined); or

[0208] A sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence.

[0209] Optionally, these may include sequences encoding sets of CDRs from the VH and VL domains described herein.

[0210] Polynucleotide variants may contain changes in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain changes that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded CAR polypeptides. In some embodiments, polynucleotide variants are included in changes that do not produce any changes in the amino acid sequence. In some embodiments, due to the degeneracy of the genetic code, polynucleotide variants contain "silent" substitutions. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize the codon expression of a particular host.

[0211] In some embodiments, the polynucleotides described herein are isolated.

[0212] The polynucleotide encoding CAR is 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.

[0213] B. Expression Constructs and Vectors

[0214] The polynucleotide encoding the CAR of the present disclosure may include a regulatory element operably connected to the nucleotide sequence encoding the CAR. For example, the polynucleotide may include one or more transcriptional regulatory elements, such as a promoter or enhancer, which cause the sequence encoding the CAR to be expressed in the cell when the polynucleotide is present in the cell.

[0215] Nucleic acids disclosed herein can be incorporated into vectors capable of transfecting cells. Such vectors include, but are not limited to, viral vectors, plasmids, and microvesicles, such as liposomes. Exemplary viral vectors are adenovirus vectors Ad, AAV, slow viruses, and vesicular stomatitis virus (VSV) and retroviruses. Slow viruses are a genus of the Retroviridae family, and include HIV, SIV, and FIV. Slow viruses can deliver large amounts of genetic material into the DNA of host cells. They can infect cells that do not divide.

[0216] IV. Cells

[0217] In some embodiments, the recombinant (host) cell has a nucleic acid molecule encoding a disclosed CAR, wherein the nucleic acid molecule may further include an expression control sequence operably connected to a nucleotide sequence encoding CAR. The assembled CAR (by synthesis, site-directed mutagenesis or another method known to those skilled in the art), the nucleic acid molecule encoding the disclosed CAR may be inserted into an expression vector and operably connected to an expression control sequence suitable for expressing the disclosed CAR in the desired host. The correct assembly can be confirmed by nucleotide sequencing, restriction maps and / or expression of CAR polypeptides in a suitable host. As is well known in the art, in order to obtain a high expression level of a transfected gene in a host, the gene must be operably connected to a transcription and translation expression control sequence that is functional in a selected expression host.

[0218] The present disclosure also provides cells (e.g., recombinant cells) comprising a nucleic acid molecule encoding a CAR and / or expressing a CAR.

[0219] The nucleic acid molecules encoding the disclosed CAR can be delivered to host cells by plasmids or viral vectors known to those skilled in the art, including but not limited to T cells, B cells, bone marrow progenitor cells, macrophages, etc. The resulting recombinant (host) cells may include but are not limited to T cells, CD4 T cells, Treg cells, CD8αT cells, CD8βT cells, T helper cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages and dendritic cells, T memory stem cells, and cells expressing class I or class II MHC known to those skilled in the art. In some embodiments, as known to those skilled in the art, the recombinant (host) cells with nucleic acid molecules encoding the disclosed CAR can be selected from the bone marrow progenitor cells of the group consisting of common bone marrow progenitor cells, granulocyte macrophage progenitor cells, megakaryocyte erythrocyte progenitor cells, granulocyte progenitor cells and monocyte progenitor cells. In some embodiments, bone marrow cells are autologous or allogeneic cells.

[0220] In some embodiments, the cells expressing the CARs of the present disclosure are Treg cells. reg T cells are cells belonging to a specific subset of T cells whose function is to suppress immune responses and thus maintain homeostasis and self-tolerance. reg It can inhibit T cell proliferation and cytokine production and play a key role in preventing autoimmunity. reg The characteristic of T is the expression of FoxP3. reg Surface markers include CD4, CD25high (high molecular weight density), and CD127low (low molecular weight density). Mouse and human Tregs express GITR / AITR and CTLA-4. Human CD4+FoxP3+Treg cells can be divided into three subsets: (1) CD45RA+CD25+FoxP3l0w resting Treg cells, (2) CD45RO+CD25highFoxP3high activated Treg cells, and (3) CD45RO+CD25+FoxP3low non-suppressive effector T cells (Teff) that produce pro-inflammatory cytokines.

[0221] The cells transformed with the nucleic acids disclosed herein can be cells taken from the subject to which the recombinant cells are administered. In this way, the problem of allogeneic immune responses can be alleviated.

[0222] The cells can be expanded in vitro prior to administration to a subject.

[0223] Treg cells incorporating nucleic acids expressing CARs of the present disclosure can express these CARs and be used in the methods of treating hidradenitis suppurativa described herein.

[0224] In addition, the protein produced by the conversion / recombination host can be purified according to any suitable method. These methods include chromatography (for example, ion exchange, affinity and fractionation column chromatography), centrifugation, differential dissolution or by any other standard techniques for protein purification. Affinity tags (such as hexa-histidine, maltose binding domain, influenza virus capsid sequence and glutathione-S-transferase) can be attached to protein to be easily purified by suitable affinity column. In some embodiments, the technology such as proteolysis, high performance liquid chromatography (HPLC), nuclear magnetic resonance and x-ray crystallography can also be used to physically characterize protein.

[0225] V. Composition

[0226] Also disclosed are pharmaceutical compositions comprising recombinant cells having nucleic acid molecules encoding the disclosed CAR polypeptides and / or expressing the disclosed CAR polypeptides and a pharmaceutically acceptable carrier, as well as methods for treating hidradenitis suppurativa.

[0227] As used herein, the term "pharmaceutical composition" refers to a composition comprising a pharmaceutical compound (eg, a drug or recombinant Treg cells as described herein) and a pharmaceutically acceptable carrier.

[0228] As used herein, the term "pharmaceutically acceptable" refers to a carrier that is compatible with the other ingredients of a pharmaceutical composition and can be safely administered to a subject. The term is used synonymously with "physiologically acceptable" and "pharmacologically acceptable". According to the present disclosure, pharmaceutical compositions and their preparation and use techniques are known to those skilled in the art. For a detailed list of suitable pharmaceutical compositions and their administration techniques, 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.

[0229] Pharmaceutically acceptable carriers are typically sterile, at least for human use. Depending on the route of administration, pharmaceutical compositions typically include agents for buffering and storage preservation, and may include buffers and carriers for appropriate delivery. Examples of pharmaceutically acceptable carriers include, but are not limited to, normal (0.9%) saline, phosphate buffered saline (PBS), Hank's balanced salt solution (HBSS), and various electrolyte solutions, such as PlasmaLyte ATM (Baxter).

[0230] The pharmaceutical compositions can be formulated for any route of administration, including mucosal (eg, nasal, sublingual, vaginal, buccal, or rectal), parenteral (eg, subcutaneous, intravenous, intramuscular, or intraarterial injection, bolus or infusion), oral, or transdermal.

[0231] Injectable (e.g., intravenous) compositions may include a solution of a composition suspended in an acceptable carrier (such as an aqueous carrier). Any of a variety of aqueous carriers may be used, such as water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% glucose, etc., and glycoproteins such as albumin, lipoprotein, globulin, etc., may be included for enhancing stability. Typically, common buffered saline (135-150 mM NaCl) will be used. The composition may contain pharmaceutically acceptable auxiliary substances to approach physiological conditions, such as pH regulators and buffers, tension regulators, wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the composition may be formulated into a kit for intravenous administration.

[0232] Preparations suitable for parenteral administration such as, for example, by intraarticular (in a joint), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes include aqueous and nonaqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents and solutes that make the preparation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions, which may include suspending agents, solubilizing agents, thickening agents, stabilizers and preservatives. Injection solutions and suspensions may also be prepared from sterile powders, granules and tablets. In the practice of the present invention, the composition may be administered, for example, by intravenous infusion, topically, intraperitoneally, intravesically or intrathecally. The preparation of the composition may be present in a sealed container, such as an ampoule and a vial, for unit dose or multiple doses.

[0233] The cells can be cryopreserved. Cryopreservation can include formulating the cells with a cryopreservative such as DMSO. Commercially available media include, for example, and

[0234] The composition can be formulated into a dosage form for administration. The term "dosage form" refers to a specific form of the drug, and depends on the route of administration. Examples of dosage forms include, but are not limited to, dispersions; suppositories; ointments; cataplasms (mud compresses); 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 patients, 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 patients; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to patients.

[0235] The terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on many factors, including the frequency of administration; the size and tolerance of the individual; the severity of the condition; the risk of side effects; the route of administration; and the imaging modality of the detectable label, if any. Those skilled in the art will recognize that the dose can be modified according to the above factors or as the treatment progresses.

[0236] Pharmaceutical preparations can be packaged or prepared into unit dosage forms. In such forms, the preparation is subdivided into unit doses containing appropriate amounts of active ingredients, for example, according to the dosage of the therapeutic agent or the concentration of the composition. The unit dosage form can be a packaged preparation that contains discrete amounts of the preparation. If desired, the composition may also contain other compatible therapeutic agents.

[0237] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a complementarity determining region (CDR) from a VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a CDR from a VL domain of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising CDRs from VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 3. In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising CDRs from VH and VL domains of SEQ ID NO: 1 and SEQ ID NO: 4. In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising CDRs from VH and VL domains of SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising CDRs from SEQ ID NO:2 and SEQ ID NO:4 VH and VL domains. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0238] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, and the CAR polypeptide comprises a complementary determining region (CDR) from a VH domain of SEQ ID NO:49. In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, and the CAR polypeptide comprises a CDR from a VL domain of SEQ ID NO:50. In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, and the CAR polypeptide comprises CDRs from VH and VL domains of SEQ ID NO:49 and SEQ ID NO:50. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0239] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising CDRs from VH and VL domains 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 an amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising an amino acid sequence of SEQ ID NO: 36, and the VL domain of the CAR polypeptide comprises a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 39, a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 comprising an 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 CD8α T cell, a CD8β 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 cell is a Treg cell.

[0240] In another embodiment, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a scFV containing one or more VH domains, the VH domain 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, which comprises an amino acid sequence having at least any one 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 the citrullinated antigen described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0241] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a scFV containing 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 any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:3 or SEQ ID NO:4, provided that the scFV domain is bound to the citrullinated antigen described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0242] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a scFV containing SEQ ID NO: 1 and SEQ ID NO: 3 VH and VL domains. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 1 or SEQ ID NO: 3, provided that the scFV domain is bound to the citrullinated antigen described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0243] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a scFV containing SEQ ID NO: 1 and SEQ ID NO: 4 VH and VL domains. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 1 and SEQ ID NO: 4, provided that the scFV domain is bound to the citrullinated antigen described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0244] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a scFV containing SEQ ID NO:2 and SEQ ID NO:3 VH and VL domains. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:2 and SEQ ID NO:3, provided that the scFV domain is bound to the citrullinated antigen described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0245] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a scFV containing SEQ ID NO:2 and SEQ ID NO:4 VH and VL domains. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any one of 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 is bound to the citrullinated antigen described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0246] In some embodiments, the composition of the present invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide, the CAR polypeptide comprising a scFV containing SEQ ID NO:49 and SEQ ID NO:50 VH and VL domains. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any one of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:49 and SEQ ID NO:50, provided that the scFV domain binds to the citrullinated antigen described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8αT cell, a CD8βT 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 cell is a Treg cell.

[0247] VI. How to use

[0248] T cells (particularly Treg cells expressing the CAR disclosed herein) can be used to treat hidradenitis suppurativa. The method of use comprises administering an effective amount of the pharmaceutical composition of the present disclosure to a subject in need thereof, such as a subject suffering from hidradenitis suppurativa.

[0249] 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 health care provider, such as a physician or nurse. Subjects include mammals, such as humans and non-human primates, such as monkeys, as well 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 treatment regimen, etc. The term "hidradenitis suppurativa subject" refers to an individual diagnosed with hidradenitis suppurativa. Hidradenitis suppurativa patients may include individuals who have not been treated, individuals who are currently being treated, individuals who have been treated, and individuals who have stopped treatment.

[0250] As used herein, the terms "effective amount", "effective dose" and "therapeutically effective amount" refer to the amount of a medicament sufficient to produce a desired response, such as reducing or eliminating the signs or symptoms of a condition or improving a condition. In some embodiments, an "effective amount" is an amount that treats (including preventing) one or more symptoms and / or potential causes of any condition or disease and / or prevents disease progression. For example, for a given parameter, a therapeutically effective amount will show an increase or decrease in the therapeutic effect by at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100%. The therapeutic efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times or more than a control.

[0251] The pharmaceutical composition can be administered by any suitable route, including but not limited to intravenous, subcutaneous, intramuscular or intraperitoneal routes. The example of administering the pharmaceutical composition includes storing the composition of 10 mg / ml in a sterile isotonic saline solution for injection at 4°C and diluting it in 100 ml or 200 ml of 0.9% sodium chloride for injection before administering it to the patient. The pharmaceutical composition is administered by intravenous infusion for a time course of 1 hour at a dosage of 0.2 mg / kg and 10 mg / kg. In other embodiments, the pharmaceutical composition is administered by intravenous infusion within a time period between 15 minutes and 2 hours. In other embodiments, the administration procedure is by subcutaneous rapid bolus injection.

[0252] The dosage of the composition is selected so that effective treatment is provided to the patient, and is less than 0.1mg / kg body weight in the range of about 25mg / kg body weight, or in the range of 1mg-2g / patient. In some cases, the dosage range is 1-100mg / kg, or about 50mg-8000mg / patient. According to the pharmacokinetics (for example, the half-life of the composition in circulation) and the pharmacodynamic response (for example, the duration of the composition therapeutic effect) of the composition, it is possible to repeat the administration with an appropriate frequency, and the frequency can be in the range of once a day to once every three months. In some embodiments, the half-life in vivo between about 7 days and about 25 days and the composition administration once a week to once every 3 months are repeated.

[0253] Administration can be cyclical. Depending on the route of administration, the dose can be administered, for example, once every 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, or 28 days or longer (e.g., once every 2 months, 3 months, 4 months, or 6 months). In some cases, administration is more frequent, for example, 2 or 3 times a day. As will be appreciated by those skilled in the art, the patient can be monitored to adjust the dose and frequency of administration based on the progress of treatment and any adverse side effects.

[0254] Thus, in some embodiments, additional administrations are dependent on the patient's progress, e.g., monitoring the patient between administrations. For example, after the first administration or first round of administrations, the patient's symptom relief rate can be monitored.

[0255] In certain embodiments, the T cells described herein are administered to a skin lesion in a subject suffering from hidradenitis suppurativa.

[0256] Exemplary methods of the present disclosure include isolating T lymphocytes from a biological sample obtained from a subject. Such T cells can be separated by immunoaffinity, for example, using solid support-derived and anti-CD4 antibodies. CD4+T regulatory cells (Treg) can be separated from non-Treg cells according to their marker spectrum. Treg cells are CD4+, CD25+, CD127lo. Non-Treg cells are: CD4+, CD25+ and CD127+. The separated Treg cells are then transfected with an expression vector encoding a chimeric antigen receptor (CAR) disclosed herein. The transfected cells are amplified. The amplified cells are administered to the subject.

[0257] VII. Kit

[0258] As used herein, the term "kit" refers to a group of articles intended to be used together. The kit may optionally include reference reagents and / or instructions for use thereof. The kit may further include a transport container suitable for accommodating a container, such as a vial containing a composition disclosed herein. The kit may include a container containing a group of articles.

[0259] The kit of the present disclosure may include a pharmaceutical composition as described herein, contained in a container, such as a bag or bottle for intravenous administration. The kit may also include a fluid conduit with a drip chamber, such as a plastic tube. The drip chamber may be communicated with an intravenous needle through the fluid conduit. The fluid conduit may also include one or more Y-shaped tees and a roller clamp.

[0260] VIII. Enumerated Embodiments

[0261] 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, wherein

[0262] The modified Treg cells express a chimeric antigen receptor (CAR), which comprises an antigen binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain,

[0263] The antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof, and

[0264] The modified Treg cells are CD4+, CD25+ and CD127lo.

[0265] 2. A method as described in embodiment 1, wherein the antigen binding domain of the CAR specifically binds to citrullinated vimentin.

[0266] 3. A method as described in embodiment 1, wherein the antigen binding domain of the CAR binds to all three of the following: (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptide fragments thereof.

[0267] 4. The method of embodiment 3, wherein the antigen binding domain of the CAR comprises a VH domain and a VL domain, wherein:

[0268] (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

[0269] (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.

[0270] 5. The method of embodiment 3, wherein the antigen binding domain of the CAR comprises a VH domain and a VL domain, wherein:

[0271] (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

[0272] (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.

[0273] 6. A method as described in any one of embodiments 1 to 5, wherein the intracellular signaling domain of the CAR is derived from CD3-ζ.

[0274] 7. A method as described in any one of embodiments 1 to 6, wherein the at least one co-stimulatory domain of the CAR comprises a co-stimulatory 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, optionally wherein the at least one co-stimulatory domain comprises a CD28 and / or 4-1BB co-stimulatory domain.

[0275] 8. A method as described in any one of embodiments 1 to 7, 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.

[0276] 9. A method as described in any one of embodiments 1 to 8, wherein the transmembrane domain of the CAR is a CD8 transmembrane domain or a CD28 transmembrane domain.

[0277] 10. A method as described in any one of embodiments 1 to 9, wherein the hinge domain of the CAR is a CD8 hinge domain or a CD28 hinge domain.

[0278] 11. A method as described in any one of embodiments 1 to 10, wherein the CAR also comprises a signal peptide.

[0279] 12. A method as described in embodiment 11, wherein the signal peptide of the CAR is a CD8 signal peptide or a GM-CSF signal peptide.

[0280] 13. A method as described in any one of embodiments 1 to 12, wherein the antigen binding domain of the CAR comprises a single-chain fragment.

[0281] 14. The method of embodiment 13, wherein the single-chain fragment comprises a single-chain variable fragment (scFv).

[0282] 15. The method of embodiment 14, wherein the scFv fragment comprises:

[0283] (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

[0284] (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.

[0285] 16. The method of embodiment 14, wherein the scFv fragment comprises:

[0286] (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

[0287] (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.

[0288] 17. The method of embodiment 14, wherein the scFv fragment comprises:

[0289] (a) a VH selected from the following:

[0290] (1)SBT01 VH(M)

[0291] or

[0292] (2) SBT01 VH(G)

[0293] and

[0294] (b) a VL selected from the following:

[0295] (1)SBT01 VL(M)

[0296] or

[0297] (2)SBT01 VL(G)

[0298] SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQA PVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQV WDSSSDHQVFGTGTKVTVL (SEQ ID NO: 4).

[0299] 18. The method of any one of embodiments 1 to 17, wherein the VH-VL fragments are connected by a linker selected from the group consisting of: (i) GGGSx3 (SEQ ID NO: 20), (ii) Whitlow 218 (SEQ ID NO: 21), and (iii) AB Pur (SEQ ID NO: 22).

[0300] 19. The method of embodiment 18, wherein the scFv comprises the following amino acid sequence:

[0301] (a) the SBT01G-VHVL-GGGSx3 linker of SEQ ID NO: 5; or

[0302] (b) the SBT01G-VHVL-Whitlow 218 linker of SEQ ID NO: 6; or

[0303] (c) the SBT01G-VHVL-AB pur linker of SEQ ID NO: 7; or

[0304] (d) U01-VHVL-AB pur linker of SEQ ID NO:51.

[0305] 20. The method of any one of embodiments 1 to 19, wherein the modified Treg cells are human T cells and the subject is a human patient.

[0306] 21. The method of any one of embodiments 1 to 20, wherein the modified Treg cells are primary T cells.

[0307] 22. The method of any one of embodiments 1 to 21, wherein the pharmaceutical composition is administered intravenously.

[0308] 23. A method of treating a subject suffering from hidradenitis suppurativa, the method comprising:

[0309] (a) isolating T cells from a biological sample obtained from the subject;

[0310] (b) T cells enriched for T regulatory cells (Treg);

[0311] (c) transfecting the enriched Treg cells with an expression vector encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain to produce modified Treg cells expressing the CAR, wherein

[0312] The antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof, and

[0313] The modified Treg cells are CD4+, CD25+ and CD127lo;

[0314] (d) expanding the modified Treg cells; and

[0315] (e) administering the modified Treg cells to the subject.

[0316] 24. A method as described in embodiment 23, wherein the amplification comprises using anti-CD3 / CD28 coated beads.

[0317] 25. The method of embodiment 23, wherein the amplification does not comprise the use of anti-CD3 / CD28 coated beads.

[0318] 26. The method of any one of embodiments 23 to 25, wherein the transfection is performed by using a viral vector, electroporation, heat shock, viral phage, ultrasonic treatment, or calcium phosphate.

[0319] 27. The method of any one of embodiments 23 to 26, further comprising administering one or more anti-inflammatory and / or therapeutic agents to the subject.

[0320] 28. The method of embodiment 27, wherein the one or more anti-inflammatory agents comprise antibodies that inhibit pro-inflammatory cytokines.

[0321] 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.

[0322] 30. The method of any one of embodiments 23 to 29, further comprising the limitations of any one of embodiments 2 to 22.

[0323] 31. Use of a plurality of modified regulatory T (Treg) cells in the manufacture of a medicament for treating hidradenitis suppurativa in a subject, wherein

[0324] The modified Treg cells express a chimeric antigen receptor (CAR), which comprises an antigen binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain,

[0325] The antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof, and

[0326] The modified Treg cells are CD4+, CD25+ and CD127lo.

[0327] 32. The use according to embodiment 31, and also including the limitations of any one of embodiments 2 to 22.

[0328] 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 the plurality of modified regulatory T (Treg) cells to the subject, wherein

[0329] The modified Treg cells express a chimeric antigen receptor (CAR), which comprises an antigen binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain,

[0330] The antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof, and

[0331] The modified Treg cells are CD4+, CD25+ and CD127lo.

[0332] 34. A plurality of modified Treg cells for use as described in embodiment 33, and further comprising the limitations of any one of embodiments 2 to 22.

[0333] Example

[0334] 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 (peptidyl arginine 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).

[0335] Example 1: Detection of citrullinated antigens at sites of inflammation in patients with hidradenitis suppurativa and rheumatoid arthritis

[0336] To assess the presence of citrullinated antigens in biopsy samples from HS patients, an immunohistochemistry (IHC) assay was developed. The IHC assay employed 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). The assay was optimized for use with fixed tissues using formalin-fixed, paraffin-embedded positive and negative control cell lines. The S01-mIgG2a Ab produced positive staining on SKNBE2 cells overexpressing PAD2 and PAD4 (positive control cell line) and did not react with the negative control cell line SKNBE2 WT. In all staining runs, the specific reaction of the S01-mIgG2a Ab was accompanied by the negative control antibody, which showed a lack of specific reaction. To detect binding in patient biopsy samples, S01-mIgG2a Ab at a concentration of 12 μg / mL was applied to HS skin sections (n=58) and normal skin sections from adjacent samples (n=10) and then visualized using the EnVision FLEX system on a Dako Autostainer Link 48. Additional sections from all tested samples were stained with hematoxylin and eosin (H&E) to assess tissue quality and determine the extent of inflammatory cell infiltration. In addition, all samples were stained with a negative control mouse IgG antibody to confirm the specificity of S01-mIgG2a Ab staining.

[0337] result

[0338] Strong reactivity to S01-mIgG2a Ab staining was observed in multiple cell types in HS skin biopsy sections, including squamous epithelial cells, synovial lining cells, and immune cells, as well as extracellular staining, particularly in areas of necrotic tissue. In contrast, little staining was observed in normal skin sections.

[0339] Figure 1A to Figure 1B showed that citrullinated protein (CitP) was present in samples of hidradenitis suppurativa (HS) lesions and synovial tissue samples from rheumatoid arthritis (RA) patients, but was absent in samples of normal skin (non-HS) and normal synovial tissue (non-RA).

[0340] FIG. 2A to FIG. 2B showed higher levels of inflammation, including higher numbers of CitP+ inflammatory cells in lesional HS skin compared with adjacent normal skin. Figure 2C As shown in , there are higher numbers of viable CitP+ non-inflammatory cells in lesional HS skin compared to adjacent normal skin. Thus, immunohistochemical evaluation showed that there is increased inflammation and increased expression of the target antigen (CitP) in lesional HS skin.

[0341] Example 2: Detection of citrullinated antigens in sera from patients with hidradenitis suppurativa and rheumatoid arthritis

[0342] To evaluate the presence of citrullinated proteins (CitP) in the serum of patients with HS and RA, cell-based assays and ELISA were performed using CV-CAR transduced cells and anti-CV-Ab, respectively. In addition, the levels of VICM in serum were measured. The levels of C-reactive protein (CRP) and anti-citrullinated protein antibodies (ACPA) were measured in HS and normal serum samples.

[0343] Jurkat-FF-luciferase transduction. Approximately 24 × 10 6 Jurkat-FF-luc cells were pelleted and resuspended in 24 ml RPMI containing protamine sulfate and virus at an MOI of 3 to express CV-CAR. The cells were mixed and 4 ml was aliquoted into each well of a 6-well plate. The plate was then spun and placed in an incubator overnight. The cells were pelleted and re-seeded in 25 ml RPMT in a T75 flask.

[0344] Stimulants. The transduced cells were pelleted and resuspended in RPMI and placed in the wells of a black / white plate. Serum samples from HS patients and RA patients were thawed, vortexed, and diluted in RPMI before being added to the plates containing the transduced cells. Normal serum samples served as controls. Alternatively, citrullinated or non-citrullinated proteins were added to the plates as stimulants. Cells were cultured at 37°C in the presence of stimulants.

[0345] 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.

[0346] result

[0347] like Figure 3 As shown in , the CV-CAR reporter cell line was activated by citrullinated vimentin, fibrinogen, and filaggrin, but not by their non-citrullinated counterparts.

[0348] like FIG. 4A to FIG. 4B As shown in , the CitP level in the serum from patients with HS or RA was higher than that in the serum from normal control subjects. Figure 4C As shown in , the levels of citrullinated and MMP-degraded vimentin (VICM) in sera from patients with HS or RA were higher than those in sera from normal control subjects.

[0349] like FIG. 5A to FIG. 5B As shown in , serum samples of HS patients had elevated CRP and ACPA levels compared with normal serum samples.

[0350] Fig. 6A A schematic diagram of a cell-based detection assay for measuring the presence of citrullinated antigens in serum is provided. Figure 6B It is shown that serum samples from HS patients have elevated levels of citrullinated antigens compared to normal serum samples. Strikingly, 42 out of 65 HS serum samples were found to have Cit-P levels above the detection limit indicated by the dotted line.

[0351] Example 3: Isolation and characterization of Treg cells from hidradenitis suppurativa patients and healthy donors

[0352] Treg cells recovered from whole blood samples obtained from healthy donors and HS patients were immunophenotyped.

[0353] Treg isolation. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. Treg cells were then isolated using FACS by gating on CD4+CD25+CD127lo cells.

[0354] 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 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 staining buffer. The stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry.

[0355] result

[0356] like FIG. 7A to FIG. 7D As shown in Figure 2, HS patients have Treg frequencies and phenotypes comparable to healthy donors. The CD4 and CD8 T cell subset profiles of HS patients were similar to those of HD controls. Although Tregs from HS patients had the same frequencies as Tregs from healthy donors, Tregs from HS patients had higher absolute counts. This difference was driven by higher pan-CD4 T cell frequencies and counts.

[0357] Example 4: Treg cell isolation, transduction, expansion and characterization

[0358] Treg cells were recovered from whole blood samples of healthy donors and HS patients.

[0359] Isolation and tissue culture of Treg. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. CD25+ cells were enriched by positive selection. Treg cells were isolated using FACS by gating CD4+CD25+CD127lo cells. After separation, 140,000 Treg cells in a 48-well tissue culture plate were expanded.

[0360] Transduction of primary Tregs: Primary Treg cells were transduced with CV-CAR constructs on day 2 of expansion in the presence of protamine sulfate.

[0361] Flow cytometry and FACS analysis: Treg-expressing CV-CARs were stained for transcription factors FoxP3 and Helios on day 14 of expansion. Cells were fixed and permeabilized using eBiosciences FoxP3 transcription factor buffer set (eBiosciences).

[0362] result

[0363] like FIG. 8A to FIG. 8BAs shown in , Treg frequencies and recovery rates during the production of CV-CAR Treg cells were comparable between HS and HD patients. This observation applies to patients with moderate and severe HS (Hurley 2 and Hurley 3). Figure 8C ) and Helios (data not shown), CV-CAR Treg cells can be successfully generated from HS patients.

[0364] Example 5: In vitro evaluation of CV-CAR Treg suppressive activity and activation of citrullinated vimentin

[0365] Treg cells were recovered from whole blood samples obtained from healthy donors (N=6) or HS patients (N=7).

[0366] Isolation and tissue culture of Treg. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. CD25+ cells were enriched by positive selection. Next, FACS was used to isolate Treg cells by gating CD4+CD25+CD127lo cells. After separation, 140,000 Treg cells in a 48-well tissue culture plate were expanded.

[0367] Transduction of primary Tregs: Primary Treg cells were transduced with CV-CAR constructs on day 2 of expansion in the presence of protamine sulfate.

[0368] Flow cytometry and FACS analysis: Treg cell cultures were collected and centrifuged at 300 × g for 5 minutes and then resuspended in 1X Flowstain buffer (Invitrogen) containing active 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 staining buffer. The stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry. The expression of Helios and FoxP3 by Treg cells was also evaluated by flow cytometry.

[0369] Treg activation: Uninduced and CV-CAR-expressing Treg cells were cultured in vitro with citrullinated vimentin beads at doses ranging from 1:243 to 1:1 pCVbead:Treg ratios. Treg activation was assessed by measuring the percentage of proliferating cells and CD71 expression.

[0370] Treg suppression assay: Tregs were co-cultured with CD3 / CD28 activated or allogeneic total 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.

[0371] result

[0372] To date, no disease-specific differences in Treg cell isolation, phenotype, or function have been observed, and Tregs from healthy donors and HS patients have similar levels of Helios and FoxP3 (data not shown). FIG. 9A to FIG. 9B and FIG. 10A to FIG. 10B It was shown that Tregs from healthy donors and HS patients had similar capacity to proliferate and activate by CAR stimulators (pCV beads) and similar suppressive activity.

[0373] It should be understood that the description and the accompanying drawings are not intended to limit the present invention to the specific form disclosed, on the contrary, the present invention is intended to cover all modifications, equivalents and substitutes falling within the spirit and scope of the present invention as defined by the appended claims. According to this description, other modifications and alternative embodiments of various aspects of the present invention will be apparent to those skilled in the art. Therefore, the description and the accompanying drawings are only interpreted as illustrative, and are intended to teach those skilled in the art to implement the general manner of the present invention. It should be understood that the forms of the present invention shown and described here are examples as embodiments. Elements and materials can replace those shown and described herein, parts and processes can be reversed or omitted, and certain features of the present invention can be used independently, all of which are apparent to those skilled in the art who benefit from the description of the present invention. Changes can be made to the various elements described herein without departing from the spirit and scope of the present invention as described in the appended claims. The titles used here are only for organizational purposes and are not meant to be used to limit the scope of the description.

[0374] 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. 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, wherein The modified Treg cells express a chimeric antigen receptor (CAR), which comprises an antigen binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain, The antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof, and The modified Treg cells are CD4+, CD25+ and CD127lo.

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

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

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. 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. The method of claim 1, wherein the intracellular signaling domain of the CAR is derived from CD3-ζ.

7. The method of claim 1, wherein the at least one co-stimulatory domain of the CAR comprises a co-stimulatory 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. 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. The method of claim 1, wherein the transmembrane domain of the CAR is a CD8 transmembrane domain or a CD28 transmembrane domain.

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

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

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. 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. The method of claim 14, wherein the scFv fragment comprises: (a) a VH selected from the following: (1)SBT01 VH(M) or (2) SBT01 VH(G) and (b) a VL selected from the following: (1)SBT01 VL(M) or (2)SBT01 VL(G) SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQA PVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQV WDSSSDHQVFGTGTKVTVL (SEQ ID NO: 4).

18. The method of claim 17, wherein the VH-VL fragments are connected by a linker selected from the group consisting of: (i) GGGSx3 (SEQ ID NO: 20), (ii) Whitlow 218 (SEQ ID NO: 21), and (iii) ABPur (SEQ ID NO: 22).

19. The method of claim 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-Whitlow 218 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 claim 1, wherein the modified Treg cells are human T cells and the subject is a human patient.

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

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

23. A method of treating a subject suffering from hidradenitis suppurativa, the method comprising: (a) isolating T cells from a biological sample obtained from the subject; (b) T cells enriched for T regulatory cells (Treg); (c) transfecting the enriched Treg cells with an expression vector encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain to produce modified Treg cells expressing the CAR, wherein The antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof, and 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 claim 23, wherein the amplification comprises the use of anti-CD3 / CD28 coated beads.

25. The method of claim 23, wherein the amplification does not comprise the use of anti-CD3 / CD28 coated beads.

26. The method of claim 23, wherein the transfection is performed by using viral vectors, electroporation, heat shock, viral phage, ultrasonic treatment or calcium phosphate.

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

28. The method of claim 27, wherein the one or more anti-inflammatory agents comprise antibodies that inhibit pro-inflammatory cytokines.

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. The method of any one of claims 23 to 29 further comprising the limitations of any one of claims 2 to 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, wherein The modified Treg cells express a chimeric antigen receptor (CAR), which comprises an antigen binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain, The antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof, and The modified Treg cells are CD4+, CD25+ and CD127lo.

32. The use according to embodiment 31, and further comprising the limitations of 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 the plurality of modified regulatory T (Treg) cells to the subject, wherein The modified Treg cells express a chimeric antigen receptor (CAR), which comprises an antigen binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular signaling domain, The antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof, and The modified Treg cells are CD4+, CD25+ and CD127lo.

34. A plurality of modified Treg cells for use as described in embodiment 33, and further comprising the limitations of any one of claims 2 to 22.

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