Chimeric antigen receptors containing an antibody-inducible domain

Abstract

The present disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular accessory domain through which T-cells expressing the CARs can be activated in an antigen-independent fashion. In particular, the extracellular accessory domain of the CARs of the present disclosure is an antibody-inducible domain that is bindable by an antibody or a polypeptide comprising an antigen-binding fragment thereof. In this way, T-cells expressing the CARs are activatable by binding of the antibody to the antibody-inducible domain.

Description

x CHIMERIC ANTIGEN RECEPTORS CONTAININGAN ANTIBODY-INDUCIBLE DOMAINCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 559,795, filed February 29, 2024, which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (237752001240SEQLIST.xml; Size: 45,978 bytes; and Date of Creation: February 26, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular accessory domain through which T-cells expressing the CARs can be activated in an antigen-independent fashion. Specifically, the extracellular accessory domain is an antibody-inducible domain by which T-cells expressing the CARs can be activated upon binding of an antibody to the antibody-inducible domain.BACKGROUND OF THE INVENTION

[0004] Chimeric antigen receptors (CARs) including portions of both an antibody and a T-cell receptor (TCR) were first described the 1980s. First generation CARs included an antigen-binding domain (ABD), a hinge, a transmembrane domain and an intracellular signal transduction domain of CD3ζ Subsequent generations of CARs have been engineered to include one or more intracellular co-stimulation domains to improve their ability to activate T-cells in which they are expressed (Smith and Shen, J. Transl. Med, 21:515, 2023).

[0005] There are now four anti-CD19 CAR T-cell immunotherapies approved by the U.S. Food and Drug Administration for treatment of various CD19+ leukemias and lymphomas. In fact, anti-CD19 CAR T-cell immunotherapies have led to remarkable clinical responses in patients with poor prognoses, including high levels of remission. However, there are short- comings in manufacture and use of CAR T-cells including the need for antigen-dependent activation, and the need for co-expression of a tag for detection and / or ablation of CAR T-cells.

[0006] Thus, what is needed in the art are CAR backbones that have been engineered to be activatable in a CAR-specific, ABD-nonspecific fashion. There is also a need in the art to identify CAR-expressing T-cells in an ABD-nonspecific fashion and in the absence of co- expression of a separate polypeptide tag.SUMMARY OF THE INVENTION

[0007] The present disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular accessory domain through which T-cells expressing the CARs can be activated in an antigen-independent fashion. In particular, the extracellular accessory domain of the CARs of the present disclosure is bindable by an antibody or a polypeptide comprising an antigenbinding fragment thereof. In this way, T-cells expressing the CARs are activatable by binding of the antibody to the extracellular accessory domain.BRIEF DESCRIPTION OF DRAWINGS

[0008] FIG. 1 shows a schematic of an expression cassette of a standard chimeric antigen receptor (CAR) (top) and an exemplary expression cassette of a CAR of the present disclosure (bottom) comprising domain 3 (D3) of vascular endothelial growth factor receptor 2 (VEGFR2). The promoter region (Prom.) and the truncated epidermal growth factor receptor (EGFRt) expression tag are shown at the 5’ and 3’ ends, respectively, of the expression cassette. TM represents a transmembrane domain.

[0009] FIG. 2 depicts a standard CAR, as well as exemplary CARs comprising D3 of VEGFR2 (VEGF2R-containing CARs).

[0010] FIG. 3A shows percentages of various CAR-expressing Jurkat cells that express CD25 after culturing without antigen or with antigen-expressing K562 cells. FIG. 3B shows percentages of various CAR-expressing Jurkat cells that express CD25 after incubation with anti- VEGFR2 antibody, anti-human epidermal growth factor receptor 2 (Her2) antibody, antiinterleukin 4 receptor (IL4Ra) antibody, or without an antibody (no-stim). In both graphs, Mock represents untransduced controls.

[0011] FIG. 4A shows a timeline of transduction and expansion of Treg cells engineered to express a CAR. On day 3, Treg cells were transduced with lentivirus. Transduced Treg cells were subsequently expanded in medium containing interleukin 2 (IL-2). On day 9, transduced Treg cells were split and re-stimulated under one of four conditions. FIG. 4B shows the foldincrease of Treg cells engineered to express a VEGFR2-containing CAR and subjected to restimulation under one of four conditions on day 9: 1) anti-CD3 / anti-CD28 beads; 2) anti- VEGFR2 beads; 3) 1 pg / mL anti-VEGFR2 antibody; and 4) 3.5 pg / mL anti-VEGFR2 antibody.

[0012] FIG. 5 shows a schematic of expression cassette of a standard CAR (top) and an exemplary CAR expression cassette of the present disclosure (bottom) comprising domain 4 (D4) of human epidermal growth factor receptor 2 (Her2). Prom, and EGFRt are shown at the 5’ and 3’ ends, respectively, of the expression cassettes.

[0013] FIG. 6 depicts a standard CAR, as well as exemplary CARs comprising D4 of Her2 (Her2-containing CARs).

[0014] FIG. 7 A shows percentages of various CAR-expressing Jurkat cells that expressCD25 after culturing without antigen or with antigen-expressing K562 cells. FIG. 7B shows percentages of various CAR-expressing Jurkat cells that express CD25 after incubation with anti- VEGFR2 antibody, anti-Her2 antibody, anti-IL4Ra antibody, or without an antibody (no stim). In both graphs, Mock represents untransduced controls.

[0015] FIG. 8 shows a schematic of expression cassette of a standard CAR (top) and an exemplary CAR expression cassette of the present disclosure (bottom) comprising domains 3 through 5 of platelet-derived growth factor receptor (PDGFR) alpha. Prom, and EGFRt are shown at the 5’ and 3’ ends, respectively, of the expression cassettes.

[0016] FIG. 9 depicts a standard CAR, as well as exemplary CARs comprising D3-D5 of PDGFRa (PDGFRa-expressing CARs).

[0017] FIG. 10 shows percentages of various CAR-expressing Jurkat cells that express CD25 after culturing without antigen or with antigen-expressing K562 cells.

[0018] FIG. 11 shows a schematic of expression cassette of a standard CAR (top) and an exemplary CAR expression cassette of the present disclosure (bottom) comprising extracellular domain (ECD) 1 and / or 2 of interleukin 4 receptor alpha (IL4Ra). Prom, and EGFRt are shown in the 5’ and 3’ ends, respectively, of the expression cassettes.

[0019] FIG. 12 depicts a standard CAR, as well as exemplary CARs comprising DI and / or D2 of IL4Ra (IL4Ra-containing CARS).

[0020] FIG. 13A shows percentages of various CAR-expressing Jurkat cells that expressing CD25 after culturing without antigen or with antigen-expressing K562 cells. FIG. 13B shows percentages of various CAR-expressing Jurkat cells that express CD25 after incubation with anti-VEGFR2 antibody, anti-Her2 antibody, anti-IL4Ra antibody, or without an antibody (no stim). Mock represents untransduced controls.

[0021] FIG. 14 shows a schematic of an expression cassette of a standard CAR (top) and an expression cassette of an exemplary CAR of the present disclosure (bottom) comprising extracellular domains (ECD) 1 and / or 2 of cluster of differentiation 4 (CD4). Prom, and EGFRt are shown in the 5’ and 3’ ends, respectively, of the expression cassettes.

[0022] FIG. 15 depicts a standard CAR, as well as exemplary CARs comprising D 1 and / or D2 of CD4 (CD4-containing CARs).

[0023] FIG. 16 shows percentages of various CAR-expressing Jurkat cells that express CD25 after culturing without antigen or with antigen-expressing K562 cells. Mock represents untransduced controls.

[0024] FIG. 17 shows a schematic of an expression cassette of a standard CAR (top) and an expression cassette of an exemplary CAR of the present disclosure (bottom) comprising . extracellular domain (ECD) 1 and / or 2 of cluster of differentiation 2 (CD2). Prom, and EGFRt are shown in the 5’ and 3’ ends, respectively, of the expression cassettes.

[0025] FIG. 18 depicts a standard CAR, as well as exemplary CARs comprising D 1 and / or D2 of CD2 (CD2-containing CARs).

[0026] FIG. 19 shows percentages of various CAR-expressing Jurkat cells that express CD25 after without antigen or with antigen-expressing K562 cells. Mock represents untransduced controls.

[0027] FIG. 20 shows viability and expansion of VEGFR domain-containing CAR-Tregs (SBT-1927) after restimulation on Day 9 under the indicated conditions.

[0028] FIG. 21 shows viability and expansion of IL4R domain-containing CAR-Tregs (SBT-1934) after restimulation on Day 9 under the indicated conditions.

[0029] FIG. 22 shows viability and expansion of IL4R domain-containing CAR-Tregs (SBT-1936) after restimulation on Day 9 under the indicated conditions.

[0030] FIG. 23 shows expansion of VEGFR domain-containing CAR-Tregs (SBT-1927) after restimulation with ramucirumab (VEGFR antibody-conjugated) beads or CD3 / CD28 beads.

[0031] FIG. 24 shows expansion of VEGFR domain-containing CAR-Tregs (SBT-1934) after restimulation with dupilumab (IL4R- antibody-conjugated) beads or CD3 / CD28 beads.

[0032] FIG. 25 shows expansion of VEGFR domain-containing CAR-Tregs (SBT-1934) after restimulation with dupilumab (IL4R- antibody-conjugated) beads or CD3 / CD28 beads.DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure relates to chimeric antigen receptors (CARs) comprising an extracellular accessory domain through which T-cells expressing the CARs can be activated in an antigen-independent fashion. Specifically, the extracellular accessory domain is an antibody-inducible domain in which T-cells expressing the CARs can be activated upon binding of an antibody to the antibody-inducible domain.Definitions

[0034] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0035] As used herein, the terms “antigen,” “immunogen,” and “antibody target,” refer to a molecule, compound, or complex that is recognized by an antibody, i.e., can be bound by the antibody. The term can refer to any molecule that can be recognized by an antibody, e.g., a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.). One of skill will understand that the term does not indicate that the molecule is immunogenic in every context, but simply indicates that it can be targeted by an antibody, or an antigen-binding domain derived therefrom.

[0036] As used herein, the term “epitope” refers to the localized site on an antigen that is recognized and bound by an antigen-binding domain of an antibody or fragment derived therefrom. Epitopes can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes non-protein components, e.g., from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is a protein, the epitope can be comprised of consecutive amino acids, or amino acids from differentparts of the protein that are brought into proximity by protein folding (e.g., a discontinuous epitope).

[0037] As used herein, the term “antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene, that specifically bind and recognize an antigen. Typically, the “variable region” contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD).

[0038] Antibodies can be of (i) any of the five major classes of immunoglobulins, based on the identity of their heavy-chain constant domains - alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) and mu (IgM), or (ii) subclasses (isotypes) thereof (E.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). The light chains can be either lambda or kappa.

[0039] The following are a non-exhaustive list of different antibody forms, all retaining antigen binding activity:(1) whole immunoglobulins (also referred to as “intact” antibodies) (two light chains and two heavy chains, e.g., a tetramer);(2) an immunoglobulin polypeptide (a light chain or a heavy chain);(3) an antibody fragment, such as Fv (a monovalent or bi-valent variable region fragment, and can encompass only the variable regions (e.g., VL and / or VH), Fab (VLCL VHCH), F(ab’)2, Fv (VLVH), SCFV (single chain Fv) (a polypeptide comprising a VL and VH joined by a linker, e.g., a peptide linker), (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(FV)2 fused to CH3 domain), diabody (noncovalent dimer of single-chain Fv (scFv) fragment that consists of the heavy chain variable (VH) and light chain variable (VL) regions connected by a small peptide linker), triabody is trivalent sc(Fv)3 or trispecific sc(Fv)3;(4) a multivalent antibody (an antibody comprising binding regions that bind two different epitopes or proteins, e.g., “scorpion” antibody;(5) a fusion protein comprising a binding portion of an immunoglobulin fused to another amino acid sequence (such as a fluorescent protein); and(6) heavy chain only antibody or antibody fragment having only two heavy chains and lacking the two light chains usually found in antibodies.

[0040] The phrase “CDR sequence set” as used herein refers to the 3 heavy chain and / or 3 light chain CDRs of a particular antibody or antigen-binding domain. A “light chain” CDR sequence set refers to the light chain CDR sequences. A “heavy chain” CDR sequence set refers to the heavy chain CDR sequences. A “full” CDR sequence set refers to both heavy chain and light chain CDR sequences. CDRs are predicted based on IMGT sequence alignment.

[0041] As used herein, the term “humanized antigen-binding domain” refers to a chimeric antigen-binding domain in which the CDRs, obtained from the VH and VL regions of a non-human antibody having the desired specificity, affinity and capability are grafted to human framework regions. In one embodiment, the framework residues of the humanized antigenbinding domain are modified to refine and optimize the specificity, affinity and capability of the antigen-binding domain.

[0042] As used herein, the term “human antigen-binding domain” refers to an antigenbinding domain of an antibody produced by a human or an antibody having an amino acid sequence corresponding thereto.

[0043] As used herein, an antigen-binding domain “preferentially binds” binds a first antigen relative to a second antigen if it binds the first antigen with greater affinity than it does the second antigen. Preferential binding can be at least any of 2-fold, 5-fold, 9-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold or 1000-fold greater affinity.

[0044] As used herein, an antigen-binding domain “specifically binds” or is “specific for” a target antigen or target group of antigens if it binds the target antigen or each member of the target group of antigens with an affinity of at least any of IxlO-6M, IxlO-7M, IxlO-8M, IxlO-9M, IxlO-10M, IxlO-11M, IxlO-12M, and, for example, binds to the target antigen or each member of the target group of antigens with an affinity that is at least two-fold greater than its affinity for non-target antigens to which it is being compared. Typically, specific binding is characterized by binding the antigen with sufficient affinity that the antigen-binding domain is useful as a diagnostic to detect the antigen or epitope and / or as a therapeutic agent in targeting the antigen or epitope.

[0045] As used herein, the term “polypeptide” refers to a molecule having a sequence of natural and / or unnatural amino acids connected through peptide bonds. The term “peptide” refers to a short polypeptide, typically no more than 30 amino acids long. The amino acidsequence of a polypeptide is referred to as its “primary structure.” The term “protein” refers to a polypeptide having a secondary, tertiary and / or quaternary structure, e.g., structures stabilized by hydrogen bonds, relationships between secondary structures and structures formed of more than one protein. Proteins can be further modified by other attached moieties such as carbohydrate (glycoproteins), lipids (lipoproteins) phosphate groups (phosphoproteins) and the like.

[0046] As used herein, an amino acid sequence “consists of’ only the amino acids in that sequence.

[0047] As used herein, a first amino acid sequence “consists essentially of’ a second amino acid sequence if the first amino acid sequence ( 1 ) comprises the second amino sequence and (2) is no more than 1 , no more than 2 or no more than 3 amino acids longer than the second amino acid sequence.

[0048] As used herein, a first amino acid sequence is a “fragment” of a second amino acid sequence if the second amino acid sequence comprises the first amino acid sequence. In certain embodiments, a first amino acid sequence that is a fragment of a second amino acid sequence may have no more than any of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fewer amino acids than the second amino acid sequence.

[0049] As used herein, a “functional equivalent” of a reference amino acid sequence is a sequence that is not identical to the reference sequence, but that contains minor alterations such as, for example, insertion, deletion or substitution of one or a few amino acids. A functionally equivalent sequence retains the function (e.g., immunogenicity) of the reference sequence to which it is equivalent. If a functionally equivalent amino acid sequence contains substitution of one or more amino acids with respect to the reference sequence, these will generally be conservative amino acid substitutions.

[0050] As used herein, a “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein’s desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. See, e.g., Watson, et al., “Molecular Biology of the Gene,” 4thEdition, 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, p. 224. Examples of conservative amino acid substitution include the following (Note, some categories are not mutually exclusive):

[0051] As used herein, the term “substantially identical” refers to identity between a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and / or common functional activity and / or common immunogenicity. For example, amino acid sequences that contain a common structural or antigenic domain having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are termed sufficiently or substantially identical. In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, or encode polypeptides having the same immunogenic properties.

[0052] As used herein, a chemical entity, such as a polypeptide, is “substantially pure” or “isolated” if it is the predominant chemical entity of its kind (e.g., of polypeptides) in a composition. This includes the chemical entity representing more than 50%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, or more than 99.99% of the chemical entities of its kind in the composition. A substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition is the purified species of interest. The object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventionaldetection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules, stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition.

[0053] The term “sequence identity” as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions multiplied by 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSIBLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSLBlast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparisonof sequences is the algorithm of Myers and Miller, 1988, CAB IOS 4: 11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0054] For antibodies and antigen-binding domains, percentage sequence identities can be determined when their sequences are maximally aligned by IMGT. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, multiplied by 100 to convert to percentage.

[0055] Percent amino acid sequence identity may also be determined using the sequence comparison program NCBIBLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBIBLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md. NCBIBLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length= 15 / 5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.

[0056] In situations where NCBIBLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBLBLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequenceidentity of A to B will not equal the % amino acid sequence identity of B to A. The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. It is understood that polynucleotides comprising non-transcribable nucleotide bases may be useful as probes in, for example, hybridization assays. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents.

[0057] The term "isolated nucleic acid" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.

[0058] Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81.5°C - 16.6 (LoglO [Na+]) + 0.41(%(G+C) - 600 / 1), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1 °C decrease in Tm, for example, if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridizationconditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt’s solution / 1.0% SDS at Tm - 5°C based on the above equation, followed by a wash of 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0059] As used herein, the term “expression construct” refers to a polynucleotide comprising an expression control sequence operatively linked with a heterologous nucleotide sequence (i.e., a sequence to which the expression control sequence is not normally connected to in nature) that is to be the subject of expression. As used herein, the term “expression vector” refers to a polynucleotide comprising an expression construct and sequences sufficient for replication in a host cell or insertion into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term “expression control sequence” refers to a nucleotide sequence that regulates transcription and / or translation of a nucleotide sequence operatively linked thereto. Expression control sequences include promoters, enhancers, repressors (transcription regulatory sequences) and ribosome binding sites (translation regulatory sequences).

[0060] As used herein, a nucleotide sequence is “operatively linked” with an expression control sequence when the expression control sequence functions in a cell to regulate transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through an interaction between a polymerase and a promoter.

[0061] The term "vector" as used herein comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and / or eukaryotic cells and / or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, lentiviral vectors, Adeno Associated viral vectors and the like. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0062] “Transfection” refers to the introduction of new genetic material into a cell. It includes transformation (the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane), transduction (the introduction of foreign DNA by a bacteriophage virus into a host cell) and conjugation.

[0063] As used herein, a “host cell” refers to a recombinant cell comprising an expression construct.

[0064] As used herein, the term “biological sample” refers to a sample containing cells (e.g., cells) or biological molecules derived from cells.

[0065] As used herein, the term terms “therapy,” “treatment,” “therapeutic intervention” and “amelioration” refer to any activity resulting in a reduction in the severity of symptoms. The terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment and prevention can be complete or partial. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects, the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment.

[0066] Compositions or methods "comprising" or "including" one or more recited elements may include other elements not specifically recited (e.g., open-ended terms meaning including but not limited to). For example, a composition that "comprises" or "includes" an antibody may contain the antibody alone or in combination with other ingredients. In contrast, the phrase “consisting of’ is closed, indicating that such embodiments do not include additional elements. The term "consisting essentially of" refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination (e.g., partially closed term). It is understood that aspects and embodiments described herein as “comprising” include “consisting of’ and “consisting essentially of’ embodiments.

[0067] As used herein, the following meanings apply unless otherwise specified. The word “may” is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.”

[0068] The phrase “at least one” includes “one”, “one or more”, “one or a plurality” and “a plurality”. The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1 , 2 or 3” means “at least 1, at least 2 or at least 3”. The term “plurality” in connection with a referent (e.g., object such as a T cell) refers to 3 or more, preferably 10 or more, preferably 100 or more, preferably 1,000 or more, preferably 10,000 or more, preferably 100,000 or more, preferably, 106or more, preferably 107or more, preferably 108or more, preferably 109or more, but not an infinite number of the referent (e.g., preferably less than 1012, preferably less than 1011, and preferably less than 1010).

[0069] The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value. For instance the phrase about 20 residues (amino acids) in length to describe a polypeptide linker refers to a polypeptide linker of from 18 to 22 amino acids in length, and includes a polypeptide linker of 20 amino acids in length.I. Chimeric Antigen Receptors (CARs)

[0070] “Chimeric antigen receptors” or “CARs” are engineered molecules comprising an optional signal peptide, a target antigen-binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain and an optional co-stimulatory domain. CARs are based on the structure of T-cell receptors, which are expressed on T-cells, and are involved in the cell-mediated immune responses. The “target-binding domain” is also referred to herein as an “antigen-binding domain” or “antigen-recognition domain”, and as such the term “target” encompasses an “antigen.”

[0071] So-called “first-generation” CARs had a targeting domain and a CD3^ signal transduction domain. So-called “second generation” CARs further included a co-stimulatory domain, such as a CD28 or a 4- IBB domain. So-called “third generation” CARs comprisemultiple co-stimulatory domains. So-called “fourth generation” CARs, also referred to as “TRUCKS” are engineered to release a transgenic cytokine upon CAR signaling.

[0072] Chimeric antigen receptors (“CARs”) include the following elements: (1) an optional signal peptide, (2) a target antigen-binding domain, (3) a linker; (4) a transmembrane region; (5) an intracellular domain comprising a signal transduction domain. Optionally, the CAR can include a co-stimulatory (signal transduction) domain. That is, these optional elements can be included in addition to required elements. The target antigen-binding domain is heterologous to at least one of the other domains. That is, the antigen-binding domain does not naturally occur on a T-cell receptor or is not in the same protein as at least one of the other domains of the CAR.

[0073] The “signal peptide” guides the CAR polypeptide through the cell membrane. The “antigen-binding domain” provides binding specificity to the CAR. The antigen-binding domain can bind to a domain of an antibody that binds to the target antigen for a so-called “Universal CAR”. The linker, which may act as a “hinge region” is a flexible connector region, e.g., a natural or synthetic polypeptide, or any other type of molecule, providing structural flexibility and spacing to flanking polypeptide regions. The “transmembrane domain” is a membranespanning protein domain. The “intracellular signaling domain” transmits a signal through a signal transduction domain into the cell upon binding of an antigen by the antigen-binding domain. Such signaling activates an activity of the cell. “Co-stimulatory domains” are accessory signaling domains that further transmit signals.

[0074] Provided herein are chimeric antigen receptors (CARs) that further comprise an extracellular accessory domain through which T-cells expressing the CARs can be activated in an antigen-independent fashion. Specifically, the extracellular accessory domain of the present disclosure is an “antibody-inducible domain” through which T-cells expressing the CARs can be activated upon binding of an antibody specific to the antibody-inducible domain. In some embodiments, the linker comprises a “first linker” located between the antigen-binding domain and the antibody-inducible domain. In some embodiments, the linker further comprises a “second linker” located between the antibody-inducible domain and the transmembrane domain.A. Signal Peptide

[0075] A signal peptide that may be present at the N-terminus of a nascent CAR of the present disclosure during expression in a cell can be a signal peptide of any human Type Itransmembrane protein or any other signal peptide suitable for translocating the nascent CAR to the surface of a human cell. In some embodiments, the signal peptide is derived from a protein of the human immunoglobulin superfamily. In some embodiments, the signal peptide is derived from a CD4, CD8, CD 19, CD28, TCR or immunoglobulin chain.

[0076] An exemplary signal peptide is the GMCSF signal peptide: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 1). Another exemplary signal peptide is the CD8alpha signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO:2). In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 1, or SEQ ID NO:2.B. Antigen-Binding Domains

[0077] The antigen-binding domain (ABD) of the CARs of the present disclosure can possess any desired specificity and can be in any suitable polypeptide form for binding the target antigen and transmitting a signal through the transmembrane domain to the intracellular domain of the CAR. In some embodiments, the antigen-binding domain (ABD) is a single chain antibody (scFV). In some embodiments, such as when the ABD is a scFV, the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises complementarity region (CDR)-Hl, CDR-H2, and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 of an antibody whose sequence is deposited in Version 13.1 (August 2023) of The ABCD (Antibodies Chemically Defined) database hosted by Expasy and operated by the Swiss Institute of Bioinformatics (see, Lima et al., Nucleic Acids Research, 48:D2610264, 2020). In some embodiments, the CDRs are as defined using Kabat nomenclature, which can easily be determined for a given sequence using online tools such as the abYsis annotate tool. In some embodiments, the antigen-binding domain comprises a light chain variable region and a heavy chain variable region of an antibody disclosed in Version 13.1 (August 2023) of The ABCD, which includes 24,485 sequence antibodies directed against 4,171 different target antigens.

[0078] In some embodiments, the antigen comprises citrullinated vimentin (CV). In exemplary embodiments, the antigen-binding domain is a CV-binding domain that comprises a heavy chain variable region (VH) comprising the heavy chain CDRs of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a light chain variable region (VL) comprising the light chain CDRsof SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the CV-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a VL comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the CV-binding domain comprises a VH comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a VL comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO: 8.

[0079] In other embodiments, the antigen comprises CD 19 (also known as B-lymphocyte surface antigen B4). In exemplary embodiments, the antigen-binding domain is a CD19-binding domain that comprises a light chain variable region (VL) comprising the light chain CDRs of SEQ ID NO:9 and a heavy chain variable region (VH) comprising the heavy chain CDRs of SEQ ID NO: 10. In some embodiments, the CD19-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO:9 and a VH comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-binding domain comprises a VL comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:9 and a VH comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 10.

[0080] In further embodiments, the antigen comprises B-cell maturation antigen (BCMA, (also known as tumor necrosis factor receptors superfamily member 17 or TNFRSF17). In exemplary embodiments, the antigen-binding domain is a BCMA-binding domain that comprises a light chain variable region (VL) comprising the light chain CDRs of SEQ ID NO: 11 and a heavy chain variable region (VH) comprising the heavy chain CDRs of SEQ ID NO: 12. In some embodiments, the BCMA-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 11 and a VH comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the BCMA-binding domain comprises a VL comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:11 and a VH comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 12.

[0081] The antigen-binding domains of the CARs of the present disclosure comprising a VH and a VL region, preferably are separated by a polypeptide linker. In some embodiments, theantigen-binding domain is arranged as VH-linker-VL. In other embodiments, the antigen-binding domain is arranged as VL-linker-VH. In some embodiments, the polypeptide linker is from about 4 to about 24 amino acids in length. In exemplary embodiments, the linker is a glycine linker comprising the amino acid sequence of: GGGGSGGGGSGGGGS (SEQ ID NO: 13). In other exemplary embodiments, the linker is a Whitlow linker comprising the amino acid sequence of: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 14). In further exemplary embodiments, the linker is a ABpur linker comprising the amino acid sequence of: ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 15).

[0082] In exemplary embodiments, the antigen-binding domain (ABD) is a CV-binding domain comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 16. In other exemplary embodiments, the ABD is a CD19-binding domain comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 17 or SEQ ID NO: 18. In further exemplary embodiments, the ABD is an BCMA-binding domain comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:20, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO: 19 or SEQ ID NO:20.C. Linker

[0083] The antigen-binding domain and the antibody-inducible domain of the CARs of the present disclosure are separated by linker (1stlinker). The antibody-inducible domain and the transmembrane domain of the CARs of the present disclosure may also be separated by a linker (2ndlinker).

[0084] The linker may be a “flexible polypeptide linker” of from 4 to 24 amino acids in length. In some embodiments, the flexible polypeptide linker comprises [XaXbXcXdXeXfXg]n, wherein Xa, Xb, Xc, and Xd, are independently selected from G and S, Xe, Xf, and Xg are independently selected from G, S, and absent, and n is 1, 2 or 3. That is, the flexible polypeptide linker is rich in glycine and serine residues and is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length and at most 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 amino acids in length.

[0085] The linker may be a “hinge region” of from about 10 to about 20, 30 40 or 50 amino acids in length. In some embodiments, the hinge region is a CD8a hinge region, a CD28 hinge region or an IgG4 hinge region. In some embodiments, the hinge region is an IgG4 hinge region.

[0086] In some embodiments, the 1stlinker is a flexible polypeptide linker. In exemplary embodiments, the 1stlinker is a flexible polypeptide linker and the 2ndlinker is a flexible polypeptide linker, an IgG4 hinge region, or absent. In some embodiments, the 1stlinker is an IgG4 hinge region. In exemplary embodiments, the 1stlinker is an IgG4 hinge region, and the 2ndlinker is an IgG4 hinge region, a flexible polypeptide linker, or absent.

[0087] In some embodiments, the hinge region is a CD8a hinge region. In exemplary embodiments, the CD8a hinge region comprises the amino acid sequence of: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:21), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:21.

[0088] In some embodiments, the hinge region is a CD28 hinge region. In exemplary embodiments, the CD28 hinge region comprises the amino acid sequence of: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:22), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:22.

[0089] In some embodiments, the hinge region is an IgG4 hinge region. In exemplary embodiments, the IgG4 hinge region comprises the amino acid sequence of: ESKYGPPCPPCP (SEQ ID NO:23), or an amino acid sequence differing by one or two amino acids from SEQ ID NO:23.D. Antibody-Inducible Domain

[0090] The antibody-inducible domain (AID) of the CARs of the present disclosure is a polypeptide of from about 60 to about 360 amino acids in length that is bindable by an antibody (e.g., comprises the epitope for the antibody). The AID is located between the antigen-binding domain and the transmembrane domain. In some embodiments the AID is flanked by a 1stlinker or a hinge domain at its N-terminus. In some embodiments the AID is flanked by a 2ndlinker or a hinge at its C-terminus. In some preferred embodiments, the AID comprises afragment of an extracellular region of a human cell surface protein, with the proviso that it does not comprise a fragment of a B-cell or T-cell antigen receptor. Specifically, the AID is at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length and at most 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, or 240 amino acids in length.

[0091] The AID of the CARs of the present disclosure is capable of transmitting a signal through the transmembrane domain to the intracellular domain of the CAR when the AID is bound by an antibody (e.g. a monoclonal antibody). In this way, the AID provides an alternative to the ABD for CAR-specific activation of a T-cell in which the CAR is expressed. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is an IgG antibody. In some embodiments, the IgG antibody is an IgGl antibody. In some preferred embodiments, the monoclonal antibody is a human antibody or a humanized antibody. In other embodiments, the monoclonal antibody is a rodent antibody, such as a mouse monoclonal antibody or a rat monoclonal antibody. In some embodiments, the monoclonal antibody is a human / rodent chimeric antibody.

[0092] In some embodiments, the antibody-inducible domain comprises a member of the group consisting of a vascular endothelial growth factor receptor 2 (VEGFR2) domain, a human epidermal growth factor receptor 2 (HER2) domain, a platelet-derived growth factor receptoralpha (PDGFRa) domain, an interleukin-4 receptor-alpha (IL-4Ra) domain, a CD4 domain, and a CD2 domain. In exemplary embodiments, the antibody-inducible domain comprises a fragment of a human protein selected from the group consisting of domain 3 of vascular endothelial growth factor receptor 2 (VEGFR2-D3), domain 4 of human epidermal growth factor receptor 2 (HER2-D4), domains 3-5 of platelet-derived growth factor receptor-alpha (PDGFRa-D3-D5), domains 1-2, domain 1 or domain 2 of interleukin-4 receptor-alpha (IL-4Ra-Dl-D2, IL-4Ra-Dl, or IL-4Ra-D2), domains 1-2, domain 1 or domain 2 of CD4 (CD4-D1-2, CD4-D1, or CD4-D2), and domains 1-2, domain 1 or domain 2 of CD2 (CD2-D1-D2, CD2-D1, or CD2-D2).

[0093] In some embodiments, the AID comprises a human VEGFR2 domain. In some embodiments, the VEGFR2 domain is bindable by an anti-VEGFR2 antibody or VEGFR2- binding fragment. In some embodiments, the anti-VEGFR2 antibody is ramucirumab. In some embodiments, the anti-VEGFR2 antibody is alacizumab. In exemplary embodiments, theVEGFR2 domain comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:31.

[0094] In some embodiments, the AID comprises a human HER2 domain. In some embodiments, the HER2 domain is bindable by an anti-HER2 antibody or HER2-binding fragment thereof. In some embodiments, the anti-HER antibody is selected from the group consisting of trastuzumab, margetuximab, pertuzumab, and timigutuzumab. In some embodiments, the anti-HER2 antibody is trastuzumab. In exemplary embodiments, the HER2 domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:32.

[0095] In some embodiments, the AID comprises a human PDGFRa domain. In some embodiments, the PDGFRa domain is bindable by an anti-PDGFRa antibody or PDGFRa- binding fragment thereof. In some embodiments the anti-PDGFRa antibody is selected from the group consisting of olaratumab and tovetumab. In exemplary embodiments, the PDGFRa domain comprises the amino acid sequence of SEQ ID NO:33 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:33.

[0096] In some embodiments, the AID comprises a human IL-4Ra domain. In some embodiments, the IL-4Ra domain is bindable by an anti-IL-4Ra antibody or IL-4Ra-binding fragment thereof. In some embodiments, the IL-4Ra antibody is dupilumab. In some embodiments, the IL-4Ra antibody is pascolizumab. In exemplary embodiments, the IL-4Ra domain comprises: i) the amino acid sequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.

[0097] In some embodiments, the AID comprises a human CD4 domain. In some embodiments, the CD4 domain is bindable by an anti-CD4 antibody or CD4-binding fragment. In some embodiments, anti-CD4 antibody is selected from the group consisting of cedelizumab, clenoliximab, ibalizumab, keliximab, priliximab, tregalizumab, zanolimumab, and semzuvolimab. In exemplary embodiments, the CD4 domain comprises: i) the amino acid sequence selected of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

[0098] In some embodiments, the AID comprises a human CD2 domain. In some embodiments, the CD2 domain is bindable by an anti-CD2 antibody or CD2-binding fragment thereof or a lymphocyte function-associated antigen-IgG fusion protein selected from the group consisting of siplizumab. In exemplary embodiments, the CD2 domain comprises: i) the amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:40 or SEQ ID NO:41.E. Transmembrane Domain

[0099] The antibody-inducible domain (and optionally a 2ndlinker or hinge) of the CARs of the present disclosure is separated from the intracellular signaling domain by a transmembrane domain of from about 20 to about 30 amino acids in length.

[0100] In some embodiments, the transmembrane domain is a CD8a transmembrane domain. In exemplary embodiments, the CD8a domain comprises the amino acid sequence of: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:24), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:24.

[0101] In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In exemplary embodiments, theCD28 transmembrane domain is a wild type CD28 transmembrane domain, which comprises the amino acid sequence of: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:25). In some embodiments the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:25.

[0102] In some embodiments, the transmembrane domain is a modified CD28 transmembrane domain. In exemplary embodiments, the amino acid sequence of the modified CD28 transmembrane domain comprises an insertion, substitution, and / or deletion relative to SEQ ID NO:25. In some embodiments, the amino acid sequence of the CD28 transmembrane domain comprises at least one substitution selected from the group consisting of: C165L, Y166L, S167L, T171L (which correspond to C13L, Y14L, S15L and T19L substitutions of the wild type CD28 TM sequence of SEQ ID NO:25). In some embodiments, the modified CD28 transmembrane domain comprises the amino acid sequence of: FWVLVVVGGVLALLLLLVLVAFIIFWV (SEQ ID NO:42). In some embodiments, the CD28 TM domain comprises the consensus amino acid sequence of:FWVLVVVGGVLAX1X2X3LLVX4VAFIIFWV (SEQ ID NO:43), wherein Xi is C or L, X2is Y or L, X3 is S or L, and X4 is T or L, and / or wherein at least one of Xi, X2, X3, and X4 is L.F. Intracellular Signaling Domain

[0103] The intracellular signaling domain of the CARs of the present disclosure comprises a CD3zeta signal transduction domain. In some preferred embodiments, the intracellular signaling domain further comprises a co-stimulatory domain.

[0104] In some embodiments, the co-stimulatory domain can be derived from, for example, CD28, 4-1BB, CD2, CD27, CD30, 0X40, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3, B7-H4 and others. In some embodiments, the CARs of the present disclosure comprise two or more co-stimulatory signaling domains (e.g., CD28 and 4- IBB).

[0105] In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain. In exemplary embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence of: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:26), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:26.

[0106] In some embodiments, the co-stimulatory domain is a 4- IBB co-stimulatory domain. In exemplary embodiments, the 4- IBB co-stimulatory domain comprises the amino acid sequence of: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:27), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:27.

[0107] In exemplary embodiments, the CD3ζ signal transduction domain comprises the amino acid sequence of: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:28), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:28.

[0108] In some embodiments, the intracellular signaling domain comprises a CD28 co- stimulatory domain and a CD3ζ signal transduction domain. In exemplary embodiments, the intracellular signaling domain comprise the amino acid sequence of SEQ ID NO:29 or an aminoacid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:29.

[0109] In some embodiments, the intracellular signaling domain comprises a 4- IBB costimulatory domain and a CD3ζ signal transduction domain. In exemplary embodiments, the intracellular signaling domain comprise the amino acid sequence of SEQ ID NO:30 or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:30.II. Nucleic Acids Encoding CARs

[0110] Nucleic acid molecules (polynucleotides) encoding CARs of the present disclosure can be isolated molecules, or can be included within a cassette or a vector.A. Nucleic Acids

[0111] The present disclosure provides nucleic acid molecules (polynucleotides) comprising a nucleotide sequence that encodes (e.g., comprises the coding region of) a CAR as described herein. The nucleic acid can be in the form of DNA or in the form of RNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or singlestranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In some embodiments, the polynucleotides as described herein are isolated.

[0112] For example, nucleic acids can comprise nucleotide sequences that encode the CAR polypeptides described herein or a portion thereof. In some embodiments, the nucleotide sequences encode a polypeptide comprising the amino acid sequence of a specific SEQ ID NO, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with a specific SEQ ID NO.

[0113] In some embodiments, the polynucleotides are variants that comprise alterations in the coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain alterations that are silent. In other embodiments, the polynucleotide variants comprise substitutions, additions, or deletions, but do not alter the binding properties of the antigen-binding domain of the encoded CAR polypeptide. In some embodiments, the polynucleotide variants contain one or more alterations that do not produce any changes in the amino acid sequence of the CAR. In some embodiments, polynucleotide variants contain “silent”substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host cell.B. Expression Cassettes and Vectors

[0114] The present disclosure further provides expression cassettes and vectors comprising a nucleic acid molecule encoding a CAR described herein. In some embodiments, the nucleic acid molecule can further comprise an expression control sequence operatively linked with the nucleotide sequence encoding the CAR. In some embodiments, the nucleic acid molecule encoding the disclosed CAR can be inserted into an expression vector in operable combination with an expression control sequence appropriate for expression of the disclosed CAR in a desired host cell. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and / or expression of the CAR polypeptide in a suitable host cell.

[0115] For example, a polynucleotide can include one or more transcription regulatory elements, such as promoters or enhancers, which, when the polynucleotide is present in a cell, cause the sequence encoding the CAR to be expressed within the cell.

[0116] Nucleic acids disclosed herein can be incorporated into vectors that can be introduced into a host cell. Such vectors include, without limitation, viral vectors and plasmids. Exemplary viral vectors include but are not limited to retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Lentiviruses are particularly desirable as they are able to deliver a large amount of genetic material into the genome of a host cell and because they are able to infect non-dividing cells.III. Host Cells

[0117] The present disclosure also provides host cells (e.g., recombinant cells) comprising a nucleic acid molecule, an expression cassette, or an expression vector encoding a CAR described herein. In some embodiments, the host cell is a eukaryotic cell, such as a mammalian cell. In other embodiments, the host cell is a prokaryotic cell, such as a bacterial cell.

[0118] The nucleic acid molecule encoding the disclosed CAR can be delivered to a host cell by transformation, transfection or transduction as is known to one of skill in the art. The resulting recombinant (host) cell can be an immune cell, including but not limited to a T-cell, such as a CD4+ T-cell, CD8alpha+ T-cell, or CD8beta+ T cell. In some embodiments, the T-cell is a Treg cell. In some embodiments, the T-cell is a T helper (Th) cell. In some embodiments, theT-cell is a cytolytic T lymphocyte (CTL). In some embodiments, the recombinant (host) cell comprising the nucleic acid molecule encoding the disclosed CAR is a lymphocyte (e.g., T-cell, B-cell or NK-cell).

[0119] In some embodiments, the cells expressing the CARs of this disclosure are Treg cells. “Regulatory T cells,” or “Treg cells,” are cells belonging to a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and selftolerance. Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. Tregs are characterized by expression of FoxP3. Surface markers for Tregs include CD4, CD25high (high molecular density) and CD1271ow (low molecular density). Mouse and human Tregs express GITR / AITR, and CTLA-4. Human CD4+FoxP3+ Treg cells can be divided into three sub-populations:(1) CD45RA+CD25+FoxP31ow resting Treg cells,(2) CD45RO+CD25highFoxP3high activated Treg cells, and(3) proinflammatory cytokine-producing CD45RO+CD25+FoxP31ow non-suppressive effector T cells (Teffs).

[0120] In some embodiments, the host cell is an autologous cell (e.g., recombinant progeny of a cell obtained from the intended recipient). That is, the cells to be transformed with the nucleic acids disclosed herein can be cells taken from a subject into whom the recombinant cells are to be administered. In this way, issues of an allogeneic immune response can be mitigated. Even so, in other embodiments, the host cell is an allogeneic cell (e.g., recombinant progeny of a cell obtained from an immunologically distinct individual than the intended recipient). Regardless of their origin, host cells can be expanded ex vivo before administration to a subject.

[0121] Also, the proteins produced by a transformed / recombinant host can be purified according to any suitable method. Such methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. In some embodiments, proteins can also be physically characterized using such techniques as proteolysis, high performance liquid chromatography, nuclear magnetic resonance and x-ray crystallography.IV. Compositions

[0122] Also disclosed are pharmaceutical compositions comprising a host cell (e.g., recombinant cell) comprising a nucleic acid molecule encoding and expressing the disclosed CAR polypeptide, and a pharmaceutically acceptable excipient, as well as methods of use in the treatment of a disease or disorder.

[0123] As used herein, the term “pharmaceutical composition” refers to a composition comprising a pharmaceutical agent (e.g., a drug or a recombinant Treg cell as described herein) and a pharmaceutically acceptable excipient.

[0124] As used herein, the term “pharmaceutically acceptable” refers to a compound that is compatible with the other ingredients of a pharmaceutical composition and can be safely administered to a subject. The term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. Pharmaceutical compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and techniques for their administration one may refer to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman’s The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

[0125] Pharmaceutical compositions will generally be sterile, at least for human use. A pharmaceutical composition will generally comprise pharmaceutically acceptable excipients for buffering and preservation in storage, and can include buffers for appropriate delivery, depending on the route of administration. Examples of pharmaceutically acceptable excipients include, without limitation, normal (0.9%) saline, phosphate-buffered saline (PBS) Hank’s balanced salt solution (HBSS) and multiple electrolyte solutions.

[0126] Pharmaceutical compositions can be formulated for any route of administration. However, in most embodiments of the present disclosure, the pharmaceutical compositions are formulated for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraarterial injection, either bolus or infusion) administration.

[0127] Injectable (e.g., intravenous) pharmaceutical compositions can comprise a solution of the pharmaceutical agent suspended in a pharmaceutically acceptable excipient, such as an aqueous excipient. Any of a variety of aqueous excipients can be used, e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Often, normal buffered saline (135-150 mM NaCl) will be used. The pharmaceutically acceptable excipients can comprise auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the composition can be formulated for intravenous administration.

[0128] Pharmaceutical compositions suitable for parenteral administration, such as intravenous administration, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the compositions isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In the practice of the present invention, pharmaceutical compositions can be administered, for example, by intravenous infusion. The pharmaceutical compositions can be presented in unit-dose or multi-dose sealed containers.

[0129] Host cells can be cryopreserved. Cryopreservation can include formulating host cells with a cryopreservation agent, such as DMSO. Commercially available media include, for example, CryoStor® and pZerve®, available from Millipore Sigma.

[0130] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of the pharmaceutical agent given to an individual at each administration. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable label (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress.

[0131] The pharmaceutical composition can be packaged or prepared in unit dosage form. In such form, the pharmaceutical composition is subdivided into unit doses containing appropriate quantities of the pharmaceutical agent, e.g., according to the dose of the pharmaceutical agent or the concentration of the pharmaceutical agent in the pharmaceutical composition. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the pharmaceutical composition. The pharmaceutical composition can, if desired, also contain other compatible therapeutic agents.V. Methods of Use

[0132] Host cells (e.g., recombinant T cells) that express the CARs disclosed herein are useful in the treatment of various diseases and disorders. Methods of use comprise administering an effective amount of a pharmaceutical composition of this disclosure comprising CAR- expressing cells, such as CAR-T cells, to a subject in need thereof (e.g., an individual suffering from a disease or disorder). In some methods of the present disclosure in which the subject has cancer, the host cells comprise CD3+ T cells expressing a CAR that binds an antigen expressed by cells of the cancer. In some methods of the present disclosure in which the subject has an autoimmune disease or inflammatory disorder, the host cells comprise Treg cells expressing a CAR that binds an antigen associated with the autoimmune disorder or the inflammatory disorder. In some embodiments, the host cells are autologous to the subject.

[0133] As used herein, the term “subject” refers to an individual animal. The term “patient” as used herein refers to a subject under the care or supervision of a health care provider such as a doctor or nurse. Subjects include mammals, such as humans and non-human primates, such as monkeys, as well as dogs, cats, horses, bovines, rabbits, rats, mice, goats, pigs, and other mammalian species. Subjects can also include avians. In some embodiments, the subject is a human patient. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc. Subjects with a disease or disorder can include individuals that have not received treatment, are currently receiving treatment, have had treatment, and those that have discontinued treatment.

[0134] As used herein, the terms “effective amount,” “effective dose,” and “therapeutically effective amount,” refer to an amount of a pharmaceutical agent that is sufficient to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or ameliorate a disorder. In some examples, an “effective amount” is one that treats (includingprophylaxis) one or more symptoms and / or underlying causes of any of a disorder or disease and / or prevents progression of a disease. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least any of 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0135] The pharmaceutical composition can be administered by any suitable route, including but not limited to intravenous, subcutaneous, intramuscular or intraperitoneal routes. An example of administration of a pharmaceutical composition includes storing the composition at 10 mg / ml in sterile isotonic aqueous saline solution for injection at 4°C, and diluting it in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient. The pharmaceutical composition is administered by intravenous infusion over the course of 1 hour at a dose of between 0.2 and 10 mg / kg. In other embodiments, the pharmaceutical composition is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via sub-cutaneous bolus injection.

[0136] The dose of the pharmaceutical composition is chosen in order to provide effective therapy for the patient and is in the range of less than 0.1 mg / kg body weight to about 25 mg / kg body weight or in the range 1 mg- 2 g per patient. In some cases, the dose is in the range 1- 100 mg / kg, or approximately 50 mg- 8000 mg / patient. The dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, depending on the pharmacokinetics of the composition (e.g., half-life of the composition in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the composition). In some embodiments, the in vivo half-life of between about 7 and about 25 days and composition dosing is repeated between once per week and once every 3 months.

[0137] Administration can be periodic. Depending on the route of administration, the dose can be administered, e.g., once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer (e.g., once every 2, 3, 4, or 6 months). In some cases, administration is more frequent, e.g., 2 or 3 times per day. The patient can be monitored to adjust the dosage and frequency of administration depending on therapeutic progress and any adverse side effects, as will be recognized by one of skill in the art.

[0138] Thus, in some embodiments, additional administration is dependent on patient progress, e.g., the patient is monitored between administrations. For example, after the first administration or round of administrations, the patient can be monitored for rate of symptom relief.VI. Kits

[0139] As used herein, the term “kit” refers to a collection of items intended for use together. In some embodiments, the kit comprises an agent and instructions for use thereof. In some embodiments, the kit further comprises a container, such as a vial that contains a composition as disclosed herein. For instance, a kit can include a container, such as a bag or bottle for intravenous administration, comprising a pharmaceutical composition comprising a plurality of recombinant cells that express a CAR of the present disclosure. In some embodiments, the kit can further comprise a fluidic conduit, such as a plastic tube, with a drip chamber. The drip chamber can communicate through a fluidic conduit with an intravenous needle. The fluidic conduit also can comprise one or more Y -sites and a roller clamp.VII. Enumerated EmbodimentsEmbodiment 1. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain, a first linker, an antibody-inducible domain, a transmembrane domain, and an intracellular signaling domain, wherein the antibody-inducible domain is a polypeptide of from about 60 to about 360 amino acids in length.Embodiment 2. The CAR of embodiment 1 , wherein the antibody-inducible domain comprises a member of the group consisting of a vascular endothelial growth factor receptor 2 (VEGFR2) domain, a human epidermal growth factor receptor 2 (HER2) domain, a platelet-derived growth factor receptor-alpha (PDGFRa) domain, an interleukin-4 receptor-alpha (IL-4Ra) domain, a CD4 domain, and a CD2 domain.Embodiment 3. The CAR of embodiment 2, wherein the antibody-inducible domain comprises a VEGFR2 domain.Embodiment 4. The CAR of embodiment 3, wherein the VEGFR2 domain is bindable by an anti-VEGFR2 antibody or VEGFR2-binding fragment thereof selected from the group consistingof ramucirumab and alacizumab, optionally wherein the VEGFR2 domain is bindable by ramucirumab.Embodiment 5. The CAR of embodiment 4, wherein the VEGFR2 domain comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:31.Embodiment 6. The CAR of embodiment 2, wherein the antibody-inducible domain comprises a HER2 domain.Embodiment 7. The CAR of embodiment 6, wherein the HER2 domain is bindable by an anti- HER2 antibody or HER2 -binding fragment thereof selected from the group consisting of trastuzumab, margetuximab, pertuzumab, and timigutuzumab, optionally wherein the HER2 domain is bindable by trastuzumab.Embodiment 8. The CAR of embodiment 7, wherein the HER2 domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:32.Embodiment 9. The CAR of embodiment 2, wherein the antibody-inducible domain comprises a PDGFRa domain.Embodiment 10. The CAR of embodiment 9, wherein the PDGFRa domain is bindable by an anti-PDGFRa antibody or PDGFRa-binding fragment thereof selected from the group consisting of olaratumab and tovetumab.Embodiment 11. The CAR of embodiment 10, wherein the PDGFRa domain comprises the amino acid sequence of SEQ ID NO:33 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:33.Embodiment 12. The CAR of embodiment 2, wherein the antibody-inducible domain comprises an IL-4Ra domain.Embodiment 13. The CAR of embodiment 12, wherein the IL-4Ra domain is bindable by an anti-IL-4Ra antibody or IL-4Ra-binding fragment thereof selected from the group consisting of dupilumab and pascolizumab.Embodiment 14. The CAR of embodiment 13, wherein the IL-4Ra domain comprises: i) the amino acid sequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.Embodiment 15. The CAR of embodiment 2, wherein the antibody-inducible domain comprises a CD4 domain.Embodiment 16. The CAR of embodiment 15, wherein the CD4 domain is bindable by an anti- CD4 antibody or CD4-binding fragment thereof selected from the group consisting of cedelizumab, clenoliximab, ibalizumab, keliximab, priliximab, tregalizumab, zanolimumab, and semzuvolimab.Embodiment 17. The CAR of embodiment 16, wherein the CD4 domain comprises: i) the amino acid sequence selected of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.Embodiment 18. The CAR of embodiment 2, wherein the antibody-inducible domain comprises a CD2 domain.Embodiment 19. The CAR of embodiment 18, wherein the CD2 domain is bindable by an anti- CD2 antibody or CD2-binding fragment thereof, wherein the anti-CD2-antibody is siplizumab.Embodiment 20. The CAR of embodiment 19, wherein the CD2 domain comprises: i) the amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:40 or SEQ ID NO:41.Embodiment 21. The CAR of any one of embodiments 1-20, wherein a T-cell in which the CAR is expressed is activatable upon binding of an antibody or fragment thereof to the antibodyinducible domain,Embodiment 22. The CAR of any one of embodiments 1-21, further comprising a second linker located between the antibody-inducible domain and the transmembrane domain.Embodiment 23. The CAR of any one of embodiments 1-21, wherein the first linker and / or the second linker is a flexible polypeptide linker of from 4 to 24 amino acids in length.Embodiment 24. The CAR of embodiment 23, wherein the flexible polypeptide linker comprises the amino acid sequence of [XaXbXcXdXeXfXg]n, wherein Xa, Xb, Xc, and Xd, are independently selected from G and S, Xe, Xf and Xg are independently selected from G, S, and absent, and n is 1, 2 or 3, optionally wherein the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:46.Embodiment 25. The CAR of any one of embodiments 1-21, wherein the first linker and / or the second linker is a hinge region of from 10 to 50 amino acids in length.Embodiment 26. The CAR of embodiment 25, wherein the first linker is the flexible polypeptide linker, and the second linker is a hinge region of from 10 to 50 amino acids in length.Embodiment 27. The CAR of embodiment 25 or embodiment 26, wherein the hinge region is selected from the group consisting of a CD8a hinge, a CD28 hinge region, and an IgG4 hinge region.Embodiment 28. The CAR of embodiment 27, wherein the hinge region is a CD8a hinge region comprising the amino acid sequence of SEQ ID NO:21.Embodiment 29. The CAR of embodiment 28, wherein the hinge region is a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:22.Embodiment 30. The CAR of embodiment 29, wherein the hinge region is an IgG4 hinge region comprising the amino acid sequence of SEQ ID NO: 23.Embodiment 31. The CAR of any one of embodiments 1-30, wherein the transmembrane domain is a CD28 transmembrane domain or a CD8a transmembrane domain.Embodiment 32. The CAR of embodiment 31, wherein the transmembrane domain is a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 43, optionally wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:42.Embodiment 33. The CAR of embodiment 31, wherein the transmembrane domain is a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24.Embodiment 34. The CAR of any one of embodiments 1-33, wherein the intracellular signaling domain comprises a CD3ζ signal transduction domain.Embodiment 35. The CAR of embodiment 34, wherein the CD3ζ signal transduction domain comprises the amino acid sequence of SEQ ID NO:28.Embodiment 36. The CAR of embodiment 34 or embodiment 35, wherein the intracellular signaling domain further comprises a co-stimulatory domain.Embodiment 37. The CAR of embodiment 36, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain or a 4- IBB co-stimulatory domain.Embodiment 38. The CAR of embodiment 37, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain, optionally wherein the CD28 costimulatory domain comprises the amino acid sequence of SEQ ID NO:26.Embodiment 39. The CAR of embodiment 38, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:29.Embodiment 40. The CAR of embodiment 37, wherein the co-stimulatory domain comprises a 4-1BB co-stimulatory domain, optionally wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:27.Embodiment 41. The CAR of embodiment 40, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:30.Embodiment 42. The CAR of any one of embodiments 1-41, further comprising a signal peptide.Embodiment 43. The CAR of embodiment 42, wherein the signal peptide is a CD8a signal peptide or a GM-CSF signal peptide.Embodiment 44. The CAR of any one of embodiments 1-43, wherein the antigen-binding domain comprises a single chain antibody fragment (scFV), or a single domain antibody (sdAb).Embodiment 45. The CAR of embodiment 44, wherein the antigen-binding domain comprises a scFv.Embodiment 46. The CAR of embodiment 44, wherein the antigen-binding domain comprises a sdAb, optionally wherein the sdAb comprises a heavy chain variable region fragment (VHH).Embodiment 47. The CAR of any one of embodiments 1-46, wherein the antigen-binding domain binds to an antigen selected from the group consisting of citrullinated vimentin (CV), CD 19, and BCMA.Embodiment 48. The CAR of embodiment 47, wherein the antigen-binding domain binds to CV, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and light chain CDRs of the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO: 8;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; or(iii) the amino acid sequence of SEQ ID NO: 16.Embodiment 49. The CAR of embodiment 47, wherein the antigen-binding domain binds to CD19, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NOTO, and light chain CDRs of the amino acid sequence of SEQ ID NO:9;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9; or(iii) the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.Embodiment 50. The CAR of embodiment 47, wherein the antigen-binding domain binds to BCMA, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NO: 12, and light chain CDRs of the amino acid sequence of SEQ ID NO: 11 ;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NOT 1; or(iii) the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:20.Embodiment 51. A nucleic acid encoding the CAR of any one of embodiments 1-50.Embodiment 52. An expression vector comprising the nucleic acid of embodiment 51 in operable combination with an expression control sequence.Embodiment 53. The expression vector of embodiment 52, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.Embodiment 54. The expression vector of embodiment 53, wherein the viral vector is a lentiviral vector.Embodiment 55. The expression vector of embodiment 52, wherein the vector is a plasmid.Embodiment 56. A host cell comprising the expression vector of any one of embodiments 52-55.Embodiment 57. A modified T-cell that has been engineered to express the CAR of any one of embodiments 1-50, wherein the modified T-cell is a mammalian T-cell.Embodiment 58. A pharmaceutical composition comprising a plurality of the modified T-cells of embodiment 57 and a pharmaceutically acceptable excipient.Embodiment 59. The pharmaceutical composition of embodiment 58, wherein the modified T- cell is a human T-cell.Embodiment 60. The pharmaceutical composition of embodiment 59, wherein human T-cell is a regulatory T-cell (Treg) that is CD4+, CD25+, and CD1271o.Embodiment 61. The pharmaceutical composition of embodiment 60, wherein the human Treg is FOXP3+ and HELIOS+.Embodiment 62. A method of treating a disease or disorder comprising administering an effective amount of the pharmaceutical composition of any one of embodiments 58-61 to a subject in need thereof.Embodiment 63. A method of treating a subject suffering from an autoimmune disease or an inflammatory disorder comprising administering an effective amount of the pharmaceutical composition of any one of embodiments 58-61 to the subject, wherein the CAR expressed by the modified T-cells comprising an antigen-binding domain that binds to an antigen expressed on cells of the autoimmune disease or the inflammatory disorder.Embodiment 64. A method of treating a subject suffering from cancer comprising administering an effective amount of the pharmaceutical composition of embodiment 58 or embodiment 59 tothe subject, wherein the CAR expressed by the modified T-cells comprises an antigen-binding domain that binds to an antigen expressed on cells of the cancer.Embodiment 65. A kit comprising a container containing the pharmaceutical composition of any one of embodiments 58-61, communicating through a fluidic conduit to a drip chamber, wherein the drip chamber is configured to communicate through a fluidic conduit with an intravenous needle.EXAMPLES

[0140] Abbreviations: Ab (antibody); ABD (antigen-binding domain); AID (antibodyinducible domain); CAR (chimeric antigen receptor); CD (cluster of differentiation); CV (citrullinated vimentin); D (domain); EGFR (epidermal growth factor receptor); EGFRt (EGFR tag); extracellular domain (EC or ECD); FACS (fluorescence activated cell sorting); HER2 (human epidermal growth factor receptor 2); IL-2 (interleukin 2); IL4Ra (interleukin 4 receptor); MOI (multiplicity of infection); PDGFRa (platelet-derived growth factor receptor alpha); Prom, (promoter); scFv (single-chain variable fragment); SR (serum replacement); TM (transmembrane domain); Treg (regulatory T-cell); VEGFR2 (vascular endothelial growth factor receptor 2); and WT (wild-type).

[0141] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention, which is defined by the claims.Example 1: Materials and Methods for Construction and Testing of T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With an Antibody-Inducible Domain (AID)

[0142] Binding of a specific antigen by the antigen-binding domain of a CAR expressed on a T-cell is required for CAR-specific activation. In addition, selective detection and ablation of CAR-expressing T-cells typically requires co-expression of a tag, such as EGFRt. In order to address one or more of these shortcomings, new CAR backbones were constructed and tested. In particular, the CARs of the present disclosure were engineered to contain an antigen-inducible domain (AID). The AID serves as a tool to activate, detect and ablate CAR-T cells.

[0143] Lentiviral construct production. A standard CAR construct and various CAR constructs containing an antibody-inducible domain were produced, with each containing a coding region of an EGFRt tag following a P2A (ribosome skip) site for co-expressing of the CAR and the EGFR5. The constructs were packaged as lentiviral particles for transduction into cells of interest. HEK 293 FT suspension cells (Invitrogen) were seeded at 4.7 x 106 / mL and transfected with transfer and packaging plasmids (Aldevron pALD-VSV-G, pALD-GagPol, pALD-Rev) using LV_MAX transfection reagent as per manufacturer's protocol (Gibco). Viral supernatant was collected and filtered through a 0.45 pm polyvinylidene difluoride filter to remove cell debris. The viral supernatant was centrifuged at 10,000 x g overnight to concentrateviral vectors. After 12-16 hours, supernatant was removed, and the viral vector pellet was resuspended. Resuspended viral vector was aliquoted and stored at -80°C.

[0144] Functional titer assay. Functional titers were assessed to identify ideal transduction conditions of T cells. SupTl cells (ATCC) were cultured in RPMI medium supplemented with 10% fetal bovine serum. On the day of transduction, 50 pL of SupTl cells at 4 x 105cells / mL were added to each well of a 96-well plate. Lentivirus stock was thawed at room temperature and diluted 1 :20 in culture medium. Subsequently, a three-fold serial dilution was performed from 60 to 393,660-fold in culture medium. 50 pL of diluted lentivirus were added to a 96-well plate containing 50 pL of cells. On day 2 of transduction, 100 pL of culture media was added. On day 3, transduced cells were harvested and stained with appropriate antibody to detect surface markers. Labeled cells were analyzed by flow cytometry. Untransduced SupTl cells were used as negative control to set a gate for flow cytometry analysis. Viral dilution that produced between 5 - 20% maker positive cells were used for the calculation of functional titer, based on the following formula:

[0145] J urkat-FF -Luciferase transduction. CARs of interest were expressed in a reporter cell line by transduction with a lentiviral vector, which was produced as described above. The reporter cell line (Jurkat-NFAT-FF-luc) expresses firefly luciferase under the control of the nuclear factor of activated T-cells (NFAT) response elements. CAR expression and Jurkat cell activation were assessed by measuring bioluminescence and by flow cytometry.

[0146] Jurkat cell activation by CAR antigen-expressing target cells. CAR-expressing Jurkat cells were co-cultured with CAR antigen-expressing K562 cells overnight at various Jurkat-to-K562 cell ratios. After co-culture, a portion of the cells was used to assess luciferase expression as a marker of activation through the CAR. Luciferase expression was detected by measuring luminescence with SpectraMax plate reader. The remaining portion of the cells was used for flow cytometry, including to assess percentages of cells expressing CD25 as another marker of activation through the CAR.

[0147] Jurkat cell activation by antibody. CAR-expressing Jurkat cells were incubated overnight in medium containing 3 pg / ml of a monoclonal antibody as a stimulus or in the absence of a stimulus. The monoclonal antibodies tested included: ramucirumab (anti-VEGFR2Ab); trastuzumab (anti-HER2 Ab); and dupilumab (anti-IL4Ra Ab). The percentages of cells expressing CD25 as marker of activation through the CAR was measured by flow cytometry.

[0148] Treg isolation and expansion. Primary human Treg cells were sourced from healthy donors from leukoreduction chamber residuals or leukopaks. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll-Paque Plus. CD25+ T-cells were enriched by positive selection. Treg cells were next isolated using FACS by gating for CD4+ / CD25+ / CD1271o cells.

[0149] Treg cell transduction and expansion. CARs of interest were expressed in Treg cells by transducing Tregs with a lentiviral vector, which was produced as described above. After isolation, Tregs were stimulated on Day 0 with CTS™ Dynabeads™ Treg Xpander reagent (Gibco) at a 1: 1 bead to cell ratio in the presence of recombinant human IL-2 (300 lU / mL). On Day 3, Tregs were transduced via spinfection by adding the appropriate volume of virus to achieve an MOI of about 12 based on the Day 0 cell count. Tregs were expanded by addition of fresh medium containing IL-2 on Day 4, Day 5 and Day 7. On Day 9, expanded CAR+ Tregs were subjected to a secondary stimulation.

[0150] Secondary stimulation ofCAR+ Tregs. Expanded CAR+ Tregs were split and subjected to one of four distinct stimulation conditions in the presence of IL-2: 1) anti-CD3 and anti-CD28 antibody activation beads at a cell: bead ratio of 1:1; 2) anti-VEGFR2 antibody beads at a cel I: bead ratio of 1:3; 3) 1.0 pg / mL anti-VEGFR2 antibody (ramucirumab); and 4) 3.5 pg / mL anti-VEGFR2 antibody (ramucirumab). Fold change in Treg numbers after culturing the expanded cells under each condition was assessed on Day 14. Treg populations (CAR+ = EGFRt+) and (CAR- = EGFRt-) were also assessed by flow cytometry.

[0151] Flow cytometry. Expression of EGFRt, CAR, and CD25 on transduced cells was assessed by staining cells with anti-EGFRt, anti-scFV (CAR), and anti-CD25 antibodies and fluorescence staining was subsequently measured by flow cytometry.Example 2: T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With a VEGFR2 Antibody-Inducible Domain (AID)

[0152] This example describes new CAR backbones comprising domain 3 of vascular endothelial growth factor receptor 2 (VEGFR2) as an antibody-inducible domain, and expressionand subsequent activation of the new CARs on recombinant Jurkat cells (immortalized human T- cells) and recombinant Tregs (primary human T-cells) by use of the materials and methods of Example 1.

[0153] Expression cassettes encoding a standard CAR or a VEGFR2-containing CAR, as well as an EGFRt tag, were designed (FIG. 1). Lentiviral vectors were subsequently generated for expression of CARs by transduced T-cells. FIG. 2 shows a cartoon of a standard CAR and four different VEGFR2-containing CARs expressed on a cell surface. The combination of a VEGFR2 domain and a first and optional second linker are shown in Table 2-la.Table 2-la. Antibody-Inducible Domain and Flanking Sequences of VEGFR2-containing CARs

[0154] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies and expression of the tag and CAR was then assessed by flow cytometry. As shown in Table 2- la and Table 2-lb, over 50% of the transduced Jurkat cell populations expressed the EGFRt tag and either the standard CAR or one of the VEGFR2-containing CARs.Table 2-lb. Cells Engineered to Express VEGFR2-containing CARs

[0155] Activation of transduced Jurkat cells was then assessed by measuring CD25 (interleukin-2 receptor alpha chain) expression by transduced cells in the presence or absence of antigen (CAR antigen-expressing K562 cells) at a target-to-effector cell ratio (K562:Jurkat) of 4: 1. As shown FIG. 3A, Jurkat cells expressing either the standard CAR or one of the VEGFR2-containing CARs were specifically activated through the common antigen-binding domain of the CARs when the CAR-expressing Jurkat cells were co-cultured with antigen-expressing K562 cells.

[0156] Activation of transduced Jurkat cells was further assessed by measuring CD25 expression by transduced cells in the presence or absence of an antibody capable of binding to the VEGFR2 domain of some CARs of the present disclosure. As shown in FIG. 3B, only very small percentages of untransduced Jurkat cells (Mock) and standard-CAR expressing Jurkat cells expressed CD25 in the presence or absence of an antibody stimulus. In contrast, Jurkat cells expressing VEGFR2-containing CARs were activated by culturing the cells in the presence of an anti-VEGFR2 antibody. Neither the anti-HER2 antibody nor the anti-IL4RA antibody induced expression of CD25 on cells expressing VEGFR2-containing CARs above background levels observed in the absence of an antibody (no stimulus). As such, FIG. 3B demonstrates that T cells expressing VEGFR2-containing CARs can be activated in a CAR-specific manner when contacted with an anti-VEGFR2 antibody.

[0157] Activation of transduced Tregs was assessed by measuring Treg expansion after subjecting the Tregs to one of four restimulation conditions as depicted in FIG. 4A. In brief, Tregs expressing a VEGFR2-containing CAR (VEGFR2 CAR 1 of FIG. 2) were stimulated in a CAR-non-specific manner by culturing the cells in the presence of CD3 / CD28 beads and IL-2 or in a CAR-specific (and antigen-binding domain-non-specific) manner by culturing the cells in the presence of anti-VEGFR2 beads or an anti-VEGFR2 antibody (ramucirumab) and IL-2. As shown in FIG. 4B, Tregs expressing a VEGFR2-containing CAR were expanded to comparable levels by Day 14 with an anti-VEGFR2 re-stimulus (beads or antibody) introduced on Day 9, which was somewhat lower than the level of expansion observed by Day 14 with anti-CD3 / anti- CD28 beads re-stimulus introduced on Day 9. Interestingly, use of an anti-VEGFR2 re-stimulus apparently resulted in the selective expansion of CAR-expressing Tregs as indicated by the increased percentages of EGFRt-positive Tregs observed in the transduced Treg populations restimulated with anti-VEGFR2 beads or anti-VEGFR2 antibody, as compared to transduced Tregs re-stimulated with anti-CD3 / anti-CD28 beads (see, Table 2-2).Table 2-2. Percentages of VEGFR2-CAR Expressing Tregs After RestimulationExample 3: T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With a HER2 Antibody-Inducible Domain (AID)

[0158] This example describes new CAR backbones comprising domain 4 of human epidermal growth factor receptor 2 (HER2) as an antibody-inducible domain, and expression and subsequent activation of the new CARs on recombinant Jurkat cells (immortalized human T- cells) by use of the materials and methods of Example 1.

[0159] Expression cassettes encoding a standard CAR or a HER2-containing CAR, as well as an EGFRt tag, were designed (FIG. 5). Lentiviral vectors were subsequently generated for expression of CARs by transduced T-cells. FIG. 6 shows a cartoon of a standard CAR and three different HER2-containing CARs expressed on a cell surface. The combination of a HER2 domain and a first and optional second linker are shown in Table 3-la.Table 3-la. Antibody-Inducible Domain and Flanking Sequences of HER2-containing CARs

[0160] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies and expression of the tag and CAR was then assessed by flow cytometry. As shown in Table 3- la and Table 3-lb, over 50% of the transduced Jurkat cell populations expressed the EGFRt tag and either the standard CAR or one of the HER2-containing CARs.Table 3-lb. Cells Engineered to Express HER2-containing CARs

[0161] Activation of transduced Jurkat cells was then assessed by measuring CD25(interleukin-2 receptor alpha chain) expression by transduced cells in the presence or absence of antigen (CAR antigen-expressing K562 cells) at a target-to-effector cell ratio (K562:Jurkat) of 4: 1. As shown FIG. 7A, Jurkat cells expressing either the standard CAR or one of the HER2- containing CARs were specifically activated through the common antigen-binding domain of the CARs when the CAR-expressing Jurkat cells were co-cultured with antigen-expressing K562 cells.

[0162] Activation of transduced Jurkat cells was further assessed by measuring CD25 expression by transduced cells in the presence or absence of an antibody capable of binding to the HER2 domain of some CARs of the present disclosure. As shown in FIG. 7B, only very small percentages of untransduced Jurkat cells (Mock) and standard-CAR expressing Jurkat cells expressed CD25 in the presence or absence of an antibody stimulus. In contrast, Jurkat cells expressing HER2-containing CARs were activated by culturing the cells in the presence of an anti-HER2 antibody. Neither the anti-VEGFR2 antibody nor the anti-IL4RA antibody induced expression of CD25 on cells expressing HER2-containing CARs above background levels observed in the absence of an antibody (no stimulus). As such, FIG. 7B demonstrates that T cells expressing HER2-containing CARs can be activated in a CAR-specific manner when contacted with an anti-HERR2 antibody.Example 4: T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With a PDGFR-alpha Antibody-Inducible Domain (AID)

[0163] This example describes new CAR backbones comprising domains 3 through 5 of platelet-derived growth factor receptor-alpha (PDGFRa) as an antibody-inducible domain, and expression and subsequent activation of the new CARs on recombinant Jurkat cells (immortalized human T-cells) by use of the materials and methods of Example 1.

[0164] Expression cassettes encoding a standard CAR or a PDGFRa-containing CAR, as well as an EGFRt tag, were designed (FIG. 8). Lentiviral vectors were subsequently generated for expression of CARs by transduced T-cells. FIG. 9 shows a cartoon of a standard CAR and three different PDGFRa-containing CARs expressed on a cell surface. The combination of a PDGFRa domain and a first and optional second linker are shown in Table 4-la.Table 4-la. Antibody-Inducible Domain and Flanking Sequences of PDGFRa-containing CARs

[0165] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies and expression of the tag and CAR was then assessed by flow cytometry. As shown in Table 4- la and Table 4-lb, over 20% of the transduced Jurkat cell populations expressed the EGFRt tag and either the standard CAR or one of the PDGFRa-containing CARs.Table 4-lb. Cells Engineered to Express PDGFRa-containing CARs

[0166] Activation of transduced Jurkat cells was then assessed by measuring CD25 (interleukin-2 receptor alpha chain) expression by transduced cells in the presence or absence of antigen (CAR antigen-expressing K562 cells) at several effector-to-target cell ratios(Jurkat: K562). As shown FIG. 10, Jurkat cells expressing either the standard CAR or one of the PDGFRa-containing CARs were specifically activated through the common antigen-binding domain of the CARs when the CAR-expressing Jurkat cells were co-cultured with antigenexpressing K562 cells.Example 5: T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With a IL4R-alpha Antibody-Inducible Domain (AID)

[0167] This example describes new CAR backbones comprising domains 1 and 2, domain 1 or domain 2 of interleukin-4 receptor-alpha (IL4Ra) as an antibody-inducible domain, and expression and subsequent activation of the new CARs on recombinant Jurkat cells (immortalized human T-cells) by use of the materials and methods of Example 1.

[0168] Expression cassettes encoding a standard CAR or an IL4Ra-containing CAR, as well as an EGFRt tag, were designed (FIG. 11). Lentiviral vectors were subsequently generated for expression of CARs by transduced T-cells. FIG. 12 shows a cartoon of a standard CAR and five different IL4Ra-containing CARs expressed on a cell surface. The combination of a IL4Ra domain and a first and optional second linker are shown in Table 5-la.Table 5-la. Antibody-Inducible Domain and Flanking Sequences of IL4Ra-containing CARs

[0169] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies and expression of the tag and CAR was then assessed by flow cytometry. As shown in Table 5- la and Table 5-lb, over 50% of the transduced Jurkat cell populations expressed the EGFRt tag and either the standard CAR or one of the IL4Ra-containing CARs.Table 5-lb. Cells Engineered to Express IL4Ra-containing CARs

[0170] Activation of transduced Jurkat cells was then assessed by measuring CD25 (interleukin-2 receptor alpha chain) expression by transduced cells in the presence or absence of antigen (CAR antigen-expressing K562 cells) at a target-to-effector cell ratio (K562:Jurkat) of 4: 1. As shown FIG. 13A, Jurkat cells expressing either the standard CAR or one of the IL4Ra- containing CARs were specifically activated through the common antigen-binding domain of the CARs when the CAR-expressing Jurkat cells were co-cultured with antigen-expressing K562 cells.

[0171] Activation of transduced Jurkat cells was further assessed by measuring CD25 expression by transduced cells in the presence or absence of an antibody capable of binding to the IL4Ra domain of some CARs of the present disclosure. As shown in FIG. 13B, only very small percentages of untransduced Jurkat cells (Mock) and standard-CAR expressing Jurkat cells expressed CD25 in the presence or absence of an antibody stimulus. In contrast, Jurkat cells expressing IL4Ra-containing CAR 1-4 (comprising domain 1 or domains 1 and 2) were activated by culturing the cells in the presence of an anti-IL4Ra antibody. Neither the anti-VEGFR2 antibody nor the anti-HER2 antibody induced expression of CD25 on cells expressing IL4Ra- containing CARs above background levels observed in the absence of an antibody (no stimulus). As such, FIG. 13B demonstrates that T cells expressing VEGFR2-containing CARs can be activated in a CAR-specific manner when contacted with an anti-VEGFR2 antibody.Example 6: T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With a CD4 Antibody-Inducible Domain (AID)

[0172] This example describes new CAR backbones comprising domains 1 and 2, domain 1 or domain 2 of cluster of differentiation 4 (CD4) as an antibody-inducible domain, and expression and subsequent activation of the new CARs on recombinant Jurkat cells (immortalized human T-cells) by use of the materials and methods of Example 1.

[0173] Expression cassettes encoding a standard CAR or a CD4-containing CAR, as well as an EGFRt tag, were designed (FIG. 14). Lentiviral vectors were subsequently generated for expression of CARs by transduced T-cells. FIG. 15 shows a cartoon of a standard CAR andseven different CD4-containing CARs expressed on a cell surface. The combination of a CD4 domain and a first and optional second linker are shown in Table 6-la.Table 6-la. Antibody-Inducible Domain and Flanking Sequences of CD4-containing CARs

[0174] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies and expression of the tag and CAR was then assessed by flow cytometry. As shown in Table 6- la and Table 6-lb, over 20% of the transduced Jurkat cell populations expressed the EGFRt tag and either the standard CAR or one of the CD4-containing CARs.Table 6-lb. Cells Engineered to Express CD4-containing CARs

[0175] Activation of transduced Jurkat cells was then assessed by measuring CD25 (interleukin-2 receptor alpha chain) expression by transduced cells in the presence or absence of antigen (CAR antigen-expressing K562 cells) at two different effector-to-target cell ratios (Jurkat: K562) ratios. As shown FIG. 16, Jurkat cells expressing either the standard CAR or one of the CD4-containing CARs were specifically activated through the common antigen-bindingdomain of the CARs when the CAR-expressing Jurkat cells were co-cultured with antigenexpressing K562 cells.Example 7: T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With a CD2 Antibody-Inducible Domain (AID)

[0176] This example describes new CAR backbones comprising domains 1 and 2 or domain 1 of cluster of differentiation 2 (CD2) as an antibody-inducible domain, and expression and subsequent activation of the new CARs on recombinant Jurkat cells (immortalized human T- cells) by use of the materials and methods of Example 1.

[0177] Expression cassettes encoding a standard CAR or a CD2-containing CAR, as well as an EGFRt tag, were designed (FIG. 17). Lentiviral vectors were subsequently generated for expression of CARs by transduced T-cells. FIG. 18 shows a cartoon of a standard CAR and four different CD2-containing CARs expressed on a cell surface. The combination of a CD2 domain and a first and optional second linker are shown in Table 7-la.Table 7-la. Antibody-Inducible Domain and Flanking Sequences of CD2-containing CARs

[0178] Transduced Jurkat cells were stained with anti-EGFR and anti-CAR antibodies and expression of the tag and CAR was then assessed by flow cytometry. As shown in Table 7- la and Table7-lb, over 30% of the transduced Jurkat cell populations expressed the EGFRt tag and either the standard CAR or one of the CD2-containing CARs.Table 7-lb. Cells Engineered to Express CD2-containing CARs

[0179] Activation of transduced Jurkat cells was then assessed by measuring CD25 (interleukin-2 receptor alpha chain) expression by transduced cells in the presence or absence of antigen (CAR antigen-expressing K562 cells) at various effector-to-target cell (K562:Jurkat) ratios. As shown FIG. 19, Jurkat cells expressing either the standard CAR or one of the CD2- containing CARs were activated through the common antigen-binding domain of the CARs when the CAR-expressing Jurkat cells were co-cultured with antigen-expressing K562 cells. However, the percentages of Jurkat cells expressing CD2 CAR1, CD2 CAR2, or CD2 CAR3 in the absence of antigen was (background level) was higher than that observed for Jurkat cells expressing CARs containing other antibody-inducible domains.Example 8: Expansion and Characterization of T-cells Engineered to Express a Chimeric Antigen Receptor (CAR) With an Antibody-Inducible Domain (AID)

[0180] This example describes the assessment of human regulatory T-cells (Tregs) engineered to express a CAR comprising an antibody-inducible domain (AID) and a first and optional second linker as shown in Table 8-1.Table 8-1. Antibody-Inducible Domain and Flanking Sequences of CARsAASEQ ID: G4Sx3 (NO:46); G3SG3 (NO:45); VEGFR2 (NO:31); IL4Ra(Dl-D2) (NO:36); IL4Ra(Dl) (NO:34); IgG4 hinge (NO:23); CD28TM (NO:25); and CD28TM(M5) (NO:42).

[0181] All of the constructs of Table 8-1 included an IL5Ra tag, and all of the constructs included a CAR with the same antigen-binding domain with the exception of the control SBT- 1026, which lacked a CAR. Tregs expressing a CAR comprising a VEGFR2 domain (SBT-1927) were stimulable with ramucirumab (anti-VEGFR) antibody. Tregs expressing a CAR comprising an IL4Ra domain (SBT-1934, SBT-1935 & SBT-1936) were stimulable with dupilumab (anti- IL4R antibody).

[0182] On Day 0, Tregs were isolated from a healthy human donor and activated with CTS™ Dynabeads™ Treg Xpander reagent (Gibco) at a 1: 1 bead to cell ratio in the presence of recombinant human IL-2 (300 lU / mL). On Day 3, Tregs were transduced with lentiviral expression vectors Transduced Tregs were expanded by addition of fresh medium containing IL- 2, restimulated on Day 9, further expanded and subsequently harvested. Various restimulation conditions were tested: IL-2 alone (i); CD3 / CD28 beads 1:1 (ii); VEGFR or IL4R Ab biotin- strep beads 1:1 (iii) or 2: 1 (iv); VEGFR or IL4R Ab biotin 3.5 mcg / ml (v) or 7 mcg / ml (vi).

[0183] As shown in FIG. 20, transduced Tregs expressing a CAR with a VEGFR2 antibody-inducible domain (SBT-1927) remained viable, and expanded nicely when stimulated with either CD3 / CD28 beads or VEGFR Ab beads at a 1: 1 cell to bead ratio. Very little expansion was observed when the Tregs were stimulated with VEGFR Ab in solution (not conjugated to beads).

[0184] There was greater variation in viability of transduced Tregs expressing a CAR with a IL4R antibody-inducible domain (SBT-1934 or SBT-1936) after restimulation with IL4R Ab (see, FIG. 21 and FIG. 22). While the Tregs did expand in the presence of IL4R beads at a 1: 1 cell to bead ratio, cell proliferation was delayed and Treg cell numbers were lower than when restimulated with CD3 / CD28 beads.

[0185] Strikingly, stimulation with bead-bound VEGFR Ab led to enrichment, as well as proliferation, of Tregs expressing a CAR with a VEGFR2 antibody-inducible domain (SBT- 1927) as shown in FIG. 23 and Table 8-2.Table 8-2. Percent Tag-positive Tregs (Indicative of VEGFR2-containing CAR Expression SBT-1927)

[0186] Stimulation with bead-bound IL4R Ab led to enrichment and expansion of Tregs expressing a CAR with an IL4R antibody-inducible domain (SBT-1934 and SBT-1936) as shown in FIG. 24, FIG. 25, Table 8-3 and Table 8-4.Table 8-3. Percent Tag-positive Tregs (Indicative of IL4R-containing CAR Expression SBT-1934)Table 8-4. Percent Tag-positive Tregs (Indicative of IL4R-containing CAR Expression SBT-1936)

[0187] Treg cells engineered to express a CAR with an antibody-inducible domain were characterized further by flow cytometry after staining for expression of CD4, CD25, CD27, CD70, CD71, IL5Ra tag (CAR Tag), G4S (flexible linker), FOXP3 and Helios.

[0188] On Day 9, prior to restimulation, expression of the G4S flexible linker and the CAR Tag were measured. In addition as shown in Table 8-5, the Tregs were immunophenotyped for expression of CD27, CD70 and CD71 (TfRl, transferrin receptor protein 1).Table 8-5. Linker and CAR Tag Expression on Tregs Prior to Restimulation

[0189] On Day 14 of the expansion study (5 days post-restimulation), Treg cells engineered to express a CAR with an antibody-inducible domain were assessed by flow cytometry. Table 8-6 shows results from SBT-1934 Tregs (expressing VEGFR domaincontaining CAR) after restimulation under various conditions. Table 8-7 shows results from SBT-1934 Tregs (expressing IL4R domain-containing CAR) after restimulation under various conditions. Table 8-8 shows results from SBT-1936 Tregs (expressing IL4R domain-containing CAR) after restimulation under various conditions.Table 8-6. Immunophenotype of Tregs Engineered to Express VEGFR Domain- Containing CAR (SBT-1927)Table 8-7. Immunophenotype of Tregs Engineered to Express IL4R Domain- Containing CAR (SBT-1934)Table 8-8. Immunophenotype of Tregs Engineered to Express IL4R Domain- Containing CAR (SBT-1936)

[0190] In order to assess the efficacy of restimulating Tregs engineered to express an IL4R domain-containing CAR (SBT-1934) with IL4R antibody beads at a 1:1 cell to bead ratio, Helios-l- Tregs were assessed for CAR (via staining for G4S linker) and activation marker (CD71) expression. As shown in Table 8-9, Tregs expressing high levels of FOXP3 were enriched in activated cells (CD71+) expressing an IL4R domain-containing CAR (G4S+). Conversely, Tregs expressing low levels of FOXP3 had far fewer activated cells (CD71+) expressing an IL4R domain-containing CAR (G4S+).Table 8-9. Correlation between Levels of CAR Expression and Activation

[0191] In conclusion, the CAR backbones of the present disclosure are useful for the production of engineered CAR-T-cells that are stimulable through the CAR via antibody binding to an antibody-inducible domain, not just through the CAR via antigen binding to an antigenbinding domain, or through the TCR via cross-linking of CD3 and CD28. While stimulation through TCR via cross-linking of anti-CD3 and CD28 did not lead to change of CAR tagpositive T-cells, stimulation through the antibody-inducible domain on the CAR backbone resulted in a highly enriched population of CAR-T-cells. As such, the data presented herein is indicative of a manufacturing benefit in using such antibodies directed to an extracellular accessory domain of a CAR for in vitro production of CAR-expressing T-cells, such as CAR- Tregs.SEQUENCES

Claims

CLAIMSWe claim:

1. A chimeric antigen receptor (CAR) comprising: an antigen-binding domain, a first linker, an antibody-inducible domain, a transmembrane domain, and an intracellular signaling domain, wherein the antibody-inducible domain is a polypeptide of from about 60 to about 360 amino acids in length.

2. The CAR of claim 1 , wherein the antibody-inducible domain comprises a member of the group consisting of a vascular endothelial growth factor receptor 2 (VEGFR2) domain, a human epidermal growth factor receptor 2 (HER2) domain, a platelet-derived growth factor receptoralpha (PDGFRa) domain, an interleukin-4 receptor-alpha (IL-4Ra) domain, a CD4 domain, and a CD2 domain.

3. The CAR of claim 2, wherein the antibody-inducible domain comprises a VEGFR2 domain.

4. The CAR of claim 3, wherein the VEGFR2 domain is bindable by an anti-VEGFR2 antibody or VEGFR2 -binding fragment thereof selected from the group consisting of ramucirumab and alacizumab, optionally wherein the VEGFR2 domain is bindable by ramucirumab.

5. The CAR of claim 4, wherein the VEGFR2 domain comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:31.

6. The CAR of claim 2, wherein the antibody-inducible domain comprises a HER2 domain.

7. The CAR of claim 6, wherein the HER2 domain is bindable by an anti-HER2 antibody or HER2-binding fragment thereof selected from the group consisting of trastuzumab,margetuximab, pertuzumab, and timigutuzumab, optionally wherein the HER2 domain is bindable by trastuzumab.

8. The CAR of claim 7, wherein the HER2 domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:32.

9. The CAR of claim 2, wherein the antibody-inducible domain comprises a PDGFRa domain.

10. The CAR of claim 9, wherein the PDGFRa domain is bindable by an anti-PDGFRa antibody or PDGFRa-binding fragment thereof selected from the group consisting of olaratumab and tovetumab.

11. The CAR of claim 10, wherein the PDGFRa domain comprises the amino acid sequence of SEQ ID NO:33 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:33.

12. The CAR of claim 2, wherein the antibody-inducible domain comprises an IL-4Ra domain.

13. The CAR of claim 12, wherein the IL-4Ra domain is bindable by an anti-IL-4Ra antibody or IL-4Ra-binding fragment thereof selected from the group consisting of dupilumab and pascolizumab.

14. The CAR of claim 13, wherein the IL-4Ra domain comprises: i) the amino acid sequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.

15. The CAR of claim 2, wherein the antibody-inducible domain comprises a CD4 domain.

16. The CAR of claim 15, wherein the CD4 domain is bindable by an anti-CD4 antibody or CD4-binding fragment thereof selected from the group consisting of cedelizumab, clenoliximab, ibalizumab, keliximab, priliximab, tregalizumab, zanolimumab, and semzuvolimab.

17. The CAR of claim 16, wherein the CD4 domain comprises: i) the amino acid sequence selected of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

18. The CAR of claim 2, wherein the antibody-inducible domain comprises a CD2 domain.

19. The CAR of claim 18, wherein the CD2 domain is bindable by an anti-CD2 antibody or CD2-binding fragment thereof, wherein the anti-CD2-antibody is siplizumab.

20. The CAR of claim 19, wherein the CD2 domain comprises: i) the amino acid sequence of SEQ ID NO:40 or SEQ ID NO:41; or ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:40 or SEQ ID NO:41.

21. The CAR of any one of claims 1-20, wherein a T-cell in which the CAR is expressed is activatable upon binding of an antibody or fragment thereof to the antibody-inducible domain,22. The CAR of any one of claims 1-21, further comprising a second linker located between the antibody-inducible domain and the transmembrane domain.

23. The CAR of any one of claim 1-21, wherein the first linker and / or the second linker is a flexible polypeptide linker of from 4 to 24 amino acids in length.

24. The CAR of claim 23, wherein the flexible polypeptide linker comprises the amino acid sequence of [XaXbXcXdXeXfXg]n, wherein Xa, Xb, Xc, and Xd, are independently selected from G and S, Xe, Xf and Xg are independently selected from G, S, and absent, and n is 1, 2 or 3, optionally wherein the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:46.

25. The CAR of any one of claims 1-21, wherein the first linker and / or the second linker is a hinge region of from 10 to 50 amino acids in length.

26. The CAR of claim 25, wherein the first linker is the flexible polypeptide linker, and the second linker is a hinge region of from 10 to 50 amino acids in length.

27. The CAR of claim 25 or claim 26, wherein the hinge region is selected from the group consisting of a CD8a hinge, a CD28 hinge region, and an IgG4 hinge region.

28. The CAR of claim 27, wherein the hinge region is a CD8a hinge region comprising the amino acid sequence of SEQ ID NO:21.

29. The CAR of claim 28, wherein the hinge region is a CD28 hinge region comprising the amino acid sequence of SEQ ID NO:22.

30. The CAR of claim 29, wherein the hinge region is an IgG4 hinge region comprising the amino acid sequence of SEQ ID NO:23.

31. The CAR of any one of claims 1-30, wherein the transmembrane domain is a CD28 transmembrane domain or a CD8a transmembrane domain.

32. The CAR of claim 31 , wherein the transmembrane domain is a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 43.

33. The CAR of claim 31, wherein the transmembrane domain is a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO:24.

34. The CAR of any one of claims 1-33, wherein the intracellular signaling domain comprises a CD3ζ signal transduction domain.

35. The CAR of claim 34, wherein the CD3ζ signal transduction domain comprises the amino acid sequence of SEQ ID NO:28.

36. The CAR of claim 34 or claim 35, wherein the intracellular signaling domain further comprises a co-stimulatory domain.

37. The CAR of claim 36, wherein the co-stimulatory domain comprises a CD28 costimulatory domain or a 4- IBB co-stimulatory domain.

38. The CAR of claim 37, wherein the co-stimulatory domain comprises a CD28 co- stimulatory domain.

39. The CAR of claim 38, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:29.

40. The CAR of claim 37, wherein the co-stimulatory domain comprises a 4- IBB costimulatory domain.

41. The CAR of claim 40, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:30.

42. The CAR of any one of claims 1-41, further comprising a signal peptide.

43. The CAR of claim 42, wherein the signal peptide is a CD8a signal peptide or a GM-CSF signal peptide.

44. The CAR of any one of claims 1-43, wherein the antigen-binding domain comprises an single chain antibody fragment (scFV), or a single domain antibody (sdAb).

45. The CAR of claim 44, wherein the antigen-binding domain comprises a scFv.

46. The CAR of claim 44, wherein the antigen-binding domain comprises a sdAb, optionally wherein the sdAb comprises a heavy chain variable region fragment (VHH).

47. The CAR of any one of claims 1-46, wherein the antigen-binding domain binds to an antigen selected from the group consisting of citrullinated vimentin (CV), CD 19, and BCMA.

48. The CAR of claim 47, wherein the antigen-binding domain binds to CV, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and light chain CDRs of the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO: 8;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; or(iii) the amino acid sequence of SEQ ID NO: 16.

49. The CAR of claim 47, wherein the antigen-binding domain binds to CD 19, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NOTO, and light chain CDRs of the amino acid sequence of SEQ ID NO:9;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9; or(iii) the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

50. The CAR of claim 47, wherein the antigen-binding domain binds to BCMA, optionally wherein the antigen-binding domain comprises:(i) heavy chain complementarity determining regions (CDRs) of the amino acid sequence of SEQ ID NO: 12, and light chain CDRs of the amino acid sequence of SEQ ID NO: 11 ;(ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NOT 1; or(iii) the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:20.

51. A nucleic acid encoding the CAR of any one of claims 1-50.

52. An expression vector comprising the nucleic acid of claim 51 in operable combination with an expression control sequence.

53. The expression vector of claim 52, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

54. The expression vector of claim 53, wherein the viral vector is a lentiviral vector.

55. The expression vector of claim 52, wherein the vector is a plasmid.

56. A host cell comprising the expression vector of any one of claims 52-55.

57. A modified T-cell that has been engineered to express the CAR of any one of claims 1-50, wherein the modified T-cell is a mammalian T-cell.

58. A pharmaceutical composition comprising a plurality of the modified T-cells of claim 57 and a pharmaceutically acceptable excipient.

59. The pharmaceutical composition of claim 58, wherein the modified T-cell is a human T- cell.

60. The pharmaceutical composition of claim 59, wherein human T-cell is a regulatory T-cell (Treg) that is CD4+, CD25+, and CD1271o.

61. The pharmaceutical composition of claim 60, wherein the human Treg is FOXP3+ and HELIOS+.

62. A method of treating a disease or disorder comprising administering an effective amount of the pharmaceutical composition of any one of claims 58-61 to a subject in need thereof.

63. A method of treating a subject suffering from an autoimmune disease or an inflammatory disorder comprising administering an effective amount of the pharmaceutical composition of any one of claims 58-61 to the subject, wherein the CAR expressed by the modified T-cells comprising an antigen-binding domain that binds to an antigen expressed on cells of the autoimmune disease or the inflammatory disorder.

64. A method of treating a subject suffering from cancer comprising administering an effective amount of the pharmaceutical composition of claim 58 or claim 59 to the subject, wherein the CAR expressed by the modified T-cells comprises an antigen-binding domain that binds to an antigen expressed on cells of the cancer.

65. A kit comprising a container containing the pharmaceutical composition of any one of claims 58-61, communicating through a fluidic conduit to a drip chamber, wherein the drip chamber is configured to communicate through a fluidic conduit with an intravenous needle.

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