Synovial extracellular matrix-specific chimeric antigen receptors for targeting regulatory T cells to treat autoimmune diseases

Abstract

Disclosed herein is a chimeric antigen receptor ("CAR") that comprises an antigen binding site that recognizes citrullinated polypeptide.Citrullinated polypeptides, such as citrullinated vimentin, fibrinogen, and filaggrin, are expressed in the synovium of subjects with rheumatoid arthritis.Furthermore, disclosed is a T cell that expresses such CAR, in particular a Treg cell.The administration of such CAR-T cell is useful in the treatment of not only rheumatoid arthritis but also other diseases associated with citrullinated peptides.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 227,320, filed July 29, 2021, and U.S. Provisional Patent Application No. 63 / 339,361, filed May 6, 2022, each of which is incorporated by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (237752000340SEQLIST.xml; size: 50,843 bytes; and creation date: July 28, 2022) are incorporated herein by reference in their entirety.

[0003] Field The present disclosure relates to chimeric antigen receptors reactive to citrullinated antigens and regulatory T cells expressing the receptors for the treatment of autoimmune diseases. [Background technology]

[0004] background Autoimmune diseases affect a significant number of people. For example, rheumatoid arthritis (RA) is a chronic inflammatory disease that targets peripheral joints, causing bone erosion, impaired mobility, and reduced quality of life. It affects 0.5-1% of the world's population, and the incidence continues to rise. The pathogenesis of RA is primarily localized in the synovial joints, where immune cells, consisting of T cells, B cells, macrophages, and dendritic cells, infiltrate the synovium. Moreover, fibroblast-like synoviocytes present in the sublining layer of the synovium proliferate and contribute to cartilage damage.

[0005] Synovial hyperplasia in rheumatoid arthritis leads to infiltration of the synovium by immune cells and subsequent cartilage damage and bone erosion.

[0006] Currently, there is no cure for RA, as well as many other autoimmune conditions. Patients with RA usually require lifelong treatment, which can be extremely expensive as well as carry the risk of severe side effects in the long term, such as infections and rheumatoid arthritis.

[0007] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate example embodiments and, together with the description, further serve to enable one of ordinary skill in the art to make and use such embodiments, as well as other embodiments that will be apparent to those skilled in the art. The present invention is described in more detail in conjunction with the following drawings: [Brief description of the drawings]

[0008] [Figure 1] Initial data involving the MND promoter and EGFRt backbone showed that the CV CARs BVCA1 and SBT01G responded most robustly to plate-bound full-length CV (n=2).

[0009] [Diagram 2] Assays with soluble full-length CV showed a dose response only for the CV CARs BVCA1 and SBT01G-HL (n=1).

[0010] [Figure 3-1] Binding specificity was demonstrated by assays using soluble bead-bound peptides (n=1). [Figure 3-2] Binding specificity was demonstrated by assays using soluble bead-bound peptides (n=1).

[0011] [Figure 4] MND-SBT01G shows a stronger response than MND-BVCA1 to plate-bound and antibody-captured CV but not to soluble CV.

[0012] [Figure 5-1]Testing of the first lot of synovial fluid from Innovative Research showed that SBT01G produced a more robust response than BVCA1. [Figure 5-2] Testing of the first lot of synovial fluid from Innovative Research showed that SBT01G produced a more robust response than BVCA1.

[0013] [Figure 6-1] Further testing of 15 synovial fluid samples from Swedish patients showed that in those samples that produced a response, SBT01G was more potent than BVCA1. [Figure 6-2] Further testing of 15 synovial fluid samples from Swedish patients showed that in those samples that produced a response, SBT01G was more potent than BVCA1.

[0014] [Figure 7] Primary Treg responses to synovial fluid (SF) demonstrate that SBT01G is more sensitive to SF from RA patients than BVCA1.

[0015] [Figure 8] SBT01G, but not BVCA1, is also capable of responding to plate-bound full-length PAD2 citrullinated fibrinogen.

[0016] [Figure 9] SBT01G CAR, but not BVCA1, as effector and Treg cells respond to citrullinated fibrinogen.

[0017] [Figure 10-1] Both the EF1A and MND promoters demonstrate a functional response by the CV CAR to soluble bead-bound peptides. Thus, the CAR promoter does not affect the Treg phenotype. [Figure 10-2]Both the EF1A and MND promoters demonstrate a functional response by the CV CAR to soluble bead-bound peptides. Thus, the CAR promoter does not affect the Treg phenotype. [Figure 10-3] Both the EF1A and MND promoters demonstrate a functional response by the CV CAR to soluble bead-bound peptides. Thus, the CAR promoter does not affect the Treg phenotype. [Figure 10-4] Both the EF1A and MND promoters demonstrate a functional response by the CV CAR to soluble bead-bound peptides. Thus, the CAR promoter does not affect the Treg phenotype.

[0018] [Figure 11] The scFv linker has little or no effect on the function based on the SBT01G CAR.

[0019] [Figure 12-1] Although SBT01G is expressed by a higher percentage of cells than BVCA1, BVCA1 and SBT01G CAR-T cells have similar profiles of FoxP3 and Helios. [Figure 12-2] Although SBT01G is expressed by a higher percentage of cells than BVCA1, BVCA1 and SBT01G CAR-T cells have similar profiles of FoxP3 and Helios. [Figure 12-3] Although SBT01G is expressed by a higher percentage of cells than BVCA1, BVCA1 and SBT01G CAR-T cells have similar profiles of FoxP3 and Helios.

[0020] [Figure 13] CV-CAR Treg cells (SBT01G), but not non-transduced Treg cells, are activated by citrullinated vimentin (CV), as demonstrated by target antigen-specific increases in proliferation, CD71 expression, and IL-10 secretion.

[0021] [Figure 14-1] CV-CAR Treg cells respond to citrullinated proteins in synovial fluid from the majority of RA patients. In contrast, CV-CAR Treg cells do not respond to synovial fluid from normal controls (subjects without RA). [Figure 14-2] CV-CAR Treg cells respond to citrullinated proteins in synovial fluid from the majority of RA patients. In contrast, CV-CAR Treg cells do not respond to synovial fluid from normal controls (subjects without RA).

[0022] [Figure 15] CV-CAR Treg cells (SBT01G), but not non-transduced Treg cells from two donors, are specifically activated by synovial fluid from RA patients.

[0023] [Figure 16-1] Evaluation of the suppressive function of CV-CAR Treg cells. Figure 16A shows that CV-CAR Treg cells are able to suppress the proliferation of CD3 / CD28 preactivated Teff cells in the presence of CV but not in the absence of CV. Figure 16B shows that CV-CAR Treg cells are able to suppress the proliferation of CD19-CAR Teff cells in the presence of CV, whereas non-transduced Treg cells are not able to. [Figure 16-2] Evaluation of the suppressive function of CV-CAR Treg cells. Figure 16A shows that CV-CAR Treg cells are able to suppress the proliferation of CD3 / CD28 preactivated Teff cells in the presence of CV but not in the absence of CV. Figure 16B shows that CV-CAR Treg cells are able to suppress the proliferation of CD19-CAR Teff cells in the presence of CV, whereas non-transduced Treg cells are not able to.

[0024] [Figure 17]A timeline of human CV-CAR Treg cell activation in vivo in a lipopolysaccharide (LPS)-induced lung inflammation mouse model is shown. Briefly, human CV-CAR Treg cells were administered intravenously (IV) on day 0, human IL-2 was administered intraperitoneally (IP) twice daily, and LPS was administered intranasally (IN) on days 0, 1, 6, and 12. Mice were sacrificed on day 13 and organs were harvested to facilitate analysis of Treg cells.

[0025] [Figure 18] Flow cytometry dot plots comparing the levels of epidermal growth factor (EGFR) expression versus Cell Trace Violet (CTV) expression by human CV-CAR Treg cells are shown. Figure 18A shows how the proliferation ratio of CV-CAR Treg (EGFR+) is determined. In detail, the proliferation ratio is equal to the % of EGFR+CTV- cells divided by the % of EGFR+CTV- cells. Figure 18B shows how the fold change in the EGFR ratio is determined.

[0026] [Figure 19-1] CV-CAR Tregs expand in an LPS-induced lung inflammation mouse model but not in PBS control recipients. Figure 19A shows the absolute number of CD45+CD3+ Tregs in the lungs of the different test groups, while Figure 19B shows the expansion ratio of Tregs in the lungs of the different test groups. [Figure 19-2] CV-CAR Tregs expand in an LPS-induced lung inflammation mouse model but not in PBS control recipients. Figure 19A shows the absolute number of CD45+CD3+ Tregs in the lungs of the different test groups, while Figure 19B shows the expansion ratio of Tregs in the lungs of the different test groups. Summary of the Invention [Means for solving the problem]

[0027] overview Regulatory T cells (Tregs) are deficient in patients and mouse models of RA. Thus, Treg-based adoptive cell therapy (ACT) has become a promising approach in RA. Indeed, Treg-based ACT reverses the disease in animal models of RA. In this study, we used antibodies isolated from RA patients to engineer CARs specific for citrullinated vimentin (CV) and other post-translationally modified proteins that are abundantly and almost exclusively found in the synovial extracellular matrix (ECM) of diseased joints.

[0028] Disclosed herein is a chimeric antigen receptor (CAR) that specifically recognizes the antigen associated with autoimmune disease.In particular, CAR can be specific to post-translationally modified antigen.In particular, CAR can be specific to binding to citrullinated polypeptide, including vimentin, citrullinated filaggrin, and citrullinated fibrinogen.

[0029] Chimeric antigen receptors (CARs) were engineered to specifically target post-translationally modified proteins expressed in the extracellular matrix of inflamed joints in patients with rheumatoid arthritis (RA), namely citrullinated vimentin, citrullinated filaggrin, and citrullinated fibrinogen. In some embodiments, the single-chain fragment variable (scFv) portion of the CAR is obtained from an antibody highly specific for citrullinated proteins isolated from peripheral blood of RA patients. In one embodiment, the specific scFv chain was inserted into a second generation CAR construct. In some embodiments, the scFv chain was inserted into a CAR construct cloned into a lentiviral vector. In the detailed description, references to antibodies are applicable to the antigen-binding domain of the CAR of the present disclosure unless the context dictates otherwise. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description I. Definition Unless otherwise specified, biochemistry, nucleic acid chemistry, molecular biology, developmental biology, and molecular genetic terms and symbols follow standard conventions and texts in the art, such as Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, 1989); Alberts and Singer, Developmental Biology, Eighth Edition (Sinauer Associates Inc., Sunderland, MA, 2006); Kornberg and Baker, DNA Replication, Second Edition (WH Freeman, New York, 1992); Gaits, ed., Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991), and the like.

[0031] As used herein, the terms "antigen", "immunogen" and "antibody target" refer to a molecule, compound or complex that is recognized by an antibody, i.e., that an antibody can bind to. The terms can refer to any molecule that an antibody can recognize, such as a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.). Those skilled in the art will understand that the terms do not indicate that a molecule is immunogenic in any context, but simply that the molecule can be targeted by an antibody.

[0032] As used herein, the term "epitope" refers to the localized site on an antigen that an antibody recognizes and binds to. An epitope can comprise a small number of amino acids or a portion of a small number of amino acids, for example, 5 or 6 or more, for example, 20 or more amino acids, or a portion of such amino acids. In some cases, an epitope comprises non-protein components, for example, 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 can be composed of consecutive amino acids, or amino acids from various parts of a protein that are in close proximity due to protein folding (for example, a non-continuous epitope).

[0033] As used herein, the term "antibody" refers to a polypeptide comprising a framework region derived from an immunoglobulin gene that specifically binds and recognizes an antigen. Typically, the "variable region" comprises the antigen-binding region of an antibody (or a functional equivalent thereof) and is most important in the specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical paired polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD).

[0034] Antibodies can be (i) any of the five major immunoglobulin classes based on the identity of the heavy chain constant domain: alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) and mu (IgM), or (ii) any of the subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The light chains can be either lambda or kappa.

[0035] The following is a non-comprehensive list of various antibody types, all of which retain antigen-binding activity: (1) Whole immunoglobulins (also called "intact" antibodies) (two light chains and two heavy chains, e.g., tetramers); (2) an immunoglobulin polypeptide (light or heavy chain); (3) Antibody fragments, such as Fv (monovalent or bivalent variable region fragments and variable region (e.g., V L and / or V H Fab(V L C L V H C H ), F(ab')2, Fv(V L V H ), scFv (single chain Fv) (V linked by a linker, e.g., a peptide linker L and V H (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(Fv)2 fused to a CH3 domain), diabody (non-covalent dimer of single chain Fv (scFv) fragments consisting of heavy chain variable (VH) and light chain variable (VL) regions linked by a small peptide linker), triabody is a trivalent sc(Fv)3 or trispecific sc(Fv)3, (4) multivalent antibodies (antibodies that contain binding regions that bind to two different epitopes or proteins, e.g., "scorpion" antibodies); (5) Fusion proteins that contain an immunoglobulin-binding moiety fused to another amino acid sequence (e.g., a fluorescent protein); and (6) A heavy-chain-only antibody or antibody fragment, which has only two heavy chains and lacks the two light chains normally found in antibodies.

[0036] The generation and properties of tandem scFvs and diabodies are described, for example, in Asano et al. (2011) J Biol. Chem. 286:1812; Kenanova et al. (2010) Prot Eng Design Sel 23:789; Asano et al. (2008) Prot Eng Design Sel 21:597.

[0037] The phrase "set of CDR sequences" as used herein refers to the three heavy and / or three light chain CDRs of a particular antibody described herein. The "light chain" CDR sequence set refers to the light chain CDR sequences. The "heavy chain" CDR sequence set refers to the heavy chain CDR sequences. The "complete" CDR sequence set refers to both the heavy and light chain CDR sequences. The CDRs are predicted based on the alignment of IMGT sequences.

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

[0039] As used herein, the term "humanized antibody" refers to a chimeric antibody in which CDRs from the VH and VL regions of a non-human antibody with desired specificity, affinity and capacity have been grafted onto human framework sequences. In one embodiment, the framework residues of the humanized antibody are modified to improve and optimize the specificity, affinity and capacity of the antibody. Humanization, i.e., substitution of non-human CDR sequences for the corresponding sequences of a human antibody, can be carried out according to the methods described, for example, in U.S. Pat. Nos. 5,545,806; 5,569,825; 5,633,425; 5,661,016; Riechmann et al., Nature 332:323-327 (1988); Marks et al., Bio / Technology 10:779-783 (1992); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996).

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

[0041] Specificity of binding can be defined in terms of the comparative dissociation constant (Kd) of an antibody (or other targeting moiety) for a target as compared to the dissociation constant (Kd) for the antibody and other substances or general unrelated molecules in its environment. When the Kd is large (high), the Kd describes a low affinity interaction. Conversely, when the Kd is small (low), the Kd describes a high affinity interaction or strong binding. By way of example only, an antibody that specifically binds to a target may have a Kd in the femtomolar, picomolar, nanomolar, or micromolar range, whereas an antibody that binds to unrelated substances may have a Kd in the millimolar range or greater. Binding affinity can be measured in the micromolar range (kD=10 -4 ~10 -6 ), nanomolar range (kD=10 -7 M~10 -9 M), picomole range (kD = 10 -10 M~10 -12 M), or femtomole range (kD=10 -13 M~10 -15 M).

[0042] As used herein, an antibody is -4An agent "binds" or "recognizes" an antigen or epitope when it binds to the antigen or epitope with a Kd of less than M (i.e., in the micromolar range). The term "binds" in reference to a cell type (e.g., an antibody that binds to cancer cells) typically indicates that the agent binds to the majority of cells in a pure population of such cells. For example, an antibody that binds to a given cell type typically binds to at least 2 / 3 (e.g., 67, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) of the cells in a population of cells specified. In some cases, binding to a polypeptide can be assayed by comparing the binding of the antibody to cells that display the polypeptide with the binding (or lack thereof) of the antibody to cells that do not express the polypeptide. Those skilled in the art will recognize that some variability occurs depending on the method and / or threshold value that determines binding. The affinity of an antibody for a target can be determined according to methods known in the art, for example, as outlined in Ernst et al. Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009).

[0043] As used herein, the term "greater affinity than" refers to the relative degree of antibody binding, where antibody X binds to target Y more strongly (Kon) and / or with a smaller dissociation constant (Koff) than to target Z, in this context, antibody X has a greater affinity for target Y than to Z. Similarly, the term "lower affinity" refers to the degree of antibody binding, where antibody X binds to target Y less strongly and / or with a larger dissociation constant than to target Z, in this context, antibody X has a lesser affinity for target Y than to Z. The binding affinity of an antibody to its target antigen can be expressed as KA=1 / KD, where KD is equal to koff / koff. koff and koff values ​​can be measured using surface plasmon resonance technology, for example, using the Molecular Affinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany). Antagonists or blocking antibodies are antibodies that partially or completely block, inhibit or neutralize the biological activity associated with the target antigen in the absence of the antibody, compared to the activity under similar physiological conditions. Antagonists can be competitive, non-competitive or irreversible. Competitive antagonists are substances that bind to the natural ligand or receptor at the same site as the natural ligand-receptor interaction, or bind allosterically to induce changes that prevent normal binding. Non-competitive antagonists bind to a site different from the natural ligand-receptor interaction, but generate a low KD or signal from this interaction. Irreversible inhibitors cause covalent modifications to the receptor to prevent any subsequent binding.

[0044] As used herein, the term "avidity" refers to the overall stability of the binding complex between antibody and target antigen.It is determined by three factors: (i) the intrinsic affinity of antibody to antigen, (2) the binding valency of antibody, and (3) the geometric arrangement of interacting components.Affinity is the strength of the interaction of antibody with a single target, while avidity is the cumulative strength of multiple affinities.In one embodiment, the antibody provided herein is bivalent.

[0045] As used herein, an antibody "preferentially binds" a first antigen compared to a second antigen if it binds the first antigen with greater affinity than it binds to 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, an antibody is defined as a concentration of 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 An antibody "specifically binds to" or is "specific for" a target antigen or target group of antigens when it binds to the target antigen or each member of the target group of antigens with at least some affinity of M, e.g., with an affinity that is at least two-fold greater than its affinity for a comparable non-target antigen. Typically, specific binding is characterized by the antibody binding to the antigen with sufficient affinity that it is useful as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent in targeting the antigen or epitope.

[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, typically 30 amino acids or less in length. The amino acid sequence of a polypeptide is referred to as the "primary structure". The term "protein" refers to a polypeptide having secondary, tertiary and / or quaternary structure, e.g., a structure stabilized by hydrogen bonds, which is the correlation between secondary structure and structures formed by more than one protein. Proteins can be further modified by other binding moieties, e.g., 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 one amino acid or less, two amino acids or less, or three amino acids or less longer than the second amino acid sequence.

[0050] As used herein, when a second amino acid sequence comprises a first amino acid sequence, the first amino acid sequence is a "fragment" of the second amino acid sequence. In certain embodiments, a first amino acid sequence that is a fragment of a second amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less amino acids than the second amino acid sequence.

[0051] As used herein, the "functional equivalent" of a reference amino acid sequence is a sequence that is not identical to the reference sequence, but contains minor changes such as, for example, the insertion, deletion or substitution of one or several amino acids.A functional equivalent sequence retains the function (e.g., immunogenicity) of the equivalent reference sequence.When a functionally equivalent amino acid sequence with respect to a reference sequence contains one or more amino acid substitutions, these can generally be conservative amino acid substitutions.

[0052] As used herein, a "conservative amino acid substitution" is one that replaces one amino acid residue with another without losing the desired properties of a protein. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R chain length for each other. See, for example, Watson, et al., "Molecular Biology of the Gene," 4th Edition, 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, p. 224. Examples of conservative amino acid substitutions include (note that some categories are not mutually exclusive): [Table 1]

[0053] As used herein, the term "substantially identical" refers to an identity with a first amino acid sequence that includes a sufficient or minimum number of amino acid residues that are i) identical to the aligned amino acid residues of the second amino acid sequence, or ii) are conservative substitutions for the aligned amino acid residues of the second amino acid sequence, such that the first and second amino acid sequences have a common structural domain and / or a common functional activity and / or a common immunogenicity. For example, amino acid sequences that include a common structural or antigenic domain that have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are referred to as being fully 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 a minimum number of nucleotides that are identical to aligned nucleotides of a second nucleic acid sequence, provided that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, or encode polypeptides having the same immunogenic properties.

[0054] As used herein, a chemical entity, e.g., a polypeptide, is "substantially pure" or "isolated" if it is the predominant chemical entity of that species (e.g., polypeptide) in a composition. This includes 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 entity of that species in the composition. A substantially purified fraction is a composition in which the species of interest comprises at least about 50% (on a molar basis) of all macromolecular species present. In general, a substantially pure composition means that about 80%-90% or more of the macromolecular species present in the composition are the purified species of interest. When a composition consists essentially of a single macromolecular species, the species of interest is purified to essentially homogeneity (no contaminants are detectable in the composition by conventional detection methods). Solvent species, small molecules, stabilizers (eg, BSA), and elemental ion species are not considered to be macromolecular species for the purposes of this definition.

[0055] The phrase "isolated antibody" refers to an in vivo or in vitro produced antibody removed from the source which produced the antibody, e.g., an animal, hybridoma or other cell line (e.g., an insect, yeast or bacterial recombinant cell that produces the antibody).

[0056] The term "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules at that position are identical. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions x 100%). In one embodiment, the two sequences are of the same length. The determination of the percent identity between two sequences can also be achieved using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the parameters of the NBLAST nucleotide program set, for example, 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 parameters of the XBLAST program set, for example, score-50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules described herein.To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform iterative searches to detect distant relationships between molecules (ibid.). When using BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm used to compare sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0057] For antibodies, the percentage sequence identity can be determined when the antibody sequences are maximally aligned by IMGT. After alignment, when a region of a subject antibody (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by identical amino acids in both the subject and reference antibody regions divided by the total number of aligned positions in the two regions, multiplied by 100 to convert to a percentage.

[0058] Percent amino acid sequence identity may 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 Institutes of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15 / 5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25, and scoring matrix=BLOSUM62.

[0059] In the context of utilizing NCBI-BLAST2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to, with, or to a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity relative to, with, or to a given amino acid sequence B) is calculated as follows: 100×(X / Y), where X is the number of amino acid residues scored as perfect matches from a program alignment of A and B by the sequence alignment program NCBI-BLAST2, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. The term "nucleic acid sequence" as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages, and includes cDNA. The term also includes modified or substituted sequences, including non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also include modified bases. Examples of such modified bases include aza and deazaadenine, aza and deazaguanine, aza and deazacytosine, aza and deazathymidine, and aza and deazauracil; and xanthine and hypoxanthine. It is understood that polynucleotides that include non-transcribeable nucleotide bases may be useful as probes, for example, in hybridization assays. Nucleic acids may be either double-stranded or single-stranded, and may be sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon-optimized or synonymous codon equivalents.

[0060] The term "isolated nucleic acid," as used herein, refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid from which it is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0061] Hybridization may occur in all or part of the nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrid, is determined by Tm, which in sodium-containing buffer is a function of sodium ion concentration and temperature (Tm=81.5°C-16.6(Log10[Na+])+0.41(%(G+C)-600 / l), or similar formula). Thus, the parameters of the washing conditions that determine the stability of the hybrid are sodium ion concentration and temperature. To identify molecules that are similar but not identical to known nucleic acid molecules, it can be assumed that 1% mismatch reduces Tm by about 1°C, for example, when a nucleic acid molecule with more than 95% identity is sought, the final washing temperature is reduced by about 5°C. Based on such considerations, those skilled in the art can easily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. By way of example, stringent hybridization may be achieved using the following conditions: hybridization with 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS at Tm-5°C based on the above formula, followed by a wash with 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a wash step with 3x SSC at 42°C. However, it is understood that equivalent stringency may be achieved using alternative buffers, salts, and temperatures. Further guidance regarding hybridization conditions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 2002, and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0062] As used herein, the term "expression construct" refers to a polynucleotide that contains an expression control sequence that is operatively linked to a heterologous nucleotide sequence to be expressed (i.e., a sequence to which the expression control sequence is not normally physically linked). As used herein, the term "expression vector" refers to a polynucleotide that contains an expression construct and a sequence sufficient for replication in a host cell or insertion into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term "expression control sequence" refers to a nucleotide sequence that regulates the transcription and / or translation of a nucleotide sequence operatively linked to it. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences), and ribosome binding sites (translational regulatory sequences).

[0063] 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, including promoting transcription of the nucleotide sequence through interaction of a polymerase with a promoter.

[0064] The term "vector", as used herein, includes any intermediate vehicle for a nucleic acid molecule that allows said nucleic acid molecule to be introduced into, for example, a prokaryotic and / or eukaryotic cell and / or integrated into a genome, including plasmids, phagemids, bacteriophages or viral vectors, such as retrovirus-based vectors, lentivirus vectors, adeno-associated virus vectors, etc. The term "plasmid", as used herein, generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, which can replicate independently of chromosomal DNA.

[0065] "Transfection" refers to the introduction of new genetic material into a cell. It includes transformation (direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane), transduction (introduction of foreign DNA into a host cell by a bacteriophage virus), and conjugation.

[0066] As used herein, a "host cell" refers to a recombinant cell that contains an expression construct.

[0067] As used herein, the term "biological sample" refers to a sample that contains cells (e.g., tumor cells) or biological molecules derived from cells.

[0068] As used herein, the term terms "therapy", "treatment", "therapeutic intervention" and "amelioration" refer to any activity that results in a reduction in the severity of symptoms. The terms "treating" and "preventing" are not intended to be absolute. Treatment and prevention can refer to either delaying onset, reversing symptoms, improving patient survival, increasing survival time or survival rate, and the like. Treatment and prevention can be complete or partial. The effect of treatment can be compared to an individual or pool of individuals not receiving treatment, or to the same patient at different times before or during treatment. In some embodiments, the severity of the disease is reduced by at least 10% compared to the individual before administration or to an individual control not receiving treatment. In some embodiments, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases is no longer detectable using standard diagnostic techniques. "Treating" and "treatment" can also mean an extension of survival compared to expected survival in the absence of treatment. "Treating" and "treatment", as used herein, also include prophylactic treatment.

[0069] A composition or method that "comprising" or "including" one or more recited elements may include other elements not specifically recited (e.g., open-ended terms meaning to include but not be limited to). For example, a composition that "comprises" or "includes" an antibody may include the antibody alone or in combination with other components. In contrast, the phrase "consisting of" is restrictive and indicates that such embodiment does not include additional elements. The term "consisting essentially of" refers to the inclusion of the recited elements and other elements (e.g., partial restrictive terms) that do not materially affect the basic and novel properties of the claimed combination. It is understood that aspects and embodiments described herein as "comprising" include the "consisting of" and "consisting essentially of" embodiments.

[0070] As used herein, the following meanings apply unless otherwise specified: The word "may" is used in a permissive sense (i.e., having the potential for) rather than a mandatory sense (i.e., meaning must). The singular forms "a," "an," and "the" include plural references. Thus, for example, a reference to an "element" includes combinations of two or more elements, notwithstanding the use of other terms and phrases about one or more elements, e.g., "one or more." The phrase "at least one" includes "one," "one or more," and "one or a plurality," as well as "plurality." The term "or" is non-exclusive, i.e., encompasses both "and" and "or," unless otherwise indicated. The term "any of" placed between a modifier and a sequence means that the modifier modifies each member of the sequence. Thus, for example, the phrase "at least any of 1, 2, or 3" means "at least 1, at least 2, or at least 3." II. Chimeric Antigen Receptors

[0071] "Chimeric antigen receptor" or "CAR" is an engineered molecule that contains an optional signal peptide, a target binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain, and an optional costimulatory domain. CAR is based on the structure of the T cell receptor, which is expressed on T cells and participates in cell-mediated immune response. "Target binding domain" is also referred to as "antigen binding domain" herein, and therefore the term "target" encompasses "antigen".

[0072] The so-called "first generation" CARs had a targeting domain and a CD3ζ signaling domain. The so-called "second generation" CARs further contained a costimulatory domain, such as a CD28 or 4-1BB domain. The so-called "third generation" CARs contained multiple costimulatory domains. The so-called "fourth generation" CARs, also called "TRUCKS", were engineered to release transgenic cytokines upon CAR signaling.

[0073] A chimeric antigen receptor ("CAR") comprises the following elements: (1) an optional signal peptide, (2) a target binding domain, (3) an optional hinge region, (4) a transmembrane region, and (5) an intracellular domain that includes a signaling domain. Optionally, a CAR may comprise either a CD3ζ signaling domain, an Fc receptor signaling domain, or a costimulatory (signaling) domain. That is, such optional elements may be included in addition to or in place of other optional elements. The target binding domain is heterologous to at least one of the other domains. That is, the target binding domain is not naturally present on the T cell receptor or in the same protein as at least one of the other domains.

[0074] The "target binding domain" provides binding specificity for the CAR. The "signal peptide" guides the polypeptide through the cell membrane. The target binding domain can bind to a domain of an antibody that binds to a target antigen for a so-called "universal CAR". The "hinge region" is a flexible connector region, e.g., a natural or synthetic polypeptide, or any other type of molecule that provides structural flexibility and spaces adjacent polypeptide regions. The "transmembrane domain" is a domain of a protein that spans a membrane, typically hydrophobic. The "signaling domain" or "signaling domain" transmits a signal through a signal transduction pathway into the cell upon binding. Such signaling activates the activity of the cell. The "costimulatory domain" is an accessory signaling domain that further transmits the signal.

[0075] In some embodiments, the CAR comprises: (i) A target binding domain (also referred to herein as an antigen binding domain) reactive with a citrullinated protein or a citrullinated fragment thereof, e.g., VH-VL or VL-VH, in which the two variable domains are separated by a flexible linker of 15-25 amino acids in length. (ii) a hinge domain, (iii) a costimulatory domain, and (iv) Intracellular signaling domain (also referred to herein as activation domain). That is, in some embodiments, the CAR comprises an antigen-binding domain fused to the CAR platform, which comprises a hinge domain, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and an activation domain. The CAR may further comprise a signal peptide (also referred to herein as a leader sequence) that directs the expression of the CAR to the surface of a cell, e.g., Treg.

[0076] In some embodiments, the CAR comprises an antigen binding domain fused in frame to a CAR platform comprising the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 28, and SEQ ID NO: 19. In other embodiments, the CAR comprises an antigen binding domain fused in frame to a CAR platform comprising the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 28, and SEQ ID NO: 19. In some embodiments, the CAR comprises an antigen binding domain fused in frame to a CAR platform comprising the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 29, and SEQ ID NO: 19. In other embodiments, the CAR comprises an antigen binding domain fused in frame to a CAR platform comprising the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 16, SEQ ID NO: 29, and SEQ ID NO: 19. A. Signal Peptides

[0077] The signal peptide may be any peptide whose function is to allow the polypeptide to cross the cell membrane. The signal peptide may be derived from CD4, CD8, CD28, TLR or the immunoglobulin family of receptors.

[0078] For example, the signal peptide may comprise the following sequence: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 18), or MALPVTALLLPLALLLHAAR (sequence number 23). B. Target Binding Domain 1. Structure

[0079] The target binding domain may comprise any polypeptide that comprises a target binding function, such as an antibody as defined herein. In one embodiment, the target binding domain may comprise an antibody type that retains the antigen binding activity as defined herein. In one embodiment, the target binding domain may comprise a single chain antibody (scFV). The scFV may be connected to the transmembrane domain via a hinge domain, whose length, flexibility, and origin provide variability in CAR design, and together with the transmembrane domain, may contribute to the interaction with antigen, the construction of the immunological synapse, and affect the association of the CAR with additional proteins required to provide a robust activation signal. 2. Target / antigen

[0080] The chimeric antigen receptor disclosed herein comprises a target binding domain (also referred to herein as an antigen binding domain) that binds to a citrullinated antigen found, for example, in the synovium of a subject with rheumatoid arthritis. In particular, the target binding domain may bind to (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) one or more of these citrullinated peptides. In some embodiments, the target binding domain may bind to a citrullinated peptide fragment of (i)-(iii), where the peptide fragment is at least 10 amino acids in length, for example, 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: (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) tenascin-C, or citrullinated peptide fragments thereof.

[0081] In some embodiments, the targeting domain is one or more citrullinated peptides selected from the following sequences: ST(Cit)SVSSSSY(Cit)(Cit)MFGG (SEQ ID NO:24); VYAT(Cit)SSAV(Cit)L(Cit)SSV(SEQ ID NO: 25); (Cit)PAPPPISGGGY(Cit)A(Cit) (SEQ ID NO:26); SHQEST(Cit)GRSRGRSGRSGS (SEQ ID NO:27).

[0082] In some embodiments, the antigen binding domain binds to one or more citrullinated peptides but not to non-citrullinated counterparts. In some embodiments, the antigen binding domain binds to the citrullinated vimentin peptide set forth as SEQ ID NO: 24, rather than STRSVSSSSYRRMFGG (SEQ ID NO: 45). In some embodiments, the antigen binding domain binds to the citrullinated vimentin peptide set forth as SEQ ID NO: 25, rather than VYATRSSAVRLRSSV (SEQ ID NO: 46). In some embodiments, the antigen binding domain binds to the citrullinated fibrinogen peptide set forth as SEQ ID NO: 26, rather than RPAPPPISGGGYRAR (SEQ ID NO: 47). In some embodiments, the antigen binding domain binds to the citrullinated filaggrin peptide set forth as SEQ ID NO: 27, rather than SHQESTRGRSRGRSGRSGS (SEQ ID NO: 48).

[0083] The target binding domain may comprise sequences from the VH and VL domains of an antibody. In some embodiments, the target binding domain may comprise sequences from a heavy chain-only antibody or antibody fragment with only two VH domains. It comprises a specific set of CDRs from the 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 domain of SEQ ID NO: 1 and the VL domain of SEQ ID NO: 3, respectively. In some embodiments, the target binding domain comprises CDRs from the VH domain of SEQ ID NO: 1 and the VL domain of SEQ ID NO: 4, respectively. In some embodiments, the target binding domain comprises CDRs from the VH domain of SEQ ID NO: 2 and the VL domain of SEQ ID NO: 3, respectively. In some embodiments, the target binding domain comprises CDRs from the VH domain of SEQ ID NO: 2 and the VL domain of SEQ ID NO: 4, respectively.

[0084] In some embodiments, the target binding domain comprises complementarity determining regions (CDRs) from the VH domain of SEQ ID NO: 1 (SBT01 VH(M)) and the VL domain of SEQ ID NO: 4 (SBT01 VL(G)). In some embodiments, the VH domain of the target binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, and the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. [Table 2]

[0085] In certain embodiments, the target binding domain comprises: (1) SBT01 VH(M) HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 1); (2) SBT01 VH(G) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 2), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. wherein the target binding domain binds to a citrullinated antigen as described herein.

[0086] The target binding region is (1) SBT01 VL(M) SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVL (SEQ ID NO:3); (2) SBT01 VL(G) SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVL (SEQ ID NO: 4), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. wherein the target binding domain binds to a citrullinated antigen as described herein.

[0087] In another embodiment, the antigen binding region comprises an scFV comprising one or more VH domains comprising the amino acid sequence of the VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the antigen binding region comprises an scFV comprising an amino acid sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 1 or SEQ ID NO: 2, provided that the scFV domain binds to a citrullinated antigen as described herein.

[0088] In some embodiments, the scFV comprises a VL domain of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the antigen binding region comprises an scFV that comprises an amino acid sequence having at least any 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 as described herein.

[0089] 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 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 disposed between the VH and VL domains.

[0090] 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 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 disposed between the VH and VL domains.

[0091] 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 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 disposed between the VH and VL domains.

[0092] 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 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 disposed between the VH and VL domains.

[0093] In another embodiment, the antigen binding region comprises: SBT01G-VHVL-GGGSx3 linker-pSB_0149 [ka] (GGGSx3 linker underlined); SBT01G-VHVL-Whitlow 218 linker-pSB_0158 [ka] (Whitlow 218 linker underlined); SBT01G-VHVL-AB pur linker-pSB_0159 [ka] (AB pur linker is underlined); or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequences. wherein the target binding domain binds to a citrullinated antigen as described herein.

[0094] For example, the linker may have the sequence: A GGGSx3 linker of GGGGSGGGGSGGGGS (SEQ ID NO: 20), or A Whitlow218 linker of GSTSGSGKPGSGEGSTKG (SEQ ID NO:21), or AB pur linker of ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 22) may include.

[0095] Optionally, any of the foregoing sequences may comprise a set of CDRs from the VH and VL domains described above. C. Hinge region

[0096] In some embodiments, the hinge region of the disclosed CARs can be selected from the CD8, CD4, or CD28 extracellular domains, the Fc region of an IgG1 antibody, or the extracellular domain of any of the TLR receptors, as known to those of skill in the art and can be found in the GenBank database.

[0097] For example, the hinge region may have the sequence: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 30) (CD28), or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 15) (CD8), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned hinge region sequence. may include. D. Transmembrane domain

[0098] The transmembrane domain can include the transmembrane domain of an immunoglobulin family receptor, such as CD8. The intracellular domain can be selected from any molecule that spans the membrane on a T cell. For example, the transmembrane (TM) domain of the disclosed CAR can include the TM domain of CD2, CD3, CD16, CD32, CD64, CD28, CD247, 4-1BBL, CD4, or CD8.

[0099] For example, the transmembrane domain may have the sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 16), or IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 17), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned hinge region sequence. may include. E. Signal Transduction Domains 1. CD3ζ signaling domain

[0100] In some embodiments, the signaling domain comprises a CD3 zeta signaling domain. The CD3 zeta signaling domain of the disclosed CAR molecule can comprise a CD3 zeta amino acid sequence, for example, the signaling domain of CD3 zeta.

[0101] For example, the CD3 ζ signaling domain has the sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 19), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned CD3 zeta signaling domain sequence. may include.

[0102] For example, the CD3 zeta signaling domain can include amino acids 21-163, 31-142, 68-89, and / or 138-158 of the sequence set forth in SEQ ID NO:19, or a functional variant thereof (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10-20 amino acid substitutions, deletions, or additions). 2. Fc receptor signaling domain

[0103] In some embodiments, the signaling domain comprises an Fc signaling domain. The Fc signaling domain can be any one of Fc alpha, Fc gamma, Fc epsilon, Fc mu, and Fc delta receptors. For example, the Fc receptor signaling domain can comprise amino acids involved in interaction with Src family kinases (e.g., Fgr, Fyn, Hck, Lyn, Yes, and Src) and ZAP-70 family kinases, e.g., 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., derived from or substantially identical to any one of Fc alpha, Fc gamma, Fc epsilon, Fc mu, and Fc delta receptors.

[0104] The sequence can also be found as follows: [Table 3] F. Costimulatory Domain

[0105] The CAR of the present disclosure may include one or more costimulatory domains in addition to the signaling domain of CD3zeta or Fc receptor. The costimulatory domain 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. The CAR construct may include two or more costimulatory signaling domains (e.g., CD28 and 4-1BB).

[0106] One or more costimulatory domains may be located between the signaling domain and the transmembrane region.

[0107] In a specific embodiment, the CD28 costimulatory domain has the sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:29), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. may include.

[0108] In certain embodiments, the 41BB costimulatory domain has the sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 28), or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. may include. III. Nucleic acids A. Nucleic Acid Encoding CAR

[0109] Disclosed herein is a nucleic acid molecule (polynucleotide) that comprises a nucleotide sequence that codes for the CAR of the present disclosure.The nucleic acid of the disclosed CAR can be in the form of DNA or RNA.DNA includes cDNA, genomic DNA, and synthetic DNA, and can be double-stranded or single-stranded, and when single-stranded, can be coding strand or non-coding (antisense) strand.RNA includes mRNA, siRNA, sRNA, ssRNA, etc.

[0110] For example, the nucleic acid can comprise a nucleotide sequence encoding the polypeptide of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. In certain embodiments, the nucleotide sequence encoding the VH domain is (1) SBT01 VH(M) [ka] (2) SBT01 VH(G) [ka] or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. Includes.

[0111] In certain embodiments, the nucleotide sequence encoding the VL domain is (1) SBT01 VL(M) [ka] (2) SBT01 VL(G) [ka] or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. Includes.

[0112] 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 sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 8 or SEQ ID NO: 9, with the proviso that the scFV domain binds to a citrullinated antigen as described herein.

[0113] In some embodiments, the nucleic acid molecule encoding the VL domain comprises SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the nucleic acid molecule encoding the VL domain comprises a nucleic acid sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 10 or SEQ ID NO: 11, with the proviso that the scFV domain binds to a citrullinated antigen as described herein.

[0114] In some embodiments, the nucleic acid molecule encoding the scFv domain comprises SEQ ID NO: 8 and SEQ ID NO: 10. In some embodiments, the nucleic acid molecule encoding the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 8 or SEQ ID NO: 10, provided that the scFV domain binds to a citrullinated antigen as described herein.

[0115] In some embodiments, the nucleic acid molecule encoding the scFv domain comprises SEQ ID NO: 8 and SEQ ID NO: 11. In some embodiments, the nucleic acid molecule encoding the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 8 or SEQ ID NO: 11, provided that the scFV domain binds to a citrullinated antigen as described herein.

[0116] In some embodiments, the nucleic acid molecule encoding the scFv domain comprises SEQ ID NO: 9 and SEQ ID NO: 10. In some embodiments, the nucleic acid molecule encoding the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 9 or SEQ ID NO: 10, provided that the scFV domain binds to a citrullinated antigen as described herein.

[0117] In some embodiments, the nucleic acid molecule encoding the scFv domain comprises SEQ ID NO: 9 and SEQ ID NO: 11. In some embodiments, the nucleic acid molecule encoding the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 9 or SEQ ID NO: 11, provided that the scFV domain binds to a citrullinated antigen as described herein.

[0118] In another embodiment, the nucleic acid molecule encodes an scFv molecule and has the nucleotide sequence: SBT01G-VHVL-GGGSx3 linker-pSB_0149 [ka] [ka] (GGGSx3 linker underlined); or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. has.

[0119] In another embodiment, the nucleic acid molecule encodes an scFv molecule and has the nucleotide sequence: SBT01G-VHVL-Whitlow 218 linker-pSB_0158 [ka] (Whitlow 218 linker underlined); or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. has.

[0120] In another embodiment, the nucleic acid molecule encodes an scFv molecule and has the nucleotide sequence: SBT01G-VHVL-AB pur linker-pSB_0159 [ka] (AB pur linker is underlined); or A sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity with the aforementioned sequence. has.

[0121] Optionally, these may include sequences encoding a set of CDRs from the VH and VL domains described herein.

[0122] Polynucleotide variants may include modifications in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants include modifications that result in silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded CAR polypeptide. In some embodiments, polynucleotide variants include modifications that do not result in any changes in the amino acid sequence. In some embodiments, polynucleotide variants include "silent" substitutions due to the degeneracy of the genetic code. Polynucleotide variants may be generated for various reasons, for example, to optimize the expression of codons for a particular host.

[0123] In some embodiments, the polynucleotides described herein are isolated.

[0124] The polynucleotide encoding the CAR can be an isolated molecule or can be contained within a vector, such as a plasmid, cosmid, artificial chromosome, or virus. Such vectors can be used to transfect target cells. B. Expression Constructs and Vectors

[0125] A polynucleotide encoding a CAR of the present disclosure can include a regulatory element operably linked to the nucleotide sequence encoding the CAR. For example, the polynucleotide can include one or more transcriptional regulatory elements, such as a promoter or enhancer, such that when the polynucleotide is present in a cell, the sequence encoding the CAR is expressed in the cell.

[0126] The 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 include adenoviral vectors Ad, AAV, lentiviruses, and vesicular stomatitis virus (VSV) and retroviruses. Lentiviruses are a genus of the Retroviridae family, including HIV, SIV, and FIV. Lentiviruses can deliver large amounts of genetic material into the DNA of host cells. They are capable of infecting non-dividing cells. IV.Cells

[0127] In some embodiments, a recombinant (host) cell harboring a nucleic acid molecule encoding a disclosed CAR, wherein the nucleic acid molecule may further comprise an expression control sequence operably linked to the nucleotide sequence encoding the CAR. The assembled CAR (by synthesis, site-directed mutagenesis or another method as known to those skilled in the art), the nucleic acid molecule encoding the disclosed CAR, can be inserted into an expression vector and operably linked to an appropriate expression control sequence for expression of the disclosed CAR in a desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and / or expression of the CAR polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0128] The disclosure also provides cells (e.g., recombinant cells) that contain a nucleic acid molecule that encodes and / or expresses a CAR.

[0129] The nucleic acid molecule encoding the disclosed CAR can be delivered to a host cell, including, but not limited to, T cells, B cells, myeloid progenitor cells, macrophages, etc., by a plasmid or viral vector as known to those skilled in the art. The resulting recombinant (host) cells can include, but are not limited to, T cells, CD4 T cells, Treg cells, CD8 alpha T cells, CD8 beta T cells, helper T cells, granulocytes (neutrophils, basophils, eosinophils), megakaryocytes, monocytes, macrophages, and dendritic cells, stem cell memory T cells, and cells expressing MHC class I or class II, as known to those skilled in the art. In some embodiments, the recombinant (host) cells carrying the nucleic acid molecule encoding the disclosed CAR can be myeloid progenitor cells selected from the group consisting of general myeloid progenitor cells, granulocyte macrophage progenitor cells, megakaryocyte erythroid progenitor cells, granulocyte progenitor cells, and monocyte progenitor cells, as known to those skilled in the art. In some embodiments, the bone marrow cells are autologous or allogeneic cells.

[0130] In some embodiments, a cell expressing a CAR of the present disclosure is a Treg cell. reg "T cells" are cells that belong to a specialized subpopulation of T cells that act to suppress immune responses, thereby maintaining homeostasis and self-tolerance. reg inhibits T cell proliferation and cytokine production and may play an important role in preventing autoimmunity. reg is characterized by expression of FoxP3. reg Surface markers for Tregs include CD4, CD25high (high molecular density), and CD127low (low molecular density). Mouse and human Tregs express GITR / AITR and CTLA-4. Human CD4+FoxP3+Treg cells can be divided into three subpopulations: (1) CD45RA+CD25+FoxP3l0w resting Treg cells, (2) CD45RO+CD25highFoxP3high activated Treg cells, and (3) proinflammatory cytokine-producing CD45RO+CD25+FoxP3low non-suppressive effector T cells (Teff).

[0131] The cells transformed with the nucleic acids disclosed herein can be cells taken from the subject to which the recombinant cells are administered, thus reducing the problem of allogeneic immune responses.

[0132] The cells can be expanded ex vivo prior to administration to a subject.

[0133] Treg cells incorporating a nucleic acid expressing a CAR of the present disclosure can be used in the methods described herein to express such CARs and treat rheumatoid arthritis.

[0134] Proteins produced by transformed / recombinant hosts can also be purified according to any suitable method, including by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Affinity tags, such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione S-transferase, can be attached to the protein to facilitate purification by passing through an appropriate affinity column. In some embodiments, proteins can be physically characterized using techniques such as proteolysis, high performance liquid chromatography (HPLC), nuclear magnetic resonance, and X-ray crystallography. V. Composition

[0135] Also disclosed are pharmaceutical compositions comprising recombinant cells harboring a nucleic acid molecule encoding and / or expressing the disclosed CAR polypeptides and a pharma- ceutically acceptable carrier, and methods of use in treating rheumatoid arthritis.

[0136] As used herein, the term "pharmaceutical composition" refers to a composition comprising a pharmaceutical compound (e.g., a drug or recombinant Treg cells described herein) and a pharma- ceutically acceptable carrier.

[0137] As used herein, the term "pharmaceutical acceptable" refers to a carrier that is compatible with other components of a pharmaceutical composition and can be safely administered to a subject. This term is used synonymously with "physiologically acceptable" and "pharmacologically acceptable". Pharmaceutical compositions and the techniques for their preparation and use are known to those skilled in the art in light of this disclosure. For detailed listings of pharmacological compositions and techniques suitable for these administrations, 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.

[0138] Pharmaceutically acceptable carriers are generally sterile, at least for human use. Pharmaceutical compositions generally include agents for buffering and preservation in reservoirs, and may include buffers and carriers for appropriate delivery depending on the route of administration. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological (0.9%) saline, phosphate buffered saline (PBS), Hank's balanced salt solution (HBSS), and polyelectrolyte solutions, such as PlasmaLyte A™ (Baxter).

[0139] The pharmaceutical compositions may be formulated for any route of administration, including mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraarterial injection, either bolus or infusion), oral, or transdermal.

[0140] Injectable (e.g., intravenous) compositions may include a solution of the composition suspended in an acceptable carrier, e.g., an aqueous carrier. Any of a variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, etc., and may include glycoproteins, e.g., albumin, lipoproteins, globulins, etc., for enhanced stability. Buffered saline (135-150 mM NaCl) is often used. The composition may include pharma- ceutically acceptable auxiliary substances to approximate physiological conditions, e.g., pH adjusting and buffering agents, osmolality adjusting agents, wetting agents, e.g., 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.

[0141] Formulations suitable for parenteral administration, such as by intraarticular (intra-articular), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions, which may include antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions may also be prepared from sterile powders, granules, and tablets. In the practice of the invention, the compositions may be administered, for example, by intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally. Formulations of the compositions may be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0142] The cells can be cryopreserved. Cryopreservation can include formulating the cells with a cryopreservative, such as DMSO. Commercially available media include, for example, CryoStor® and pZerve® available from Millipore Sigma.

[0143] The composition can be formulated as a dosage form for administration. The term "dosage form" refers to the particular form of a pharmaceutical product and depends on the route of administration. Examples of dosage forms include, but are not limited to, liquid dispersions; suppositories; ointments; poultices; pastes; powders; dressings; creams; plasters; liquids; patches; aerosols (e.g., nasal sprays or inhalants); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0144] The terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual at each administration. Dose can vary depending on several factors, including frequency of administration; size and tolerance of the individual; severity of condition; risk of side effects; route of administration; and imaging mode of detectable label (if any). Those skilled in the art will recognize that dose can be modified depending on the above factors or based on the progress of treatment.

[0145] Pharmaceutical preparations can be packaged or prepared in unit dosage form.In this form, the preparation is divided into unit dosages containing appropriate amounts of active ingredient, for example, according to the dose of therapeutic agent or the concentration of composition.The unit dosage form can be a packaged preparation, and the package contains a discrete amount of the preparation.If desired, the composition can also contain other compatible therapeutic agents.

[0146] In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a complementarity determining region (CDR) from the VH domain of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a CDR from the VL domain of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a VH domain of SEQ ID NO: 1 and a CDR from the VL domain of SEQ ID NO: 3. In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a VH domain of SEQ ID NO: 1 and a CDR from the VL domain of SEQ ID NO: 4. In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a VH domain of SEQ ID NO: 2 and a CDR from the VL domain of SEQ ID NO: 3. In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising a VH domain of SEQ ID NO: 2 and a CDR from the VL domain of SEQ ID NO: 4. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a helper T cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a stem cell memory T cell. In some embodiments, the cell is a Treg cell.

[0147] In some embodiments, compositions of the invention comprise recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising CDRs from the VH domain of SEQ ID NO: 1 (SBT01 VH(M)) and the VL domain of SEQ ID NO: 4 (SBT01 VL(G)). In some embodiments, the VH domain of the CAR polypeptide comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, and the VL domain of the CAR polypeptide comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a helper T cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a stem cell memory T cell. In some embodiments, the cells are Treg cells.

[0148] In another embodiment, the composition of the invention comprises a recombinant cell having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising one or more VH domains comprising the amino acid sequence of the VH domain of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the nucleic acid molecule encoding a CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:1 or SEQ ID NO:2, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a helper T cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a stem cell memory T cell. In some embodiments, the cell is a Treg cell.

[0149] In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising a VL domain of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding a CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:3 or SEQ ID NO:4, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a helper T cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a stem cell memory T cell. In some embodiments, the cell is a Treg cell.

[0150] In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO:1 and SEQ ID NO:3. In some embodiments, the nucleic acid molecule encoding a CAR polypeptide comprises an scFV comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of SEQ ID NO:1 or SEQ ID NO:3, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a helper T cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a stem cell memory T cell. In some embodiments, the cell is a Treg cell.

[0151] In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO:1 and SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any 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, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a helper T cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a stem cell memory T cell. In some embodiments, the cell is a Treg cell.

[0152] In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO:2 and SEQ ID NO:3. In some embodiments, the nucleic acid molecule encoding a CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:2 and SEQ ID NO:3, provided that the scFV domain binds to a citrullinated antigen as described herein. In some embodiments, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a helper T cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a stem cell memory T cell. In some embodiments, the cell is a Treg cell.

[0153] In some embodiments, the compositions of the invention include recombinant cells having a nucleic acid molecule encoding a CAR polypeptide comprising an scFV comprising the VH and VL domains of SEQ ID NO:2 and SEQ ID NO:4. In some embodiments, the nucleic acid molecule encoding the CAR polypeptide comprises an scFV comprising an amino acid sequence having at least any 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, the cell is a T cell, a CD4 T cell, a Treg cell, a CD8 alpha T cell, a CD8 beta T cell, a helper T cell, a granulocyte (neutrophil, basophil, eosinophil), a megakaryocyte, a monocyte, a macrophage, and a dendritic cell, or a stem cell memory T cell. In some embodiments, the cell is a Treg cell. VI. Method of Use

[0154] The T cells, and in particular Treg cells, expressing the CAR disclosed herein are useful in the treatment of rheumatoid arthritis.The method of use includes administering an effective amount of the pharmaceutical composition of the present disclosure to a subject in need thereof, for example, a subject suffering from rheumatoid arthritis.

[0155] As used herein, the term "subject" refers to an individual animal. As used herein, the term "patient" refers to a subject under the medical care or supervision of a medical provider, e.g., a doctor or a nurse. Subjects include mammals, e.g., humans and non-human primates, e.g., 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 who is seeking treatment, monitoring, adjustment or modification of an existing treatment regimen, etc. The term "rheumatoid arthritis subject" refers to an individual who has been diagnosed with rheumatoid arthritis. Rheumatoid arthritis patients may include individuals who are not receiving treatment, individuals who are currently receiving treatment, individuals who have been receiving treatment, and individuals who have discontinued treatment.

[0156] As used herein, the terms "effective amount", "effective dose" and "therapeutically effective amount" refer to an amount of an agent sufficient to produce a desired response, for example, to reduce or eliminate the signs or symptoms of a condition or to reverse a disorder. In some examples, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or the underlying cause of any of the disorders or diseases, and / or prevents the progression of the disease. For example, for a given parameter, a therapeutically effective amount indicates an increase or decrease in therapeutic efficacy of 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. The therapeutic efficacy may also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount may have an effect of 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more than that of the control.

[0157] The pharmaceutical composition may be administered by any suitable route, including, but not limited to, intravenous, subcutaneous, intramuscular, or intraperitoneal routes. An example of administration of the pharmaceutical composition includes storing the composition at 10 mg / ml in sterile isotonic aqueous saline for injection at 4° C. and diluting it in either 100 ml or 200 ml of 0.9% sodium chloride for injection prior to administration to the patient. The pharmaceutical composition is administered by intravenous infusion over 1 hour at a dose between 0.2-10 mg / kg. In other embodiments, the pharmaceutical composition is administered by intravenous infusion over a period of 15 minutes to 2 hours. In yet other embodiments, the administration procedure is via subcutaneous bolus injection.

[0158] The dose of the composition is selected to provide an effective treatment to the patient and ranges from 0.1 mg / kg body weight to less than about 25 mg / kg body weight or from 1 mg to 2 g per patient. In some cases, the dose ranges from 1 to 100 mg / kg, or from approximately 50 mg to 8000 mg per patient. The dose may be repeated at an appropriate frequency, which may range from once a day to once every three months, depending on the pharmacokinetics (e.g., half-life of the composition in circulation) and pharmacodynamic response (e.g., duration of therapeutic effect of the composition) of the composition. In some embodiments, the in vivo half-life is about 7 to about 25 days and the composition administration is repeated from once a week to once every three months.

[0159] Administration can be regular.Depending on the route of administration, for example, the dose can be administered once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer (for example, once every 2, 3, 4, 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 and the dosage and frequency of administration can be adjusted according to the progress of treatment and any adverse side effects.

[0160] Thus, in some embodiments, further administrations will depend on the patient's progress, e.g., the patient will be monitored between administrations. For example, after the initial administration or repeated administrations, the patient may be monitored for the rate of tumor growth, recurrence (e.g., in the case of post-operative patients), or systemic symptoms associated with the disease, e.g., weakness, pain, nausea, etc.

[0161] In certain embodiments, the T cells described herein are administered to the synovium of a joint of a subject with rheumatoid arthritis. Such administration can be by direct injection into the joint.

[0162] An exemplary method of the present disclosure includes isolating T lymphocytes from a biological sample obtained from a subject. Such T cells can be isolated, for example, by immunoaffinity using a derivatized solid support and an anti-CD4 antibody. CD4+ regulatory T cells (Treg) can be separated from non-Treg cells based on marker profiles. Treg cells are CD4+, CD25+, CD127lo. Non-Treg cells are CD4+, CD25+, and CD127+. The isolated Treg cells are then transfected with an expression vector encoding the chimeric antigen receptor (CAR) of the present disclosure. The transfected cells are expanded. The expanded cells are administered to a subject. VII. Kit

[0163] As used herein, the term "kit" refers to a group of items that are intended to be used together. The kit may optionally include a reference agent and / or instructions for its use. The kit may further include a shipping container configured to hold a container such as a vial that contains the composition disclosed herein. The kit may include a container that contains a group of items.

[0164] The kit of the present disclosure may include the pharmaceutical composition described herein in a container such as a bag or bottle for intravenous administration.The kit may also include a fluid conduit, such as a plastic tube, with a drip chamber.The drip chamber may communicate with an intravenous needle through the fluid conduit.The fluid conduit may also include one or more Y-sections and a roller clamp. EXAMPLES

[0165] Abbreviations: CAR (chimeric antigen receptor), CF (citrullinated fibrinogen), CFSE (carboxyfluorescein succinimidyl ester), CV (citrullinated vimentin), EGFR (epidermal growth factor receptor), IN (intranasal), IV (intravenous), LPS (lipopolysaccharide), PAD2 (peptidylarginine deiminase 2), PBMC (peripheral blood mononuclear cells), PBS (phosphate buffered saline), RA (rheumatoid arthritis), scFv (single-chain variable fragment), SF (synovial fluid), Teff (effector T cell), Treg (regulatory T cell), and UTD (non-transduced). Example 1 SBT01G consistently performed as well as or better than BVCA1 in luciferase systems

[0166] Jurkat FF luciferase transduction - 50,000 cells per well were plated in RPMI plus 2x protamine sulfate in each of two flat-bottom 96-well plates. Virus was diluted in RPMI to an approximate MOI of 1 in 100 μl. 100 μl was then added to one row of each 96-well plate. Plates were then rotated and placed in the incubator. Samples were pooled the next day. Transduced cells were resuspended in media and 100 μl transferred in duplicate to U-bottom plates. Cells were stained with CV-AF488 and CV-AF647 at 4°C for 20 minutes with 1:100 anti-EGFR-PE, washed once, and then analyzed by Novocyte. Samples were then scaled up to 6-well plates by adding 3 ml of fresh media.

[0167] Plate Coating - Citrullinated Vimentin (CV) was first diluted 1:100 in 4 ml PBS. Five 4-fold dilutions were made by transferring 1 ml to 3 ml. 100 μl was then added to a 96-well plate. Plates were placed at 4°C to coat overnight.

[0168] Luciferase Assay - Normalized transduced cells were pelleted and resuspended in 1 ml RPMI. CV coated plates were washed 3 times with PBS. 50 μl of cells were added to the plate. 25 μl of 3xPMA / Iono was added and the plate was placed in a 37°C incubator. After approximately 24 hours, luciferase plates were read by adding 75 μl of BioGlo reagent, incubating for 2-3 minutes in the dark and reading the plate.

[0169] Figure 1 shows the response of virus-transduced Jurkat FF luciferase cells to various concentrations of full-length CV coated plates. The results show that BVCA1 and STB01 responded most strongly to plate-bound full-length CV.

[0170] Figure 2 shows the response of virally transduced Jurkat FF luciferase cells to various concentrations of soluble CV. The results show that only BVCA1 and SBT01G-HL responded to soluble full-length CV in a dose-dependent manner.

[0171] FIG. 3 shows that BVCA1 and SBT01 demonstrated binding specificity for CV in assays using soluble bead-bound peptides.

[0172] Figure 4 shows the response of BVCA1 and SBT01G to plate-bound CV, antibody (V9) captured CV, and soluble CV at various protein concentrations. The results show that SBT01G responded more strongly than BVCA1 to plate-bound CV and antibody (V9) captured CV, but not to soluble CV. Example 2 SBT01 and BVCA1 respond to synovial fluid from rheumatoid arthritis patients

[0173] Jurkat FF luciferase transduction - 24 x 10 6Jurkat FF-luc cells were pelleted and resuspended in 24 ml of RPMI with protamine sulfate and virus at an MOI of 3 to express CARs of pSB_0147, pSB-0149, and pSB0139. Cells were mixed and aliquoted into each well of a 6-well plate. Plates were then rotated and placed in an incubator overnight. Cells were pelleted and reseeded in 25 ml of RPMT in a T75 flask.

[0174] Synovial fluid stimulation - Transduced cells were pelleted, resuspended in RPMI and placed into wells of black / white plates. Synovial fluid (SF) samples from RA patients were thawed, vortexed, diluted in RPMI and added to plates containing transduced cells. Cells were cultured with SF at 37°C.

[0175] 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.

[0176] Treg isolation and tissue culture - Primary human Treg cells were sourced from leukoreduction chamber residues or leukopaks of healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus by density gradient centrifugation. CD25+ cells were enriched by positive selection. Treg cells were then isolated by gating on CD4+CD25+CD127lo cells using FACS. After isolation, cells were stimulated with CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead-to-cell ratio and cultured at a density of 250,000–300,000 cells / mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and beta-mercaptoethanol with 300 IU / mL recombinant human IL-2. On day 9 of expansion, fresh CTS Dynabeads Treg Xpander was added at a 1:1 bead-to-cell ratio.

[0177] Transduction of primary Tregs - On day 2 of expansion, primary Tregs were transduced with the CV-CAR construct by spinoculation in the presence of protamine sulfate.

[0178] Treg activation – CV-CAR-expressing Treg cells were cultured in vitro with dilutions of synovial fluid samples ranging from 1:5 to 1:160. Treg activation was assessed by measuring CD71 expression.

[0179] Flow cytometry and FACS analysis - Activated cultures were harvested, centrifuged at 300xg for 5 min, and then resuspended in 1x Flowstain Buffer (Invitrogen) supplemented with viability dye (Invitrogen), anti-EGFR surface staining antibody, and CD71 surface staining antibody. Tregs were incubated at 4°C for 30 min, then centrifuged and washed with 1x Flow stain Buffer. Stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry.

[0180] Figure 5 shows the response of SBT01G and BVCA1 to synovial fluid from RA patients. The data show that SBT01G produces a stronger response than BVCA1 to synovial fluid from several RA patients.

[0181] FIG. 6 shows that in synovial fluid samples from Swedish RA patients in which a response was elicited, SBT01G was also more potent than BVCA1.

[0182] Figure 7 shows the response of primary Treg cells transduced with CV-CAR to synovial fluid from multiple RA patients. Primary Treg cells expressing SBT01G CAR are more sensitive to synovial fluid from RA patients than primary Treg cells expressing BVCA1 CAR. Example 3 Response of SBT01 and BVCA1 to citrullinated fibrinogen

[0183] Plate Coating - Citrullinated proteins were diluted in PBS and added to the wells of a black / white Isoplate. The plate was left at 4°C overnight to coat the plate wells.

[0184] Luciferase assay - Stably transduced Jurkat FF-luc cell lines and untransduced (UTD) Jurkat FF-luc cell lines were pelleted and resuspended in RPMI with 10% FBS. Citrullinated protein coated plates were washed 3 times with PBS. 50,000 cells in 75 μl per well were added to the coated plates. 5 μl of 15×PMA / Iono in RPMI was added to each well and the plates were placed in a 37°C incubator. After approximately 24 hours, 75 μl of BioGlo reagent was added to each well and the plates were incubated in the dark for 2-3 minutes. Luminescence was then read on a plate reader.

[0185] Treg Isolation and Tissue Culture - Primary human Treg cells were sourced from leukoreduction chamber residues or leukopacks of healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus by density gradient centrifugation. CD25+ cells were enriched by positive selection. Treg cells were then isolated by gating on CD4+CD25+CD127lo cells using FACS, and Teff were isolated using FACS gating on CD4+CD25loCD127pos. After isolation, cells were stimulated with CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead-to-cell ratio and cultured at a density of 250,000–300,000 cells / mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and b-mercaptoethanol with 300 IU / mL recombinant human IL-2. On day 9 of expansion, fresh CTS Dynabeads Treg Xpander was added at a 1:1 bead-to-cell ratio.

[0186] Transduction of primary Tregs and Teffs - On day 2 of expansion, primary Tregs and Teffs were transduced with CD19-CAR or CV-CAR constructs by spinoculation in the presence of protamine sulfate.

[0187] Treg activation - CAR-expressing Tregs and Teffs were labeled with the proliferation dye CFSE prior to the start of activation cultures. CD19-CAR and CV-CAR expressing Treg cells were cultured in vitro with citrullinated vimentin (CV) or citrullinated fibrinogen (CF) coated onto plates over a dose range from 30ng / ml to 10μg / ml. Treg activation was assessed by measuring the percentage of proliferating cells and by expression of CD71.

[0188] Flow cytometry and FACS analysis - Activated cultures were harvested, centrifuged at 300xg for 5 min, then resuspended in 1x Flowstain Buffer (Invitrogen) supplemented with viability dye (Invitrogen), anti-EGFR and CD71 surface staining antibodies. Tregs were incubated at 4°C for 30 min, then centrifuged and washed with 1x Flowstain Buffer. Stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry.

[0189] 8 shows the response of SBT01G and BVCA1 to plate-bound full-length peptidylarginine deiminase 2 (PAD2) citrullinated fibrinogen. The results show that SBT01G, but not BVCA1, is also capable of responding to plate-bound full-length PAD2 citrullinated fibrinogen.

[0190] Figure 9 shows that SBT01G CAR expressed on Teff and Treg cells responds to citrullinated vimentin (CV) and citrullinated fibrinogen (CF). In contrast, BVCA1 CAR expressed on Teff and Treg cells responds to CV but not CF.

[0191] Thus, Figures 1-9 show that SBT01G consistently performed comparably to or better than BVCA1 in all assay systems. Example 4 CV CAR demonstrates functional response when using different promoters and linkers

[0192] Treg Isolation and Tissue Culture - Primary human Treg cells were sourced from leukoreduction chamber residues or leukopacks of healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus by density gradient centrifugation. CD25+ cells were enriched by positive selection. Treg cells were then isolated by gating on CD4+CD25+CD127lo cells using FACS. After isolation, cells were stimulated with CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead-to-cell ratio and cultured at a density of 250,000–300,000 cells / mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and b-mercaptoethanol with 300 IU / mL recombinant human IL-2. On day 9 of expansion, fresh CTS Dynabeads Treg Xpander was added at a 1:1 bead-to-cell ratio.

[0193] Transduction of primary Tregs - On day 2 of expansion, primary Tregs were transduced with the CV-CAR construct by spinoculation in the presence of protamine sulfate.

[0194] Treg activation - CAR-expressing Tregs were labeled with the proliferation dye CFSE prior to the start of activation cultures. CV-CAR-expressing Treg cells were cultured in vitro with CV peptide captured on beads. Treg activation was assessed by measuring the percentage of proliferating cells.

[0195] Flow cytometry and FACS analysis Activation assay - Activation cultures were harvested, centrifuged at 300 x g for 5 min, then resuspended in 1x Flowstain Buffer (Invitrogen) with viability dye (Invitrogen) and anti-EGFR surface staining antibody. Tregs were incubated at 4°C for 30 min, then centrifuged and washed with 1x Flowstain Buffer. Stained cells were fixed with CytoFix (BD Biosciences) and then analyzed by flow cytometry.

[0196] Flow cytometry and FACS analysis of Treg phenotype - On day 14 of expansion, untransduced (UTD) Tregs, Tregs expressing CD19-CAR and CV-CAR were stained for transcription factors FoxP3 and Helios. Cells were fixed and permeabilized using eBiosciences FoxP3 transcription factor buffer set (eBiosciences).

[0197] On day 14 of resting-expanding Treg cultures, Tregs expressing CD19-CAR and CV-CAR were harvested, the beads were removed using a magnet, and cultured at 500,000 cells / ml with 300 IU / ml IL-2 alone.

[0198] Flow cytometry and FACS analysis of CAR expression - CD19-CAR and CV-CAR expressing Tregs after 14 days of expansion and 2 or 5 days of rest were stained with viability dye (Invitrogen) and anti-EGFR surface staining antibody. CV-CAR expression was detected by incubating cells with 1 μg / ml CV followed by FITC-labeled anti-vimentin (clone V9, Invitrogen).

[0199] Plate Coating - Citrullinated Vimentin (CV) was diluted in PBS and added to the wells of a black / white Isoplate. The plate was left at 4°C overnight to coat the plate wells.

[0200] Luciferase Assay - Stably transduced Jurkat FF-luc cell lines and untransduced (UTD) Jurkat FF-luc cell lines were pelleted and resuspended in RPMI with 10% FBS. Citrullinated protein coated plates were washed 3 times with PBS. 50,000 cells per well in 75 μl were added to the coated plates. 5 μl of 15×PMA / Iono in RPMI was added to each well and the plates were placed in a 37°C incubator. After approximately 24 hours, 75 μl of BioGlo reagent was added to each well and the plates were incubated in the dark for 2-3 minutes. Luminescence was then read on a plate reader.

[0201] Figure 10A shows that both EF1A and MND promoters drive expression and functional responses by CV-CAR T cells to soluble bead-bound citrullinated peptides (pCV). Figure 10B shows that the use of different promoters does not alter the phenotype of CV-CAR Treg cells.

[0202] Figure 11 shows that the choice of scFv linker has little or no effect on SBT01G CAR T cell responses to full-length CV.

[0203] Figure 12A shows that transduction with the SBT01G CAR vector resulted in CV-CAR expression in a higher percentage of cells than transduction with the BVCA1 CAR vector. Figure 12B shows that SBT01G CAR and BVCA1 CAR Treg cells have similar FoxP3 and Helios profiles.

[0204] Figures 10-12 show that SBT01G exhibited a high percentage of CAR-positive Tregs, and SBT01G exhibited high levels of CAR expression. Example 5 Evaluation of CV-CAR Treg activation by citrullinated vimentin in vitro

[0205] Treg Isolation and Tissue Culture - Primary human Treg cells were sourced from leukoreduction chamber residues or leukopacks of healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus by density gradient centrifugation. CD25+ cells were enriched by positive selection. Treg cells were then isolated by gating on CD4+CD25+CD127lo cells using FACS. After isolation, cells were stimulated with CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead-to-cell ratio and cultured at a density of 250,000–300,000 cells / mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and penicillin / streptomycin with 300 IU / mL recombinant human IL-2.

[0206] Treg activation - Non-transduced and CV-CAR-expressing Treg cells were cultured in vitro with citrullinated vimentin antigen over a dose range from 10 ng / mL to 10 μg / mL. Treg activation was assessed by measuring the percentage of proliferating cells and the levels of CD71 expression and IL-10 secretion.

[0207] Flow cytometry and FACS analysis: Non-transduced and CV-CAR-expressing Treg cells were harvested, centrifuged at 300×g for 5 min, and then resuspended in 1× RoboSep Buffer (StemCell Technologies) with CD71 surface staining antibody and Cell Trace Violet (CTV) viability dye cocktail (Invitrogen). Tregs were incubated at 4° C. for 30 min, then centrifuged and washed with 1× RoboSep Buffer. Stained cells were then analyzed by flow cytometry. result

[0208] Activation of Tregs in response to citrullinated vimentin (CV) was evaluated in vitro. Figure 13 shows dose-dependent CV-induced activation of CV-CAR Treg cells as demonstrated by increased proliferation cells, CD71 expression, and IL-10 secretion. In contrast, non-transduced Treg cells were not activated by CV. These results demonstrate that Treg activation upon stimulation with CV is specific to Tregs expressing CV-CAR.

[0209] The amount of CV present in the synovial fluid of healthy donors and rheumatoid arthritis (RA) patients was assessed by ELISA as shown in Figure 14. Jurkat reporter cells expressing one of three different CV-CARs or a control CD19-CAR were incubated with increasing amounts of synovial fluid and the response was measured as an indicator of CV-CAR binding and signaling for T cell activation. Figure 14 shows that Jurkat cells (immortalized human T lymphocytes) expressing CV-CAR1 (BVCA1) and CV-CAR2 (SBT01G), but not CV-CAR3 (C03) or CD19-CAR, showed an increase in response to synovial fluid (SF). Figure 14 also shows that CV-CAR2 (SBT01G) T cells were more activated than CV-CAR1 (BVCA1) T cells by SF samples from RA patients.

[0210] To demonstrate that CV-CAR-expressing Tregs are activated by citrullinated antigens in the synovial fluid of RA patients, non-transduced and CV-CAR2 Tregs were incubated with RA synovial fluid or vehicle, and the levels of proliferation and CD71 expression were measured. Figure 15 shows that CV-CAR2 Treg cells from two donors were specifically activated by RA synovial fluid.

[0211] Collectively, these results establish that CV-CAR Treg cells are able to bind citrullinated antigens present in RA synovial fluid and induce the activation required for suppressive function. Example 6 Evaluation of suppression of CV induction in Teff cells by CV-CAR Tregs

[0212] Treg suppression assay - Treg cells expressing CV-CAR were prepared as above from fresh human leukopacks in 96-well U-bottom plates. Tregs were incubated with CV or vehicle control as antigenic stimulation. Tregs were co-cultured with either CD3 / CD28-activated Teff cells or CD19-CAR-specific Teff cells to assess Treg suppression of target cells. Teff cells were co-cultured at the following Treg:Teff ratios: (CD3 / CD28-activated) 1:4=0.25, 1:2=0.5, 1:1=1, 2:1=2, and 4:1=4, or (CD19-specific) 1:4, 1:2, 1:1, 2:1, and 4:1. result

[0213] Figure 16A shows that CV-CAR Tregs stimulated with CV were able to suppress the proliferation of CD3 / CD28-activated Teff cells at low Treg:Teff ratios. Figure 16B shows that CV-CAR Tregs stimulated with CV were also able to suppress the proliferation of CD19-CAR Teff cells at low Treg:Teff ratios. Example 7 Analysis of CV-CAR Tregs in rodent models of CV-associated lung inflammation

[0214] In human rheumatoid arthritis (RA) patients, consistently high concentrations of citrullinated vimentin (CV) in synovial fluid identify CV as a relevant biomarker for human RA. However, current RA mouse models exhibit inconsistent CV levels, which complicates CV-CAR Treg analysis in vivo. To induce CV production and release into the extracellular matrix, we developed an LPS-induced pulmonary inflammation mouse model. This model is characterized by an acute inflammatory response including pulmonary neutrophilia, increased secretion of the inflammatory cytokines interleukin-6 (IL-6), interleukin-1 beta (IL-1b), and tumor necrosis factor alpha (TNF alpha), and CV accumulation in lung tissue within days after intranasal exposure to LPS. A chimeric antigen receptor (CAR) specific for CV has been shown to respond to CV protein accumulated in lung tissue, which results in increased antigen-specific survival, proliferation, and suppressive activity of the respective CV-CAR Treg cells. method

[0215] Immunocompromised NCG mice aged 6–8 weeks were obtained from Charles River Laboratories. Mice were randomized by body weight and divided into groups of 5–10 animals. Mice were subjected to intranasal (IN) treatment with LPS (5 mg / kg) on ​​days 0, 1, 6, and 12 to induce pulmonary inflammation and release and accumulate citrullinated vimentin (CV) in affected tissues. CV-CAR Treg cells were harvested from actively growing cultures on day 0, stimulatory beads were magnetically removed, and Tregs were subsequently labeled with Cell Trace Violet (CTV) (Invitrogen). Approximately 4 hours after the first LPS challenge, CTV-labeled Tregs were diluted to 25 × 10 6 The cells were adjusted to 10 cells / mL and a volume of 200 μl was injected intravenously (IV) to deliver a total of 5 × 10 cells / mL to each mouse. 6Cells were administered. After adoptive cell transfer of CV-CAR Tregs or control CD19-CAR Tregs, all mice received intraperitoneal (IP) injections of IL-2 (160,000 IU) on days 1, 2, 5, 6, 7, 8, 9, and 12. Mice were monitored daily for any clinical signs of distress and weighed twice weekly. On day 13, mice were euthanized and spleens and lungs were harvested and processed into single cell suspensions for flow cytometry analysis of human CV-CAR Tregs and CD19-CAR Tregs. result

[0216] Figure 17 shows an exemplary timeline for induction of CV-associated lung inflammation and activation of CV-CAR Treg cells in vivo in mice. result

[0217] FIG. 18A shows how the proliferation ratio was calculated from the flow cytometry data, while FIG. 18B shows how the fold change in EGFR ratio (CAR marker) was calculated from the same data.

[0218] Figure 19A shows that the absolute number of CV-CAR Tregs is significantly enriched in the lung tissue from mice with LPS-induced pulmonary inflammation. Figure 19B shows that the proliferation ratio of CV-CAR Tregs is significantly increased in the lung tissue from mice with LPS-induced pulmonary inflammation. These results demonstrate that CV-CAR Tregs have improved ability to cultivate and proliferate in inflamed lung tissues characterized by the accumulation of citrullinated vimentin compared to control Tregs. Therefore, this mouse lung inflammation model allows the examination of human CV-CAR Tregs in vivo. Example Embodiments 1. A chimeric antigen receptor (CAR) comprising an antigen binding domain, a hinge domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain, wherein the antigen binding domain binds to a citrullinated polypeptide, and optionally, the antigen binding domain specifically binds to three or more different citrullinated proteins or citrullinated fragments thereof. 2. The CAR of embodiment 1, wherein the antigen-binding domain binds to a plurality of different citrullinated proteins or citrullinated fragments thereof. 3. The CAR of embodiment 1, wherein the antigen-binding domain binds to one or more of, for example, 10, 12, 14 or 16 amino acids of at least any of: (i) citrullinated vimentin, (ii) citrullinated filaggrin, (iii) citrullinated fibrinogen, and (iv) citrullinated peptide fragments thereof. 4. The CAR of embodiment 1, wherein the antigen-binding domain binds to at least two of: (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptide fragments thereof. 5. The CAR of embodiment 1, wherein the antigen-binding domain binds to all three of: (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptide fragments thereof. 6. The CAR of embodiment 5, which further binds to citrullinated tenascin C. 7. The antigen-binding domain comprises a VH domain and a VL domain; (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 36; (ii) A CAR as described in embodiment 1, wherein the VL domain of the target binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. 8. The CAR of embodiment 1, wherein the intracellular signaling domain is derived from CD3 zeta. 9. The CAR of embodiment 1, wherein at least one costimulatory domain is derived from a member of the group consisting of FceR1g, Fcg, CD28, 4-1BB, CTLA-4, CTLA-4 / CD-28 hybrid, DAP10, CD27, and 2B4, and optionally wherein at least one costimulatory domain comprises a CD28 and / or a 4-1BB costimulatory domain. 10. The CAR of embodiment 1, wherein the antigen-binding domain comprises an antibody, an antibody fragment, a camelid nanobody, a heavy chain-only antibody or an aptamer. 11. The CAR of any one of embodiments 1-10, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain, the hinge domain is a CD8 hinge domain or a CD28 hinge domain, and / or further comprises a signal peptide, optionally wherein the signal peptide is a CD8 signal peptide or a GM-CSF signal peptide. 12. The CAR of embodiment 11, wherein the antigen-specific binding domain comprises a single chain fragment. 13. The single chain fragment comprises a single chain variable fragment (scFv), and optionally the scFv fragment comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO:1, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:4 13. The CAR of embodiment 12, comprising: 14. The scFv fragment, (a)(1)SBT01 VH(M) HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 1), or (2) SBT01 VH(G) A VH selected from QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 2), and (b)(1)SBT01 VL(M) SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVL (SEQ ID NO: 3), or (2) SBT01 VL(G) SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVL (SEQ ID NO: 4) VL selected from 14. The CAR of embodiment 13, comprising: 15. The CAR of embodiment 14, wherein the VH-VL fragments are joined by a linker selected from the group consisting of: (i) GGGSx3 (SEQ ID NO: 20), (ii) Whitlow218 (SEQ ID NO: 21), and (iii) AB Pur (SEQ ID NO: 22). 16. scFv, (a) SBT01G-VHVL-GGGS×3 linker-pSB_0149 [ka] (GGGSx3 linker underlined), or (b) SBT01G-VHVL-Whitlow218 linker-pSB_0158 [ka] (Whitlow 218 linker underlined), or (c)SBT01G-VHVL-AB pur linker-pSB_0159 [ka] (AB pur linker is underlined) 17. The CAR of embodiment 16, comprising the amino acid sequence of: 17. The CAR of any of embodiments 1-16, wherein the costimulatory domain comprises a CD28, 41BB, OX40, OX40, CD40L, MyD88, CD40, CD27, ICOS, or RANK / TRANCE-R costimulatory domain. 18. A nucleic acid encoding a CAR according to any one of embodiments 1 to 17. 19. The nucleic acid of embodiment 18, comprising DNA or RNA. 20. VH, [ka] and wherein the nucleic acid sequence is VL, [ka] 20. The nucleic acid of embodiment 18, wherein the nucleic acid is encoded by a nucleic acid sequence comprising: 21. The nucleic acid of embodiment 20, wherein the VH and VL fragments are joined by a linker and comprise the nucleic acid sequence of (i) SEQ ID NO: 12, (ii) SEQ ID NO: 13, or (iii) SEQ ID NO: 14. 22. An expression vector comprising an expression control sequence operably linked to the nucleic acid sequence of any one of embodiments 16 to 21. 23. The expression vector of embodiment 22, wherein the expression control sequence comprises a regulatory region, the regulatory region being selected from the group consisting of promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions, transcription initiation sites, termination sequences, polyadenylation sequences, nuclear localization, signals, and introns. 24. The expression vector of embodiment 23, which is an adenoviral vector, a lentiviral vector or a plasmid. 25. A host cell comprising the expression vector of embodiment 22, 23 or 24. 26. A modified T cell modified to express a chimeric antigen receptor (CAR) according to any one of embodiments 1 to 17. 27. The modified T cell of embodiment 26, which is a mammalian T cell. 28. The modified T cell of embodiment 27, which is a Treg cell. 29. The modified T cell of embodiment 28, which is a human T cell. 30. The modified T cell of embodiment 29, which is a primary T cell. 31. The modified T cell of embodiment 30, which is CD4+, CD25+, and CD127lo. 32. A pharmaceutical composition comprising a plurality of the modified T cells according to any of embodiments 26-31 and a pharma- ceutically acceptable carrier. 33. A method for treating a subject suffering from rheumatoid arthritis, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 32. 34. The method of embodiment 33, wherein the subject is a human. 35. The method of embodiment 33 or 34, wherein the pharmaceutical composition is administered to the synovium of the subject. 36. The method according to embodiment 33 or 34, wherein the pharmaceutical composition is administered intravenously. 37A. The administering step comprises: (a) isolating T cells from a biological sample obtained from a subject; (b) enriching the T cells for regulatory T cells (Treg); (c) transfecting the enriched Treg cells with an expression vector comprising an expression control sequence operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain that binds to a citrullinated polypeptide; (d) expanding the transfected Treg cells; (e) administering the expanded Treg cells to a subject. 34. The method of embodiment 33, comprising: 37B. A method of treating a subject suffering from rheumatoid arthritis, comprising: (a) isolating T cells from a biological sample obtained from a subject; (b) enriching the T cells for regulatory T cells (Treg); (c) transfecting the enriched Treg cells with an expression vector encoding a CAR according to any one of embodiments 1 to 17; (d) expanding the transfected Treg cells; (e) administering the expanded Treg cells to a subject. A method comprising: 38. The method of embodiment 37, wherein the expansion comprises using anti-CD3 / CD28 coated beads. 39. The method of embodiment 37, wherein the expansion does not use anti-CD3 / CD28 coated beads. 40. The method according to any of embodiments 37 to 40, wherein transfection is carried out by use of a viral vector, electroporation, heat shock, bacteriophage, sonication, or calcium phosphate. 41. The method of embodiment 37, further comprising administering to the subject one or more anti-inflammatory and / or therapeutic agents. 42. The method of embodiment 41, wherein the anti-inflammatory agent comprises an antibody that inhibits inflammatory cytokines. 43. The method of embodiment 42, wherein the anti-inflammatory agent is an anti-TNF antibody, an anti-IL-6 antibody, or a combination thereof. 44. A kit comprising a container containing the pharmaceutical composition of embodiment 32 and in communication with a drip chamber via a fluid conduit, the drip chamber in communication with an intravenous needle via a fluid conduit. 45. The kit of embodiment 44, wherein the container comprises a bag. 46. ​​The kit of embodiment 44, wherein the fluid conduit between the container and the needle comprises one or more Y-sites and a roller clamp.

[0219] It should be understood that the description and drawings are not intended to limit the invention to the particular forms disclosed, but on the contrary, are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of the present description. Thus, the description and drawings should be interpreted as merely illustrative and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It should be understood that the forms of the invention shown and described herein are interpreted as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as will become apparent to those skilled in the art after having the benefit of the description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as set forth in the following claims. The headings used herein are for organizational purposes only and are not intended to be used to limit the scope of the description.

[0220] 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

**Claim 1** A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain, wherein the antigen-binding domain specifically binds to three or more different citrullinated proteins or citrullinated fragments thereof. **Claim 2** The CAR according to claim 1, wherein the antigen-binding domain binds to all three of (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or to citrullinated peptide fragments thereof. **Claim 3** The CAR according to claim 2, wherein the antigen-binding domain further binds to citrullinated tenascin C. **Claim 4** The antigen-binding domain comprises a VH domain and a VL domain, (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, (ii) the VL domain of the antigen-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. The CAR according to claim 1. **Claim 5** The CAR according to claim 1, wherein the intracellular signaling domain is derived from CD3 zeta. **Claim 6** At least one of the co-stimulatory domains comprises a co-stimulatory domain of a member of the group consisting of FcεR1γ, Fcγ, CD28, CD134 (OX40), CD137 (4-1BB), CTLA-4, CTLA-4 / CD-28 hybrid, DAP10, CD27, 2B4, and combinations thereof, and optionally, at least one of the co-stimulatory domains comprises a CD28 and / or 4-1BB co-stimulatory domain. The CAR according to claim 1. **Claim 7** The CAR according to claim 1, wherein the antigen-binding domain comprises an antibody, an antibody fragment, a camelid nanobody, a heavy chain-only antibody, or an aptamer. **Claim 8** The CAR according to claim 1, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain. **Claim 9** The CAR according to claim 1, wherein the hinge domain is a CD8 hinge domain or a CD28 hinge domain. **Claim 10** The CAR according to claim 1, further comprising a signal peptide.

11. The CAR according to claim 10, wherein the signal peptide is a CD8 signal peptide or a GM-CSF signal peptide.

12. The CAR according to claim 1, wherein the antigen-specific binding domain comprises a single-chain fragment.

13. The CAR according to claim 12, wherein the single-chain fragment comprises a single-chain variable fragment (scFv).

14. The scFv fragment comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 1, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 4 The CAR according to claim 13, comprising.

15. The scFv fragment comprises (a) (1) SBT01 VH(M) HLHLQESGPGLVKPSETLSLTCTVSGGGSINDTTYYWGWIREQPPGKGL EWIGSIYYRGNTHYNSSLRSRVTMSSVDTSKNRFSLKVTSSVTAA DTAYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 1), or (2) SBT01 VH(G) QLQLQESGPGLVKPSETLSLTCTVSGGGSISSSSSYYWGWIREQPPGKGL EWIGSIYYSGSYYNPSLKSRVTISVDTSKNQFSLLSSVTAA DTAYYCARLDPFDYWGRGTLVTVSS (SEQ ID NO: 2) Selected VH, and (b) (1) SBT01 VL(M) SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTAANLTITRVEAGDDADYFCQVYDRKTDHQVF GPGTTVTVL (SEQ ID NO: 3), or (2) SBT01 VL(G) SYVLTQPPSVSVAPGK TARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYD SDSRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSD HQVFGTGTKVTVL (SEQ ID NO: 4) Selected VL The CAR according to claim 13, comprising.

16. The VH-VL fragment is joined by a linker selected from the group consisting of (i) GGGGS×3 (SEQ ID NO: 20), (ii) Whitlow218 (SEQ ID NO: 21), and (iii) AB Pur (SEQ ID NO: 22). The CAR according to claim 15.

17. The scFv is a) the SBT01G-VHVL-GGGS×3 linker of SEQ ID NO: 5, or b) the SBT01G-VHVL-Whitlow218 linker of SEQ ID NO: 6, or c) the SBT01G-VHVL-AB pur linker of SEQ ID NO: 7, and the CAR according to claim 16 comprises the amino acid sequence thereof.

18. A nucleic acid encoding the CAR according to any one of claims 1 to 17.

19. The nucleic acid according to claim 18, comprising DNA or RNA.

20. The nucleic acid according to claim 18, wherein the VH is encoded by a nucleic acid sequence comprising SEQ ID NO: 8 or SEQ ID NO: 9, and the VL is encoded by a nucleic acid sequence comprising SEQ ID NO: 10 or SEQ ID NO:

11.

21. The nucleic acid according to claim 20, wherein the VH and VL fragments are joined by a linker and comprise the nucleic acid sequence of SEQ ID NO: 12, or (ii) SEQ ID NO: 13, or (iii) SEQ ID NO:

14.

22. An expression vector comprising an expression control sequence operably linked to the nucleic acid sequence according to claim 21.

23. The expression vector according to claim 22, wherein the expression control sequence comprises a regulatory region, and the regulatory region is selected from the group consisting of a promoter sequence, an enhancer sequence, a response element, a protein recognition site, an inducible element, a protein binding sequence, 5' and 3' untranslated regions, a transcription start site, a termination sequence, a polyadenylation sequence, a nuclear localization signal, and an intron.

24. The expression vector according to claim 23, which is an adenovirus vector, a lentivirus vector or a plasmid.

25. A host cell comprising the expression vector according to claim 24.

26. A modified T cell modified to express the chimeric antigen receptor (CAR) according to any one of claims 1 to 17.

27. The modified T cell according to claim 26, wherein the T cell is a mammalian T cell.

28. The modified T cell according to claim 27, wherein the T cell is a Treg cell.

29. The modified T cell according to claim 28, wherein the T cell is a human T cell.

30. The modified T cell according to claim 29, wherein the T cell is a primary T cell.

31. The modified T cell according to claim 30, wherein the T cell is CD4+, CD25+, and CD127lo.

32. A pharmaceutical composition comprising the modified T cells according to claim 31 and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition according to claim 32 for treating a subject suffering from rheumatoid arthritis.

34. The pharmaceutical composition according to claim 33, wherein the subject is a human.

35. The pharmaceutical composition according to claim 33, wherein the pharmaceutical composition is administered into the synovium of the subject.

36. The pharmaceutical composition according to claim 33, wherein the pharmaceutical composition is administered intravenously.

37. A method for preparing a composition for treating a subject suffering from rheumatoid arthritis, comprising an increased amount of Treg cells, the method comprising: (a) isolating T cells from a biological sample obtained from the subject; (b) enriching the T cells for regulatory T cells (Tregs); (c) transfecting the enriched Treg cells with an expression vector encoding the CAR according to any one of claims 1 to 17; [[ID=...]] (d) expanding the transfected Treg cells; (e) preparing the composition using the expanded Treg cells.

38. The method according to claim 37, wherein the expansion comprises using anti-CD3 / CD28 coated beads.

39. The method according to claim 37, wherein the expansion does not comprise using anti-CD3 / CD28 coated beads.

40. The method according to claim 37, wherein the transfection is performed by using a viral vector, electroporation, heat shock, bacteriophage, sonication, or calcium phosphate.

41. The method according to claim 37, wherein the composition is administered to the subject in combination with one or more anti-inflammatory agents and / or therapeutic agents.

42. The method according to claim 41, wherein the anti-inflammatory agent comprises an antibody that inhibits an inflammatory cytokine.

43. The method according to claim 42, wherein the anti-inflammatory agent is an anti-TNF antibody, an anti-IL-6 antibody, or a combination thereof.

44. A kit comprising the pharmaceutical composition according to claim 32 and a container communicating with a drip chamber via a fluid conduit, wherein the drip chamber communicates with an intravenous injection needle via a fluid conduit.

45. The kit according to claim 44, wherein the container comprises a bag. **Claim 46** The kit according to claim 44, wherein the fluid conduit between the container and the needle comprises one or more Y-sites and roller clamps.

Images