Chimeric antigen receptors with atypical transmembrane domains and / or hinge domains

Abstract

The present disclosure relates to chimeric antigen receptors (CARs) that do not result in a substantial reduction of endogenous CD28 protein expression in CAR-expressing T cells.

Description

Cross-reference to related applications

[0001] This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 620,690, filed January 12, 2024, the entire contents of which are incorporated herein by reference.

[0002] Reference to the electronic sequence list The contents of the electronic serial number (237752000940SEQLIST.xml; size: 44,566 bytes; and creation date: January 9, 2025) are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to a chimeric antigen receptor (CAR) that causes a small to no reduction in the expression of endogenous CD28 protein in T cells expressing the CAR. Background Technology

[0004] Chimeric antigen receptors (CARs), comprising portions of both antibodies and T-cell receptors (TCRs), were first described in the 1980s. First-generation CARs contained an antigen-binding domain, hinge, transmembrane domain, and intracellular signal transduction domain of CD3ζ. Subsequent generations of CARs have been engineered to include one or more intracellular co-stimulatory domains to enhance their ability to activate T cells expressing them (Smith and Shen, J. Transl. Med, 21:515, 2023).

[0005] Currently, four anti-CD19 CAR T-cell immunotherapies have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of various CD19+ leukemias and lymphomas. In fact, anti-CD19 CAR T-cell immunotherapy has resulted in significant clinical responses, including high levels of remission, in patients with poor prognoses. However, several challenges limit the therapeutic efficacy of cancer antigen-reactive CAR T-cell therapy, including toxicity and limited antitumor activity against non-hematologic malignancies and other hematologic malignancies.

[0006] While CAR development has primarily focused on antigen-binding domains and intracellular co-stimulatory domains, the effects of other CAR domains (such as hinge and transmembrane domains) on the function of CAR-expressing T cells remain unclear. Therefore, there is a need in the art for CAR scaffolds that have been engineered to reduce their potentially detrimental effects in T cells expressing them. Summary of the Invention

[0007] This disclosure relates to a chimeric antigen receptor (CAR) that results in a small to no reduction in the expression of endogenous CD28 protein in T cells expressing the CAR. Specifically, the CAR of this disclosure contains hinge and / or transmembrane domains different from those of the four currently approved anti-CD19 CAR T-cell therapies by the U.S. Food and Drug Administration (tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, and lisocabtagene maraleucel). Attached Figure Description

[0008] Figure 1A A schematic diagram of a chimeric antigen receptor (CAR) with a wild-type CD28 transmembrane domain and a wild-type CD28 hinge region and its corresponding expression construct is presented. The promoter region (Prom.) and the truncated epidermal growth factor receptor (EGFRt) transduction tag are shown at the 5' and 3' ends of the expression construct, respectively. Figure 1B This is a representative fluorescence-activated cell sorting (FACS) plot of regulatory T (Treg) cells expressing CARs with wild-type CD28 transmembrane domains and wild-type CD28 hinge regions. Cells were 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 parental 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 quadrant. EGFRt is a truncated epidermal growth factor receptor transduction tag. Figure 1C This is a bar chart showing CD28 expression in mean fluorescence intensity (MFI) form for untransduced Treg cells (CAR-) and Treg cells (CAR+) expressing CARs with wild-type CD28 transmembrane domains and wild-type CD28 hinge regions. Figure 1D This is a histogram of normalized cell counts versus CFSE signal intensity for untransduced Treg cells (CAR-) and Treg cells (CAR+) expressing CARs with wild-type CD28 transmembrane domains and wild-type CD28-hinge regions. CFSE is a fluorescent cell staining dye (carboxyfluorescein succinimide ester) whose staining intensity decreases with each cell division.

[0009] Figure 2A-2BSchematic diagrams are shown of a CAR having a wild-type CD28 transmembrane domain and a wild-type CD28 hinge region (referred to herein as the WT backbone) and an exemplary CAR having modifications in the transmembrane domain and / or hinge region (referred to herein as the modified backbone). Abbreviations: Mut (mutant); TRa (T cell receptor α chain); TMD (transmembrane domain); and WT (wild-type).

[0010] Figures 3A-3B Schematic diagrams of various CAR expression constructs are shown, each containing 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 as in SEQ ID NO:42.

[0011] Figure 4A Representative FACS plots of Treg cells expressing various CARs are shown. Cells were stained with EGFRt and CAR antibodies to identify EGFRt+ and CAR+ cells. Each quadrant (Q1, Q2, Q3, and Q4) distinguishes various Treg cell populations within the parental population: Q1 = 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 each quadrant. Figure 4B This is a bar graph showing CAR expression in Treg cells expressing various CARs in the form of mean fluorescence intensity (MFI). Figure 4C This is a bar graph showing CD28 expression in mean fluorescence intensity (MFI) form for untransduced Treg cells (blank control) and Treg cells expressing various CARs.

[0012] Figure 5A Representative FACS plots of Treg cells expressing various CARs are shown. Cells were 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 parental 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 each quadrant. Figure 5B Representative FACS plots of Treg cells expressing various CARs are shown. Cells were 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 parental 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 each quadrant. Figure 5C Representative FACS plots of Treg cells expressing various CARs are shown. Cells were stained with Helios and FoxP3 antibodies to identify Helios+ and FoxP3+ cells. Each quadrant (Q9, Q10, Q11, and Q12) distinguishes various Treg cell populations within the parental population: Q9 = Helios+, FoxP3- Treg cells; Q10 = Helios+, FoxP3+ Treg cells; Q11 = Helios-, FoxP3+ Treg cells; and Q12 = Helios-, FoxP3- Treg cells. The percentage of each Treg cell population is shown within the quadrant. UTD represents Figures 5A-5C Untransduced Treg cells were used as a control (blank control).

[0013] Figure 6A This is a bar chart showing CAR expression in untransduced Jurkat cells (blank control) and transduced Jurkat cells expressing various CARs. Figure 6B This is a bar chart showing CD28 expression in untransduced Jurkat cells (blank control) and transduced Jurkat cells expressing various CARs. Figure 6C This is a bar chart showing CD28 expression in transduced Jurkat cells that are positive or negative for the expression of transduction markers associated with CAR expression.

[0014] Figure 7A This paper presents a histogram showing normalized cell counts versus CFSE signal intensity for untransduced Treg cells (CAR-) and Treg cells expressing various CARs (CAR+). CFSE is a fluorescent cell staining dye (carboxyfluorescein succinimide ester) whose staining intensity decreases with each round of cell division. Each row represents Treg cells isolated from different donors. Figure 7BThe percentage of proliferation of CAR-expressing Treg cells (CAR+=EGFR+) was shown as a function of the cell-to-CD3 / CD28 ratio compared to Treg cells that do not express CAR (CAR-=EGFR-).

[0015] Figure 8A The luminescence indicating cell activation was shown as a function of the ratio of CAR+Jurkat cells to CAR antigen+K562 cells. Figure 8B The percentage of various CAR+Jurkat cells expressing high levels of CD69 is shown as a function of their ratio to CAR antigen+K562 cells. Figure 8C The percentages of various CAR+Jurkat cells expressing CD25 after activation with CAR antigen-positive K562 cells at different Jurkat cell to K562 cell ratios are shown. The blank control represents untransduced (CAR-) control cells. Detailed Implementation

[0016] This disclosure relates to a chimeric antigen receptor (CAR) that does not result in a substantial reduction in the expression of endogenous CD28 protein in CAR-expressing T cells. Specifically, the CAR of this disclosure contains a hinge domain and / or transmembrane domain that differ from four well-known anti-CD19 CARs (tisagen, axicabtagene, brexucabtagene, and lisocabtagene). That is, the CAR of this disclosure does not contain a combination of a CD8a hinge region and a CD8a transmembrane domain, a combination of a wild-type CD28 hinge region and a wild-type CD28 transmembrane domain, or a combination of an IgG4 hinge region and a wild-type CD28 transmembrane domain. The CAR of this disclosure also does not contain a combination of a wild-type CD28 hinge region and a modified CD28 transmembrane domain.

[0017] Expression of the CARs disclosed herein in T cells results in a reduction in the cell surface expression of endogenous CD28 protein to a lesser extent than that of a control CAR of the same nature when expressed in control T cells. As used herein, the term "substantial reduction" means a reduction in properties of at least 25%. In contrast, the term "minor reduction to no reduction" means a reduction in properties of less than 25%, 20%, 15%, 10%, or 5%. The CARs disclosed herein are advantageous because their expression in T cells results in a reduction (MFI) of endogenous CD28 protein of less than 25%, 20%, 15%, 10%, or 5%, as determined by flow cytometry.

[0018] definition Unless otherwise stated, the practice of this disclosure will employ conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, which are within the skill level of those skilled in the art.

[0019] As used herein, the terms “antigen,” “immunogen,” and “antibody target” refer to a molecule, compound, or complex that is recognized (i.e., can be bound by an antibody). The term can refer to any molecule that can be recognized by an antibody, such as peptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations thereof (e.g., phosphorylated or glycosylated peptides). Those skilled in the art will understand that this term does not imply that the molecule is immunogenic in every case, but only that it can be targeted by an antibody or an antigen-binding domain derived therefrom.

[0020] As used herein, the term "epitope" refers to a local site on an antigen that is recognized and bound by an antigen-binding domain of an antibody or a fragment derived therefrom. An epitope may comprise a portion of several amino acids, such as five or six, or more, such as 20 or more amino acids, or portions of those amino acids. In some cases, an epitope includes non-protein components, such as components derived from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional portion. Thus, for example, when the target is a protein, an epitope may consist of consecutive amino acids or amino acids from different portions of a protein that are adjacent through protein folding (e.g., a discontinuous epitope).

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

[0022] Antibodies can belong to (i) any of the five major classes of immunoglobulins based on the identity of their heavy chain constant domains: α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), or (ii) their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The light chain can be λ or κ.

[0023] The following is a non-exhaustive list of different antibody forms, all of which retain antigen-binding activity: (1) Whole immunoglobulin (also known as "intact" antibody) (two light chains and two heavy chains, such as tetramer); (2) Immunoglobulin polypeptides (light chain or heavy chain); (3) Antibody fragments, such as Fv (monovalent or bivalent variable region fragments, and may only cover the variable region (e.g., V). L and / or V H ), Fab (V L C L V H C H ), F(ab')2, Fv (V L V H ), scFv (single-chain Fv) (containing V linked by a linker (e.g., a peptide linker) L and V H The peptides, (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, microantibodies (sc(FV)2 fused with the CH3 domain), bimeric antibodies (non-covalent dimers of single-chain Fv(scFv) fragments composed of heavy chain variable regions (VH) and light chain variable regions (VL) linked by small peptide linkers), and trimeric antibodies (trivalent sc(Fv)3 or trispecific sc(Fv)3); (4) Multivalent antibodies (including antibodies that bind to the binding regions of two different epitopes or proteins, such as "scorpion" antibodies); (5) Fusion proteins (such as fluorescent proteins) that include a binding portion of an immunoglobulin fused to another amino acid sequence; and (6) Antibodies or antibody fragments that have only heavy chains, which have only two heavy chains and lack the two light chains that are usually found in antibodies.

[0024] As used in this article, the phrase "CDR sequence set" refers to the three heavy chain and / or three light chain CDRs of a specific antibody or antigen-binding domain. The "light chain" CDR sequence set refers to light chain CDR sequences. The "heavy chain" CDR sequence set refers to heavy chain CDR sequences. The "complete" CDR sequence set refers to both heavy 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 into a human frame region by transplanting a CDR obtained from the VH and VL regions of a nonhuman antibody having the desired specificity, affinity, and capability. In one embodiment, the frame residues of the humanized antigen-binding domain are modified to improve and optimize the specificity, affinity, and capability of the antigen-binding domain.

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

[0027] As used herein, if an antigen-binding domain binds to a first antigen with a greater affinity than the second antigen, then it "preferentially binds" to the first antigen relative to the second antigen. Preferential binding can be at least one of 2, 5, 9, 10, 20, 30, 40, 50, 100, 500, or 1000 times greater affinity.

[0028] As used in this article, if the antigen-binding domain is 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 If at least one of M binds to each member of the target antigen or the target antigen group with an affinity at least twice that of a non-target antigen used as a comparison, then it "specifically binds to the target antigen or the target antigen group" or "is specific to the target antigen or the target antigen group". Typically, specific binding is characterized by binding to the antigen with sufficient affinity such that the antigen-binding domain can be used as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent targeting the antigen or epitope.

[0029] As used herein, the term "polypeptide" refers to a molecule having a sequence of natural and / or non-natural amino acids linked by peptide bonds. The term "peptide" refers to a short polypeptide, typically no more than 30 amino acids in length. The amino acid sequence of a polypeptide is referred to as its "primary structure." The term "protein" refers to a polypeptide having secondary, tertiary, and / or quaternary structures, such as structures stabilized by hydrogen bonds, relationships between secondary structures, and structures formed by more than one protein. Proteins can be further modified by other attached components, such as carbohydrates (glycoproteins), lipids (lipoproteins), phosphate groups (phosphoproteins), etc.

[0030] As used in this article, an amino acid sequence is "composed" only of the amino acids in that sequence.

[0031] As used herein, if a first amino acid sequence (1) contains a second amino acid sequence and (2) is longer than the second amino acid sequence by no more than one, two, or three amino acids, then the first amino acid sequence is "substantially composed" of the second amino acid sequence.

[0032] As used herein, if a second amino acid sequence contains a first amino acid sequence, then the first amino acid sequence is a “fragment” of the second amino acid sequence. In some embodiments, the first amino acid sequence, which is a fragment of the second amino acid sequence, may be no more than 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 differs from the reference sequence but contains minor alterations, such as, for example, the insertion, deletion, or substitution of one or more amino acids. The functionally equivalent sequence retains the function (e.g., immunogenicity) of its equivalent reference sequence. If the functionally equivalent amino acid sequence contains substitutions of one or more amino acids relative to the reference sequence, these are typically conserved amino acid substitutions.

[0034] As used herein, “conservative amino acid substitution” refers to the substitution of one amino acid residue for another without compromising the desired properties of the protein. Suitable conservative amino acid substitutions can be achieved by substituting amino acids with similar hydrophobicity, polarity, and R-chain length. See, for example, Watson et al., “Molecular Biology of the Gene,” 4th ed., 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, p. 224. Examples of conserved amino acid substitutions include the following (note that some categories are not mutually exclusive):

[0035] As used herein, the term "substantially identical" refers to the identity between first amino acid sequences containing a sufficient or minimum number of amino acid residues that i) are identical to the matched amino acid residues in the second amino acid sequence, or ii) are conserved substitutions of the matched amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences have common structural domains and / or common functional activities and / or common immunogenicity. For example, amino acid sequences containing common structural or antigenic domains with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity are described as sufficiently or substantially identical. In the context of nucleotide sequences, the term "substantially identical" herein refers to a first nucleic acid sequence containing a sufficient or minimum number of nucleotides identical to the matched nucleotides in the second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides with common functional activities, or encode common structural polypeptide domains or common functional polypeptide activities, or encode polypeptides with the same immunogenic properties.

[0036] As used herein, a chemical entity (such as a polypeptide) is considered "substantially pure" or "isolated" if it is the dominant chemical entity of its class (e.g., a polypeptide) in the composition. This includes chemical entities representing more than 50%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99% of their class in the composition. A substantially purified fraction is a composition in which the target class constitutes at least about 50% (on a molar basis) of all macromolecular classes present. Typically, a substantially pure composition means that about 80% to 90% or more of the macromolecular classes present in the composition are the class of interest to be purified. If the composition consists substantially of a single macromolecular class, the target class is purified to substantially homogeneity (contaminant classes are not detectable in the composition by conventional detection methods). Solvents, small molecules, stabilizers (e.g., BSA), and elemental ions are not considered macromolecular classes in this definition.

[0037] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid or nucleic acid sequence to allow for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. 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 molecule is identical at that position. The percentage of identity between two sequences is a function of the number of shared positions (i.e., identity % = number of overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length. Mathematical algorithms can also be used to determine the percentage of identity between two sequences. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, adapted as described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set (e.g., score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of this disclosure. BLAST protein searches can be performed using the XBLAST procedure parameter set (e.g., up to score 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gap alignments for comparative purposes, gap BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, can be used. Alternatively, PSI-BLAST can be used for iterative searches to detect distance relationships (Id.) between molecules. When using BLAST, gap BLAST, and PSI-Blast procedures, the default parameters of each procedure (e.g., the default parameters for XBLAST and NBLAST) can be used (see, for example, the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4 can be used. The percentage of identity between two sequences can be determined using techniques similar to those described above, allowing or disallowing gaps. Typically, only exact matches are calculated when determining the percentage of identity.

[0038] For antibody and antigen-binding domains, the percentage of sequence identity can be determined when their sequences are aligned to the maximum extent via IMGT. After alignment, if the subject antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared with the same region of the reference antibody, the percentage of sequence identity between the subject antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acids in both the subject antibody region and the reference antibody region by the total number of aligned positions in both regions, multiplied by 100, and then converted to a percentage.

[0039] The percentage of amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program is available from the National Institutes of Health in Bethesda, Maryland. NCBI-BLAST2 uses several search parameters, all of which are set to their default values, including, for example, demasking = yes, chain = all, expected occurrences = 10, minimum low complexity length = 15 / 5, multiple pass e-value = 0.01, multiple pass constant = 25, final gap alignment decay = 25, and score matrix = BLOSUM62.

[0040] When using NCBI-BLAST2 for amino acid sequence comparison, the amino acid sequence identity % between a given amino acid sequence A and a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing specific amino acid sequence identity with a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program NCBI-BLAST2 in the A and B alignments of this program, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % between A and B is not equal to the amino acid sequence identity % between B and A. As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of natural bases, sugars, and inter-sugar (backbone) bonds, and includes cDNA. This term also includes modified or substituted sequences containing monomers or portions thereof that are not naturally occurring. The nucleic acid sequence disclosed herein may be a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence, and may include naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequence may also contain modified bases. Examples of such modified bases include aza- and deza-adenine, guanine, cytosine, thymidine, and uracil; and xanthine and hypoxanthine. It should be understood that polynucleotides containing non-transcribed nucleotide bases can be used as probes, for example, in hybridization assays. Nucleic acids may be double-stranded or single-stranded, and represent sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon optimizations or synonymous codon equivalents.

[0041] As used herein, the term "isolated nucleic acid" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA technology, or that is substantially free of chemical precursors or other chemical substances when chemically synthesized. Isolated nucleic acids also substantially do not contain the naturally occurring sequence flanking the nucleic acid (i.e., the sequence located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid originates.

[0042] Hybridization can occur in whole or in part with nucleic acid sequences. The length of the hybridized portion is typically at least 15 nucleotides (e.g., 20, 25, 30, 40, or 50). Those skilled in the art will recognize that the stability of the nucleic acid duplex or hybrid is determined by Tm, which, in a sodium-containing buffer, is a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C) - 600 / l), or a similar equation). Therefore, the parameters determining the stability of the hybrid in the washing conditions are sodium ion concentration and temperature. To identify molecules similar to but not identical to known nucleic acid molecules, it can be assumed that a 1% mismatch results in a decrease in Tm of approximately 1°C; for example, if searching for nucleic acid molecules with >95% identity, the final washing temperature will be reduced by approximately 5°C. Based on these considerations, those skilled in the art will be able to readily select suitable hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. As an example, stringent hybridization can be achieved under the following conditions: based on the equation above, hybridization is performed at Tm –5°C in 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt solution / 1.0% SDS, followed by washing at 60°C with 0.2x SSC / 0.1% SDS. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. However, it is understood that equivalent stringency can be achieved using alternative buffers, salts, and temperatures. Further guidance on hybridization conditions can be found in: Current Protocols in Molecular Biology, John Wiley & Sons, NY, 2002, and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0043] As used herein, the term "expression construct" refers to a polynucleotide containing an expression control sequence operatively linked to a heterologous nucleotide sequence that is the object of expression (i.e., a sequence that is not normally linked in nature). As used herein, the term "expression vector" refers to a polynucleotide containing an expression construct and sequence sufficient to replicate in a host cell or insert into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term "expression control sequence" refers to a nucleotide sequence that regulates transcription and / or translation of a nucleotide sequence operatively linked to it. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences), and ribosome-binding sites (translational regulatory sequences).

[0044] As used in this article, when the expression control sequence functions to regulate the transcription of nucleotide sequences in the cell, the nucleotide sequence is "operably linked" to the expression control sequence. This includes promoting the transcription of the nucleotide sequence through the interaction between the polymerase and the promoter.

[0045] As used herein, the term "vector" includes any intermediate medium for nucleic acid molecules that enables said nucleic acid molecules to be introduced, for example, into prokaryotic and / or eukaryotic cells and / or integrated into the genome, and includes plasmids, phage particles, viral phages, or viral vectors, such as retrovirus-based vectors, lentiviral vectors, adeno-associated virus vectors, etc. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, typically a circular DNA double helix, which can replicate independently of chromosomal DNA.

[0046] "Transfection" refers to the introduction of new genetic material into cells. It includes transformation (the direct uptake and incorporation of foreign genetic material from the surrounding environment through the cell membrane), transduction (the introduction of foreign DNA into host cells through viruses, bacteriophages, and conjugation).

[0047] As used in this article, “host cell” refers to a recombinant cell containing the expression construct.

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

[0049] As used herein, the terms “therapy,” “treatment,” “therapeutic intervention,” and “improvement” refer to any activity that results in a reduction in the severity of symptoms. The terms “treatment” and “prevention” are not absolute terms. Treatment and prevention can refer to any delay in onset, improvement in symptoms, increase in patient survival, increase in survival time or survival rate, etc. Treatment and prevention can be complete or partial. Treatment effects can be compared to untreated individuals or groups, or to the same patient before treatment or at different times during treatment. In some respects, the severity of the disease is reduced by at least 10% compared to, for example, individuals before administration or untreated controls. In some respects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, it is no longer detectable using standard diagnostic techniques. “Treatment” or “treatment” can also mean prolonged survival compared to expected survival without treatment. The term “treatment” as used herein also includes preventative treatment.

[0050] A composition or method that “comprising” or “includes” one or more of the listed elements may include other elements not specifically listed (e.g., an open-ended term means including, but not limited to). For example, a composition that “comprising” or “includes” an antibody may contain an antibody, alone or in combination with other components. Conversely, the phrase “composed of” is closed, indicating that such an embodiment does not include additional elements. The term “substantially composed of” refers to including the listed elements and other elements that do not materially affect the essential and novel features of the claimed combination (e.g., a partially closed term). It should be understood that aspects and embodiments described herein as “comprising” include embodiments “composed of” and “substantially composed of”.

[0051] As used herein, unless otherwise stated, the following meanings apply. The word “may” is used in a permissive sense (i.e., implying a possibility), not in a mandatory sense (i.e., implying a requirement). The singular forms “a / an” and “the” include plural pronouns. Thus, for example, a submission of “one element” includes a combination of two or more elements, although other terms and phrases such as “one or more” are used for one or more elements.

[0052] The phrase “at least one” includes “one,” “one or more,” “one or plurality,” and “multiple.” Unless otherwise stated, the term “or” is non-exclusive, meaning it encompasses both “and” and “or.” The term “any” refers to each member of the sequence modified by the modifier. Thus, for example, the phrase “at least one of 1, 2, or 3” means “at least 1, at least 2, or at least 3.” The term “multiple” in relation to a reference object (e.g., an object such as T cells) refers to 3 or more, preferably 10 or more, preferably 100 or more, preferably 1,000 or more, preferably 10 ... 6 One or more, preferably 10 7 One or more, preferably 10 8 One or more, preferably 10 9 One or more, but not an unlimited number of, references (e.g., preferably less than 10) 12 10 or less, preferably less than 10 11 10 or less, preferably less than 10 10 indivual).

[0053] As used in this document, the term “about” covers 90% to 110% of the value. For example, the phrase “about 20 residues (amino acids) long” used to describe a peptide linker refers to a peptide linker of 18 to 22 amino acids in length, and includes peptide linkers of 20 amino acids in length.

[0054] I. Chimeric antigen receptor (CAR) A chimeric antigen receptor (CAR) is an engineered molecule comprising an optional signal peptide, a target antigen-binding domain, an optional hinge region, a transmembrane domain, an intracellular signal transduction domain, and an optional co-stimulatory domain. CARs are based on the structure of T-cell receptors, which are expressed on T cells and participate in cell-mediated immune responses. The target-binding domain is also referred to herein as an antigen-binding domain or antigen recognition domain, and therefore the term "target" encompasses "antigen."

[0055] So-called "first-generation" CARs have a targeting domain and a CD3ξ signaling domain. So-called "second-generation" CARs further include co-stimulatory domains, such as CD28 or 4-1BB domains. So-called "third-generation" CARs contain multiple co-stimulatory domains. So-called "fourth-generation" CARs (also known as "TRUCKS") are engineered to release transgenic cytokines during CAR signaling.

[0056] A chimeric antigen receptor (“CAR”) comprises the following elements: (1) an optional signal peptide, (2) a target antigen-binding domain, (3) an optional hinge region, (4) a transmembrane region, and (5) an intracellular domain containing a signal transduction domain. Optionally, the CAR may include a co-stimulatory (signal transduction) domain. That is, in addition to the desired elements, these optional elements may be included. The target antigen-binding domain is heterologous to at least one of the other domains. That is, the antigen-binding domain is not natively present 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 across the cell membrane. The antigen-binding domain provides the CAR with binding specificity. This domain can bind to the domain of an antibody, which then binds to the target antigen, forming a so-called "universal CAR." The hinge region is a flexible connecting region, such as a natural or synthetic polypeptide, or any other type of molecule, providing structural flexibility and spacing for the side-attached polypeptide regions. The transmembrane domain is a transmembrane protein domain. The intracellular signal transduction domain, after the antigen-binding domain binds to the antigen, transmits the signal into the cell via the signal transduction domain. This signal transduction activates cellular activity. The co-stimulatory domain is an auxiliary signal transduction domain that further transmits the signal.

[0058] This document provides a chimeric antigen receptor (CAR) that results in a small to no reduction in the expression of endogenous CD28 protein in T cells expressing the CAR. Specifically, the CAR disclosed herein contains a hinge domain and / or transmembrane domain different from the four anti-CD19 CAR T-cell therapies currently approved by the U.S. Food and Drug Administration (tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, and lisocabtagene maraleucel). That is, compared to a control CAR expressed in control T cells (containing the wild-type CD28 hinge region of SEQ ID NO:31 and the wild-type CD28 transmembrane domain of SEQ ID NO:40), the CAR disclosed herein is believed to exhibit reduced dimerization with endogenous CD28 protein when expressed in T cells.

[0059] A. Signal peptide The signal peptide that may be present at the N-terminus of the nascent CAR of this disclosure during cellular expression can be the signal peptide of any human type I transmembrane protein or any other signal peptide suitable for transferring the nascent CAR to the surface of human cells. 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 CD4, CD8, CD19, CD28, TCR, or an immunoglobulin chain.

[0060] One example signal peptide is the GMCSF signal peptide: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:1). Another example signal peptide is the CD8α 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 with SEQ ID NO:1 or SEQ ID NO:2.

[0061] B. Antigen-binding domain The antigen-binding domain (ABD) of the CAR disclosed herein can have any desired specificity and can be any suitable polypeptide form for binding the target antigen and transmitting the 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 implementations, such as when ABD is scFV, the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody, wherein the heavy chain variable region comprises complementary regions (CDR)-H1, CDR-H2, and CDR-H3 of the antibody, and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 of the antibody. The sequence of the antibody is stored in version 13.1 (August 2023) of the ABCD (Antibody Chemical Definition) database hosted by Expasy and operated by the Swiss Institute of Bioinformatics (see Lima et al., Nucleic Acids Research, 48:D2610264, 2020). In some implementations, the CDR, as defined using Kabat nomenclature, can be easily determined using online tools such as the abYsis annotation tool for a given sequence. In some implementations, the antigen-binding domain includes the light chain variable region and heavy chain variable region of the antibody disclosed in ABCD version 13.1 (August 2023), which includes 24,485 sequence antibodies against 4,171 different target antigens.

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

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

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

[0065] The antigen-binding domain of the CAR disclosed herein comprises a VH region and a VL region, preferably separated by a peptide linker. In some embodiments, the antigen-binding domain is arranged as VH-linker-VL. In other embodiments, the antigen-binding domain is arranged as a VL-linker-VH structure. In some embodiments, the peptide linker is about 4 to about 24 amino acids in length. In an exemplary embodiment, the linker is a glycine linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 13). In other exemplary embodiments, the linker is a Whitlow linker comprising the following amino acid sequence: GSTGSGKPGSGEGSTKG (SEQ ID NO: 14). In yet another exemplary embodiment, the linker is an ABpur linker comprising the following amino acid sequence: ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 15).

[0066] In an exemplary embodiment, 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 with 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 with SEQ ID NO:17 or SEQ ID NO:18. In another exemplary embodiment, ABD is a 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 with SEQ ID NO:19 or SEQ ID NO:20.

[0067] C. Hinge area The antigen-binding domain (ABD) and transmembrane domain of the CAR disclosed herein are separated by hinge regions of about 10 to about 20, 30, 40 or 50 amino acids in length.

[0068] In some embodiments, the hinge region is a CD8a hinge region. In an exemplary embodiment, the CD8a hinge region comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 21), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 21. In some embodiments, when the GAI CAR contains 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 an exemplary embodiment, the CD28 hinge region comprises the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 22), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 22. In a preferred embodiment, when the CAR comprises a wild-type CD28 hinge region, the transmembrane domain is not a wild-type CD28 transmembrane domain (SEQ ID NO: 25).

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

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

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

[0073] In some embodiments, the hinge region is a CD11a hinge region. In an exemplary embodiment, the CD11a hinge region comprises: an amino acid sequence of VDVVYEKQML (SEQ ID NO: 35), or an amino acid sequence that differs from SEQ ID NO: 35 by one or two amino acids.

[0074] In some embodiments, the hinge region is a CD2 hinge region. In an exemplary embodiment, the CD2 hinge region comprises the amino acid sequence of SKESSVEPVSCPEKGLD (SEQ ID NO: 7), or an amino acid sequence that differs from SEQ ID NO: 37 by one or two amino acids.

[0075] In some embodiments, the hinge region is a TCRα hinge region. In an exemplary embodiment, the TCRα hinge region comprises the amino acid sequence PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO: 38), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 38.

[0076] In some embodiments, the hinge region is an IgG4 hinge region. In an exemplary embodiment, the IgG4 hinge region comprises: the amino acid sequence of ESKYGPPCPPCP (SEQ ID NO: 39), or an amino acid sequence that differs from SEQ ID NO: 39 by one or two amino acids.

[0077] D. Transmembrane domain The hinge region and intracellular signal transduction domain of the CAR disclosed herein are separated by transmembrane domains of about 20 to about 30 amino acids in length.

[0078] In some embodiments, the transmembrane domain is a CD8a transmembrane domain. In an exemplary embodiment, the CD8a domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:24), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:24. In a preferred embodiment, when the CAR comprises a CD8a transmembrane domain, the hinge region is not a CD8a hinge region (SEQ ID NO:21).

[0079] In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In an exemplary embodiment, the CD28 transmembrane domain is a wild-type CD28 transmembrane domain comprising the following amino acid sequence: 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 with SEQ ID NO:25. In a preferred embodiment, when the CAR comprises a wild-type CD28 transmembrane domain, the hinge region is neither a wild-type CD28 hinge region (SEQ ID NO:22) nor an IgG4 hinge region (SEQ ID NO:23 or SEQ ID NO:36).

[0080] In some embodiments, the transmembrane domain is a modified CD28 transmembrane domain. In an exemplary embodiment, the amino acid sequence of the modified CD28 transmembrane domain comprises insertions, substitutions, and / or deletions 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 (corresponding to C13L, Y14L, S15L, and T19L as shown in the amino acid sequence of the modified CD28™ as in SEQ ID NO:42), and any combination thereof. In some embodiments, the modified CD28 transmembrane domain comprises the following amino acid sequence: FWVLVVVGGVLALLLLLLLVLVAFIIFWV (SEQ ID NO:41). In some embodiments, the transmembrane domain sequence comprises the amino acid sequence of SEQ ID NO:11, and the hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-9.

[0081] In some embodiments, the transmembrane domain is a CD4 transmembrane domain. In an exemplary embodiment, the CD4 transmembrane domain comprises the amino acid sequence MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO:42), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:42.

[0082] In some embodiments, the transmembrane domain is a CD11a transmembrane domain. In an exemplary embodiment, the CD11a transmembrane domain comprises the amino acid sequence YLYVLSGIGGLLLLLLIFIVLYKV (SEQ ID NO:43), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:43.

[0083] In some embodiments, the transmembrane domain is a CD18 transmembrane domain. In some embodiments, the CD18 transmembrane domain comprises the amino acid sequence IAAIVGGTVAGIVLIGILLLVIW (SEQ ID NO:44), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:44.

[0084] In some embodiments, the transmembrane domain is a CD2 transmembrane domain. In an exemplary embodiment, the CD2 transmembrane domain comprises the amino acid sequence IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO:45), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:45.

[0085] In some embodiments, the transmembrane domain is the HER2 transmembrane domain. In an exemplary embodiment, the HER2 transmembrane domain comprises the amino acid sequence SIISAVVGILLVVVLGVVFGILI (SEQ ID NO:46), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:46.

[0086] E. Intracellular signal transduction domains The intracellular signal transduction domain of the CAR disclosed herein includes a CD3ζ signal transduction domain. In some preferred embodiments, the intracellular signal transduction domain further includes a co-stimulatory domain.

[0087] In some embodiments, the co-stimulatory domain may be derived from, for example, CD28, 4-1BB, CD2, CD27, CD30, OX40, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3, B7-H4, etc. In some embodiments, the CAR of this disclosure comprises two or more co-stimulatory signal transduction domains (e.g., CD28 and 4-1BB).

[0088] In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain. In an exemplary embodiment, the CD28 co-stimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:26), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:26.

[0089] In some embodiments, the co-stimulatory domain is a 4-1BB co-stimulatory domain. In an exemplary embodiment, the 4-1BB co-stimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:27), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:27.

[0090] In an exemplary embodiment, the CD3ζ signal transduction domain comprises the amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:28), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:28.

[0091] In some embodiments, the intracellular signal transduction domain comprises a CD28 co-stimulatory domain and a CD3ζ signal transduction domain. In an exemplary embodiment, the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:29, or amino acids having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:29.

[0092] In some embodiments, the intracellular signal transduction domain comprises a 4-1BB co-stimulatory domain and a CD3ζ signal transduction domain. In an exemplary embodiment, the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:30, or amino acids having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:30.

[0093] II. Nucleic acids encoding CAR The nucleic acid molecule (polynucleotide) encoding the CAR disclosed herein can be a separate molecule or can be contained in a cassette or vector.

[0094] A. Nucleic acid This disclosure provides nucleic acid molecules (polynucleotides) comprising a nucleotide sequence encoding a CAR as described herein (e.g., containing a coding region of a CAR as described herein). The nucleic acid may be in the form of DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotides as described herein are isolated.

[0095] For example, the nucleic acid may contain a nucleotide sequence encoding the CAR polypeptide described herein or a portion thereof. In some embodiments, the nucleotide sequence encodes a polypeptide containing an 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.

[0096] In an exemplary implementation, the nucleic acid encoding is as follows: Figure 2A , Figure 2B , Figure 3A or Figure 3BThe CAR described herein comprises an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is the truncated CD28 hinge region of SEQ ID NO:32 and the transmembrane domain is the mutant CD28 transmembrane domain of SEQ ID NO:41. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is the truncated CD4 hinge region of SEQ ID NO:34 and the transmembrane domain is the CD4 transmembrane domain of SEQ ID NO:42. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is the CD11a hinge region of SEQ ID NO:35 and the transmembrane domain is the CD11a transmembrane domain of SEQ ID NO:43. In some embodiments, the nucleic acid encodes a CAR in which the hinge region is the wild-type CD28 hinge region of SEQ ID NO:31 and the transmembrane domain is the CD18 transmembrane domain of SEQ ID NO:44. In some embodiments, the nucleic acid encodes a CAR wherein the hinge region is the TCRα hinge region of SEQ ID NO:38 and the transmembrane domain is the CD4 transmembrane domain of SEQ ID NO:42. In some embodiments, the nucleic acid encodes a CAR wherein the hinge region is the CD2 hinge region of SEQ ID NO:37 and the transmembrane domain is the CD2 transmembrane domain of SEQ ID NO:45. In some embodiments, the nucleic acid encodes a CAR wherein the hinge region is the truncated CD28 hinge region of SEQ ID NO:32 and the transmembrane domain is the wild-type CD28 transmembrane domain of SEQ ID NO:40. In some embodiments, the nucleic acid encodes a CAR wherein the hinge region is the wild-type CD28 hinge region of SEQ ID NO:31 and the transmembrane domain is the CD4 transmembrane domain of SEQ ID NO:42. In some embodiments, the nucleic acid encodes a CAR wherein the hinge region is the wild-type CD28 hinge region of SEQ ID NO:31 and the transmembrane domain is the CD11a transmembrane domain of SEQ ID NO:43. In some implementations, the nucleic acid encodes a CAR in which the hinge region is the wild-type CD28 hinge region of SEQ ID NO:31 and the transmembrane domain is the HER2 transmembrane domain of SEQ ID NO:46.

[0097] In further exemplary embodiments, the nucleic acid encodes a CAR comprising an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain, wherein the transmembrane domain is a mutant CD28 transmembrane domain. In some embodiments, the nucleic acid encodes a CAR wherein the hinge region is the truncated CD4 hinge region of SEQ ID NO:34 and the transmembrane domain is the mutant CD28 transmembrane domain of SEQ ID NO:41. In some embodiments, the nucleic acid encodes a CAR wherein the hinge region is the CD11a hinge region of SEQ ID NO:35 and the transmembrane domain is the mutant CD28 transmembrane domain of SEQ ID NO:41. In some embodiments, the nucleic acid encodes a CAR wherein the hinge region is the CD2 hinge region of SEQ ID NO:37 and the transmembrane domain is the mutant CD28 transmembrane domain of SEQ ID NO:41. In some implementations, the nucleic acid encodes a CAR in which the hinge region is the TCRα hinge region of SEQ ID NO:38 and the transmembrane domain is the mutant CD28 transmembrane domain of SEQ ID NO:41.

[0098] In some embodiments, the polynucleotide is a variant comprising changes to a coding region, a non-coding region, or both. In some embodiments, the polynucleotide variant comprises a silent change. In other embodiments, the polynucleotide variant comprises substitutions, additions, or deletions, but does not alter the binding properties of the antigen-binding domain of the encoded CAR polypeptide. In some embodiments, the polynucleotide variant comprises one or more changes that do not alter the amino acid sequence of the CAR. In some embodiments, the polynucleotide variant contains a “silent” substitution due to the degeneracy of the genetic code. Polynucleotide variants can arise for a variety of reasons, such as to optimize codon expression in a particular host cell.

[0099] B. Expression box and carrier This disclosure further provides expression cassettes and vectors comprising a nucleic acid molecule encoding the CAR described herein. In some embodiments, the nucleic acid molecule may further comprise an expression control sequence operatively linked to a nucleotide sequence encoding the CAR. In some embodiments, the nucleic acid molecule encoding the disclosed CAR may be inserted into an expression vector in a operative combination with an expression control sequence suitable for expressing the disclosed CAR in desired host cells. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and / or expression of the CAR peptide in suitable host cells.

[0100] For example, a polynucleotide may contain one or more transcriptional regulatory elements (such as promoters or enhancers) that, when present in a cell, cause the expression of a sequence encoding a CAR in the cell.

[0101] The nucleic acids disclosed herein can be incorporated into vectors that can be introduced into host cells. Such vectors include, but are not limited to, viral vectors and plasmids. Exemplary viral vectors include, but are not limited to, retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Lentivirals are particularly desirable because they are capable of delivering large amounts of genetic material into the host cell's genome and because they are capable of infecting non-dividing cells.

[0102] III. Host Cell This disclosure also provides a host cell (e.g., a recombinant cell) comprising a nucleic acid molecule, expression cassette, or expression vector encoding the 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.

[0103] As is well known to those skilled in the art, the nucleic acid molecule encoding the disclosed CAR can be delivered to host cells via transformation, transfection, or transduction. The resulting recombinant (host) cells can be immune cells, including but not limited to T cells, such as CD4+ T cells, CD8α+ T cells, or CD8β+ T cells. In some embodiments, the T cells are Treg cells. In some embodiments, the T cells are T helper (Th) cells. In some embodiments, the T cells are cytotoxic T lymphocytes (CTLs). In some embodiments, the recombinant (host) cell containing the nucleic acid molecule encoding the disclosed CAR is a lymphocyte (e.g., T cells, B cells, or NK cells).

[0104] In some embodiments, the cells expressing the CAR of this disclosure are Treg cells. "Regulatory T cells" or "Treg cells" are cells belonging to a specific subset of T cells that suppress immune responses, thereby maintaining homeostasis and self-tolerance. Tregs can suppress T cell proliferation and cytokine production and play a key role in preventing autoimmunity. Tregs are characterized by the expression of FoxP3. Surface markers of Tregs include high CD4 and CD25 (high molecular density) and low CD127 (low molecular density). Mouse and human Tregs express GITR / AITR and CTLA-4. Human CD4+FoxP3+ Treg cells can be divided into three subsets: (1) CD45RA+CD25+FoxP3low resting Treg cells (2) CD45RO+CD25highFoxP3high activates Treg cells, and (3) CD45RO+CD25+FoxP3low non-inhibitory effector T cells (Teff) that produce pro-inflammatory cytokines.

[0105] In some embodiments, the host cells are autologous cells (e.g., recombinant progeny of cells obtained from the intended recipient). That is, the cells to be transformed with the nucleic acids disclosed herein can be cells taken from the subject receiving the recombinant cells. This mitigates the problem of allogeneic immune responses. Even so, in other embodiments, the host cells are allogeneic cells (e.g., recombinant progeny of cells obtained from an individual immunologically different from the intended recipient). Regardless of the source of the host cells, they can be expanded in vitro prior to administration to the subject.

[0106] Furthermore, proteins derived from transformed / recombinant hosts can be purified using any suitable method. These methods include chromatography (e.g., ion exchange, affinity and fractionation column chromatography), centrifugation, differential dissolution, or any other standard technique used for protein purification. Affinity tags (such as hexahistidine, maltose-binding domains, influenza virus capsid sequences, and glutathione S-transferases) can be attached to proteins to facilitate purification by passing them through a suitable affinity column. In some embodiments, the proteins can also be physically characterized using techniques such as proteolysis, high-performance liquid chromatography, nuclear magnetic resonance, and X-ray crystallography.

[0107] IV. Composition The pharmaceutical composition and method of use for treating diseases or conditions are also disclosed, the pharmaceutical composition comprising a host cell (e.g., a recombinant cell) containing a nucleic acid molecule encoding and expressing the disclosed CAR polypeptide and a pharmaceutically acceptable excipient.

[0108] As used herein, the term "pharmaceutical composition" refers to a composition comprising a pharmaceutical agent (e.g., a drug as described herein or recombinant Treg cells) and a pharmaceutically acceptable excipient.

[0109] As used herein, the term “pharmaceutical acceptable” means a compound that is compatible with the other components of a pharmaceutical composition and can be safely administered to a subject. This term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable.” Based on this disclosure, pharmaceutical compositions and techniques for their preparation and use are known to those skilled in the art. For a detailed list of suitable pharmaceutical compositions and techniques for their administration, reference may be made to texts such as: Remington's Pharmaceutical Sciences, 17th edition, 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.

[0110] Pharmaceutical compositions are generally sterile, at least for human use. They typically contain pharmaceutically acceptable excipients for buffering and preservation during storage, and may include buffers for appropriate delivery, depending on the route of administration. Examples of pharmaceutically acceptable excipients include, but are not limited to, plain (0.9%) saline, phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), and various electrolyte solutions.

[0111] The pharmaceutical composition can be formulated for any route of administration. However, in most embodiments of this disclosure, the pharmaceutical composition is formulated for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intra-arterial injection, whether by bolus or infusion) administration.

[0112] Injectable (e.g., intravenous) pharmaceutical compositions may comprise a solution of the drug suspended in a pharmaceutically acceptable excipient, such as an aqueous excipient. Any of a variety of aqueous excipients may be used, such as water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% glucose, etc., and may include glycoproteins for enhancing stability, such as albumin, lipoproteins, globulins, etc. Typically, a common buffered saline (135-150 mM NaCl) will be used. Pharmaceutically acceptable excipients may contain adjuvants for mimicking physiological conditions, such as pH adjusters and buffers, tonicotinic agents, wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the composition may be formulated for intravenous administration.

[0113] Pharmaceutical compositions suitable for parenteral administration (such as intravenous administration) include aqueous and non-aqueous isotonic sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the composition isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Injectable solutions and suspensions may also be prepared from sterile powders, granules, and tablets. In the practice of this disclosure, pharmaceutical compositions may be administered, for example, by intravenous infusion. Pharmaceutical compositions may be provided in single-dose or multi-dose sealed containers.

[0114] The host cells may be cryopreserved. Cryopreservation may involve preparing the host cells with a cryopreservation agent such as DMSO. Commercially available culture media include, for example, CryoStor® and pZerve®, available from Millipore Sigma.

[0115] The terms “dosage” and “volume” are used interchangeably herein. A dosage refers to the amount of a drug administered to an individual at each administration. Dosages will vary depending on many factors, including frequency of administration; individual size and tolerability; severity of disease; risk of side effects; route of administration; and the form of imaging of detectable markers (if present). Those skilled in the art will recognize that dosages can be modified based on the foregoing factors or on treatment progress.

[0116] Pharmaceutical compositions can be packaged or prepared in unit dose form. In this form, the pharmaceutical composition is subdivided into unit doses containing appropriate amounts of the pharmaceutical agent, for example, based on the dosage or concentration of the agent in the pharmaceutical composition. The unit dose form can be a packaged formulation containing discrete amounts of the pharmaceutical composition. If desired, the pharmaceutical composition may also contain other compatible therapeutic agents.

[0117] V. Usage Instructions Host cells expressing the CARs disclosed herein (e.g., recombinant T cells) can be used to treat a variety of diseases and conditions. Methods of use include administering an effective amount of the pharmaceutical composition of this disclosure containing cells expressing CARs (such as CAR-T cells) to a subject in need (e.g., an individual suffering from a disease or condition). In some methods of this disclosure where the subject has cancer, the host cells comprise CD3+ T cells expressing a CAR that binds to an antigen expressed by the cells of the cancer. In some methods of this disclosure where the subject has an autoimmune disease or inflammatory condition, the host cells comprise Treg cells expressing a CAR that binds to an antigen associated with the autoimmune disease or inflammatory condition. In some embodiments, the host cells are the subject's own cells.

[0118] As used herein, the term "subject" refers to an individual animal. As used herein, the term "patient" refers to a subject under the care or supervision of a healthcare provider, such as a doctor or nurse. Subjects include mammals, such as humans, and non-human primates, such as monkeys, as well as dogs, cats, horses, cattle, rabbits, rats, mice, goats, pigs, and other mammal species. Subjects may also include birds. In some embodiments, the subject is a human patient. A patient can be an individual seeking treatment, monitoring, adjustment, or modification of an existing treatment regimen, etc. Subjects suffering from a disease or condition can include individuals who have not yet received treatment, individuals who are currently receiving treatment, individuals who have previously received treatment, and individuals who have discontinued treatment.

[0119] As used herein, the terms “effective amount,” “effective dose,” and “therapeutic effective amount” refer to the amount of a drug sufficient to produce a desired response, such as reducing or eliminating signs or symptoms of a disease or improving the condition. In some instances, an “effective amount” is an amount that treats (including prevents) one or more symptoms and / or underlying causes of any condition or disease and / or prevents disease progression. For example, for a given parameter, a therapeutic effective amount will show an increase or decrease in therapeutic effect of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as an increase or decrease of “-fold.” For example, a therapeutic effective amount may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more than a control.

[0120] The pharmaceutical composition may be administered via any suitable route, including but not limited to intravenous, subcutaneous, intramuscular, or intraperitoneal routes. Examples of administration include storing 10 mg / ml of the composition in a sterile isotonic saline solution at 4°C and diluting it in 100 ml or 200 ml of 0.9% sodium chloride for injection prior to administration to the patient. The pharmaceutical composition is administered via intravenous infusion over a 1-hour timeframe at a dose between 0.2 mg / kg and 10 mg / kg. In other embodiments, the pharmaceutical composition is administered via intravenous infusion over a period between 15 minutes and 2 hours. In still other embodiments, the administration procedure is a rapid subcutaneous concentration.

[0121] The dosage of the pharmaceutical composition is selected to provide an effective therapy to 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 of 1 mg to 2 g / patient. In some cases, the dosage is in the range of 1-100 mg / kg, or about 50 mg to 8000 mg / patient. Depending on the pharmacokinetics of the composition (e.g., the half-life of the composition in circulation) and pharmacodynamic response (e.g., the duration of the therapeutic effect of the composition), the dosage may be repeated at an appropriate frequency, ranging from once daily to once every three months. In some embodiments, the in vivo half-life is between about 7 and about 25 days, and the composition is administered between once weekly and once every three weeks, or between once weekly and once every three months.

[0122] Application can be periodic. Depending on the route of administration, the dosage can be given 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, such as twice or three times daily. As those skilled in the art will recognize, patients can be monitored to adjust the dosage and frequency of administration based on treatment progress and any adverse side effects.

[0123] Therefore, in some implementations, additional administration is based on patient progress, for example, by monitoring the patient between administrations. For instance, the patient's symptom relief rate can be monitored after the first administration or after the first round of administration.

[0124] VI. Reagent Kit As used herein, the term "kit" refers to a group of items intended to be used together. In some embodiments, the kit includes a reagent and instructions for use thereof. In some embodiments, the kit further includes a container, such as a vial, containing a composition as disclosed herein. For example, the kit may include a container containing a pharmaceutical composition, such as a bag or bottle for intravenous administration, the pharmaceutical composition containing multiple recombinant cells expressing the CAR of this disclosure. In some embodiments, the kit may further include a fluid conduit, such as a plastic tube, having an infusion lumen. The infusion lumen may be communicated with an intravenous injection needle via the fluid conduit. The fluid conduit may also include one or more Y-shaped tees and roller clamps.

[0125] VII. Listed Implementation Schemes 1. A chimeric antigen receptor (CAR) comprising: Antigen-binding domain; CD28 hinge area; Transmembrane domains, selected from the group consisting of: CD4 transmembrane domains, CD11a transmembrane domains, CD18 transmembrane domains, CD2 transmembrane domains, and HER2 transmembrane domains; and Intracellular signal transduction domains.

[0126] 2. The CAR as described in embodiment 1, wherein when the CAR is expressed in T cells, the CAR does not result in a substantial reduction in the expression of endogenous CD28 protein on the cell surface.

[0127] 3. The CAR of embodiment 1, wherein the cell surface expression of endogenous CD28 protein in T cells expressing the CAR is substantially higher than that in control T cells expressing the control CAR, wherein the control CAR comprises the wild-type CD28 hinge region of SEQ ID NO:31 and the wild-type D28 transmembrane domain of SEQ ID NO:40, and optionally wherein the control CAR is otherwise identical to the implemented CAR.

[0128] 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.

[0129] 5. A chimeric antigen receptor (CAR) comprising: Antigen-binding domain; The hinge area of ​​CD28 has been shortened; Transmembrane domains; and Intracellular signal transduction domains The truncated CD28 hinge region comprises a truncation of about 10 to about 30 amino acids relative to the amino acid sequence of SEQ ID NO:31.

[0130] 6. The CAR as described in embodiment 5, wherein the truncated CD28 hinge region is truncated at the N end.

[0131] 7. The CAR as described in embodiment 6, wherein the truncated CD28 hinge region comprises the amino acid sequence of SEQ ID NO:32.

[0132] 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.

[0133] 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.

[0134] 10. The CAR of any one of embodiments 5-7, wherein the transmembrane domain is selected from the group consisting of: CD4 transmembrane domain, CD11a transmembrane domain, CD18 transmembrane domain, CD2 transmembrane domain and HER2 transmembrane domain.

[0135] 11. A chimeric antigen receptor (CAR) comprising: Antigen-binding domain; The hinge region is selected from the group consisting of: the truncated CD4 hinge region, the CD11a hinge region, the CD2 hinge region, and the TCRα hinge region. Transmembrane domains; and Intracellular signal transduction domains.

[0136] 12. The CAR of embodiment 11, wherein the hinge region is a truncated CD4 hinge region comprising a truncation of 1 to 12 amino acids relative to the amino acid sequence of SEQ ID NO:33.

[0137] 13. The CAR of embodiment 12, wherein the truncated CD4 hinge region comprises the amino acid sequence of SEQ ID NO:34.

[0138] 14. The CAR of embodiment 11, wherein the hinge region is a CD11a hinge region, and optionally wherein the CD11a hinge region comprises the amino acid sequence of SEQ ID NO:35.

[0139] 15. The CAR of embodiment 11, wherein the hinge region is a CD2 hinge region, and optionally wherein the CD2 hinge region comprises the amino acid sequence of SEQ ID NO:37.

[0140] 16. The CAR of embodiment 11, wherein the hinge region is a TCRα hinge region, optionally wherein the TCRα hinge region comprises the amino acid sequence of SEQ ID NO:38.

[0141] 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.

[0142] 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.

[0143] 19. The CAR of any one of embodiments 11-16, wherein the transmembrane domain is selected from the group consisting of: CD4 transmembrane domain, CD11a transmembrane domain, CD18 transmembrane domain, CD2 transmembrane domain and HER2 transmembrane domain.

[0144] 20. The CAR as described in 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.

[0145] 21. The CAR as described in embodiments 1, 10, or 19, wherein the transmembrane domain is the CD11a transmembrane domain, optionally wherein the CD11a transmembrane domain comprises the amino acid sequence of SEQ ID NO:43.

[0146] 22. The CAR as described in embodiment 1, 10 or 19, wherein the transmembrane domain is the CD18 transmembrane domain, optionally wherein the CD18 transmembrane domain comprises the amino acid sequence of SEQ ID NO:44.

[0147] 23. The CAR as described in embodiments 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.

[0148] 24. The CAR as described in 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.

[0149] 25. The CAR as described in any one of embodiments 5-24, wherein when the CAR is expressed in T cells, the CAR does not result in a substantial reduction in the cell surface expression of endogenous CD28 protein.

[0150] 26. The CAR of any one of embodiments 5-24, wherein the cell surface expression of endogenous CD28 protein in T cells expressing the CAR is substantially higher than that in control T cells expressing the control CAR, wherein the control CAR comprises the wild-type CD28 hinge region of SEQ ID NO:31 and the wild-type D28 transmembrane domain of SEQ ID NO:40, and optionally wherein the control CAR is otherwise identical to the implemented CAR.

[0151] 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 NO:42-46.

[0152] 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 NO:40-46.

[0153] 29. The CAR of embodiment 11, wherein the hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:33-38, and the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:40-46.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 47. The CAR of any one of embodiments 1-46, wherein the intracellular signal transduction domain comprises the CD3ζ signal transduction domain.

[0172] 48. The CAR of embodiment 47, wherein the CD3ζ signal transduction domain comprises the amino acid sequence of SEQ ID NO:28.

[0173] 49. The CAR as described in embodiment 47 or embodiment 48, wherein the intracellular signal transduction domain further comprises a co-stimulatory domain.

[0174] 50. The CAR of embodiment 49, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain.

[0175] 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.

[0176] 52. The CAR of embodiment 51, wherein the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:29.

[0177] 53. The CAR of embodiment 50, wherein the co-stimulatory domain comprises a 4-1BB co-stimulatory domain, optionally wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:27.

[0178] 54. The CAR of embodiment 53, wherein the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:30.

[0179] 55. The CAR as described in any one of embodiments 1-54, further comprising a signal peptide.

[0180] 56. The CAR as described in embodiment 55, wherein the signal peptide is a CD8a signal peptide or a GM-CSF signal peptide.

[0181] 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).

[0182] 58. The CAR as described in embodiment 57, wherein the antigen-binding domain comprises scFv.

[0183] 59. The CAR of embodiment 57, wherein the antibody-binding domain comprises an sdAb, and optionally the sdAb comprises a heavy chain variable region fragment (VHH).

[0184] 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), CD19 and BCMA.

[0185] 61. The CAR of embodiment 60, wherein the antigen-binding domain binds to CV, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 and the light chain CDR of the amino acid sequences 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.

[0186] 62. The CAR of embodiment 60, wherein the antigen-binding domain binds to CD19, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequence of SEQ ID NO:10 and the light chain CDR of the amino acid sequence of SEQ ID NO:9; (ii) The heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10 and the 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.

[0187] 63. The CAR of embodiment 60, wherein the antigen-binding domain binds to BCMA, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequence of SEQ ID NO:12 and the light chain CDR of the amino acid sequence of SEQ ID NO:11; (ii) The heavy chain variable region comprising the amino acid sequence of SEQ ID NO:12 and the 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.

[0188] 64. A nucleic acid encoding a CAR according to any one of embodiments 1-63.

[0189] 65. An expression vector comprising an operative combination of the nucleic acid described in embodiment 64 and an expression control sequence.

[0190] 66. The expression vector as described in embodiment 65, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector, a retroviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0191] 67. The expression vector as described in embodiment 66, wherein the viral vector is a lentiviral vector.

[0192] 68. The expression vector as described in embodiment 65, wherein the vector is a plasmid.

[0193] 69. A host cell comprising the expression vector described in any one of embodiments 65-68.

[0194] 70. A modified T cell engineered to express the CAR of any one of embodiments 1-65, wherein the modified T cell is a mammalian T cell.

[0195] 71. A pharmaceutical composition comprising a plurality of modified T cells as described in embodiment 70 and a pharmaceutically acceptable excipient.

[0196] 72. The pharmaceutical composition of embodiment 71, wherein the modified T cells are human T cells.

[0197] 73. The pharmaceutical composition of embodiment 72, wherein the human T cells are regulatory T cells (Tregs) as CD4+, CD25+ and CD12710.

[0198] 74. The pharmaceutical composition of embodiment 73, wherein the human Treg is FOXP3+ and HELIOS+.

[0199] 75. A method of treating a disease or ailment, comprising administering to a subject in need an effective amount of the pharmaceutical composition described in any one of embodiments 71-74.

[0200] 76. A method of treating a subject suffering from an autoimmune disease or inflammatory condition, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of embodiments 71-74, wherein the CAR expressed by modified T cells comprises an antigen-binding domain that binds to an antigen expressed on cells of the autoimmune disease or the inflammatory condition.

[0201] 77. A method of treating a subject with cancer, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 71 or embodiment 72, wherein the CAR expressed by modified T cells comprises an antigen-binding domain that binds to an antigen expressed on the cells of the cancer.

[0202] 78. A kit comprising a container containing a pharmaceutical composition according to any one of embodiments 71-74, the container being in communication with an infusion chamber via a fluid conduit, wherein the infusion chamber is configured to be in communication with an intravenous injection needle via a fluid conduit.

[0203] Example Abbreviations: CAR (Chimeric Antigen Receptor); CFSE (Carboxyfluorescein Succinimide); CV (Citrus-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 (Mutant); PBMC (Peripheral Blood Mononuclear Cells); PBS (Phosphate-Buffered Saline); Prom. (Promoter); scFv (Single-Strand Variable Fragment); TRa (T Cell Receptor α); Teff (Effective T Cells); TM or TMD (Transmembrane Domain); Treg (Regulatory T Cells); Trun (Truncation); and UTD (Untransduced), WT (Wild Type).

[0204] Although this disclosure has been described in 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 made. Therefore, the following embodiments should not be construed as limiting the scope of this disclosure, which is defined by the claims.

[0205] Example 1: T cells engineered to express a chimeric antigen receptor (CAR) with a CD28 hinge region and a CD28 transmembrane domain CD28 is a co-stimulatory molecule essential for T cell activation and proliferation. Surprisingly, expression of CARs with wild-type CD28 transmembrane domains and wild-type CD28 hinge regions (hereinafter referred to as the wild-type scaffold) on regulatory T cells leads to reduced endogenous CD28 expression. Furthermore, regulatory T cells expressing CARs with the wild-type scaffold (referred to as Treg cells or Tregs in this paper) exhibit lower proliferative levels upon activation.

[0206] Generation of Lentiviral Constructs A construct containing a CAR with a wild-type backbone and an EGFRt tag was packaged into lentiviral particles for transduction into cells of interest. HEK 293 FT suspension cells (Invitrogen) were packaged at 4.7 x 10⁻⁶ cells per cell. 6Inoculate at / mL and transfect with LV_MAX transfection reagents using transfer and packaging plasmids (Aldevron pALD-VSV-G, pALD-GagPol, pALD-Rev) according to the manufacturer's protocol (Gibco). Collect viral supernatant and filter through a 0.45 µm polyvinylidene fluoride filter to remove cell debris. Centrifuge the viral supernatant at 10,000 xg overnight to concentrate the viral vector. After 12–16 hours, remove the supernatant and resuspend the viral vector pellet in Opti-MEM medium. Aliquot the resuspended viral vector and store at -80°C.

[0207] Functional titer determination Functional titers were assessed to identify optimal T cell transduction conditions. SupT1 cells (ATCC) were cultured in RPMI medium supplemented with 10% fetal bovine serum. On the day of transduction, 50 µL of SupT1 cells were inoculated at 4 x 10⁻⁶ cells / mL. 5 Cells / mL were added to each well of a 96-well plate. The lentiviral stock solution was thawed at room temperature and diluted 1:20 in medium. Subsequently, three-fold serial dilutions were performed in medium, from 60 to 393,660 times. 50 µL of the diluted lentiviral solution was added to a 96-well plate containing 50 µL of cells. On day 2 of transduction, 100 µL of medium was added. On day 3, transduced cells were harvested and stained with an appropriate antibody to detect surface markers. The labeled cells were analyzed by flow cytometry. Untransduced SupT1 cells were used as a negative control to set the gating for flow cytometry analysis. The functional titer was calculated using a viral dilution that produced 5%–20% marker-positive cells according to the following formula: .

[0208] Treg isolation and amplification.Primary human Treg cells were derived from leukoreduction chamber residuals or leukopaks from healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Ficoll-PaquePlus. CD25+ cells were enriched by positive selection. Treg cells were then isolated using FACS with gated CD4+ / CD25+ / CD127lo cells. After isolation, cells were stimulated with CTSDynabeads Treg Xpander (Gibco) at a 1:1 bead-to-cell ratio and expanded for 14 days, followed by restimulation on day 9. Cells were cultured at a density of 0.25–0.3 million cells / mL in RPMI medium supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, and penicillin / streptomycin, along with 300 IU / mL of recombinant human IL-2.

[0209] Treg cell transduction. Treg cells were transduced using the lentiviral vector described above to express a CAR with a wild-type backbone. On day 0, Treg cells were loaded at a rate of 2.5 x 10⁻⁶ cells / cells. 5个 Cells were spread in 1 mL / well of medium in 24-well plates. On day 2, Treg cells were transduced via rotational transfection by adding an appropriate volume of virus to each well to achieve an MOI of 5–10 and adding protamine sulfate to a final concentration of 100 µg / mL. Cells were centrifuged at 1200 xg for 30 min at 30 °C with the lentivirus. Shortly after rotation or immediately thereafter, fresh medium containing 2X recombinant human IL-2 was added at a 1:1 ratio to conditioned medium.

[0210] Flow cytometry and FACS analysis The presence of EGFR, CAR with a wild-type backbone, and CD28 on Tregs was assessed on day 14 post-transduction. Treg cells were collected and centrifuged at 300 xg for 5 min, then resuspended in 1X RoboSep buffer (StemCell Technologies) with surface staining antibody. The Tregs were then incubated at 4°C for 30 min, centrifuged, and washed with 1X RoboSep buffer. The stained cells were then analyzed by flow cytometry.

[0211] Treg cell antibody activation assay. Treg cells expressing a wild-type CAR backbone were prepared using the method described above. Cells were rested for 72 hours on day 14 and then activated with CFSE-labeled beads and anti-CD3 and anti-CD28 antibodies. Proliferation of transduced Treg cells was measured by FACS 72 hours post-stimulation.

[0212] Figure 1A A schematic diagram of a CAR with a wild-type backbone and an EGFRt tag, and the corresponding expression construct are presented. The CAR with the wild-type backbone was transduced into Treg cells. After transduction, the Treg cells were stained with anti-EGFRt and anti-CD28 antibodies and analyzed by FACS. Figure 1B-1C As shown, Treg cells transduced with CARs having a wild-type scaffold exhibited reduced expression of endogenous CD28. Figure 1B Representative scatter plots depicting EGFRt and CD28 signal intensities in transduced Treg cell populations were presented. Each quadrant (Q5, Q6, Q7, and Q8) distinguished various Treg cell populations within the parental population. The majority of Treg cells (66.1%) were found in quadrant Q5, which identifies untransduced Treg cells. Quadrants Q6 and Q7 represent populations of transduced Treg cells expressing a CAR with a wild-type scaffold co-expressed with the EGFRt tag. Quadrants Q6 and Q7 were distinguished by CD28 expression levels, with Q6 identifying CD28(+) Treg cells and Q7 identifying CD28(-) Treg cells. Surprisingly, a significant proportion of the transduced Treg cell populations (Q7) showed reduced endogenous CD28 expression levels compared to the untransduced Treg cell population. Figure 1C A bar chart depicting CD28 expression levels in CAR-expressing Treg cells (CAR+) and untransduced Treg cells (CAR-) was created. It was observed that CD28 expression was reduced in CAR-expressing Treg cells compared to untransduced Treg cells.

[0213] The ability to activate CAR-expressing Treg cells via antibody transduction was also evaluated. After transduction, Treg cells were labeled with CFSE and activated with anti-CD3 and anti-CD28 antibodies. Proliferation of transduced Treg cells was assessed using FACS, such as... Figure 1D As shown in the figure, this plot depicts a histogram of normalized cell counts versus CFSE signal intensity for CAR-expressing Treg cells (CAR+) and untransduced Treg cells (CAR-). CFSE signal intensity is indirectly correlated with cell proliferation. As Treg cells proliferate, CFSE fluorescence intensity halves with each cell division, causing the histogram to shift to the left. Figure 1D As shown, due to reduced CD28 expression, CAR-expressing Treg cells cannot proliferate like untransduced Treg cells.

[0214] Example 2: CAR engineered to avoid loss of endogenous CD28 expression in transduced T cells As described in Example 1, expression of CARs with a wild-type scaffold (CD28 hinge region and CD28 transmembrane domain) in Treg cells leads to reduced endogenous CD28 expression. To avoid the loss of endogenous CD28 expression in transduced Treg cells, novel CAR scaffolds modified in the transmembrane domain and / or hinge region were constructed. Table 2-1 lists... Figure 2A-2B The novel CAR described in the paper combines a hinge region, a transmembrane domain, and an intracellular domain.

[0215] Table 2-1. CAR skeleton

[0216] Table 2-1 also lists Figures 3A-3B The exemplary CAR expression cassette shown is a combination of its hinge region, transmembrane domain, and intracellular domain. Each construct includes an EGFRt tag for confirming successful transduction into T cells.

[0217] Example 3: CAR expression on transduced T cells The CAR of Example 2 was expressed in two cell types: Treg cells and Jurkat cells. Endogenous CD28 expression was measured to determine if the novel CAR lacked the defects observed in CARs with the wild-type scaffold (CD28 hinge region and CD28 transmembrane domain). Helios and FoxP3 expression on transduced Treg cells was also evaluated to determine if the Treg phenotype was preserved.

[0218] Expression on Treg cells Following the method in Example 1, a vector encoding a CAR with a wild-type (WT) backbone or a modified backbone was transduced into Treg cells. After transduction, the Treg cells were stained with anti-CAR and anti-EGFRt antibodies. Figure 4A As shown in -B, Treg cells have been successfully transduced with various CARs. Figure 4ARepresentative scatter plots illustrating the levels of EGFRt tag and CAR expression in transduced Treg cells were depicted. Each plot shows Treg cells expressing different CARs, either with a wild-type scaffold or a modified scaffold. Two main Treg cell populations were observed in each scatter plot. The first cell population, located in quadrant Q4, represents untransduced Treg cells that are CAR-negative and therefore also negative for the co-expressed EGFRt tag. The second cell population, located in quadrant Q2, represents transduced Treg cells expressing both CAR and EGFRt tags. Notably, for each scatter plot, the transduced Treg cell population constituted the largest proportion of the parental Treg cell population. For each expressed CAR, transduced Treg cells comprised at least 60% of the parental population, indicating durable and effective transduction. The percentages of different Treg cell populations are shown in each quadrant. Figure 4B A bar chart depicting CAR expression levels in transduced Treg cells was created.

[0219] After demonstrating CAR transduction into Treg cells, CAR-expressing Treg cells were then tested to determine whether endogenous CD28 expression was preserved due to the engineering of the CAR scaffold. CAR-expressing Treg cells were stained with an anti-CD28 antibody and then analyzed using flow cytometry. Figure 4C It was demonstrated that endogenous CD28 expression was restored in Treg cells expressing CARs with a modified scaffold compared to Treg cells expressing CARs with a wild-type scaffold. Figure 4C A bar chart depicting CD28 expression levels in various CAR-expressing Treg cells compared to the blank control (Mock) (untransduced) sample is presented. Notably, CD28 expression is increased in Treg cells expressing CARs with a modified scaffold compared to Treg cells expressing CARs with a wild-type scaffold. Furthermore, CD28 expression in Treg cells expressing CARs with a modified scaffold is comparable to that in the blank control (untransduced) sample.

[0220] like Figure 5A As shown in Figure -B, other CAR expression vectors were transduced into Treg cells, and their effect on the restoration of endogenous CD28 expression was tested. After transduction, Treg cells were stained with anti-CAR and anti-EGFRt antibodies. Figure 5A This demonstrates the successful transduction and persistent expression of various CARs in Treg cells. Figure 5ARepresentative scatter plots illustrating the levels of EGFRt tag and CAR expression in transduced Treg cells are depicted. The scatter plot in the upper left corner shows Treg cells that were not transduced using a lentiviral vector (UTD). The remaining scatter plots show Treg cells expressing different CARs, either with a wild-type CAR backbone or a modified CAR backbone. Each quadrant of the scatter plot distinguishes different Treg cell populations within the parental population. Untransduced Treg cells are located in quadrant Q8; these cells are CAR-negative and therefore also negative for the co-expressed EGFRt tag. As expected, most cells from the UTD control are located in Q8. Quadrant Q7 identifies transduced Treg cells that express the EGFRt tag but not CAR. Quadrant Q6 identifies transduced Treg cells that express both CAR and the EGFRt tag (thus demonstrating successful transduction).

[0221] After demonstrating that the CAR expression vector was transduced into Treg cells, the CAR-expressing Treg cells were then tested to determine whether endogenous CD28 expression was restored due to the engineering of the CAR scaffold. CAR-expressing Treg cells were stained with an anti-CD28 antibody and then analyzed using flow cytometry. Figure 5B It was demonstrated that CD28 expression was restored in Treg cells expressing CARs with a modified scaffold compared to Treg cells expressing CARs with a wild-type scaffold. Figure 5BRepresentative scatter plots depicting the levels of EGFRt tag and CD28 expression in transduced Treg cells are presented. The scatter plot in the upper left corner shows Treg cells not transduced with a lentiviral vector (UTD). The remaining scatter plots show Treg cells expressing CARs, either with a wild-type CAR backbone or a modified CAR backbone. Each quadrant (Q13, Q14, Q15, and Q8) distinguishes various Treg cell populations within the parental population. Q13 identifies untransduced Treg cells that are negative for the EGFRt tag and therefore also negative for CARs. As expected, these cells are positive for endogenous CD28 expression. Most cells from the UTD control are located in Q13. Quadrants Q14 and Q15 identify transduced Treg cells that express the EGFRt tag and therefore express CARs. The transduced Treg cells could be further distinguished into CD28-positive Treg cells (Q14) and CD28-negative Treg cells (Q15). Similar to what was previously demonstrated in Example 1, a significant proportion of the transduced Treg cells expressing CARs with a wild-type scaffold showed reduced CD28 expression. In contrast, Treg cells expressing CARs with a modified scaffold were predominantly CD28(+) and were located within Q14, demonstrating successful restoration of endogenous CD28 expression.

[0222] Finally, the Helios and FoxP3 phenotypes of Treg cells expressing various CARs were evaluated. CAR-expressing Treg cells were fixed and permeabilized. The Treg cells were then stained with anti-Helios and anti-FoxP3 antibodies and analyzed by flow cytometry. Figure 5C It was demonstrated that Treg cells expressing CAR expressed both Helios and FoxP3 markers. Figure 5C A representative scatter plot showing the expression levels of Helios and FoxP3 in transduced Treg cells was created.

[0223] Expression on Jurkat cells The expression of CAR and CD28 was assessed by flow cytometry using anti-scFv and anti-CD28 antibodies, respectively.

[0224] Jurkat cell transduction. Jurkat cells were transduced using a lentiviral vector. After transduction, Jurkat cells were stained with anti-CAR antibody. Figure 6A As shown, Jurkat cells have been successfully transduced with various CARs. Figure 6A A bar chart depicting CAR expression levels in transduced Jurkat cells compared to blank control (untransduced) cells is presented.

[0225] Jurkat cells expressing CAR were then tested to determine whether endogenous CD28 expression was restored due to engineering of the CAR scaffold. Jurkat cells expressing CAR were stained with an anti-CD28 antibody and then analyzed using flow cytometry. Figure 6B As shown, endogenous CD28 expression was restored in Jurkat cells expressing CARs with a modified backbone. Figure 6B A bar chart depicting the CD28 signal intensity of various CAR-expressing Jurkat cells compared to untransduced Jurkat cells (UTD) was presented. Notably, Jurkat cells expressing CARs with modified scaffolds exhibited significantly higher CD28 signal intensity and therefore higher CD28 expression compared to Jurkat cells expressing CARs with wild-type scaffolds. Furthermore, CD28 expression in Jurkat cells expressing CARs with modified scaffolds was comparable to that in untransduced Jurkat cells. Figure 6C As shown, additional CARs with a modified backbone were expressed in Jurkat cells, and their effect on the restoration of endogenous CD28 expression was tested. Figure 6C A bar chart depicting the CD28 signal intensity of various CAR-expressing Jurkat (CAR+) cells compared to CAR-Jurkat cells was presented. Similar to what was previously demonstrated in Example 1, Jurkat cells expressing CARs with a wild-type scaffold exhibited reduced CD28 expression compared to untransduced Jurkat cells. Jurkat cells expressing CARs with a modified scaffold showed higher CD28 expression compared to Jurkat cells expressing CARs with a wild-type scaffold. Furthermore, the CD28 expression of Jurkat cells expressing CARs with a modified scaffold was comparable to that of untransduced Jurkat cells.

[0226] Example 4: Activation of CAR+ T cells In Example 3, modification of the CAR backbone resulted in the preservation of endogenous CD28 expression in CAR-expressing T cells. In this example, we tested the ability of T cells expressing various CARs to be activated upon stimulation.

[0227] Activation of Treg cells expressing CARs with modified scaffolds Activation of Treg cells using CD3 and CD28 antibodies.Treg cells expressing CARs with wild-type or modified scaffolds were prepared using the method described in Example 1. Cells were rested for 72 hours on day 14, then labeled with CFSE and activated with anti-CD3 and anti-CD28 antibody activation beads. The proliferation of engineered Treg cells was measured by FACS 72 hours post-stimulation.

[0228] The CAR expression vector was transduced into Treg cells, then labeled with CFSE, and activated with anti-CD3 and anti-CD28 antibodies. Figure 7A This study demonstrates an improvement in activation and proliferation of Treg cells expressing CARs with a modified scaffold compared to Treg cells expressing CARs with a wild-type scaffold. As Treg cells proliferate, the CSFE fluorescence intensity halves with each cell division, causing a leftward shift in the histogram. Similar to what was previously demonstrated in Example 1, Treg cells expressing CARs with a wild-type scaffold do not proliferate as well as CAR-Treg cells. In contrast, Treg cells expressing CARs with a modified scaffold exhibit comparable proliferation to CAR-Treg cells. Figure 7B The study demonstrated improved proliferation of Treg cells expressing CAR with a modified scaffold compared to Treg cells expressing CAR with a wild-type scaffold. Figure 7B The percentage of proliferation of CAR-expressing Treg cells (EGFR+) compared to CAR-Treg cells (EGFR-) was depicted as a function of the cell-to-CD3 / CD28 ratio. Treg cells expressing CARs with a wild-type scaffold did not proliferate as well as CAR-Treg cells. Treg cells expressing CARs with a modified scaffold exhibited improved activation and proliferation compared to Treg cells expressing CARs with a wild-type scaffold.

[0229] Activation of Jurkat cells expressing CARs with modified scaffolds CARs with wild-type or modified scaffolds were expressed on the Jurkat reporter cell line. This reporter cell line (Jurkat-NFAT-FF-luc) expresses firefly luciferase under the control of the nuclear factor of activated T-cell (NFAT) response element.

[0230] Activation of Jurkat cells using target cells expressing CAR antigens.Jurkat cells expressing CAR were prepared using the method described in Example 3 and subsequently co-cultured with CAR antigen-positive K562 cells. Following co-culture, a fraction of these cells was used to assess luciferase expression as a biomarker for activation via CAR. Luciferase expression was detected by measuring luminescence using a SpectraMax plate reader. The remaining fractions of these cells were used to assess the expression of activation biomarkers by staining with anti-CD69 and anti-CD25 antibodies and FACS analysis.

[0231] Activate CAR-expressing Jurkat cells using K562 cells expressing CAR antigen. Figure 8A The luminescence was depicted as a function of the Jurkat to K562 cell ratio. CAR-expressing Jurkat cells were stained with anti-CD69 antibody. Figure 8B The percentage of Jurkat cells expressing the CD69 activation marker was depicted as a function of the Jurkat to K562 cell ratio. Figure 8C The percentage of Jurkat cells expressing the CD25 activation marker was depicted. With a near 1:1 ratio of CAR+Jurkat cells to CAR antigen+K562 cells, CAR-expressing Jurkat cells exhibited increased CD25 expression. Furthermore, Jurkat cells expressing CARs with a modified backbone showed comparable upregulation of the activation marker CD25 to Jurkat cells expressing CARs with a wild-type backbone.

[0232] sequence >SEQ ID NO:1 (GMCSF signal peptide) Homo sapiens MLLLVTSLLL CELPHPAFLLIP >SEQ ID NO:2 (CD8a signal peptide) Homo sapiens MALPVTALLLPLALLLHAARP >SEQ ID NO:3 (anti-CV VH1) Homo sapiens-116aa HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS >SEQ ID NO:4 (anti-CV VH2) Homo sapiens-116aa QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS >SEQ ID NO:5 (Anti-CV VH3) Homo sapiens - 119aa EVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSS >>SEQ ID NO:6 (Anti-CV VL1) Homo sapiens - 109aa SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQVFGPGTTVTVLR >SEQ ID NO:7 (Anti-CV VL2) Homo sapiens - 109aa SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR >SEQ ID NO:8 (Anti-CV VL3) Homo sapiens - 111aa AIQMTQSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIKRAAA >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 NO:14 Synthetic Whitlow linker GSTSGSGKPGSGEGSTKG >SEQ ID NO:15 Synthetic ABpur linker ASSGGSTSGSGKPGSGEGSSGSAR >SEQ ID NO:16 – Anti-CV ABD (254aa) Synthetic EVKLIESGGGLVEPGRSLRLACTTSGFTFADYGLSWFRQGPGKGLEWVGFTGPKHLGETTECAPSVEDRCTISRDDSKSTVYLQMHRLQHEDTAVYFCVGPWFGDLLMWGQGTLVTVSSASSGGSTSGSGKPGSGEGSSGSARAIQMTQSPSSLSASVGDRVSITCRATQDISTSLGWYHQRPGKAPRLLIYGASKVQTGVPSRFSGNGSGTEFTLTISSLQPEDIGTYYCLQDDGFPFTVGQGTKLDIKRAAA >Synthesis of SEQ ID NO:17 - tisa CD19 ABD (242aa) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS >Synthesis of SEQ ID NO:18 - liso CD19 ABD (245aa) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS >Synthesis of SEQ ID NO:19 - ida BCMA ABD (from 246aa) DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS >Synthesis of SEQ ID NO:20 - cilta BCMA ABD (242aa) QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSGG GGSEVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSS >SEQ ID NO:21-CD8a Hinge - Homo sapiens TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD >SEQ ID NO:22 – CD28 Hinge - Homo sapiens IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP >SEQ ID NO:23 – IgG4 hinge - Homo sapiens ESKYGPPCPPCP >SEQ ID NO:24 - CD8a transmembrane – Homo sapiens IYIWAPLAGTCGVLLLSLVITLYC >SEQ ID NO:25 – CD28 transmembrane-Homo sapiens [X]FWVLVVVGGVLACYSLLVTVAFIIFWV X = M or missing >SEQ ID NO:26 (CD28 costimulatory domain) Homo sapiens RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS >SEQ ID NO:27 (41BB costimulatory domain) Homo sapiens KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL >SEQ ID NO:28 (CD3ζ intracellular signal transduction domain) Homo sapiens RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Synthesis of SEQ ID NO:29 (CD28+CD3ζ) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Synthesis of SEQ ID NO:30 (41BB+CD3ζ) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:31 (CD28 hinge region amino acid sequence, wild-type reference) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP SEQ ID NO:32 (CD28 truncated hinge region amino acid sequence) SPLFPGPSKP SEQ ID NO:33 (CD4 hinge region amino acid sequence, wild-type reference) SGQVLLESNIKVLPTWSTPVQP SEQ ID NO:34 (CD4 truncated hinge region amino acid sequence) LPTWSTPVQP SEQ ID NO:35 (CD11a hinge region amino acid sequence) VDVVYEKQML SEQ ID NO:36 (IgG4 hinge region amino acid sequence “X”) ESKYGPPCP[X]CP X = P or S SEQ ID NO:37 (CD2 hinge region amino acid sequence) SKESSVEPVSCPEKGLD SEQ ID NO:38 (TCRα hinge region amino acid sequence) PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS SEQ ID NO:39 (IgG4 hinge region amino acid sequence) ESKYGPPCPPCP SEQ ID NO:40 (CD28 transmembrane domain amino acid sequence, wild-type reference) FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO:41 (CD28 modified transmembrane domain amino acid sequence) FWVLVVVGGVLALLLLLVLVAFIIFWV SEQ ID NO:42 (CD4 transmembrane domain amino acid sequence) MALIVLGGVAGLLLFIGLGIFF SEQ ID NO:43 (CD11a transmembrane domain amino acid sequence) YLYVLSGIGGLLLLLLIFIVLYKV SEQ ID NO:44 (CD18 transmembrane domain amino acid sequence) IAAIVGGTVAGIVLIGILLLVIW SEQ ID NO:45 (CD2 transmembrane domain amino acid sequence) IYLIIGICGGGSLLMVFVALLVFYIT SEQ ID NO:46 (HER2 transmembrane domain amino acid sequence) SIISAVVGILLVVVLGVVFGILI

Claims

1. A chimeric antigen receptor (CAR) comprising: Antigen-binding domain; CD28 hinge area; Transmembrane domains, selected from the group consisting of: CD4 transmembrane domains, CD11a transmembrane domains, CD18 transmembrane domains, CD2 transmembrane domains, and HER2 transmembrane domains; and Intracellular signal transduction domains.

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

3. The CAR of claim 1, wherein the cell surface expression of endogenous CD28 protein in T cells expressing the CAR is substantially higher than that in control T cells expressing the control CAR, wherein the control CAR comprises the wild-type CD28 hinge region of SEQ ID NO:31 and the wild-type D28 transmembrane domain of SEQ ID NO:40, and 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: Antigen-binding domain; The hinge area of ​​CD28 has been shortened; Transmembrane domains; and Intracellular signal transduction domains The truncated CD28 hinge region comprises a truncation of 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 end.

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 insertions, substitutions, and / or deletions relative to SEQ ID NO:

40.

10. The CAR of claim 5, wherein the transmembrane domain is selected from the group consisting of: CD4 transmembrane domain, CD11a transmembrane domain, CD18 transmembrane domain, CD2 transmembrane domain and HER2 transmembrane domain.

11. A chimeric antigen receptor (CAR) comprising: Antigen-binding domain; The hinge region is selected from the group consisting of: the truncated CD4 hinge region, the CD11a hinge region, the CD2 hinge region, and the TCRα hinge region. Transmembrane domains; and Intracellular signal transduction domains.

12. The CAR of claim 11, wherein the hinge region is a truncated CD4 hinge region comprising a truncation of 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 the CD11a hinge region.

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

16. The CAR of claim 11, wherein the hinge region is a TCRα 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 insertions, substitutions, and / or deletions relative to SEQ ID NO:

40.

19. The CAR of claim 11, wherein the transmembrane domain is selected from the group consisting of: CD4 transmembrane domain, CD11a transmembrane domain, CD18 transmembrane domain, CD2 transmembrane domain and HER2 transmembrane domain.

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

21. The CAR of claim 1, wherein the transmembrane domain is the CD11a transmembrane domain.

22. The CAR of claim 1, wherein the transmembrane domain is the CD18 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 when the CAR is expressed in T cells, the CAR does not result in a substantial reduction in the cell surface expression of endogenous CD28 protein.

26. The CAR of claim 5, wherein the cell surface expression of endogenous CD28 protein in T cells expressing the CAR is substantially higher than that in control T cells expressing the control CAR, wherein the control CAR comprises the wild-type CD28 hinge region of SEQ ID NO:31 and the wild-type D28 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 NO: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 NO: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 NO:33-38, and the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: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 signal transduction domain comprises the CD3ζ signal transduction domain.

48. The CAR of claim 47, wherein the CD3ζ signal transduction domain comprises the amino acid sequence of SEQ ID NO:

28.

49. The CAR of claim 47, wherein the intracellular signal transduction domain further comprises a co-stimulatory domain.

50. The CAR of claim 49, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain.

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

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

29.

53. The CAR of claim 50, wherein the co-stimulatory domain comprises a 4-1BB co-stimulatory domain.

54. The CAR of claim 53, wherein the intracellular signal transduction 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 a single-chain antibody fragment (scFV) or a single-domain antibody (sdAb).

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

59. The CAR of claim 57, wherein the antibody-binding domain comprises an sdAb, and 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), CD19, and BCMA.

61. The CAR of claim 60, wherein the antigen-binding domain binds to CV, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 and the light chain CDR of the amino acid sequences 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 CD19, optionally wherein the antigen-binding domain comprises: (i) The heavy chain complementarity-determining region (CDR) of the amino acid sequence of SEQ ID NO:10 and the light chain CDR of the amino acid sequence of SEQ ID NO:9; (ii) The heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10 and the 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) The heavy chain complementarity-determining region (CDR) of the amino acid sequence of SEQ ID NO:12 and the light chain CDR of the amino acid sequence of SEQ ID NO:11; (ii) The heavy chain variable region comprising the amino acid sequence of SEQ ID NO:12 and the 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 an operative combination of the nucleic acid of claim 64 and 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 adenovirus vector, or an adeno-associated virus 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 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 modified T cells as described in claim 70 and a pharmaceutically acceptable excipient.

72. The pharmaceutical composition of claim 71, wherein the modified T cells are human T cells.

73. The pharmaceutical composition of claim 72, wherein the human T cells are regulatory T cells (Tregs) that are CD4+, CD25+ and CD12710.

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

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

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

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

78. A kit comprising a container containing the pharmaceutical composition of claim 71, the container being in communication with an infusion chamber via a fluid conduit, wherein the infusion chamber is configured to be in communication with an intravenous injection needle via a fluid conduit.

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