Chimeric antigen receptors with atypical transmembrane and / or hinge domains

Abstract

The present disclosure relates to chimeric antigen receptors (CARs) that do not cause a substantial a reduction in expression of endogenous CD28 protein in T-cells in which the CARs are expressed.

Description

CHIMERIC ANTIGEN RECEPTORS WITH ATYPICAL TRANSMEMBRANEAND / OR HINGE DOMAINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 620,690, filed January 12, 2024, which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (237752000940SEQLIST.xml; Size: 44,566 bytes; and Date of Creation: January 9, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to chimeric antigen receptors (CARs) that cause little to no reduction in expression of endogenous CD28 protein in T-cells in which the CARs are expressed.BACKGROUND OF THE INVENTION

[0004] Chimeric antigen receptors (CARs) including portions of both an antibody and a T-cell receptor (TCR) were first described the 1980s. First generation CARs included an antigen-binding domain, a hinge, a transmembrane domain and an intracellular signal transduction domain of CD3Q Subsequent generations of CARs have been engineered to include one or more intracellular co-stimulation domains to improve their ability to activate T-cells in which they are expressed (Smith and Shen, J. Transl. Med, 21:515, 2023).

[0005] There are now four anti-CD19 CAR T-cell immunotherapies approved by the U.S. Food and Drug Administration for treatment of various CD19+ leukemias and lymphomas. In fact, anti-CD19 CAR T-cell immunotherapies have led to remarkable clinical responses in patients with poor prognoses, including high levels of remission. However, several challenges limit the therapeutic efficacy of cancer antigen-reactive CAR T-cells including toxicities and modest anti-tumor activity against non-hematological and other hematological malignancies.

[0006] While CAR development has focused predominantly on the antigen-binding and intracellular co-stimulation domains, the effect of other CAR domains, such as the hinge andtransmembrane domains, on functions of T-cells expressing the CAR remains unclear. Thus, what is needed in the art are CAR backbones that have been engineered to reduce potentially deleterious effects in T-cells in which they are expressed.SUMMARY OF THE INVENTION

[0007] The present disclosure relates to chimeric antigen receptors (CARs) that cause little to no reduction in expression of endogenous CD28 protein in T-cells in which the CARs are expressed. In particular, CARs of the present disclosure comprise different hinge and / or transmembrane domains than the four anti-CD19 CAR T-cell therapies that are currently approved by the U.S. Food and Drug Administration (tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel and lisocabtagene maraleucel).BRIEF DESCRIPTION OF DRAWINGS

[0008] FIG. 1A presents a schematic of a chimeric antigen receptor (CAR) with a wildtype CD28 transmembrane domain and a wild-type CD28 hinge region and the corresponding expression construct. The promoter region (Prom.) and the truncated epidermal growth factor receptor (EGFRt) transduction tag are shown in the 5’ and 3’ ends, respectively, of the expression construct. FIG. IB is a representative fluorescence activated cell sorting (FACS) plot of regulatory T (Treg) cells expressing a CAR with a wild-type CD28 transmembrane domain and a wild-type CD28 hinge region. Cells are stained with EGFRt and CD28 antibodies to identify EGFRt+ and CD28+ cells. Each quadrant (Q5, Q6, Q7, and Q8) distinguishes various Treg cell populations within the parent population: Q5 = EGFRt-, CD28+ Treg cells; Q6 = EGFRt+, CD28+ Treg cells; Q7 = EGFRt+, CD28- Treg cells; and Q8 = EGFRt-, CD28- Treg cells. The percentage of each Treg cell population is shown within the quadrants. EGFRt is the truncated epidermal growth factor receptor transduction tag. FIG. 1C is a bar graph showing CD28 expression as mean fluorescence intensity (MFI) for untransduced Treg cells (CAR-) and Treg cells expressing a CAR with a wild-type CD28 transmembrane domain and a wild-type CD28 hinge region (CAR+). FIG. ID is a histogram of normalized cell count versus CFSE signal intensity of untransduced Treg cells (CAR-) and Treg cells expressing a CAR with a wildtype CD28 transmembrane domain and a wild-type CD28 hinge region (CAR+). CFSE is a fluorescent cell staining dye (carboxyfluorescein succinimidyl ester) whose staining intensity decreases with each round of cell division.

[0009] FIG. 2A-2B show schematics of a CAR with a wild-type CD28 transmembrane domain and a wild-type CD28 hinge region (referred to herein as WT backbone) and exemplary CARs with modifications in the transmembrane domain and / or hinge region (referred to herein as modified backbone). Abbreviations: Mut (mutated); TCRa (T-cell receptor alpha chain); TMD (transmembrane domain); and WT (wild-type).

[0010] FIGS. 3A-3B show schematics of various CAR expression constructs, with each construct comprising an EGFRt transduction tag. Abbreviations: H (hinge region); IC (intracellular domain); and TM (transmembrane domain). Substitutions in the TM of CAR 3 include: C165L, Y166L, S167L, and T171L, which correspond to C13L, Y14L, S15L and T19L as shown in the amino acid sequence of the modified CD28 TM of SEQ ID NO:42.

[0011] FIG. 4A shows representative FACS plots of Treg cells expressing various CARs. Cells are stained with EGFRt and CAR antibodies to identify EGFRt+ and CAR+ cells. Each quadrant (QI, Q2, Q3, and Q4) distinguishes various Treg cell populations within the parent population: QI = EGFRt+, CAR- Treg cells; Q2 = EGFRt+, CAR+ Treg cells; Q3 = EGFRt-, and CAR+ Treg cells; Q4 = EGFRt-, CAR- Treg cells. The percentage of each Treg cell population is shown within the quadrants. FIG. 4B is a bar graph showing CAR expression as mean fluorescence intensity (MFI) for Treg cells expressing various CARs. FIG. 4C is a bar graph showing CD28 expression as mean fluorescence intensity (MFI) for untransduced Treg cells (Mock) and Treg cells expressing various CARs.

[0012] FIG. 5A shows representative FACS plots of Treg cells expressing various CARs. Cells are stained with EGFRt and CAR antibodies to identify EGFRt+ and CAR+ cells. Each quadrant (Q5, Q6, Q7, and Q8) distinguishes various Treg cell populations within the parent population: Q5 = EGFRt-, CAR+ Treg cells; Q6 = EGFRt+, CAR+ Treg cells; Q7 = EGFRt+, CAR- Treg cells; and Q8 = EGFRt-, CAR- Treg cells. The percentage of each Treg cell population is shown within the quadrants. FIG. 5B shows representative FACS plots of Treg cells expressing various CARs. Cells are stained with EGFRt and CD28 antibodies to identify EGFRt+ and CD28+ cells. Each quadrant (Q13, Q14, Q15, and Q16) distinguishes various Treg cell populations within the parent population: Q13 = EGFRt-, CD28+ Treg cells; Q14 = EGFRt+, CD28+ Treg cells; Q15 = EGFRt+, CD28- Treg cells; and Q16 = EGFRt-, CD28- Treg cells. The percentage of each Treg cell population is shown within the quadrants. FIG. 5C shows representative FACS plots of Treg cells expressing various CARs. Cells are stained with Heliosand FoxP3 antibodies to identify Helios+ and FoxP3+ cells. Each quadrant (Q9, Q10, QI 1 , and Q12) distinguishes various Treg cell populations within the parent population: Q9 = Helios+, FoxP3- Treg cells; Q10 = Helios+, FoxP3+ Treg cells; Ql l = Helios-, FoxP3+ Treg cells; and Q12 = Helios-, FoxP3- Treg cells. The percentage of each Treg cell population is shown within the quadrants. UTD represents the untransduced Treg cell control (Mock) in FIGS. 5A-5C.

[0013] FIG. 6A is a bar graph showing CAR expression by untransduced (Mock) and transduced Jurkat cells expressing various CARs. FIG. 6B is a bar graph showing CD28 expression by untransduced (Mock) and transduced Jurkat cells expressing various CARs. FIG. 6C is a bar graph showing CD28 expression by transduced Jurkat cells that were positive or negative for expression of the transduction marker associated with CAR expression.

[0014] FIG. 7 A shows a histogram of normalized cell count versus CFSE signal intensity of untransduced Treg cells (CAR-) and Treg cells expressing various CARs (CAR+). CFSE is a fluorescent cell staining dye (carboxyfluorescein succinimidyl ester) whose staining intensity decreases with each round of cell division. Each row represents Treg cells isolated from a different donor. FIG. 7B shows % proliferation as a function of cell to CD3 / CD28-bead ratio for CAR-expressing Treg cells (CAR+ = EGFR+) compared to Treg cells not expressing a CAR (CAR- = EGFR-).

[0015] FIG. 8A shows luminescence, which is indicative of cell activation, as a function of the ratio of CAR+ Jurkat cells to CAR antigen-l- K562 cells. FIG. 8B shows percentages of various CAR+ Jurkat cells expressing high levels of CD69 as a function of their ration to CAR antigen-l- K562 cells. FIG. 8C shows percentages of various CAR+ Jurkat cells expressing CD25 after activation with CAR antigen positive K562 cells at varying Jurkat cell to K562 cell ratios. Mock designates untransduced (CAR-) control cells.DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure relates to chimeric antigen receptors (CARs) that do not cause a substantial reduction in expression of endogenous CD28 protein in T-cells in which the CARs are expressed. In particular, CARs of the present disclosure comprise different hinge and / or transmembrane domains than the four well known anti-CD19 CARs, tisagen, axicabtagene, brexucabtagene and lisocabtagene. That is, the CARs of the present disclosure do not comprise a CD8a hinge region in combination with a CD8a transmembrane domain, a wildtype CD28 hinge region in combination with a wild type CD28 transmembrane domain, or an IgG4 hinge region in combination with a wild type CD28 transmembrane domain. The CARs of the present disclosure also do not comprise a wild type CD28 hinge region in combination with a modified CD28 transmembrane domain.

[0017] Expression of the CARs of the present disclosure in a T-cell results in less of a reduction in cell surface expression of endogenous CD28 protein than does a control CAR comprising a wild type CD28 hinge region and a wild type CD28 transmembrane domain, when the otherwise identical control CAR is expressed in a control T-cell. As used herein, the term “substantial reduction” refers to an at least 25% decrease in a property. In contrast, the term “little to no reduction” refers to a decrease in a property of less than a 25%, 20%, 15%, 10% or 5%. The CARs of the present disclosure are advantageous in that their expression in a T-cell causes less than a 25%, 20%, 15%, 10% or 5% reduction (MFI) in cell surface expression of endogenous CD28 protein as determined by flow cytometry.Definitions

[0018] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0019] As used herein, the terms “antigen,” “immunogen,” and “antibody target,” refer to a molecule, compound, or complex that is recognized by an antibody, i.e., can be bound by the antibody. The term can refer to any molecule that can be recognized by an antibody, e.g., a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.). One of skill will understand that the term does not indicate that the molecule is immunogenic in every context, but simply indicates that it can be targeted by an antibody, or an antigen-binding domain derived therefrom.

[0020] As used herein, the term “epitope” refers to the localized site on an antigen that is recognized and bound by an antigen-binding domain of an antibody or fragment derived therefrom. Epitopes can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes non-protein components, e.g., from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is aprotein, the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous epitope).

[0021] As used herein, the term “antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene, that specifically bind and recognize an antigen. Typically, the “variable region” contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD).

[0022] Antibodies can be of (i) any of the five major classes of immunoglobulins, based on the identity of their heavy-chain constant domains - alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) and mu (IgM), or (ii) subclasses (isotypes) thereof (E.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). The light chains can be either lambda or kappa.

[0023] The following are a non-exhaustive list of different antibody forms, all retaining antigen binding activity:(1) whole immunoglobulins (also referred to as “intact” antibodies) (two light chains and two heavy chains, e.g., a tetramer);(2) an immunoglobulin polypeptide (a light chain or a heavy chain);(3) an antibody fragment, such as Fv (a monovalent or bi-valent variable region fragment, and can encompass only the variable regions (e.g., VL and / or VH), Fab (VLCL VHCH), F(ab’)2, Fv (VLVH), SCFV (single chain Fv) (a polypeptide comprising a VL and VH joined by a linker, e.g., a peptide linker), (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(FV)2 fused to CH3 domain), diabody (noncovalent dimer of single-chain Fv (scFv) fragment that consists of the heavy chain variable (VH) and light chain variable (VE) regions connected by a small peptide linker), triabody is trivalent sc(Fv)3 or trispecific sc(Fv)3;(4) a multivalent antibody (an antibody comprising binding regions that bind two different epitopes or proteins, e.g., “scorpion” antibody;(5) a fusion protein comprising a binding portion of an immunoglobulin fused to another amino acid sequence (such as a fluorescent protein); and(6) heavy chain only antibody or antibody fragment having only two heavy chains and lacking the two light chains usually found in antibodies.

[0024] The phrase “CDR sequence set” as used herein refers to the 3 heavy chain and / or 3 light chain CDRs of a particular antibody or antigen-binding domain. A “light chain” CDR sequence set refers to the light chain CDR sequences. A “heavy chain” CDR sequence set refers to the heavy chain CDR sequences. A “full” CDR sequence set refers to both heavy chain and light chain CDR sequences. CDRs are predicted based on IMGT sequence alignment.

[0025] As used herein, the term “humanized antigen-binding domain” refers to a chimeric antigen-binding domain in which the CDRs, obtained from the VH and VL regions of a non-human antibody having the desired specificity, affinity and capability are grafted to human framework regions. In one embodiment, the framework residues of the humanized antigenbinding domain are modified to refine and optimize the specificity, affinity and capability of the antigen-binding domain.

[0026] As used herein, the term “human antigen-binding domain” refers to an antigenbinding domain of an antibody produced by a human or an antibody having an amino acid sequence corresponding thereto.

[0027] As used herein, an antigen-binding domain “preferentially binds” binds a first antigen relative to a second antigen if it binds the first antigen with greater affinity than it does 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.

[0028] As used herein, an antigen-binding domain “specifically binds” or is “specific for” a target antigen or target group of antigens if it binds the target antigen or each member of the target group of antigens with an affinity of at least any of IxlO-6M, IxlO-7M, IxlO-8M, IxlO-9M, IxlO-10M, IxlO-11M, IxlO-12M, and, for example, binds to the target antigen or each member of the target group of antigens with an affinity that is at least two-fold greater than its affinity for non-target antigens to which it is being compared. Typically, specific binding is characterized by binding the antigen with sufficient affinity that the antigen-binding domain is useful as a diagnostic to detect the antigen or epitope and / or as a therapeutic agent in targeting the antigen or epitope.

[0029] As used herein, the term “polypeptide” refers to a molecule having a sequence of natural and / or unnatural amino acids connected through peptide bonds. The term “peptide” refers to a short polypeptide, typically no more than 30 amino acids long. The amino acid sequence of a polypeptide is referred to as its “primary structure.” The term “protein” refers to a polypeptide having a secondary, tertiary and / or quaternary structure, e.g., structures stabilized by hydrogen bonds, relationships between secondary structures and structures formed of more than one protein. Proteins can be further modified by other attached moieties such as carbohydrate (glycoproteins), lipids (lipoproteins) phosphate groups (phosphoproteins) and the like.

[0030] As used herein, an amino acid sequence “consists of’ only the amino acids in that sequence.

[0031] 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 sequence and (2) is no more than 1 , no more than 2 or no more than 3 amino acids longer than the second amino acid sequence.

[0032] As used herein, a first amino acid sequence is a “fragment” of a second amino acid sequence if the second amino acid sequence comprises the first amino acid sequence. In certain embodiments, a first amino acid sequence that is a fragment of a second amino acid sequence may have no more than any of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fewer amino acids than the second amino acid sequence.

[0033] As used herein, a “functional equivalent” of a reference amino acid sequence is a sequence that is not identical to the reference sequence, but that contains minor alterations such as, for example, insertion, deletion or substitution of one or a few amino acids. A functionally equivalent sequence retains the function (e.g., immunogenicity) of the reference sequence to which it is equivalent. If a functionally equivalent amino acid sequence contains substitution of one or more amino acids with respect to the reference sequence, these will generally be conservative amino acid substitutions.

[0034] As used herein, a “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein’s desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. See, e.g.,Watson, et al., “Molecular Biology of the Gene,” 4thEdition, 1987, The Benjamin / CummingsPub. Co., Menlo Park, CA, p. 224. Examples of conservative amino acid substitution include the following (Note, some categories are not mutually exclusive):

[0035] As used herein, the term “substantially identical” refers to identity between a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and / or common functional activity and / or common immunogenicity. For example, amino acid sequences that contain a common structural or antigenic domain having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are termed sufficiently or substantially identical. In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, or encode polypeptides having the same immunogenic properties.

[0036] As used herein, a chemical entity, such as a polypeptide, is “substantially pure” or “isolated” if it is the predominant chemical entity of its kind (e.g., of polypeptides) in a composition. This includes the chemical entity representing more than 50%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, or more than 99.99% of the chemical entities of its kind in the composition. A substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantiallypure composition means that about 80% to 90% or more of the macromolecular species present in the composition is the purified species of interest. The object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules, stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition.

[0037] 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 of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions. times.100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 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 NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSLBLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). Whenutilizing BLAST, Gapped BLAST, and PSLBlast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CAB IOS 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 utilizing the ALIGN program for comparing 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.

[0038] For antibodies and antigen-binding domains, percentage sequence identities can be determined when their sequences are maximally aligned by IMGT. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, multiplied by 100 to convert to percentage.

[0039] Percent amino acid sequence identity may also be determined using the sequence comparison program NCBLBLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBLBLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md. NCBLBLAST2 uses several search parameters, wherein all of those search parameters 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.

[0040] In situations where NCBLBLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignmentprogram NCBI-BLAST2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal 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 comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present disclosure 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 contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. It is understood that polynucleotides comprising non-transcribable nucleotide bases may be useful as probes in, for example, hybridization assays. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term “nucleic acid” includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents.

[0041] The term “isolated nucleic acid” as used herein refers to a nucleic acid 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 (i.e. sequences located at the 5’ and 3’ ends of the nucleic acid) from which the nucleic acid is derived.

[0042] Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81.5°C - 16.6 (LoglO [Na+]) + 0.41(%(G+C) - 600 / 1), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed toresult in about a 1 °C decrease in Tm, for example, if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt’s solution / 1.0% SDS at Tm - 5°C based on the above equation, followed by a wash of 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0043] As used herein, the term “expression construct” refers to a polynucleotide comprising an expression control sequence operatively linked with a heterologous nucleotide sequence (i.e., a sequence to which the expression control sequence is not normally connected to in nature) that is to be the subject of expression. As used herein, the term “expression vector” refers to a polynucleotide comprising an expression construct and sequences sufficient for replication in a host cell or insertion into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term “expression control sequence” refers to a nucleotide sequence that regulates transcription and / or translation of a nucleotide sequence operatively linked thereto. Expression control sequences include promoters, enhancers, repressors (transcription regulatory sequences) and ribosome binding sites (translation regulatory sequences).

[0044] As used herein, a nucleotide sequence is “operatively linked” with an expression control sequence when the expression control sequence functions in a cell to regulate transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through an interaction between a polymerase and a promoter.

[0045] The term “vector” as used herein comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and / or eukaryotic cells and / or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, lentiviral vectors,Adeno Associated viral vectors and the like. The term “plasmid” as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0046] “Transfection” refers to the introduction of new genetic material into a cell. It includes transformation (the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane), transduction (the introduction of foreign DNA by a bacteriophage virus into a host cell) and conjugation.

[0047] As used herein, a “host cell” refers to a recombinant cell comprising an expression construct.

[0048] As used herein, the term “biological sample” refers to a sample containing cells (e.g., cells) or biological molecules derived from cells.

[0049] As used herein, the term terms “therapy,” “treatment,” “therapeutic intervention” and “amelioration” refer to any activity resulting in a reduction in the severity of symptoms. The terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment and prevention can be complete or partial. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects, the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques. “Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treating” and “treatment” as used herein also include prophylactic treatment.

[0050] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited (e.g., open-ended terms meaning including but not limited to). For example, a composition that “comprises” or “includes” an antibody may contain the antibody alone or in combination with other ingredients. In contrast, the phrase “consisting of’ is closed, indicating that such embodiments do not include additional elements. The term “consisting essentially of’ refers to the inclusion of recited elements andother elements that do not materially affect the basic and novel characteristics of a claimed combination (e.g., partially closed term). It is understood that aspects and embodiments described herein as “comprising” include “consisting of’ and “consisting essentially of’ embodiments.

[0051] As used herein, the following meanings apply unless otherwise specified. The word “may” is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.”

[0052] The phrase “at least one” includes “one”, “one or more”, “one or a plurality” and “a plurality”. The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1 , 2 or 3” means “at least 1, at least 2 or at least 3”. The term “plurality” in connection with a referent (e.g., object such as a T-cell) refers to 3 or more, preferably 10 or more, preferably 100 or more, preferably 1,000 or more, preferably 10,000 or more, preferably 100,000 or more, preferably, 106or more, preferably 107or more, preferably 108or more, preferably 109or more, but not an infinite number of the referent (e.g., preferably less than 1012, preferably less than 1011, and preferably less than 1010).

[0053] The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value. For instance the phrase about 20 residues (amino acids) in length to describe a polypeptide linker refers to a polypeptide linker of from 18 to 22 amino acids in length, and includes a polypeptide linker of 20 amino acids in length.I. Chimeric Antigen Receptors (CARs)

[0054] “Chimeric antigen receptors” or “CARs” are engineered molecules comprising an optional signal peptide, a target antigen-binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain and an optional co-stimulatory domain. CARs are based on the structure of T-cell receptors, which are expressed on T-cells, and are involved in the cell-mediated immune responses. The “target-binding domain” is alsoreferred to herein as an “antigen-binding domain” or “antigen-recognition domain”, and as such the term “target” encompasses an “antigen.”

[0055] So-called “first-generation” CARs had a targeting domain and a CD3^ signal transduction domain. So-called “second generation” CARs further included a co-stimulatory domain, such as a CD28 or a 4- IBB domain. So-called “third generation” CARs comprise multiple co-stimulatory domains. So-called “fourth generation” CARs, also referred to as “TRUCKS” are engineered to release a transgenic cytokine upon CAR signaling.

[0056] Chimeric antigen receptors (“CARs”) include the following elements: (1) an optional signal peptide, (2) a target antigen-binding domain, (3) an optional hinge region; (4) a transmembrane region; (5) an intracellular domain comprising a signal transduction domain. Optionally, the CAR can include a co-stimulatory (signal transduction) domain. That is, these optional elements can be included in addition to required elements. The target antigen-binding domain is heterologous to at least one of the other domains. That is, the antigen-binding domain does not naturally occur on a T-cell receptor or is not in the same protein as at least one of the other domains of the CAR.

[0057] The “signal peptide” guides the CAR polypeptide through the cell membrane. The “antigen-binding domain” provides binding specificity to the CAR. The antigen-binding domain can bind to a domain of an antibody that binds to the target antigen for a so-called “Universal CAR”. The “hinge region” is a flexible connector region, e.g., a natural or synthetic polypeptide, or any other type of molecule, providing structural flexibility and spacing to flanking polypeptide regions. The “transmembrane domain” is a membrane-spanning protein domain. The “intracellular signaling domain” transmits a signal through a signal transduction domain into the cell upon binding of an antigen by the antigen-binding domain. Such signaling activates an activity of the cell. “Co-stimulatory domains” are accessory signaling domains that further transmit signals.

[0058] Provided herein are chimeric antigen receptors (CARs) that cause little to no reduction in expression of endogenous CD28 protein in T-cells in which the CARs are expressed. In particular, CARs of the present disclosure comprise different hinge and / or transmembrane domains than the four anti-CD19 CAR T-cell therapies that are currently approved by the U.S. Food and Drug Administration (tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel and lisocabtagene maraleucel). That is, the CARs of the presentdisclosure are thought to display decreased dimerization with endogenous CD28 proteins when the CARs are expressed in a T-cell compared to a control CAR (comprising a wild type CD28 hinge region of SEQ ID NO:31 and a wild type CD28 transmembrane domain of SEQ ID NO:40) expressed in a control T-cell.A. Signal Peptide

[0059] A signal peptide that may be present at the N-terminus of a nascent CAR of the present disclosure during expression in a cell can be a signal peptide of any human Type I transmembrane protein or any other signal peptide suitable for translocating the nascent CAR to the surface of a human cell. In some embodiments, the signal peptide is derived from a protein of the human immunoglobulin superfamily. In some embodiments, the signal peptide is derived from a CD4, CD8, CD 19, CD28, TCR or immunoglobulin chain.

[0060] An exemplary signal peptide is the GMCSF signal peptide: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 1). Another exemplary signal peptide is the CD8alpha signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO:2). In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 1, or SEQ ID NO:2.B. Antigen-Binding Domains

[0061] The antigen-binding domain (ABD) of the CARs of the present disclosure can possess any desired specificity and can be in any suitable polypeptide form for binding the target antigen and transmitting a signal through the transmembrane domain to the intracellular domain of the CAR. In some embodiments, the antigen-binding domain (ABD) is a single chain antibody (scFV). In some embodiments, such as when the ABD is a scFV, the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises complementarity region (CDR)-Hl, CDR-H2, and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 of an antibody whose sequence is deposited in Version 13.1 (August 2023) of The ABCD (Antibodies Chemically Defined) database hosted by Expasy and operated by the Swiss Institute of Bioinformatics (see, Lima et al., Nucleic Acids Research, 48:D2610264, 2020). In some embodiments, the CDRs are as defined using Kabat nomenclature, which can easily bedetermined for a given sequence using online tools such as the abYsis annotate tool. In some embodiments, the antigen-binding domain comprises a light chain variable region and a heavy chain variable region of an antibody disclosed in Version 13.1 (August 2023) of The ABCD, which includes 24,485 sequence antibodies directed against 4,171 different target antigens.

[0062] In some embodiments, the antigen comprises citrullinated vimentin (CV). In exemplary embodiments, the antigen-binding domain is a CV-binding domain that comprises a heavy chain variable region (VH) comprising the heavy chain CDRs of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a light chain variable region (VL) comprising the light chain CDRs of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the CV-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a VL comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the CV-binding domain comprises a VH comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a VL comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO: 8.

[0063] In other embodiments, the antigen comprises CD 19 (also known as B-lymphocyte surface antigen B4). In exemplary embodiments, the antigen-binding domain is a CD19-binding domain that comprises a light chain variable region (VL) comprising the light chain CDRs of SEQ ID NO:9 and a heavy chain variable region (VH) comprising the heavy chain CDRs of SEQ ID NOTO. In some embodiments, the CD19-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO:9 and a VH comprising the amino acid sequence of SEQ ID NOTO. In some embodiments, the CD19-binding domain comprises a VL comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:9 and a VH comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 10.

[0064] In further embodiments, the antigen comprises B-cell maturation antigen (BCMA, (also known as tumor necrosis factor receptors superfamily member 17 or TNFRSF17). In exemplary embodiments, the antigen-binding domain is a BCMA-binding domain that comprises a light chain variable region (VL) comprising the light chain CDRs of SEQ ID NO: 11 and a heavy chain variable region (VH) comprising the heavy chain CDRs of SEQ ID NO: 12. In someembodiments, the BCMA-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 11 and a VH comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the BCMA-binding domain comprises a VL comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:11 and a VH comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 12.

[0065] The antigen-binding domains of the CARs of the present disclosure comprising a VH and a VL region, preferably are separated by a polypeptide linker. In some embodiments, the antigen-binding domain is arranged as VH-linker-VL. In other embodiments, the antigen-binding domain is arranged as VL-linker-VH. In some embodiments, the polypeptide linker is from about 4 to about 24 amino acids in length. In exemplary embodiments, the linker is a glycine linker comprising the amino acid sequence of: GGGGSGGGGSGGGGS (SEQ ID NO: 13). In other exemplary embodiments, the linker is a Whitlow linker comprising the amino acid sequence of: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 14). In further exemplary embodiments, the linker is a ABpur linker comprising the amino acid sequence of: ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 15).

[0066] In exemplary embodiments, the antigen-binding domain (ABD) is a CV-binding domain comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 16. In other exemplary embodiments, the ABD is a CD19-binding domain comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 17 or SEQ ID NO: 18. In further exemplary embodiments, the ABD is an BCMA-binding domain comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:20, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 19 or SEQ ID NO:20.C. Hinge Region

[0067] The antigen-binding domain (ABD) and the transmembrane domain of the CARs of the present disclosure are separated by a hinge region of from about 10 to about 20, 3040 or 50 amino acids in length.

[0068] In some embodiments, the hinge region is a CD8a hinge region. In exemplary embodiments, the CD8a hinge region comprises the amino acid sequence of: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:21), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:21. In some embodiments, when the CAR comprises a CD8a hinge region, the transmembrane domain is not a CD8a transmembrane domain.

[0069] In some embodiments, the hinge region is a CD28 hinge region. In exemplary embodiments, the CD28 hinge region comprises the amino acid sequence of: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:22), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:22. In preferred embodiments, when the CAR comprises a wild type CD28 hinge region, the transmembrane domain is not a wildtype CD28 transmembrane domain (SEQ ID NO:25).

[0070] In some embodiments, the hinge region is an IgG4 hinge region. In exemplary embodiments, the IgG4 hinge region comprises the amino acid sequence of: ESKYGPPCPPCP (SEQ ID NO:23), or an amino acid sequence differing by one or two amino acids from SEQ ID NO:23. In some embodiments, the IgG4 hinge region comprises the amino acid sequence of: ESKYGPPCP[X]CP, in which X is P or S (SEQ ID NO:36). In preferred embodiments, when the CAR comprises an IgG4 hinge region, the transmembrane domain is not a wild type CD28 transmembrane domain (SEQ ID NO:25).

[0071] In some embodiments, the hinge region is a truncated CD28 hinge region, which has a truncation of from about 10 to about 30 amino acids relative to the wild type amino acid sequence of SEQ ID NO:31. In exemplary embodiments, the truncated CD28 hinge region comprises the amino acid sequence of: SPLFPGPSKP (SEQ ID NO:32), or an amino acid sequence differing by one or two amino acids from SEQ ID NO:32.

[0072] In some embodiments, the hinge region is a CD4 hinge region. In exemplary embodiments, the CD4 hinge region comprises the amino acid sequence of: SGQVLLESNIKVLPTWSTPVQP (SEQ ID NO:33), or an amino acid sequence differing by one or two amino acids from SEQ ID NO:33. In some embodiments, the CD4 hinge region is a truncated CD4 hinge region, which has a truncation of from 1 to about 15 amino acids or from 1 to 12 amino acids relative to the wild type amino acid sequence of SEQ ID NO:33. In exemplary embodiments, the truncated CD4 hinge region comprises the amino acid sequence of:LPTWSTPVQP (SEQ ID NO:34), or an amino acid sequence differing by one or two amino acids from SEQ ID NO:34.In some embodiments, the hinge region is a CD1 la hinge region. In exemplary embodiments, the CD1 la hinge region comprises the amino acid sequence of: VDVVYEKQML (SEQ ID NO:35); or an amino acid sequence differing by one or two amino acids from SEQ ID NO:35.

[0073] In some embodiments, the hinge region is a CD2 hinge region. In exemplary embodiments, the CD2 hinge region comprises the amino acid sequence of: SKESSVEPVSCPEKGLD (SEQ ID NO:7), or an amino acid sequence differing by one or two amino acids from SEQ ID NO:37.

[0074] In some embodiments, the hinge region is a TCRa hinge region. In exemplary embodiments, the TCRa hinge region comprises the amino acid sequence of: PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO:38), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:38.In some embodiments, the hinge region is an IgG4 hinge region. In exemplary embodiments, the IgG4 hinge region comprises the amino acid sequence of: ESKYGPPCPPCP (SEQ ID NO:39), or an amino acid sequence differing by one or two amino acids from SEQ ID NO:39.D. Transmembrane Domain

[0075] The hinge region and the intracellular signaling domain of the CARs of the present disclosure are separated by a transmembrane domain of from about 20 to about 30 amino acids in length.

[0076] In some embodiments, the transmembrane domain is a CD8a transmembrane domain. In exemplary embodiments, the CD8a domain comprises the amino acid sequence of: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:24), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:24. In preferred embodiments, when the CAR comprises a CD 8 a transmembrane domain, the hinge region is not a CD8a hinge region (SEQ ID NO:21).

[0077] In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In exemplary embodiments, theCD28 transmembrane domain is a wild type CD28 transmembrane domain, which comprises the amino acid sequence of:FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:25). In some embodiments the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:25. In preferred embodiments, when the CAR comprises a wildtype CD28 transmembrane domain, the hinge region is not a wild type CD28 hinge region (SEQ ID NO:22) or an IgG4 hinge region (SEQ ID NO:23 or SEQ ID NO:36).

[0078] In some embodiments, the transmembrane domain is a modified CD28 transmembrane domain. In exemplary embodiments, the amino acid sequence of the modified CD28 transmembrane domain comprises an insertion, substitution, and / or deletion relative to SEQ ID NO:40. In some embodiments, the amino acid sequence of the CD28 transmembrane domain comprises at least one substitution selected from the group consisting of: C165L, Y166L, S167L, T171L (which correspond to C13L, Y14L, S15L and T19L as shown in the amino acid sequence of the modified CD28 TM of SEQ ID NO:42), and any combination thereof. In some embodiments, the modified CD28 transmembrane domain comprises the amino acid sequence of: FWVLVVVGGVLALLLLLVLVAFIIFWV (SEQ ID NO:41). In some embodiment, the transmembrane domain sequence comprises the amino acid sequence of SEQ ID NO: 11, and the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:l-9.

[0079] In some embodiments, the transmembrane domain is a CD4 transmembrane domain. In exemplary embodiments, the CD4 transmembrane domain comprises the amino acid sequence of: MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO:42), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:42.

[0080] In some embodiments, the transmembrane domain is a CD1 la transmembrane domain. In exemplary embodiments, the CD1 la transmembrane domain comprises the amino acid sequence of: YLYVLSGIGGLLLLLLIFIVLYKV (SEQ ID NO:43), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:43.

[0081] In some embodiments, the transmembrane domain is CD 18 transmembrane domain. In some embodiments, the CD 18 transmembrane domain comprises the amino acid sequence of: IAAIVGGTVAGIVLIGILLLVIW (SEQ ID NO:44), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:44.

[0082] In some embodiments, the transmembrane domain is CD2 transmembrane domain. In exemplary embodiments, the CD2 transmembrane domain comprises the amino acid sequence of: IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO:45), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:45.

[0083] In some embodiments, the transmembrane domain is HER2 transmembrane domain. In exemplary embodiments, the HER2 transmembrane domain comprises the amino acid sequence of: SIISAVVGILLVVVLGVVFGILI (SEQ ID NO:46), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:46.E. Intracellular Signaling Domain

[0084] The intracellular signaling domain of the CARs of the present disclosure comprises a CD3zeta signal transduction domain. In some preferred embodiments, the intracellular signaling domain further comprises a co-stimulatory domain.

[0085] In some embodiments, the co-stimulatory domain can be derived from, for example, CD28, 4-1BB, CD2, CD27, CD30, 0X40, 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 and others. In some embodiments, the CARs of the present disclosure comprise two or more co-stimulatory signaling domains (e.g., CD28 and 4- IBB).

[0086] In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain. In exemplary embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence of: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:26), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:26.

[0087] In some embodiments, the co-stimulatory domain is a 4- IBB co-stimulatory domain. In exemplary embodiments, the 4- IBB co-stimulatory domain comprises the amino acid sequence of: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:27), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:27.

[0088] In exemplary embodiments, the CD3^ signal transduction domain comprises the amino acid sequence of: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:28), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:28.

[0089] In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory domain and a CD3^ signal transduction domain. In exemplary embodiments, the intracellular signaling domain comprise the amino acid sequence of SEQ ID NO:29 or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:29.

[0090] In some embodiments, the intracellular signaling domain comprises a 4- IBB costimulatory domain and a CD3^ signal transduction domain. In exemplary embodiments, the intracellular signaling domain comprise the amino acid sequence of SEQ ID NO:30 or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:30.II. Nucleic Acids Encoding CARs

[0091] Nucleic acid molecules (polynucleotides) encoding CARs of the present disclosure can be isolated molecules, or can be included within a cassette or a vector.A. Nucleic Acids

[0092] The present disclosure provides nucleic acid molecules (polynucleotides) comprising a nucleotide sequence that encodes (e.g., comprises the coding region of) a CAR as described herein. The nucleic acid can be in the form of DNA or in the form of RNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or singlestranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In some embodiments, the polynucleotides as described herein are isolated.

[0093] For example, nucleic acids can comprise nucleotide sequences that encode the CAR polypeptides described herein or a portion thereof. In some embodiments, the nucleotide sequences encode a polypeptide comprising the amino acid sequence of a specific SEQ ID NO,or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with a specific SEQ ID NO.

[0094] In exemplary embodiments, the nucleic acid encodes a CAR comprising an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain, as depicted in FIG. 2A, FIG. 2B, FIG. 3A or FIG. 3B. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a truncated CD28 hinge region of SEQ ID NO:32, and the transmembrane domain is a mutated CD28 transmembrane domain of SEQ ID NO:41. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a truncated CD4 hinge region of SEQ ID NO:34, and the transmembrane domain is a CD4 transmembrane domain of SEQ ID NO:42. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a CD1 la hinge region of SEQ ID NO:35, and the transmembrane domain is a CD1 la transmembrane domain of SEQ ID NO:43. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a wildtype CD28 hinge region of SEQ ID NO:31, and the transmembrane domain is a CD18 transmembrane domain of SEQ ID NO:44. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a TCRa hinge region of SEQ ID NO:38, and the transmembrane domain is a CD4 transmembrane domain of SEQ ID NO:42. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a CD2 hinge region of SEQ ID NO:37, and the transmembrane domain is a CD2 transmembrane domain of SEQ ID NO:45. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a truncated CD28 hinge region of SEQ ID NO:32, and the transmembrane domain is a wildtype CD28 transmembrane domain of SEQ ID NO:40. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a wildtype CD28 hinge region of SEQ ID NO:31, and the transmembrane domain is a CD4 transmembrane domain of SEQ ID NO:42. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a wildtype CD28 hinge region of SEQ ID NO:31, and the transmembrane domain is a CD1 la transmembrane domain of SEQ ID NO:43. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a wildtype CD28 hinge region of SEQ ID NO:31, and the transmembrane domain is a HER2 transmembrane domain of SEQ ID NO:46.

[0095] In a further exemplary embodiment, the nucleic acid encodes a CAR comprising an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellularsignaling domain in which the transmembrane domain is a mutated CD28 transmembrane domain. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a truncated CD4 hinge region of SEQ ID NO:34, and the transmembrane domain is a mutated CD28 transmembrane domain of SEQ ID NO:41. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a CD1 la hinge region of SEQ ID NO:35, and the transmembrane domain is a mutated CD28 transmembrane domain of SEQ ID NO:41. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a CD2 hinge region of SEQ ID NO:37, and the transmembrane domain is a mutated CD28 transmembrane domain of SEQ ID NO:41. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is a TCRa hinge region of SEQ ID NO:38, and the transmembrane domain is a mutated CD28 transmembrane domain of SEQ ID NO:41.

[0096] In some embodiments, the polynucleotides are variants that comprise alterations in the coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain alterations that are silent. In other embodiments, the polynucleotide variants comprise substitutions, additions, or deletions, but do not alter the binding properties of the antigen-binding domain of the encoded CAR polypeptide. In some embodiments, the polynucleotide variants contain one or more alterations that do not produce any changes in the amino acid sequence of the CAR. In some embodiments, polynucleotide variants contain “silent” substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host cell.B. Expression Cassettes and Vectors

[0097] The present disclosure further provides expression cassettes and vectors comprising a nucleic acid molecule encoding a CAR described herein. In some embodiments, the nucleic acid molecule can further comprise an expression control sequence operatively linked with the nucleotide sequence encoding the CAR. In some embodiments, the nucleic acid molecule encoding the disclosed CAR can be inserted into an expression vector in operable combination with an expression control sequence appropriate for expression of the disclosed CAR in a desired host cell. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and / or expression of the CAR polypeptide in a suitable host cell.

[0098] For example, a polynucleotide can include one or more transcription regulatory elements, such as promoters or enhancers, which, when the polynucleotide is present in a cell, cause the sequence encoding the CAR to be expressed within the cell.

[0099] Nucleic acids disclosed herein can be incorporated into vectors that can be introduced into a host cell. Such vectors include, without limitation, viral vectors and plasmids. Exemplary viral vectors include but are not limited to retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Lentiviruses are particularly desirable as they are able to deliver a large amount of genetic material into the genome of a host cell and because they are able to infect non-dividing cells.III. Host Cells

[0100] The present disclosure also provides host cells (e.g., recombinant cells) comprising a nucleic acid molecule, an expression cassette, or an expression vector encoding a CAR described herein. In some embodiments, the host cell is a eukaryotic cell, such as a mammalian cell. In other embodiments, the host cell is a prokaryotic cell, such as a bacterial cell.

[0101] The nucleic acid molecule encoding the disclosed CAR can be delivered to a host cell by transformation, transfection or transduction as is known to one of skill in the art. The resulting recombinant (host) cell can be an immune cell, including but not limited to a T-cell, such as a CD4+ T-cell, CD8alpha+ T-cell, or CD8beta+ T-cell. In some embodiments, the T-cell is a Treg cell. In some embodiments, the T-cell is a T helper (Th) cell. In some embodiments, the T-cell is a cytolytic T lymphocyte (CTL). In some embodiments, the recombinant (host) cell comprising the nucleic acid molecule encoding the disclosed CAR is a lymphocyte (e.g., T-cell, B-cell or NK-cell).

[0102] In some embodiments, the cells expressing the CARs of this disclosure are Treg cells. “Regulatory T-cells,” or “Treg cells,” are cells belonging to a specialized subpopulation of T-cells that act to suppress immune response, thereby maintaining homeostasis and selftolerance. Tregs are able to inhibit T-cell proliferation and cytokine production and play a critical role in preventing autoimmunity. Tregs are characterized by expression of FoxP3. Surface markers for Tregs include CD4, CD25high (high molecular density) and CD1271ow (low molecular density). Mouse and human Tregs express GITR / AITR, and CTLA-4. Human CD4+FoxP3+ Treg cells can be divided into three sub-populations:(1) CD45RA+CD25+FoxP31ow resting Treg cells,(2) CD45RO+CD25highFoxP3high activated Treg cells, and(3) proinflammatory cytokine-producing CD45RO+CD25+FoxP31ow non-suppressive effector T-cells (Teffs).

[0103] In some embodiments, the host cell is an autologous cell (e.g., recombinant progeny of a cell obtained from the intended recipient). That is, the cells to be transformed with the nucleic acids disclosed herein can be cells taken from a subject into whom the recombinant cells are to be administered. In this way, issues of an allogeneic immune response can be mitigated. Even so, in other embodiments, the host cell is an allogeneic cell (e.g., recombinant progeny of a cell obtained from an immunologically distinct individual than the intended recipient). Regardless of their origin, host cells can be expanded ex vivo before administration to a subject.

[0104] Also, the proteins produced by a transformed / recombinant host can be purified according to any suitable method. Such methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. In some embodiments, proteins can also be physically characterized using such techniques as proteolysis, high performance liquid chromatography, nuclear magnetic resonance and x-ray crystallography.IV. Compositions

[0105] Also disclosed are pharmaceutical compositions comprising a host cell (e.g., recombinant cell) comprising a nucleic acid molecule encoding and expressing the disclosed CAR polypeptide, and a pharmaceutically acceptable excipient, as well as methods of use in the treatment of a disease or disorder.

[0106] As used herein, the term “pharmaceutical composition” refers to a composition comprising a pharmaceutical agent (e.g., a drug or a recombinant Treg cell as described herein) and a pharmaceutically acceptable excipient.

[0107] As used herein, the term “pharmaceutically acceptable” refers to a compound that is compatible with the other ingredients of a pharmaceutical composition and can be safelyadministered to a subject. The term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. Pharmaceutical compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and techniques for their administration one may refer 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.

[0108] Pharmaceutical compositions will generally be sterile, at least for human use. A pharmaceutical composition will generally comprise pharmaceutically acceptable excipients for buffering and preservation in storage, and can include buffers for appropriate delivery, depending on the route of administration. Examples of pharmaceutically acceptable excipients include, without limitation, normal (0.9%) saline, phosphate-buffered saline (PBS) Hank’s balanced salt solution (HBSS) and multiple electrolyte solutions.

[0109] Pharmaceutical compositions can be formulated for any route of administration. However, in most embodiments of the present disclosure, the pharmaceutical compositions are formulated for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraarterial injection, either bolus or infusion) administration.

[0110] Injectable (e.g., intravenous) pharmaceutical compositions can comprise a solution of the pharmaceutical agent suspended in a pharmaceutically acceptable excipient, such as an aqueous excipient. Any of a variety of aqueous excipients can be used, e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Often, normal buffered saline (135-150 mM NaCl) will be used. The pharmaceutically acceptable excipients can comprise auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity 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 can be formulated for intravenous administration.

[0111] Pharmaceutical compositions suitable for parenteral administration, such as intravenous administration, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the compositions isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In the practice of the present disclosure, pharmaceutical compositions can be administered, for example, by intravenous infusion. The pharmaceutical compositions can be presented in unit-dose or multi-dose sealed containers.

[0112] Host cells can be cryopreserved. Cryopreservation can include formulating host cells with a cryopreservation agent, such as DMSO. Commercially available media include, for example, CryoStor® and pZerve®, available from Millipore Sigma.

[0113] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of the pharmaceutical agent given to an individual at each administration. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable label (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress.

[0114] The pharmaceutical composition can be packaged or prepared in unit dosage form. In such form, the pharmaceutical composition is subdivided into unit doses containing appropriate quantities of the pharmaceutical agent, e.g., according to the dose of the pharmaceutical agent or the concentration of the pharmaceutical agent in the pharmaceutical composition. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the pharmaceutical composition. The pharmaceutical composition can, if desired, also contain other compatible therapeutic agents.V. Methods of Use

[0115] Host cells (e.g., recombinant T-cells) that express the CARs disclosed herein are useful in the treatment of various diseases and disorders. Methods of use comprise administering an effective amount of a pharmaceutical composition of this disclosure comprising CAR-expressing cells, such as CAR-T-cells, to a subject in need thereof (e.g., an individual suffering from a disease or disorder). In some methods of the present disclosure in which the subject has cancer, the host cells comprise CD3+ T-cells expressing a CAR that binds an antigen expressed by cells of the cancer. In some methods of the present disclosure in which the subject has an autoimmune disease or inflammatory disorder, the host cells comprise Treg cells expressing a CAR that binds an antigen associated with the autoimmune disorder or the inflammatory disorder. In some embodiments, the host cells are autologous to the subject.

[0116] As used herein, the term “subject” refers to an individual animal. The term “patient” as used herein refers to a subject under the care or supervision of a health care provider such as a doctor or nurse. Subjects include mammals, such as humans and non-human primates, such as monkeys, as well as dogs, cats, horses, bovines, rabbits, rats, mice, goats, pigs, and other mammalian species. Subjects can also include avians. In some embodiments, the subject is a human patient. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc. Subjects with a disease or disorder can include individuals that have not received treatment, are currently receiving treatment, have had treatment, and those that have discontinued treatment.

[0117] As used herein, the terms “effective amount,” “effective dose,” and “therapeutically effective amount,” refer to an amount of a pharmaceutical agent that is sufficient to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or ameliorate a disorder. In some examples, an “effective amount” is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of any of a disorder or disease and / or prevents progression of a disease. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least any of 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0118] The pharmaceutical composition can be administered by any suitable route, including but not limited to intravenous, subcutaneous, intramuscular or intraperitoneal routes. An example of administration of a pharmaceutical composition includes storing the composition at 10 mg / ml in sterile isotonic aqueous saline solution for injection at 4°C, and diluting it in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient.The pharmaceutical composition is administered by intravenous infusion over the course of 1 hour at a dose of between 0.2 and 10 mg / kg. In other embodiments, the pharmaceutical composition is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via sub-cutaneous bolus injection.

[0119] The dose of the pharmaceutical composition is chosen in order to provide effective therapy for the patient and is in the range of less than 0.1 mg / kg body weight to about 25 mg / kg body weight or in the range 1 mg to 2 g per patient. In some cases, the dose is in the range 1- 100 mg / kg, or approximately 50 mg- 8000 mg / patient. The dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, depending on the pharmacokinetics of the composition (e.g., half-life of the composition in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the composition). In some embodiments, the in vivo half-life is between about 7 and about 25 days and composition dosing is repeated between once per week and once every 3 weeks, or once per week and once every 3 months.

[0120] Administration can be periodic. Depending on the route of administration, the dose can be administered, e.g., once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer (e.g., once every 2, 3, 4, or 6 months). In some cases, administration is more frequent, e.g., 2 or 3 times per day. The patient can be monitored to adjust the dosage and frequency of administration depending on therapeutic progress and any adverse side effects, as will be recognized by one of skill in the art.

[0121] Thus, in some embodiments, additional administration is dependent on patient progress, e.g., the patient is monitored between administrations. For example, after the first administration or round of administrations, the patient can be monitored for rate of symptom relief.VI. Kits

[0122] As used herein, the term “kit” refers to a collection of items intended for use together. In some embodiments, the kit comprises an agent and instructions for use thereof. In some embodiments, the kit further comprises a container, such as a vial that contains a composition as disclosed herein. For instance, a kit can include a container, such as a bag orbottle for intravenous administration, comprising a pharmaceutical composition comprising a plurality of recombinant cells that express a CAR of the present disclosure. In some embodiments, the kit can further comprise a fluidic conduit, such as a plastic tube, with a drip chamber. The drip chamber can communicate through a fluidic conduit with an intravenous needle. The fluidic conduit also can comprise one or more Y -sites and a roller clamp.VII. Enumerated Embodiments1. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain; a CD28 hinge region; a transmembrane domain selected from the group consisting of a CD4 transmembrane domain, a CD1 la transmembrane domain, a CD 18 transmembrane domain, a CD2 transmembrane domain, and a HER2 transmembrane domain; and an intracellular signaling domain.2. The CAR of embodiment 1 , wherein the CAR does not cause a substantial reduction in cell surface expression of endogenous CD28 protein when the CAR is expressed in a T-cell.3. The CAR of embodiment 1, wherein cell surface expression of endogenous CD28 protein in a T-cell expressing the CAR is substantially higher than that of a control T-cell expressing a control CAR, wherein the control CAR comprises a wild type CD28 hinge region of SEQ ID NO:31 and a wild type CD28 transmembrane domain of SEQ ID NO:40, optionally wherein the control CAR is otherwise identical to the embodied CAR.4. The CAR of any one of embodiments 1-3, wherein the CD28 hinge region comprises the amino acid sequence of SEQ ID NO:31.5. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain; a truncated CD28 hinge region; a transmembrane domain; and an intracellular signaling domain, wherein the truncated CD28 hinge region comprises a truncation of from about 10 to about 30 amino acids relative to the amino acid sequence of SEQ ID NO:31.6. The CAR of embodiment 5, wherein the truncated CD28 hinge region is truncated at the N-terminus.7. The CAR of embodiment 6, wherein the truncated CD28 hinge region comprises the amino acid sequence of SEQ ID NO:32.8. The CAR of any one of embodiments 5-7, wherein the transmembrane domain is a CD28 transmembrane domain, optionally wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:40.9. The CAR of any one of embodiments 5-7, wherein the transmembrane domain is a modified CD28 transmembrane domain comprising an insertion, substitution, and / or deletion relative to SEQ ID NO:40, optionally wherein the modified CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:41.10. The CAR of any one of embodiments 5-7, wherein the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD1 la transmembrane domain, CD 18 transmembrane domain, CD2 transmembrane domain, and a HER2 transmembrane domain.11. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain; a hinge region selected from the group consisting of a truncated CD4 hinge region, a CD1 la hinge region, a CD2 hinge region, and a TCRa hinge region; a transmembrane domain; and an intracellular signaling domain.12. The CAR of embodiment 11, wherein the hinge region is a truncated CD4 hinge region comprising a truncation of from 1 to 12 amino acids relative to the amino acid sequence of SEQ ID NO:33.13. The CAR of embodiment 12, wherein the truncated CD4 hinge region comprises the amino acid sequence of SEQ ID NO:34.14. The CAR of embodiment 11, wherein the hinge region is a CD1 la hinge region, optionally wherein the CD1 la hinge region comprises the amino acid sequence of SEQ ID NO:35.15. The CAR of embodiment 11, wherein the hinge region is a CD2 hinge region, optionally wherein the CD2 hinge region comprises the amino acid sequence of SEQ ID NO:37.16. The CAR of embodiment 11, wherein the hinge region is a TCRa hinge region, optionally wherein the TCRa hinge region comprises the amino acid sequence of SEQ ID NO:38.17. The CAR of any one of embodiments 11-16, wherein the transmembrane domain is a CD28 transmembrane domain, optionally wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:40.18. The CAR of any one of embodiments 11-16, wherein the transmembrane domain is a modified CD28 transmembrane domain comprising an insertion, substitution, and / or deletion relative to SEQ ID NO:40, optionally wherein the modified CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:41.19. The CAR of any one of embodiments 11-16, wherein the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD1 la transmembrane domain, a CD 18 transmembrane domain, a CD2 transmembrane domain, and a HER2 transmembrane domain.20. The CAR of embodiment 1, 10 or 19, wherein the transmembrane domain is the CD4 transmembrane domain, optionally wherein the CD4 transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.21. The CAR of embodiment 1, 10 or 19, wherein transmembrane domain is the CD1 la transmembrane domain, optionally wherein the Cl la transmembrane domain comprises the amino acid sequence of SEQ ID NO:43.22. The CAR of embodiment 1, 10 or 19, wherein the transmembrane domain is the CD18 transmembrane domain, optionally wherein the CD 18 transmembrane domain comprises the amino acid sequence of SEQ ID NO:44.23. The CAR of embodiment 1, 10 or 19, wherein the transmembrane domain is the CD2 transmembrane domain, optionally wherein the CD2 transmembrane domain comprises the amino acid sequence of SEQ ID NO:45.24. The CAR of embodiment 1, 10 or 19, wherein the transmembrane domain is the HER2 transmembrane domain, optionally wherein the HER2 transmembrane domain comprises the amino acid sequence of SEQ ID NO:46.25. The CAR of any one of embodiments 5-24, wherein the CAR does not cause a substantial reduction in cell surface expression of endogenous CD28 protein when the CAR is expressed in a T-cell.26. The CAR of any one of embodiments 5-24, wherein cell surface expression of endogenous CD28 protein in a T-cell expressing the CAR is substantially higher than that of a control T-cell expressing a control CAR, wherein the control CAR comprises a wild type CD28 hinge region of SEQ ID NO:31 and a wild type CD28 transmembrane domain of SEQ ID NO:40, optionally wherein the control CAR is otherwise identical to the embodied CAR.27. The CAR of embodiment 4, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:42-46.28. The CAR of embodiment 7, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:40-46.29. The CAR of embodiment 11, wherein the hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:33-38, and the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:40-46.30. The CAR of embodiment 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.31. The CAR of embodiment 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 43.32. The CAR of embodiment 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:44.33. The CAR of embodiment 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 45.34. The CAR of embodiment 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:46.35. The CAR of embodiment 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:40.36. The CAR of embodiment 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:41.37. The CAR of embodiment 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.38. The CAR of embodiment 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 43.39. The CAR of embodiment 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:44.40. The CAR of embodiment 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 45.41. The CAR of embodiment 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:46.42. The CAR of embodiment 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:34, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.43. The CAR of embodiment 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:35, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 43.44. The CAR of embodiment 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 36, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:44.45. The CAR of embodiment 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:37, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 45.46. The CAR of embodiment 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:38, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.47. The CAR of any one of embodiments 1-46, wherein the intracellular signaling domain comprises a CD3^ signal transduction domain.48. The CAR of embodiment 47, wherein the CD3^ signal transduction domain comprises the amino acid sequence of SEQ ID NO:28.49. The CAR of embodiment 47 or embodiment 48, wherein the intracellular signaling domain further comprises a co-stimulatory domain.50. The CAR of embodiment 49, wherein the co-stimulatory domain comprises a CD28 costimulatory domain or a 4- IBB co-stimulatory domain.51. The CAR of embodiment 50, wherein the co-stimulatory domain comprises a CD28 co- stimulatory domain, optionally wherein the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:26.52. The CAR of embodiment 51 , wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:29.53. The CAR of embodiment 50, wherein the co-stimulatory domain comprises a 4- IBB costimulatory domain, optionally wherein the 4- IBB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:27.54. The CAR of embodiment 53, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:30.55. The CAR of any one of embodiments 1-54, further comprising a signal peptide.56. The CAR of embodiment 55, wherein the signal peptide is a CD8a signal peptide or a GM-CSF signal peptide.57. The CAR of any one of embodiments 1-56, wherein the antigen-binding domain comprises a single chain antibody fragment (scFV), or a single domain antibody (sdAb).58. The CAR of embodiment 57, wherein the antigen-binding domain comprises a scFv.59. The CAR of embodiment 57, wherein the antibody-binding domain comprises a sdAb, optionally wherein the sdAb comprises a heavy chain variable region fragment (VHH).60. The CAR of any one of embodiments 1-59, wherein the antigen-binding domain binds to an antigen selected from the group consisting of citrullinated vimentin (CV), CD 19, and BCMA.61. The CAR of embodiment 60, wherein the antigen-binding domain binds to CV, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and light chain CDRs of the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO: 8;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; or(iii) the amino acid sequence of SEQ ID NO: 16.62. The CAR of embodiment 60, wherein the antigen-binding domain binds to CD 19, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NOTO, and light chain CDRs of the amino acid sequence of SEQ ID NO:9;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, anda light chain variable region comprising the amino acid sequence of SEQ ID NO:9; or(iii) the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.63. The CAR of embodiment 60, wherein the antigen-binding domain binds to BCMA, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NO: 12, and light chain CDRs of the amino acid sequence of SEQ ID NO: 11 ;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11; or(iii) the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:20.64. A nucleic acid encoding the CAR of any one of embodiments 1-63.65. An expression vector comprising the nucleic acid of embodiment 64 in operable combination with an expression control sequence.66. The expression vector of embodiment 65, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.67. The expression vector of embodiment 66, wherein the viral vector is a lentiviral vector.68. The expression vector of embodiment 65, wherein the vector is a plasmid.69. A host cell comprising the expression vector of any one of embodiments 65-68.70. A modified T-cell that has been engineered to express the CAR of any one of embodiments 1-65, wherein the modified T-cell is a mammalian T-cell.71. A pharmaceutical composition comprising a plurality of the modified T-cells of embodiment 70 and a pharmaceutically acceptable excipient.72. The pharmaceutical composition of embodiment 71, wherein the modified T-cell is a human T-cell.73. The pharmaceutical composition of embodiment 72, wherein human T-cell is a regulatory T-cell (Treg) that is CD4+, CD25+, and CD1271o.74. The pharmaceutical composition of embodiment 73, wherein the human Treg is FOXP3+ and HELIOS+.75. A method of treating a disease or disorder comprising administering an effective amount of the pharmaceutical composition of any one of embodiments 71-74 to a subject in need thereof.76. A method of treating a subject suffering from an autoimmune disease or an inflammatory disorder comprising administering an effective amount of the pharmaceutical composition of any one of embodiments 71-74 to the subject, wherein the CAR expressed by the modified T-cells comprising an antigen-binding domain that binds to an antigen expressed on cells of the autoimmune disease or the inflammatory disorder.77. A method of treating a subject suffering from cancer comprising administering an effective amount of the pharmaceutical composition of embodiment 71 or embodiment 72 to the subject, wherein the CAR expressed by the modified T-cells comprises an antigen-binding domain that binds to an antigen expressed on cells of the cancer.78. A kit comprising a container containing the pharmaceutical composition of any one of embodiments 71-74, communicating through a fluidic conduit to a drip chamber, wherein the drip chamber is configured to communicate through a fluidic conduit with an intravenous needle.EXAMPLES

[0123] Abbreviations: CAR (chimeric antigen receptor); CFSE (carboxyfluorescein succinimidyl ester); CV (citrullinated vimentin); EGFR (epidermal growth factor receptor); EGFRt (EGFR tag); FACS (fluorescence activated cell sorting); H (hinge region); IC (intracellular domain); IV (intravenous); MFI (mean fluorescence intensity); Mut (mutated); PBMC (peripheral blood mononuclear cells); PBS (phosphate buffered saline); Prom, (promoter); scFv (single-chain variable fragment); TCRa (T-cell receptor alpha); Teff (effector T-cell); TM or TMD (transmembrane domain); Treg (regulatory T-cell); Trun (truncated); and UTD (untransduced), WT (wild type).

[0124] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the following examples should not be construed as limiting the scope of the present disclosure, which is defined by the claims.Example 1: T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With a CD28 Hinge Region and a CD28 Transmembrane Domain

[0125] CD28 is a costimulatory molecule required for activation and proliferation of T- cells. Surprisingly, expression of CAR with a wild-type CD28 transmembrane domain and a wild-type CD28 hinge region (hereafter referred to as a wild-type backbone) on regulatory T- cells results in a decrease in endogenous CD28 expression. Furthermore, regulatory T-cells (referred to herein as Treg cells or Tregs) expressing a CAR with a wild-type backbone demonstrated lower levels of proliferation upon activation.

[0126] Lentiviral construct production. A construct comprising a CAR with a wild-type backbone and an EGFRt tag was packaged as lentiviral particles for transduction into cells of interest. HEK 293 FT suspension cells (Invitrogen) were seeded at 4.7 x 106 / mE and transfected with transfer and packaging plasmids (Aldevron pAED-VSV-G, pAED-GagPol, pAED-Rev) using LV_MAX transfection reagent as per manufacturer's protocol (Gibco). Viral supernatant was collected and filtered through a 0.45 pm polyvinylidene difluoride filter to remove cell debris. The viral supernatant was centrifuged at 10,000 x g overnight to concentrate viral vectors.After 12-16 hours, supernatant was removed, and the viral vector pellet was resuspended in Opti- MEM media. Resuspended viral vector was aliquoted and stored at -80°C.

[0127] Functional titer assay. Functional titers were assessed to identify optimal T-cell transduction conditions. SupTl cells (ATCC) were cultured in RPMI medium supplemented with 10% fetal bovine serum. On the day of transduction, 50 pL of SupTl cells at 4 x 105cells / mL were added to each well of a 96-well plate. Lentivirus stock was thawed at room temperature and diluted 1:20 in culture medium. Subsequently, a three-fold serial dilution was performed from 60 to 393,660-fold in culture medium. 50 pL of diluted lentivirus were added to a 96-well plate containing 50 pL of cells. On day 2 of transduction, 100 pL of culture media was added. On day 3, transduced cells were harvested and stained with appropriate antibody to detect surface markers. Labeled cells were analyzed by flow cytometry. Untransduced SupTl cells were used as negative control to set a gate for flow cytometry analysis. Viral dilution that produced between 5 - 20% maker positive cells were used for the calculation of functional titer, based on the following formula:[Cell #]transduced x [(%) cells]transduced x [dilution factor][(mL) volume]transduced

[0128] Treg isolation and expansion. Primary human Treg cells were sourced from healthy donors from leukoreduction chamber residuals or leukopaks. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll-Paque Plus. CD25+ cells were enriched by positive selection. Treg cells were next isolated using FACS by gating for CD4+ / CD25+ / CD1271o cells. After isolation, cells were stimulated with CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead to cell ratio and expanded for 14 days, with restimulation on day 9. Cells were cultured in RPMI medium supplemented with 10% FBS, non- essential amino acids, sodium pyruvate, and penicillin / streptomycin with recombinant human IL- 2 at 300 lU / mL at a density of 0.25-0.3 million cells / mL.

[0129] Treg cell transduction. A CAR with a wild-type backbone was expressed on Treg cells by transducing Tregs with the lentiviral vector described above. Treg cells were plated in a 24-well plate on Day 0 at 2.5xl05cells / mL in 1 mL of media per well. On Day 2, Tregs were transduced via spinfection by adding the appropriate volume of virus to achieve an MOI of 5-10 to each well and protamine sulfate at a final concentration of 100 pg / mL. Cells were centrifugedwith lentivirus at 1200 x g for 30 min at 30°C. Shortly or immediately after spin, fresh media was added with 2X recombinant human IL-2 at 1 : 1 ratio with conditioned media.

[0130] Flow cytometry and FACS analysis. Presence of EGFR, CAR with a wild-type backbone, and CD28 on Tregs was assessed at day 14 post-transduction. Treg cells were collected and centrifuged at 300 x g for 5 min and then resuspended in IX RoboSep Buffer (StemCell Technologies) with a surface staining antibody. Tregs were then incubated for 30 min at 4°C, then centrifuged and washed with IX RoboSep Buffer. Stained cells were then analyzed by flow cytometry.

[0131] Treg cell antibody activation assay. Treg cells expressing CAR with a wild-type backbone were prepared using the methods as described above. Cells rested for 72 hrs on Day 14, and then were labeled with CFSE and activated with anti-CD3 and anti-CD28 antibody activation beads. Proliferation of transduced Treg cells was determined 72 hrs post stimulation via FACS.

[0132] FIG. 1A presents a schematic and corresponding expression construct of a CAR having a wild-type backbone and an EGFRt tag. A CAR with a wild-type backbone was transduced into Treg cells. After transduction, Treg cells were stained with anti-EGFRt and anti- CD28 antibodies and analyzed by FACS. As shown in FIGS. 1B-1C, Treg cells transduced with a CAR having a wild-type backbone demonstrated a reduction in endogenous CD28 expression. FIG. IB depicts a representative dot plot of EGFRt and CD28 signal intensity of Treg cells population after transduction. Each quadrant (Q5, Q6, Q7, and Q8) distinguishes various Treg cell populations within the parent population. The majority of the Treg cells, 66.1%, are found in quadrant Q5, which identifies untransduced Treg cells. Quadrants Q6 and Q7 show populations of transduced Treg cells expressing a CAR with a wild-type backbone which is co-expressed with the EGFRt tag. Quadrants Q6 and Q7 are distinguished by expression levels of CD28, with Q6 identifying Treg cells that are CD28(+) and Q7 identifying CD28(-) Treg cells. Surprisingly, a substantial portion of the transduced Treg cell population showed reduced levels of endogenous CD28 expression (Q7) compared to the untransduced Treg cell population. FIG. 1C depicts a bar chart showing levels of CD28 expression by CAR-expressing Treg cells (CAR+) and untransduced Treg cells (CAR-). A decrease in CD28 expression was observed for CAR- expressing Treg cells as compared to untransduced Treg cells.

[0133] The ability to activate the CAR-expressing Treg cells via antibodies was also assessed. After transduction, Treg cells were labeled with CFSE and activated with anti-CD3 and anti-CD28 antibody. Proliferation of transduced Treg cells was assessed using FACS as shown in FIG. ID. The figure depicts a histogram of normalized cell count versus CFSE signal intensity of CAR-expressing Treg cells (CAR+) and untransduced Treg cells (CAR-). CFSE signal intensity is indirectly correlated with extent of cell proliferation. As Treg cells proliferate, CSFE fluorescence intensity is halved with each cell division resulting in the shift of the histogram to the left. As shown in FIG. ID, as a result of reduced CD28 expression, CAR-expressing Treg cells were not able to proliferate as well as untransduced Treg cells.Example 2: CARs Engineered to Avoid Loss of Endogenous CD28 Expression by Transduced T-cells

[0134] As described in Example 1 , expression of a CAR with a wild-type backbone (CD28 hinge region and CD28 transmembrane domain) in Treg cells resulted in a decrease in endogenous CD28 expression. In order to avoid loss of endogenous CD28 expression on transduced Treg cells, new CAR backbones were constructed with modifications in the transmembrane domain and / or the hinge region. Table 2-1 lists the hinge region, transmembrane domain and intracellular domain combinations of new CARs depicted in FIGS. 2A-2B.Table 2-1. CAR Backbones

[0135] Table 2-1 also lists the hinge region, transmembrane domain and intracellular domain combinations of exemplary CAR expression cassettes shown in FIGS. 3A-3B . Each construct comprises an EGFRt tag for confirmation of successful transduction into T-cells.Example 3: CAR Expression on Transduced T-cells

[0136] The CARs of Example 2 were expressed in two cell types, Treg cells and Jurkat cells. Expression of endogenous CD28 was measured to determine whether the new CARs were free of the defect observed in CARs having a wild type backbone (CD28 hinge region and CD28 transmembrane domain). Expression of Helios and FoxP3 on transduced Treg cells was also assessed to determine with the Treg phenotype was retained.Expression on Treg cells

[0137] Following the methods of Example 1 , vectors encoding CARs with wild-type (WT) or modified backbones were transduced into Treg cells. After transduction, Treg cells with stained with anti-CAR and anti-EGFRt antibody. As shown in FIGS. 4A-B, Treg cells were successfully transduced with the various CARs. FIGS. 4A depicts representative dot plots showing levels of EGFRt tag and CAR expression by Treg cells after transduction. Each plots shows Treg cells expressing different CARs, either a CAR with a wild-type backbone or a CAR with a modified backbone. Primarily two Treg cell populations were observed in each dot plot. The first cell population located in quadrant Q4 identifies untransduced Treg cells which were negative for CAR and thus also negative for the co-expressed EGFRt tag. The second cell population located in quadrant Q2 identifies transduced Treg cells expressing CAR and the EGFRt tag. It is notable that for each dot plot, the transduced Treg cell population represents the largest portion of the parent Treg cell population. For each CAR expressed, the transduced Treg cells make up at least 60% of the parent population indicating durable transduction. The percentages of the different Treg cell populations are shown in each quadrant. FIG. 4B depicts a bar chart showing levels of CAR expression by transduced Treg cells.

[0138] After demonstrating transduction of CARs into Treg cells, the CAR-expressing Treg cells were then tested to determine whether endogenous CD28 expression was retained as a result of engineering the CAR backbone. CAR-expressing Treg cells were stained with anti- CD28 antibody and then analyzed using flow cytometry. FIG. 4C demonstrates that endogenous CD28 expression was restored in Treg cells expressing a CAR with a modified backbonecompared to the Treg cells expressing a CAR with a wild-type backbone. FIG. 4C depicts a bar chart showing levels of CD28 expression for various CAR-expressing Treg cells compared to the Mock (untransduced) sample. It is notable that CD28 expression increased in Treg cells expressing CARs with modified backbones compared to Treg cells expressing a CAR with a wild-type backbone. Furthermore, CD28 expression by Treg cells expressing a CAR with a modified backbone was comparable to that of the Mock (untransduced) sample.

[0139] As shown in FIG. 5A-B, other CAR expression vectors were transduced into Treg cells and tested for restoration of endogenous CD28 expression. After transduction, Treg cells were stained with anti-CAR and anti-EGFRt antibody. FIG. 5A shows successful transduction and durable expression of various CARs on Treg cells. FIGS. 5A depicts representative dot plots showing levels of EGFRt tag and CAR expression by Treg cells after transduction. The dot plot in the upper left hand corner shows Treg cells that were not transduced with a lentiviral vector (UTD). The remaining dot plots show Treg cells expressing different CARs, either a CAR with a wild-type backbone or a CAR with a modified backbone. Each quadrant of the dot plot distinguishes a different Treg cell population within the parent population. Untransduced Treg cells were located in quadrant Q8 which were negative for CAR and thus also negative for the co-expressed EGFRt tag. As expected, majority of cells from the UTD control were located in Q8. Quadrant Q7 identifies transduced Treg cells expressing the EGFRt tag but not CAR. Quadrant Q6 identifies transduced Treg cells expressing CAR and the EGFRt tag thus demonstrating successful transduction.

[0140] After demonstrating transduction of CAR expression vectors into Treg cells, the CAR-expressing Treg cells were then tested to determine whether endogenous CD28 expression is restored as a result of engineering the CAR backbone. CAR-expressing Treg cells were stained with anti-CD28 antibody and then analyzed using flow cytometry. FIG. 5B demonstrates that CD28 expression was restored in Treg cells expressing a CAR with a modified backbone compared to the Treg cells expressing a CAR with a wild-type backbone. FIG. 5B depicts representative dot plots showing levels of EGFRt tag and CD28 expression by Treg cells after transduction. The dot plot in the upper left hand corner shows Treg cells that were not transduced with a lentiviral vector (UTD). The remaining dot plots show Treg cells expressing CARs, either a CAR with a wild-type backbone or a CAR with a modified backbone. Each quadrant (QI 3, QI 4, QI 5, and Q8) distinguishes various Treg cell populations within the parent population. Q13identifies untransduced Treg cells which were negative for the EGFRt tag and thus also negative for CAR. As expected, these cells were positive for endogenous CD28 expression. Majority of cells from the UTD control were located in QI 3. Quadrants Q14 and Q15 identify transduced Treg cells expressing the EGFRt tag and thus CAR. Transduced Treg cells can be distinguished further between Treg cells that were positive for CD28 expression (QI 4) and those that were negative for CD28 expression (QI 5). Similar to what was previously demonstrated in Example 1 , a substantial portion of the transduced Treg cells expressing a CAR with a wild-type backbone showed reduced CD28 expression. In contrast, Treg cells expressing CARs with a modified backbone were primarily CD28(+) and located within QI 4, thus demonstrating successful restoration of endogenous CD28 expression.

[0141] Finally, Treg cells expressing various CARs were assessed for Helios and FoxP3 phenotypes. CAR-expressing Treg cells were fixed and permeabilized. The Treg cells were then stained with anti-Helios and anti-FoxP3 antibodies and analyzed using flow cytometry. FIG. 5C demonstrates that CAR-expressing Treg cells expressed both Helios and FoxP3 markers.FIG. 5C depicts representative dot plots showing levels of Helios and FoxP3 expression by Treg cells after transduction.Expression on Jurkat cells

[0142] CAR and CD28 expression were assessed by flow cytometry using anti-scFv and anti-CD28 antibodies, respectively.

[0143] Jurkat cell transduction. Jurkat cells were transduced with a lentiviral vector. After transduction, Jurkat cells with stained with anti-CAR antibody. As shown in FIG. 6A, Jurkat cells were successfully transduced with the various CARs. FIG. 6A depicts a bar chart showing levels of CAR expression by transduced Jurkat cells compared to the Mock (untransduced) cells.

[0144] The CAR-expressing Jurkat cells were then tested to determine whether endogenous CD28 expression is restored as a result of engineering the CAR backbone. CAR- expressing Jurkat cells were stained with anti-CD28 antibody and then analyzed using flow cytometry. As shown in FIG. 6B, endogenous CD28 expression was restored in Jurkat cells expressing a CAR with a modified backbone. FIG. 6B depicts a bar chart of CD28 signal intensity for various CAR-expressing Jurkat cells compared to untransduced Jurkat cells (UTD).It is notable that CD28 signal intensity, thus expression CD28 expression, was greater for Jurkat cells expressing CARs with a modified backbone compared to that of Jurkat cells expressing a CAR with a wild-type backbone. Furthermore, CD28 expression by Jurkat cells expressing CARs with a modified backbone was comparable to that of the untransduced Jurkat cells. As shown in FIG. 6C, additional CARs with modified backbones were expressed in Jurkat cells and tested for restoration of endogenous CD28 expression. FIG. 6C depicts a bar chart of CD28 signal intensity for various CAR-expressing Jurkat (CAR+) cells compared to CAR- Jurkat cells. Similar to what was previously demonstrated in Example 1 , Jurkat cells expressing a CAR with a wild-type backbone demonstrated reduced CD28 expression compared to untransduced Jurkat cells. CD28 expression was greater for Jurkat cells expressing CARs with modified backbones compared to that Jurkat cells expressing a CAR with a wild-type backbone. Furthermore, CD28 expression by Jurkat cells expressing CARs with modified backbones was comparable to that of the untransduced Jurkat cells.Example 4: Activation of CAR+ T-cells

[0145] In Example 3, modifications of the CAR backbone led to a retention of endogenous CD28 expression in CAR-expressing T-cells. In this example, T-cells expressing various CARs were tested for their ability to be activated after stimulation.Activation of Treg cells expressing CARs with modified backbones

[0146] Treg cell activation with CDS and CD28 antibodies. Treg cells expressing a CAR with a wild-type backbone or a modified backbone were prepared using the methods described in Example 1. Cells rested for 72 hrs on Day 14, and then were labeled CFSE and activated with anti-CD3 and anti-CD28 antibody activation beads. Proliferation of engineered Treg cells was determined 72 hrs post stimulation via FACS.

[0147] CAR expression vectors were transduced into Treg cells, which were then labeled with CFSE, and activated with anti-CD3 and anti-CD28 antibodies. FIG. 7A demonstrates improved activation and proliferation of Treg cells expressing a CAR with a modified backbone compared to the Treg cells expressing a CAR with a wild-type backbone. As Treg cells proliferate, CSFE fluorescence intensity is halved with each cell division resulting in the shift of the histogram to the left. Similar to what was previously demonstrated in Example 1 , Treg cells expressing a CAR with a wild-type backbone did not proliferate as well as CAR- Treg cells. Incontrast, Treg cells expressing CARs with a modified backbone demonstrated comparable proliferation to CAR- Treg cells. FIG. 7B shows improved proliferation of Treg cells expressing a CAR with a modified backbone compared to Treg cells expressing a CAR with a wild-type backbone. FIG. 7B depicts % proliferation as a function of cell to CD3 / CD28-bead ratio for CAR-expressing Treg cells (EGFR+) compared to CAR- Treg cells (EGFR-). Treg cells expressing a CAR with a wild-type backbone did not proliferate as well as CAR- Treg cells. Treg cells expressing CARs with modified backbones demonstrated improved activation and proliferation compared to that of the Treg cells expressing a CAR with a wild-type backbone.Activation of Jurkat-cells expressing CARs with modified backbones

[0148] CARs with wild-type or modified backbones were expressed on cells of a Jurkat reporter cell line. The reporter cell line (Jurkat-NFAT-FF-luc) expresses firefly luciferase under the control of the nuclear factor of activated T-cells (NF AT) response elements.

[0149] Jurkat cell activation with CAR antigen-expressing target cells. CAR-expressing Jurkat cells were prepared using the methods described in Example 3, and subsequently cocultured with CAR antigen positive K562 cells. After co-culture, a portion of the cells was used to assess luciferase expression as a marker of activation through the CAR. Luciferase expression was detected by measuring luminescence with SpectraMax plate reader. The remaining portion of the cells was used to assess expression of activation markers by staining with anti-CD69 and anti-CD25 antibodies and FACS analysis.

[0150] CAR-expressing Jurkat cells were activated with CAR antigen-expressing K562 cells. FIG. 8A depicts luminescence as a function of the Jurkat to K562 cell ratio. CAR- expressing Jurkat cells were also stained with an anti-CD69 antibody. FIG. 8B depicts the percentage of Jurkat cells expressing the CD69 activation marker as a function of the Jurkat to K562 cell ratio. FIG. 8C depicts the percentage of Jurkat cells expressing the CD25 activation marker. As the ratio of CAR+ Jurkat cells to CAR antigen-l- K562 cells approached 1:1, CAR- expressing Jurkat cells demonstrated an increase in CD25 expression. Furthermore, Jurkat cells expressing CARs with modified backbones displayed comparable upregulation of the activation marker CD25 to that of Jurkat cells expressing a CAR with a wild-type backbone.SEQUENCES>SEQ ID NO:1 (GMCSF signal peptide) Homo sapiensMLLLVTSLLL CELPHPAFLLIP>SEQ ID NO:2 (CD8a signal peptide) Homo sapiensMALPVTALLLPLALLLHAARP>SEQ ID NOS (anti-CV VHl) Homo sapiens - 116aaHLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS>SEQ ID NO:4 (anti-CV VH2) Homo sapiens - 116aaQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR LD P FDYWG RGTLVTVSS>SEQ ID NOS (anti-CV VH3) Homo sapiens - 119aaEVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSS»SEQ ID NO:6 (anti-CV VL1) Homo sapiens - 109aaSYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVLR>SEQ ID NO:7 (anti-CV VL2) Homo sapiens - 109aaSYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR>SEQ ID NO:8 (anti-CV VL3) Homo sapiens - lllaaAIQMTQSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIKRAAA>SEQ ID NO:9 (FM63VL)DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEIT>SEQ ID NO:10 (FM63VH)EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS>SEQ ID NO:11 (ida VL)DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK>SEQ ID NO:12 (ida VH)QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS>SEQ ID NO:13 synthetic glycine linker GGGGSGGGGSGGGGS>SEQ ID N0:14 synthetic Whitlow linkerGSTSGSGKPGSGEGSTKG>SEQ ID NO:15 synthetic ABpur linkerASSGGSTSGSGKPGSGEGSSGSAR>SEQ ID NO:16 - anti-CV ABD (254aa) syntheticEVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKST VYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARAIQMTQSPSSLSA SVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGF PFTVGQGTKLDIKRAAA>SEQ ID NO:17 -tisa CD19 ABD (242aa) syntheticDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQE DIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSV TVSS>SEQ ID NO:18 - liso CD19 ABD (245aa) syntheticDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQE DIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVSS>SEQ ID NO:19 - ida BCMA ABD (246aa) syntheticDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPV EEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSIN WVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTS VTVSS>SEQ ID NO:20 - cilta BCMA ABD (242aa) syntheticQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTL YLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFTM GWFRQAPGKEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWG QGTQVTVSS>SEQ ID NO:21 - CD8a Hinge - Homo sapiensTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD>SEQ ID NO:22 - CD28 Hinge - Homo sapiensIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP>SEQ ID NO:23 - lgG4 Hinge - Homo sapiensESKYGPPCPPCP>SEQ ID NO:24 - CD8a transmembrane - Homo sapiensIYIWAPLAGTCGVLLLSLVITLYC>SEQ ID NO:25 - CD28 transmembrane - Homo sapiens[X]FWVLVWGGVLACYSLLVTVAFIIFWVX = M or absent>SEQ ID NO:26 (CD28 costimulatory domain) Homo sapiensRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS>SEQ ID NO:27 (41BB costimulatory domain) Homo sapiensKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL>SEQ ID NO:28 (CD3zeta intracellular signal transduction domain) Homo sapiensRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR>SEQ ID NO:29 (CD28+CD3zeta) syntheticRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR>SEQ ID NO:30 (41BB+CD3zeta) syntheticKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO:31 (CD28 hinge region amino acid sequence, wild-type reference) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPSEQ ID NO:32 (CD28 truncated hinge region amino acid sequence) SPLFPGPSKPSEQ ID NO:33 (CD4 hinge region amino acid sequence, wild-type reference)SGQVLLESNIKVLPTWSTPVQPSEQ ID NO:34 (CD4 truncated hinge region amino acid sequence)LPTWSTPVQPSEQ ID NO:35 (CDlla hinge region amino acid sequence)VDVVYEKQMLSEQ ID NO:36 (lgG4 hinge region amino acid sequence "X")ESKYGPPCP[X]CPX = P or SSEQ ID NO:37 (CD2 hinge region amino acid sequence) SKESSVEPVSCPEKGLDSEQ ID NO:38 (TCRa hinge region amino acid sequence)PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSSEQ ID NO:39 ( lgG4 hinge region amino acid sequence) ESKYGPPCPPCPSEQ ID NO:40 (CD28 transmembrane domain amino acid sequence, wild-type reference) FWVLWVGGVLACYSLLVTVAFIIFWVSEQ ID NO:41 (CD28 modified transmembrane domain amino acid sequence)FWVLWVGGVLALLLLLVLVAFIIFWVSEQ ID NO:42 (CD4 transmembrane domain amino acid sequence) MALIVLGGVAGLLLFIGLGIFFSEQ ID NO:43 (CDlla transmembrane domain amino acid sequence)YLYVLSGIGGLLLLLLIFIVLYKVSEQ ID NO:44 (CD18 transmembrane domain amino acid sequence) IAAIVGGTVAGI LIGILLLVIWSEQ ID NO:45 (CD2 transmembrane domain amino acid sequence) IYLIIGICGGGSLLMVFVALLVFYITSEQ ID NO:46 (HER2 transmembrane domain amino acid sequence)SIISAWGILLVWLGVVFGILI

Claims

CLAIMSWe claim:

1. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain; a CD28 hinge region; a transmembrane domain selected from the group consisting of a CD4 transmembrane domain, a CD1 la transmembrane domain, a CD 18 transmembrane domain, a CD2 transmembrane domain, and a HER2 transmembrane domain; and an intracellular signaling domain.

2. The CAR of claim 1 , wherein the CAR does not cause a substantial reduction in cell surface expression of endogenous CD28 protein when the CAR is expressed in a T-cell.

3. The CAR of claim 1, wherein cell surface expression of endogenous CD28 protein in a T- cell expressing the CAR is substantially higher than that of a control T-cell expressing a control CAR, wherein the control CAR comprises a wild type CD28 hinge region of SEQ ID NO:31 and a wild type CD28 transmembrane domain of SEQ ID NO:40, wherein the control CAR is otherwise identical to the claimed CAR.

4. The CAR of claim 1 , wherein the CD28 hinge region comprises the amino acid sequence of SEQ ID NO:31.

5. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain; a truncated CD28 hinge region; a transmembrane domain; and an intracellular signaling domain, wherein the truncated CD28 hinge region comprises a truncation of from about 10 to about 30 amino acids relative to the amino acid sequence of SEQ ID NO:31.

6. The CAR of claim 5, wherein the truncated CD28 hinge region is truncated at the N- terminus.

7. The CAR of claim 6, wherein the truncated CD28 hinge region comprises the amino acid sequence of SEQ ID NO:32.

8. The CAR of claim 5, wherein the transmembrane domain is a CD28 transmembrane domain.

9. The CAR of claim 5, wherein the transmembrane domain is a modified CD28 transmembrane domain comprising an insertion, substitution, and / or deletion relative to SEQ ID NO:40.

10. The CAR of claim 5, wherein the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD1 la transmembrane domain, CD 18 transmembrane domain, CD2 transmembrane domain, and a HER2 transmembrane domain.

11. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain; a hinge region selected from the group consisting of a truncated CD4 hinge region, a CD1 la hinge region, a CD2 hinge region, and a TCRa hinge region; a transmembrane domain; and an intracellular signaling domain.

12. The CAR of claim 11, wherein the hinge region is a truncated CD4 hinge region comprising a truncation of from 1 to 12 amino acids relative to the amino acid sequence of SEQ ID NO:33.

13. The CAR of claim 12, wherein the truncated CD4 hinge region comprises the amino acid sequence of SEQ ID NO:34.

14. The CAR of claim 11, wherein the hinge region is a CD1 la hinge region.

15. The CAR of claim 11, wherein the hinge region is a CD2 hinge region.

16. The CAR of claim 11, wherein the hinge region is a TCRa hinge region.

17. The CAR of claim 11, wherein the transmembrane domain is a CD28 transmembrane domain.

18. The CAR of claim 11, wherein the transmembrane domain is a modified CD28 transmembrane domain comprising an insertion, substitution, and / or deletion relative to SEQ ID NO:40.

19. The CAR of claim 11, wherein the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD1 la transmembrane domain, a CD 18 transmembrane domain, a CD2 transmembrane domain, and a HER2 transmembrane domain.

20. The CAR of claim 1 , wherein the transmembrane domain is the CD4 transmembrane domain.

21. The CAR of claim 1, wherein transmembrane domain is the CD1 la transmembrane domain.

22. The CAR of claim 1 , wherein the transmembrane domain is the CD 18 transmembrane domain.

23. The CAR of claim 1, wherein the transmembrane domain is the CD2 transmembrane domain.

24. The CAR of claim 1 , wherein the transmembrane domain is the HER2 transmembrane domain.

25. The CAR of claim 5, wherein the CAR does not cause a substantial reduction in cell surface expression of endogenous CD28 protein when the CAR is expressed in a T-cell.

26. The CAR of claim 5, wherein cell surface expression of endogenous CD28 protein in a T- cell expressing the CAR is substantially higher than that of a control T-cell expressing a control CAR, wherein the control CAR comprises a wild type CD28 hinge region of SEQ ID NO:31 and a wild type CD28 transmembrane domain of SEQ ID NO:40.

27. The CAR of claim 4, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:42-46.

28. The CAR of claim 7, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:40-46.

29. The CAR of claim 11, wherein the hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:33-38, and the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:40-46.

30. The CAR of claim 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.

31. The CAR of claim 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:43.

32. The CAR of claim 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:44.

33. The CAR of claim 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:45.

34. The CAR of claim 27, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:31, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:46.

35. The CAR of claim 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:40.

36. The CAR of claim 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:41.

37. The CAR of claim 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.

38. The CAR of claim 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:43.

39. The CAR of claim 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:44.

40. The CAR of claim 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:45.

41. The CAR of claim 28, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:32, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:46.

42. The CAR of claim 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:34, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.

43. The CAR of claim 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:35, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:43.

44. The CAR of claim 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:36, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:44.

45. The CAR of claim 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:37, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:45.

46. The CAR of claim 29, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:38, and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:42.

47. The CAR of claim 1, wherein the intracellular signaling domain comprises a CD3^ signal transduction domain.

48. The CAR of claim 47, wherein the CD3^ signal transduction domain comprises the amino acid sequence of SEQ ID NO:28.

49. The CAR of claim 47, wherein the intracellular signaling domain further comprises a costimulatory domain.

50. The CAR of claim 49, wherein the co-stimulatory domain comprises a CD28 costimulatory domain or a 4- IBB co-stimulatory domain.

51. The CAR of claim 50, wherein the co-stimulatory domain comprises a CD28 co- stimulatory domain.

52. The CAR of claim 51 , wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:29.

53. The CAR of claim 50, wherein the co-stimulatory domain comprises a 4- IBB co- stimulatory domain.

54. The CAR of claim 53, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:30.

55. The CAR of claim 1, further comprising a signal peptide.

56. The CAR of claim 55, wherein the signal peptide is a CD8a signal peptide or a GM-CSF signal peptide.

57. The CAR of claim 1, wherein the antigen-binding domain comprises an single chain antibody fragment (scFV), or a single domain antibody (sdAb).

58. The CAR of claim 57, wherein the antigen-binding domain comprises a scFv.

59. The CAR of claim 57, wherein the antibody-binding domain comprises a sdAb, optionally wherein the sdAb comprises a heavy chain variable region fragment (VHH).

60. The CAR of claim 1 , wherein the antigen-binding domain binds to an antigen selected from the group consisting of citrullinated vimentin (CV), CD 19, and BCMA.

61. The CAR of claim 60, wherein the antigen-binding domain binds to CV, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and light chain CDRs of the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO: 8;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; or(iii) the amino acid sequence of SEQ ID NO: 16.

62. The CAR of claim 60, wherein the antigen-binding domain binds to CD 19, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NOTO, and light chain CDRs of the amino acid sequence of SEQ ID NO:9;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9; or(iii) the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

63. The CAR of claim 60, wherein the antigen-binding domain binds to BCMA, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NO: 12, and light chain CDRs of the amino acid sequence of SEQ ID NO: 11 ;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, anda light chain variable region comprising the amino acid sequence of SEQ ID NO:11; or (iii) the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:20.

64. A nucleic acid encoding the CAR of claim 1.

65. An expression vector comprising the nucleic acid of claim 64 in operable combination with an expression control sequence.

66. The expression vector of claim 65, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

67. The expression vector of claim 66, wherein the viral vector is a lentiviral vector.

68. The expression vector of claim 65, wherein the vector is a plasmid.

69. A host cell comprising the expression vector of claim 65.

70. A modified T-cell that has been engineered to express the CAR of claim 1, wherein the modified T-cell is a mammalian T-cell.

71. A pharmaceutical composition comprising a plurality of the modified T-cells of claim 70 and a pharmaceutically acceptable excipient.

72. The pharmaceutical composition of claim 71, wherein the modified T-cell is a human T- cell.

73. The pharmaceutical composition of claim 72, wherein human T-cell is a regulatory T-cell (Treg) that is CD4+, CD25+, and CD1271o.

74. The pharmaceutical composition of claim 73, wherein the human Treg is FOXP3+ and HELIOS+.

75. A method of treating a disease or disorder comprising administering an effective amount of the pharmaceutical composition of claim 71 to a subject in need thereof.

76. A method of treating a subject suffering from an autoimmune disease or an inflammatory disorder comprising administering an effective amount of the pharmaceutical composition of claim 71 to the subject, wherein the CAR expressed by the modified T-cells comprising an antigen-binding domain that binds to an antigen expressed on cells of the autoimmune disease or the inflammatory disorder.

77. A method of treating a subject suffering from cancer comprising administering an effective amount of the pharmaceutical composition of claim 71 to the subject, wherein the CAR expressed by the modified T-cells comprises an antigen-binding domain that binds to an antigen expressed on cells of the cancer.

78. A kit comprising a container containing the pharmaceutical composition of claim 71, communicating through a fluidic conduit to a drip chamber, wherein the drip chamber is configured to communicate through a fluidic conduit with an intravenous needle.

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