WO2019241549A1

WO2019241549A1 - Foxp3-expressing car-t regulatory cells

Info

Publication number: WO2019241549A1
Application number: PCT/US2019/037038
Authority: WO
Inventors: James Johnston; Mark DARIS; Jiajia Cui; Aaron Daniel MARTIN
Original assignee: A2 Biotherapeutics, Inc.
Priority date: 2018-06-15
Filing date: 2019-06-13
Publication date: 2019-12-19

Abstract

Chimeric-antigen-receptor regulatory T cells (CAR-Tregs) generally exhibit limited persistence after administration to a patient. The present disclosure provides vectors, cells, and methods of use thereof related to chimeric-antigen-receptor regulatory T cells (CAR-Tregs) having a persistent Treg phenotype. In particular, the disclosure provides various embodiments of vectors and cells in which Foxp3 and/or IL-2 pathway signaling is controlled to increase the persistence of CAR-Tregs in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ, or in a patient being treated for autoimmune disease, allergic disease, or inflammatory disease.

Description

FOXP3-EXPRESSING CAR-T REGULATORY CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority to U.S. provisional patent application serial No. 62/685,562, filed on June 15, 2019, the contents of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

[002] The invention relates generally to cellular and gene therapy for patients undergoing stem cell or solid-organ transplant, or being treated for autoimmune, allergic disease, or inflammatory disorders. In particular, the invention relates to compositions and methods for generating regulatory T cells having a persistent Treg phenotype.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[003] The contents of the text file named“A2BI-0030lWO_SeqList.txt,” which was created on June 12, 2019 and is 174 Kilobytes in size, are hereby incorporated by reference in their entirety.

BACKGROUND

[004] Regulatory T cells (Treg cells) represent a fundamental T-cell capable of promoting self-tolerance and balancing excessive inflammation. These cells have a central role in preventing autoimmunity and curtailing the pro-inflammatory responses of myeloid cells, T cells, and B cells. Much emerging clinical data show that Treg therapy has promise in providing an alternative to current pharmacological immunosuppressive therapies. However, the lack of sustained benefit to date has limited their therapeutic utility. Thus approaches to redirect, enhance and sustain Treg function are needed to further advance their therapeutic potential.

[005] The importance of Tregs in immune homeostasis is exemplified by Foxp3 deficiency that results in a severe inflammatory condition, IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked) syndrome. The condition, which is due to a complete absence of CD4 Foxp3+ Treg cells, develops early in the first year of life and is usually fatal without bone marrow transplantation. Current therapies for autoimmune disease and organ transplant dampen the body’s immune response but leave patients vulnerable to other life-threatening conditions such as infection, ocular defects, hypertension and weight gain. Moreover, T-cell targeted therapies such as tacrolimus are blunt instruments that inhibit all T- cell function and can cause severe side effects such as posterior reversible encephalopathy syndrome (PRES).

[006] Treg therapy could provide general immunosuppression, without these side effects. Indeed animal models demonstrate that Tregs can suppress GvHD and recently a number of phase I clinical studies have shown that polyclonal autologous Treg cellular therapies are safe even at relatively high Tregs doses (5 ^c 108 Treg/kg), with no evidence of opportunistic infections in these early studies. While clinical studies are underway to determine whether polyclonal Tregs can preventing alio- and autoimmune complications, hurdles need to be overcome. These include required Treg dose for solid organ transplant, Treg persistence following therapy, and maintenance of the Treg suppressive function.

[007] There remains a need in the art for compositions and methods related to generation of regulatory T cells for use in stem-cell and solid-organ transplant, autoimmune disease, allergic disease, and inflammatory disorders. For example, methods that could prevent the emergence of antigen-specific“ex-Foxp3” Treg in the clinic should avoid exacerbation of disease. The present disclosure provides such compositions and methods, and more.

SUMMARY OF THE DISCLOSURE

[008] Chimeric -antigen-receptor regulatory T cells (CAR-Tregs) generally exhibit limited persistence after administration to a patient. The present disclosure provides vectors, cells, and methods of use thereof related to chimeric-antigen-receptor regulatory T cells (CAR-Tregs) having a persistent Treg phenotype. In particular, the disclosure provides various embodiments of vectors and cells in which Foxp3 and/or IL-2 pathway signaling is controlled to increase the persistence of CAR-Tregs in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ, or in a patient being treated for autoimmune disease, allergic disease, or inflammatory disease.

[009] The disclosure provides chimeric-antigen-receptor regulatory T cells (CAR-Tregs) comprising: (a) a first trangene encoding a chimeric antigen receptor (CAR) polypeptide targeting a human leukocyte antigen (HLA), operatively linked to a first promoter; (b) second transgene encoding Fox3p operably linked a second promoter, which is optionally the same promoter; and (c) the CAR polypeptide is expressed on the surface of the CAR-Treg; wherein the CAR-Treg is not a natural regulatory T cell (nTreg).

[010] In another aspect, the disclosure provides methods for generating chimeric-antigen- receptor regulatory T cells (CAR-Tregs) having a persistent Treg phenotype, comprising: (a) providing a population of T cells; and (b) contacting the population of T cells with one or more vectors under conditions sufficient for transduction of the vector; wherein the one or more vectors comprise a first trangene encoding a chimeric antigen receptor (CAR) polypeptide operatively linked to a first promoter and a second transgene encoding a constitutively active Fox3p operably linked a second promoter, which is optionally the same promoter.

[Oil] In another aspect, the disclosure provides vectors for use in modifying a chimeric- antigen-receptor T cell (CAR-Treg cell) to have a persistent Treg phenotype for therapeutic use in a transplant patient, comprising: (a) a sequence encoding a CAR; and (b) sequence encoding a constitutively active Foxp3.

[012] In another aspect, the disclosure provides methods of treating or inhibiting autoimmune disease, allergic disease, or inflammatory disease in a patient in need thereof, comprising: (a) providing a population of the regulatory T cell (Treg cell) of the disclosure; and (b) administering the population of Treg cells to the patient. In some embodiments, the autoimmune disease, allergic disease, or inflammatory disease is graft-versus-host disease.

[013] In another aspect, the disclosure provides methods for generating chimeric-antigen- receptor regulatory T cells (CAR-Tregs) capable of exhibiting a persistent Treg phenotype, comprising providing a regulatory T cell (Treg cell); and contacting the Treg cell with one or more vectors; wherein the one or more vectors comprise a polynucleotide encoding a chimeric antigen receptor (CAR) having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2R-beta cytoplasmic domain).

[014] In another aspect, the disclosure provides methods for generating chimeric-antigen- receptor regulatory T cells (CAR-Tregs) having a persistent Treg phenotype, comprising providing a regulatory T cell (Treg cell); and contacting the Treg cell with one or more vectors; wherein the one or more vectors comprise a polynucleotide encoding a constitutively active Foxp3.

[015] In another aspect, the disclosure provides a chimeric-antigen-receptor regulatory T cells

(CAR-Treg cells) designed to have a persistent Treg phenotype after transplantation into a patient, comprising a polynucleotide encoding a chimeric antigen receptor having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2R-beta).

[016] In another aspect, the disclosure provides vectors for use in modifying a chimeric- antigen-receptor regulatory T cell (CAR-Treg cell) to have a persistent Treg phenotype for therapeutic use in a transplant patient, comprising a polynucleotide encoding a chimeric antigen receptor having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2R- beta); and a polynucleotide encoding a constitutively active Foxp3; wherein the two polynucleotides are either the same polynucleotide or different polynucleotides.

[017] In another aspect, the disclosure provides methods of treating or inhibiting autoimmune disease, allergic disease, or inflammatory disease in a patient in need thereof, comprising providing a regulatory T cell (Treg cell); contacting the Treg cell with a vector of the disclosure; and administering the Treg cell to the patient.

[018] In another aspect, the disclosure provides methods for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ; comprising contacting the Treg cell with a vector of the disclosure; and administering the Treg cell to the patient.

[019] Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

[020] FIG. 1 shows gene cassette GG.05 CAR-T v2 CD28 (SEQ ID NO: 1), which encodes the C-terminal region of a chimeric antigen receptor beginning at the CD8 extracellular domain (ECD), the CD28 transmembrane region (TM), and the Oϋ3z intracellular domain (ICD).

[021] FIG. 2 shows gene cassette GG.12 CAR-T v2 CD28 CD3z FoxP3 wt (SEQ ID NO: 2).

[022] FIG. 3 shows gene cassette GG.13 CAR-T v2 CD28 CD3z FoxP3 S4l8E (SEQ ID NO:

3)·

[023] FIG. 4 shows gene cassette GG.14 CAR-T v2 CD28 CD3z FoxP3 NC (SEQ ID NO:

4).

[024] FIG. 5 shows gene cassette GG.15 CAR-T v2 CD28 CD3z STAT5 2x (SEQ ID NO:

5)· [025] FIG. 6 shows gene cassete GG.16 CAR-T v2 CD28 CD3z STAT5 2x FoxP3 wt (SEQ ID NO: 6).

[026] FIG. 7 shows gene cassete GG.17 CAR-T v2 CD28 CD3z STAT5 2x FoxP3 S418E (SEQ ID NO: 7).

[027] FIG. 8 shows gene cassete GG.18 CAR-T v2 CD28 CD3z STAT5 2x FoxP3 NC (SEQ ID NO: 8).

[028] FIG. 9 shows a diagram of FOXP3 stability mutants.

[029] FIG. 10A-10B show that bead-based enrichment of transduced T cells yield > 80% CAR expression. FIG. 10A is a plot showing CD4+ T cells that were activated with CD3/28 beads at a ratio of 1 : 1 and transduced with lentivirus at a MOI of 5. Chimeric antigen receptor (CAR+) cells were enriched using an optimized method developed in house to obtain > 80% FOXP3/CAR+ T cells. The Y-axis shows percentage of CAR+ T cells, the X-axis shows CAR+ cells, CAR and wild type (WT) FOXP3 cells, CAR and S418E mutant FOXP3 cells, and CAR and NC FOXP3 cells, blue bars (left) are pre-enrichment and red bars (right) are post enrichment percentages. FIG. 10B is a plot showing cell count (x 106) versus days post activation. Cells were cultured in XVIVO-15 media supplemented with 5% human A/B serum and cell proliferation was quantified using Vi-CELL. Blue circles show control (CTRL) Treg cells, yellow diamonds show control (CRTL) CD4+ cells, and green circles show CAR+ CD4+ cells.

[030] FIG. 11A-11C shows the characterization of FOXP3/CAR-T cells. FIG. 11A is a diagram showing a CAR construct with wild type (WT) FOXP3 that was used in this experiment. FIG. 11B is a series of three fluorescence activated cell sorting (FACS) graphs showing transduced and enriched CD4+ FOXP3/CAR-T cells that were cultured in X-Vivo media supplemented with 100 U/mL of hIL-2 for 8 days. From left to right, cells were phenotyped using antibodies against FOXP3 (PE), CD25 (APC), and CD 127 (APC). Dark blue indicates unstained cells, red control (CTRL) CD4+ cells and turquoise CAR+ CD4+ cells. FIG. 11C is a pair of FACS plots showing cells expressing intracellular IFN-gamma. Left, CD4+ cells; right CAR/FOXP3+ CD4+ cells. Cells were activated with CD3/28 beads for 2 days and Brefeldin A was added for 24 hours before cells were stained for intracellular IEN-g.

[031] FIG. 12 is a plot showing FOXP3/CAR-T cells suppress autologous CD3+ proliferation in a mixed lymphocyte reaction assay (MLR). CD4+ T cells were transduced with WT and mutated FOXP3/CAR and enriched to obtain >80% CAR expression. 7 days post enrichment and expansion, FOXP3/CAR-T cells were co-cultured with CFSE-labeled autologous CD3 cells (A*02:0l-) and growth arrested LCL721 cells (A*02:0l+) in an MLR assay. Cells were co-cultured for 4 day and CD3 proliferation was quantified using flow cytometry. FOXP3/CAR-T cells that recognize A* 02 demonstrated robust suppression of autologous CD3 proliferation in the presence of A*02:0l antigen (LCL721 cells) compared to CAR-T cells without FOXP3 and untransduced CD4+ T-cells.

[032] FIG. 13A-13C is a series of plots showing cytokine analysis from mixed lymphocyte reaction (MLR) assays. Supernatants from the MLR experiment were harvested and cytokine production was quantified using cytometric bead array (CBA). FIG. 13A shows that quantification of the suppressive cytokine secretion (IL-10) was increased for groups co cultured with WT FOXP3 and S418E FOXP3 CAR-T cells. FIG. 13B shows that similarly, proinflammatory cytokine secretion IL-2 was reduced in groups co-cultured with WT FOXP3 and S418E FOXP3 CAR-T cells. FIG. 13C shows that proinflammatory cytokine secretion TNF-alpha was also reduced in groups co-cultured with WT FOXP3 and S418E FOXP3 CAR- T cells.

[033] FIG. 14 is an experimental timeline and table for a graft-versus-host disease (GvHD) experiment.

[034] FIG. 15 is a Kaplan-Meier survival curve showing the survival of mice that received peripheral blood mononuclear cells (PBMCs) (blue), PBMCs and polyclonal Tregs (orange), PBMCs and CAR Tregs (green) and PBMCs and CAR CD4 cells (red).

[035] FIG. 16 is a plot showing PBMC engraftment in a mouse model of GvHD. Blood samples were taken at week 2, 3, 4, 5, 6, 8, 9 and cells used for flow cytometric analysis of engraftment of PBMCs in peripheral blood by looking at the percentage of hCD45+ cells (y- axis). Blue circles indicate percentage of hCD45+ cells from mice transplanted PBMCs, red squares = mice transplanted with BMCs and polyclonal Tregs, green triangles = mice transplanted with PBMCs and CAR Tregs and purple inverted triangle = mice transplanted with PBMCs and CAR CD4 cells.

[036] FIG. 17A-17B is a pair of plots showing that CD4+ CAR-T cells attenuate inflammatory cytokines in serum. For serum cytokine analysis samples were incubated with CBA beads and were read on an FACS CANTO (BD Biosciences) and results analyzed using FlowJo Software (Tree Star). FIG. 17A shows levels of IFN-gamma in serum at 2, 4, 6 and 8 weeks. FIG. 17B shows levels of TNF-alpha in serum at 2, 4, 6 and 8 weeks. Blue circles indicate percentage of hCD45+ cells from mice transplanted PBMCs, red squares = mice transplanted with BMCs and polyclonal Tregs, green triangles = mice transplanted with PBMCs and CAR Tregs and purple inverted triangle = mice transplanted with PBMCs and CAR CD4 cells.

[037] FIG. 18A-18F shows splenic pathology of mice transplanted with PBMCs, PMBCs, PBMCS and polyclonal Tregs, PBMCs and CAR-Tregs, PBMCs and CAR-CD4, and untreated controls. FIG. 18A shows a summary of the degree inflammation observed in each group of animals. FIG. 18B shows moderate inflammation in a representative spleen sample from a PBMC only mouse. FIG. 18C shows minimal inflammation in a representative spleen sample from a PBMC and polyclonal Treg mouse. FIG. 18D shows moderate inflammation in a representative spleen sample from a PBMC and CAR-Treg mouse. FIG. 18E shows moderate inflammation in a representative spleen sample from a PBMC and CAR-CD4 mouse. FIG. 18F shows minimal inflammation in a representative spleen sample from an untreated control mouse. The Groups cannot be segregated reliably using splenic architecture.

[038] FIG. 19A-19F shows liver pathology of mice transplanted with PBMCs, PMBCs, PBMCS and polyclonal Tregs, PBMCs and CAR-Tregs, PBMCs and CAR-CD4, and untreated controls. FIG. 19A shows a summary of the degree inflammation observed in each group of animals. FIG. 19B shows mild inflammation in a representative liver sample from a PBMC only mouse. FIG. 19C shows minimal inflammation in a representative liver sample from a PBMC and polyclonal Treg mouse. FIG. 19D shows moderate inflammation in a representative liver sample from a PBMC and CAR-Treg mouse. FIG. 19E shows minimal inflammation in a representative liver sample from a PBMC and CAR-CD4 mouse. FIG. 19F shows inflammation within normal limits in a representative liver sample from an untreated control mouse. Treatment Groups 2 (PBMC and polyclonal Treg) and 4 (PBMC and CAR-CD4) appear to have relatively reduced liver changes relative to other non-control groups.

[039] FIG. 20A-20E shows lung pathology at low magnification of mice transplanted with PBMCs, PMBCs, PBMCS and polyclonal Tregs, PBMCs and CAR-Tregs, PBMCs and CAR- CD4, and untreated controls. FIG. 20A shows mild inflammation in a representative lung sample from a PBMC only mouse. FIG. 20B shows marked inflammation in a representative lung sample from a PBMC and polyclonal Treg mouse. FIG. 20C shows severe inflammation in a representative lung sample from a PBMC and CAR-Treg mouse. FIG. 20D shows minimal inflammation in a representative lung sample from a PBMC and CAR-CD4 mouse. FIG. 20E shows inflammation within normal limits in a representative lung sample from an untreated control mouse. Treatment Group 4 (PBMC and CAR-CD4) appears to have reduced pulmonary inflammation relative to other non-control groups. [040] FIG. 21A-21F shows lung pathology at high magnification of mice transplanted with PBMCs, PMBCs, PBMCS and polyclonal Tregs, PBMCs and CAR-Tregs, PBMCs and CAR- CD4, and untreated controls. FIG. 21A shows a summary of the degree inflammation observed in each group of animals. FIG. 21B shows mild inflammation in a representative lung sample from a PBMC only mouse. FIG. 21C shows marked inflammation in a representative lung sample from a PBMC and polyclonal Treg mouse. FIG. 21D shows severe inflammation in a representative lung sample from a PBMC and CAR-Treg mouse. FIG. 21E shows minimal inflammation in a representative lung sample from a PBMC and CAR-CD4 mouse. FIG. 21F shows inflammation within normal limits in a representative lung sample from an untreated control mouse. Treatment Group 4 (PBMC and CAR-CD4) appears to have reduced pulmonary inflammation relative to other non-control groups.

[041] FIG. 22 shows a comparison of FOXP3 turnover in CAR+ CD4+ T cells. FOXP3 turnover was analyzed following cycloheximide treatment. For analysis of FOXP3 levels after 18 hours of treatment, cells were fixed and permeabilized, incubated with anti-FOXP3 antibody, and read on a FACS CANTO (BD Biosciences). Results analyzed using FlowJo Software (Tree Star) and compared via GraphPad (Prism).

[042] FIG. 23 is a diagram showing CAR designs with different ITAM multiplicity.

[043] FIG. 24A is a plot showing that ITAM multiplicity assayed with a CD8 hinge influences nFAT activation in Jurkat Cells. H = hinge, TM = transmembrane domain, ICD’s = intracellular domain, CD3z= CD3z domain with the ITAM configuration as specified.

[044] FIG. 24B is a plot showing that ITAM multiplicity assayed with a CD28 hinge influences nFAT activation in Jurkat Cells. H = hinge, TM = transmembrane domain, ICD’s = intracellular domain, CD3z= CD3z domain with the ITAM configuration as specified.

DETAILED DESCRIPTION

[045] Successful immunosuppressive therapy with regulatory T (Treg) cells requires persistence and suppressive function. Phenotypic plasticity may be a necessary homeostatic adaptive function of Tregs, but in the setting of transplant, autoimmunemallergic or inflammation disease it raises clinical safety concerns. The inventors have recognized that there are several ways by which Tregs maintain Foxp3 expression and function. The inventors have further recognized that IL-2 is crucial for the maintenance of Foxp3 expression in patients and in murine models. This highlights an important mechanism by which Treg stability is maintained in the setting of inflammation when there is increased IL-2 signaling, particularly via STAT5 phosphorylation. Therefore approaches that mimic STAT5 activation might be desirable in antigen-specific Treg therapy.

[046] The present disclosure enables the generation of long-lived (T cell Receptor) TCR-Treg or (Chimeric Antigen receptor) CAR-Tregs with enduring cell viability and prolonged suppressor function both in in vitro assays and in vivo. Some embodiments the methods of the present disclosure use vectors that encode TCR-Ts or CAR-Ts that contain an extracellular target binding module, a transmembrane domain, and an intracellular region that is capable of eliciting activation or suppressive signals. In CAR-Ts, the intracellular signaling regions in some cases consists of the TCR z chain and one, two, or more further signaling domains from CD28 or other costimulatory module(s), an NF-Kb activation module, or a JAK/STAT activation module. In some embodiments, the method comprises using sequences encoding the FOXP3 open reading frame either with or without nucleotides encoding the human amino acid residue serine 418 modified to aspartic acid or glutamic acid. In some embodiments the method comprising contact cells with distinct vectors that encode TCR-Ts or CAR-Ts separately from the either natural or modified FOXP3 gene. In some embodiments the method comprising contacting cells with distinct vectors that encode STAT binding modules as CAR-Ts separately from the either natural or modified FOXP3 gene.

[047] The present disclosure addresses at least four problems: First, the disclosure addresses the lack of stability of Tregs that have been and may be used in adoptive cell therapy, as these cells exhibit plasticity and can differentiate into pro-inflammatory effector T cells either in vitro or in vivo. Without the use of the presently disclosed compositions and methods, adoptively transferred Treg cells can be plastic, whether the autologous or allogeneic, and may become pro-inflammatory and thus cause exacerbation of inflammatory disease.

[048] Second, the disclosure addresses the ease of selecting CD4+ T cells for Treg adoptive cell therapy. To ensure all cells selected are Treg cells, one must select Treg cells using intracellular staining for Foxp3, and this requires cell permeability to stain the intracellular transcription factor. In some embodiments, the disclosed compositions and methods overcome this by enabling enrichment of Treg cells with the surface markers CD4+ and CD25+ and then expressing the modified transcription factor.

[049] Third, the disclosure addresses the fact that Interleukin-2 (IL-2) is an essential cytokine required to maintain Treg stability and longevity. In Treg cells IL-2 mainly functions by activating STAT5, the transcription factor responsible for activating IL-2 target gene transcription. In some embodiments, the addition of a STAT5 binding site to the intracellular domain of the chimeric receptor enables the CAR-Treg to mimic IL-2 signaling.

[050] Fourth, the disclosure provides methods of treatment that address graft-versus-host- disease by targeting CAR-Tregs to human leukocyte antigens of a donor.

[051] In some embodiments, the disclose provides DNA constructs (vectors) that encode proteins including a transcription factor that can maintain suppressive function independently of normal T cell signaling pathways, and/or a chimeric signaling domain that activates key pathway(s) that can sustain the growth and function of Treg cells.

[052] In particular embodiments, the disclosure provides a vector that contains the FOXP3 gene (Forkhead box protein P3) modified to encode aspartic acid or glutamic acid at amino acid residue 418 to mimic phospho-serine and thus maintain transcriptional activity. In some embodiments, the disclosure provides a chimeric signaling domain with includes nucleotides encoding some or all of the amino acid residues adjacent to tyrosine 381 and tyrosine 436 of human CD 122 (the IL-2R beta chain) that, when phosphorylated, comprise the two STAT5 SH2 binding domains.

[053] In some embodiments, the disclosure provides compositions and methods that result in increased longevity and persistence of Treg phenotype for transfected cells. In some embodiments, the disclosure provides Treg cells that are resistant to Foxp3 loss-of-function due to dephosphorylation. In some embodiments, the disclosure provides Treg cells and methods that combine persistence and longevity. In some embodiments, adoptively transferred Treg cells are maintained in the suppressive state.

Definitions

[054] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure. [056] The articles“a,”“an,” and“the” are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or one or more elements.

[057] The use of the alternative (e.g.,“or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[058] The term“and/or” should be understood to mean either one, or both of the alternatives.

[059] Throughout this specification, unless the context requires otherwise, the words “comprise”,“comprises” and“comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By“consisting of’ is meant including, and limited to, whatever follows the phrase“consisting of.” Thus, the phrase“consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By“consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase“consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are present that materially affect the activity or action of the listed elements.

[060] Reference throughout this specification to“one embodiment,”“an embodiment,”“a particular embodiment,”“a related embodiment,”“a certain embodiment,”“an additional embodiment,” or“a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in a particular embodiment.

[061] As used herein, the term“about” or“approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term“about” or“approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[062] As used herein, the term“isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term“obtained” or“derived” is used synonymously with isolated.

[063] The terms“subject,”“patient” and“individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,”“patient” or“individual” as used herein, includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[064] As used herein“treatment” or“treating,” includes any beneficial or desirable effect, and may include even minimal improvement in symptoms.“Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[065] As used herein,“prevent,” and similar words such as“prevented,”“preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein,“prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of disease prior to onset or recurrence.

[066] As used herein, the term“amount” refers to“an amount effective” or“an effective amount” of a virus to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[067] A“prophylactically effective amount” refers to an amount of a virus effective to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.

[068] A“therapeutically effective amount” of a virus or cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell are outweighed by the therapeutically beneficial effects. The term“therapeutically effective amount” includes an amount that is effective to“treat” a subject (e.g., a patient).

[069] An “increased” or “enhanced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a“statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[070] A ‘‘decrease” or “reduced” amount of a physiological response, e.g., electrophysiological activity or cellular activity, is typically a“statistically significant” amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[071] By“maintain,” or“preserve,” or“maintenance,” or“no change,” or“no substantial change,” or“no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule/composition. A comparable response is one that is not significantly different or measurable different from the reference response.

[072] In general,“sequence identity” or“sequence homology” refers to an exact nucleotide- to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their“percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403- 410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and

Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG fdter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[073] The term“exogenous” is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term“endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[074] The term“MOI” is used herein to refer to multiplicity of infection, which is the ratio of agents (e.g. viral particles) to infection targets (e.g. cells).

[075] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[076] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.

Chimeric Antigen Receptors (CARs)

[077] The present invention provides chimeric antigen receptors (CARs) comprising an extracellular and intracellular domain. The extracellular domain comprises a target-specific binding element, sometimes otherwise referred to herein as an antigen binding moiety. In some embodiments, the extracellular domain comprises a hinge. The intracellular domain comprises at least on activation domain. In some embodiments, the intracellular domain further comprises at least one co-stimulatory domain.

Extracellular Domain

[078] In some embodiments, the CARs of the present disclosure comprise a target-specific binding element otherwise referred to as an antigen binding moiety. The choice of moiety depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as ligands for the antigen moiety domain in the CAR of the invention include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.

[079] In some embodiments, the CARs of the present disclosure can be engineered to target specific alleles of components of the Major Histocompatibility Complex (MHC) through the extracellular domain. In some embodiments, for example when the CARs of the present disclosure are engineered to target alleles of components of the MHC I complex and are expressed by regulatory T cells (Tregs), the resulting CAR-Treg cells an downregulate or suppress the immune response to alloantigens.

[080] As used herein,“an alloantigen” refers to a genetically determined antigen present in some but not all subjects of a species and which is capable of inducing the production of an alloantibody by subjects which lack it. Lack of precise matching of alloantigens between donor and host can cause rejection of transplanted organs or cells, or graft-versus-host disease. Exemplary alloantigens that can lead to transplant rejection include, but are not limited to, ABO blood group and Rh blood group. Alloantigens can be presented through either MHC class I or MHC class II complexes, and allele mismatches in MHC I or MHC II are significant risk factors for transplant rejection or graft-versus-host disease.

[081] As used herein, graft-versus-host disease (GvHD) refers to a life threatening complication of cell and/or tissue transplants in which the immune system of the donor regards host as foreign, and attacks host tissues.

[082] As used herein, transplant rejection refers to a life threatening condition of organ, tissue and or cell transplants in which the immune system transplant recipient (host) attacks the transplanted donor tissue. [083] In some embodiments, the CARs of the present disclosure can be engineered to target specific alleles of components of the Major Histocompatibility Complex (MHC) e.g., HLA class 1 alleles HLA-A1, HLA-A2, HLA-A11, HLA-B44, HLA-B27, HLA-C07 and HLA-C04. In some embodiments, the MHC is MHC class I or MHC class II. In some embodiments, the MHC is MHC I. In some embodiments, the extracellular domain of the CARs of the disclosure target a human leukocyte antigen. In some embodiments, the extracellular domain of the CARs of the disclosure target an allele of human leukocyte antigen A (HLA-A, major histocompatibility complex, class I, A), human leukocyte antigen B (HLA-B, major histocompatibility complex, class I, B) or human leukocyte antigen C (HLA-C, major histocompatibility complex, class I, C).

[084] In some embodiments, the extracellular domain of the CARs of the disclosure target an allele of HLA-A. There are about 2,900 known variants of HLA-A, all of which are envisaged as within the scope of the disclosure. In some embodiments, the HLA-A allele is an HLA-A 1, HLA-A2, HLA -Al l, HLA-A24, HLA-A26, HLA-A30, HLA-A31, HLA-A34, HLA-A36, HLA-A66, HLA-A68 or HLA-A69 allele. In some embodiments, the HLA-A allele is an HLA- A2 allele. Exemplary HLA-A2 alleles include, but are not limited to, HLA-A*2:0l, an HLA- A*2:02, HLA-A* 2: 03, HLA-A*2:05, HLA-A*2:06, HLA-A*2:07 or HLA-A*2:0l l . In some embodiments, the extracellular domain of the CAR targets HLA-A*2:0l, an HLA-A* 2: 02, HLA-A* 2: 03, HLA-A*2:05, HLA-A*2:06, HLA-A*2:07 or HLA-A*2:0l l . In some embodiments, the extracellular domain of the CAR targets HLA-A*02:0l .

[085] In some embodiments, the extracellular domain of the CARs of the disclosure target an allele of HLA-B. There are about 3,600 known variants of HLA-B, all of which are envisaged as within the scope of the disclosure. In some embodiments, the extracellular domain of the CARs of the disclosure target an allele of HLA-C. There are about 2,400 known variants of HLA-B, all of which are envisaged as within the scope of the disclosure. In some embodiments, the extracellular domain of the CARs of the disclosure target an allele of human leukocyte antigen A (HLA-A, major histocompatibility complex, class I, A), human leukocyte antigen B (HLA-B, major histocompatibility complex, class I, B). In some embodiments, the allele of HLA-A, HLA-B or HLA-C is an allele selected from the group disclosed in Table 1.

Table 1. Percentage of most common HLA alleles (U.S.)

[086] In some embodiments, the CARs of the present disclosure can be engineered to target the Major Histocompatibility Complex bound to a peptide antigen of interest. The peptide antigen of interest can be a specific peptide antigen, for example a peptide antigen isolated or derived from a specific protein. In some embodiments, the peptide antigen is a host, or self, peptide antigen. In some embodiments, the peptide antigen is a host, or self, peptide antigen that is expressed in a particular tissue, and targeting the MHC bound to the peptide antigen targets the activity of the CARs of the disclosure to that particular tissue. For example, if a subject is suffering from inflammation of the kidneys, the CARs of the disclosure can engineered to target MHC bound to a kidney specific peptide antigen, thereby targeting the CARs of the disclosure to the kidneys of the subject to reduce inflammation. In some embodiments, the CARs of the present disclosure can be engineered to target specific alleles the Major Histocompatibility Complex bound to a peptide antigen of interest.

[087] An exemplary extracellular domain of a CAR of the disclosure targeting HLA-A* 02: 01 comprises a sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 11. In some embodiments, an extracellular domain of a CAR of the disclosure targeting HLA-A*02:0l comprises or consists essentially of SEQ ID NO: 11 .

[088] An exemplary sequence encoding an extracellular domain of a CAR of the disclosure targeting HLA-A*02:0l comprises a sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 12. In some embodiments, a sequence encoding an extracellular domain of a CAR of the disclosure targeting HLA-A*02:0l comprises or consists essentially of SEQ ID NO: 12.

Hinge Region

[089] In some embodiments, the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-Treg cells and improve expansion of CAR-Treg cells in vivo. Exemplary hinges can be isolated or derived from IgD and CD8 domains, for example IgGl .

[090] In some embodiments, the hinge is isolated or derived from CD8a or CD28. In some embodiments, the CD8a hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 13). In some embodiments, the CD8a hinge comprises SEQ ID NO: 13. In some embodiments, the CD8a hinge consists essentially of SEQ ID NO: 13. In some embodiments, the CD8a hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcg cccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat

( SEQ ID NO : 14 ) . In some embodiments, the CD8a hinge is encoded by SEQ ID NO: 14. In some embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:

15). In some embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO:

15. In some embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of tgtaccattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatcca tgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO:

16 ) . In some embodiments, the CD28 hinge is encoded by SEQ ID NO: 16.

Transmembrane Domain

[091] The CARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. For example, a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[092] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane -bound or transmembrane protein. Transmembrane regions may be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[093] In some embodiments of the CARs of the disclosure, the CARs comprise a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

FWVLVVVGGVLACY SLLVTVAFIIFWV (SEQ ID NO: 17). In some embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 17. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

ttctgggtgctggtcgttgtgggcggcgtgctggcctgctacagcctgctggtgacagtggccttcat catcttttgggtg (SEQ ID NO: 18). In some embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 18.

[094] In some embodiments of the CARs of the disclosure, the CARs comprise an IL- 2Rbeta transmembrane domain. In some embodiments, the IL-2Rbeta transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

i PWLGHLLVGLSGAFGFI i LVYLLI (SEQ ID NO: 19). In some embodiments, the IL-2Rbeta transmembrane domain comprises or consists essentially of SEQ ID NO: 19. In some embodiments, the IL-2Rbeta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

attccgtggc tcggccacct cctcgtgggc ctcagcgggg cttttggctt catcatctta gtgtacttgc tgatc (SEQ ID NO: 20) . In some embodiments, the IL-2Rbeta transmembrane domain is encoded by SEQ ID NO: 20.

Cytoplasmic Domain [095] The cytoplasmic domain or otherwise the intracellular signaling domain of the CARs of the instant invention is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. The term“effector function” refers to a specialized function of a cell. Effector functions of a Treg cell, for example, include the suppression or downregulation of induction or proliferation of effector T cells. Thus the term“intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired functions of the CAR-Treg cells of the instant disclosure. The term intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.

[096] Examples of intracellular signaling domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[097] Accordingly, the intracellular domain of CARs of the instant disclosure comprises at least one cytoplasmic activation domain. In some embodiments, the intracellular activation domain ensures that there is T-cell receptor (TCR) signaling necessary to activate the effector functions of the CAR T-cell. In some embodiments, the at least one cytoplasmic activation is a CD247 molecule (0O3z) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX-activating protein of 12 kDa (DAP12) activation domain. In some embodiments, the CD3z activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

RVKF SRS AD APAYKQGQN QLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 21). In some embodiments, the 0O3z activation domain comprises or consists essentially of SEQ ID NO: 21. In some embodiments, the 0O3z activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacga gctcaatctaggacgaagagaggagtacgatgttttggacaagcgtagaggccgggaccctgagatgg ggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcg gaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttacca gggactcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

(SEQ ID NO: 22). In some embodiments, the Oϋ3z activation domain is encoded by SEQ ID

NO: 22.

[098] It is known that signals generated through the TCR alone are often insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[099] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. In some embodiments, the ITAM contains a tyrosine separated from a leucine or an isoleucine by any two other amino acids (YxxL) (SEQ ID NO: 45).

[100] In some embodiments, the cytoplasmic domain contains 1, 2, or 3 ITAMs. In some embodiments, the cytoplasmic domain contains 1 ITAM. In some embodiments, the cytoplasmic domain contains 2 ITAMs. In some embodiments, the cytoplasmic domain contains 3 ITAMs. In some embodiments, the cytoplasmic domain contains 4 ITAMs. In some embodiments, the cytoplasmic domain contains 5 ITAMs.

[101] In some embodiments, the cytoplasmic domain is a 0O3z activation domain. In some embodiments, 0O3z activation domain comprises a single ITAM. In some embodiments, 0Ό3z activation domain comprises two ITAMs. In some embodiments, 0Ό3z activation domain comprises three ITAMs.

[102] In some embodiments, the 0O3z activation domain comprising a single ITAM comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR ( SEQ I D NO : 23 ) . In Some embodiments, the Oϋ3z activation domain comprises SEQ ID NO: 23. In some embodiments, the 0Ό3z activation domain comprising a single ITAM consists essentially of an amino acid sequence of RVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALP PR ( S EQ I D NO : 2 3 ) . In some embodiments, the Oϋ3z activation domain comprising a single ITAM is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttgcacat gcaggccctg

ccccctcgc ( SEQ ID NO: 24). In some embodiments, the 0Ό3z activation domain is encoded by SEQ ID NO: 24.

[103] Further examples of ITAM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from TOTIz. FcRy, FcR , CD3y, CD35, CD3s, Oϋ3z. CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the instant invention comprises a cytoplasmic signaling sequence derived from Oϋ3z.

Co-Stimulatory Domain

[104] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise the CD3z signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the instant disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3z chain portion and a co-stimulatory domain. The co stimulatory domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include the co-stimulatory domain is selected from the group consisting of IF-2R , Fc Receptor gamma (FcRy), Fc Receptor beta (FcR ), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-l), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-l (FFA-l), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (FIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional fragments thereof.

[105] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker.

[106] In some embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In some embodiments, the co-stimulatory domain is isolated or derived from CD28. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 25). In some embodiments, the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 25. In some embodiments, the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

aggagcaagcggagcagactgctgcacagcgactacatgaacatgaccccccggaggcctggccccacccggaag cactaccagccctacgcccctcccagggatttcgccgcctaccggagc (SEQ ID NO: 26). In some embodiments, the CD28 co-stimulatory domain is encoded by SEQ ID NO: 26.

[107] In some embodiments, the intracellular domain of the CARs of the instant disclosure comprises an interleukin-2 receptor beta-chain (IL-2Rbeta or IL-2R-beta) cytoplasmic domain. In some embodiments, the IL-2Rbeta domain is truncated. In some embodiments, the IL- 2Rbeta cytoplasmic domain comprises one or more STAT5-recruitment motifs. In some embodiments, the CAR comprises one or more STAT5-recruitment motifs outside the IL- 2Rbeta cytoplasmic domain.

[108] In some embodiments, the IL-2Rbeta intracellular domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

NCRNTGPWLKKVLKCNTPDPSKFFSQLS SEHGGDVQKWLS S PFPSS S FS PGGLAPEI S PLEVLERDKVTQLLPLN

TDAYLS LQELQGQDPTHLV (SEQ ID NO: 27). In some embodiments, the IL2Rbeta intracellular domain comprises or consists essentially of SEQ ID NO: 27. In some embodiments, the IL-2R- beta intracellular domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of 1 aactgcagga acaccgggcc atggctgaag aaggtcctga agtgtaacac cccagacccc

61 tcgaagttct tttcccagct gagctcagag catggaggcg acgtccagaa gtggctctct

121 tcgcccttcc cctcatcgtc cttcagccct ggcggcctgg cacctgagat ctcgccacta

181 gaagtgctgg agagggacaa ggtgacgcag ctgctccccc tgaacactga tgcctacttg

241 tctctccaag aactccaggg tcaggaccca actcacttgg tg ( SEQ ID NO: 28). In some embodiments, the IL-2Rbeta intracellular domain is encoded by SEQ ID NO: 28.

[109] In an embodiment, the IL-2R-beta cytoplasmic domain comprises one or more STAT5- recruitment motifs. Exemplary STAT5 -recruitment motifs are provided by Passerini et al. (2008) STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25+ effector T cells. International Immunology, Vol. 20, No. 3, pp. 421-431, and by Kagoya et al. (2018) A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects. Nature Medicine doi: l0. l038/nm.4478.

[110] In some embodiments, the STAT5-recruitment motif(s) consists of the sequence Tyr- Leu-Ser-Leu (SEQ ID NO: 46).

Cleavage Sites

[111] In some embodiments, of the vectors or CAR-Tregs of the disclosure, the CAR and an additional transgene are encoded by a single polynucleotide, for example as a single open reading frame under control of the same promoter, and separated by a cleavage site. Cleavage sites include the 2A family of“self-cleaving” polypeptides, which will divide a polypeptide into two parts using a ribosome skipping mechanism. Exemplary 2A peptides include T2A, E2A, F2A and P2A peptides. In some embodiments, the cleavage site comprises a T2A peptide. In some embodiments, the T2A peptide comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 30). In some embodiments, the T2A peptide comprises or consists essentially of SEQ ID NO: 30. In some embodiments, the T2A peptide is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of gagggcagaggcagcctgctgacatgtggcgacgtggaagagaaccctggcccc (SEQ ID NO: 29). In some embodiments, the T2A peptide is encoded by SEQ ID NO: 29.

[112] In some embodiments, the cleavage site comprises P2A polypeptide. In some embodiments, the P2A peptide comprises an amino acid sequence of

ATNFSLLKQAGDVEENPGP (SEQ ID NO: 31). In some embodiments, the P2A peptide comprises or consists essentially of SEQ ID NO: 31. In some embodiments, the cleavage site comprises E2A peptide. In some embodiments, the E2A peptide comprises an amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 32). In some embodiments, the E2A peptide comprises or consists essentially of SEQ ID NO: 32. In some embodiments, the cleavage site comprises F2A peptide. In some embodiments, the F2A peptide comprises an amino acid sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 33). In some embodiments, the F2A peptide comprises or consists essentially of SEQ ID NO: 33.

[113] An exemplary 2A peptide and related vectors are described in W02017040815A1, which is incorporated herein by reference in its entirety. forkhead boxP3 (Foxp3)

[114] The disclosure provides compositions, such as vector compositions, and methods of making CAR-Tregs with a persistent phenotype when transplanted in a patient. In some embodiments, this persistent phenotype is due to the expression of forkhead box P3 (Foxp3) in a CD4+ T cell, Treg cell, CD4+ CAR-T cell, or CAR-Treg cell.

[115] In some embodiments of the compositions and methods of the disclosure, the Foxp3 is wild-type (WT) Foxp3. Exemplary wild type human Foxp3 sequences are described in NP_054728.2, the contents of which are incorporated herein by reference. In some embodiments, the wild type Foxp3 comprises an amino acid sequence of having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

1 MPNPRPGKPS APSLALGPS P GAS PSWRAAP KASDLLGARG PGGTFQGRDL RGGAHAS S S S

61 LNPMPPSQLQ LPTLPLVMVA PSGARLGPLP HLQALLQDRP HFMHQLSTVD AHART PVLQV

121 HPLESPAMI S LTPPTTATGV FSLKARPGLP PGINVASLEW VSRE PALLCT FPNPSAP RKD

181 STLSAVPQS S YPLLANGVCK WPGCEKVFEE PEDFLKHCQA DHLLDEKGRA QCLLQREMVQ

241 SLEQQLVLEK EKLSAMQAHL AGKMALTKAS SVAS SDKGSC CIVAAGSQGP WPAWSGPRE

301 APDSLFAVRR HLWGSHGNST FPEFLHNMDY FKFHNMRPPF TYATLI RWAI LEAPEKQRTL

361 NEI YHWFTRM FAFFRNHPAT WKNAIRHNLS LHKCFVRVES EKGAVWTVDE LEFRKKRSQR

421 PSRCSNPTPG p ( S EQ I D NO : 9 ) . In some embodiments, the wild type Foxp3 comprises or consists essentially of SEQ ID NO: 9.

[116] In the above SEQ ID NO: 9, two residues, S418 and S422, which are mutated in some embodiments to make constitutively activated Foxp3 as described herein are bolded and underlined. In some embodiments, the wild type Foxp3 is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

1 atgcccaacc ccaggcctgg caagccctcg gccccttcct tggcccttgg cccatcccca 61 ggagcctcgc ccagctggag ggctgcaccc aaagcctcag acctgctggg ggcccggggc

121 ccagggggaa ccttccaggg ccgagatctt cgaggcgggg cccatgcctc ctcttcttcc

181 ttgaacccca tgccaccatc gcagctgcag ctgcccacac tgcccctagt catggtggca

241 ccctccgggg cacggctggg ccccttgccc cacttacagg cactcctcca ggacaggcca

301 catttcatgc accagctctc aacggtggat gcccacgccc ggacccctgt gctgcaggtg

361 caccccctgg agagcccagc catgatcagc ctcacaccac ccaccaccgc cactggggtc

421 ttctccctca aggcccggcc tggcctccca cctgggatca acgtggccag cctggaatgg

481 gtgtccaggg agccggcact gctctgcacc ttcccaaatc ccagtgcacc caggaaggac

541 agcacccttt cggctgtgcc ccagagctcc tacccactgc tggcaaatgg tgtctgcaag

601 tggcccggat gtgagaaggt cttcgaagag ccagaggact tcctcaagca ctgccaggcg

661 gaccatcttc tggatgagaa gggcagggca caatgtctcc tccagagaga gatggtacag

721 tctctggagc agcagctggt gctggagaag gagaagctga gtgccatgca ggcccacctg

781 gctgggaaaa tggcactgac caaggcttca tctgtggcat catccgacaa gggctcctgc

841 tgcatcgtag ctgctggcag ccaaggccct gtcgtcccag cctggtctgg cccccgggag

901 gcccctgaca gcctgtttgc tgtccggagg cacctgtggg gtagccatgg aaacagcaca

961 ttcccagagt tcctccacaa catggactac ttcaagttcc acaacatgcg accccctttc

1021 acctacgcca cgctcatccg ctgggccatc ctggaggctc cagagaagca gcggacactc

1081 aatgagatct accactggtt cacacgcatg tttgccttct tcagaaacca tcctgccacc

1141 tggaagaacg ccatccgcca caacctgagt ctgcacaagt gctttgtgcg ggtggagagc

1201 gagaaggggg ctgtgtggac cgtggatgag ctggagttcc gcaagaaacg gagccagagg

1261 cccagcaggt gttccaaccc tacacctggc ccc (SEQ ID NO: 34) .

[ 117 ] In some embodiments, the Foxp3 is a minimal Foxp3. In an embodiment, the one or more vectors further comprises a polynucleotide encoding a Foxp3. In an embodiment, the Foxp3 is a minimal Foxp3. Others have shown Foxp3 may be processed by proteolytic cleavage upon cell activation. In some embodiments of the present disclosure, a“minimal Foxp3” is engineered to mimic N-terminally, C-terminally, or N-and C-terminally cleaved Foxp3 forms. Minimal Foxp3 is, in some contexts, more active than wild-type (WT) Foxp3, as demonstrated by its superiority to WT Foxp3 in preventing experimental colitis. (See Zoeten et al. J. Bio. Chem. 284(9):5709-57l6 (2009)). In some embodiments, the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C-terminally truncated. In some embodiments, the N- and C-terminally truncated Foxp3 comprises an amino acid sequence of having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

1 GGAHASSSSL NPMPPSQLQL PTLPLVMVAP SGARLGPLPH LQALLQDRPH FMHQLSTVDA

61 HARTPVLQVH PLESPAMISL TPPTTATGVF SLKARPGLPP GINVASLEWV SREPALLCTF

121 PNPSAPRKDS TLSAVPQSSY PLLANGVCKW PGCEKVFEEP EDFLKHCQAD HLLDEKGRAQ

181 CLLQREMVQS LEQQLVLEKE KLSAMQAHLA GKMALTKASS VASSDKGSCC IVAAGSQGPV

241 VPAWSGPREA PDSLFAVRRH LWGSHGNSTF PEFLHNMDYF KFHNMRPPFT YATLIRWAIL

301 EAPEKQRTLN ElYHWFTRMF AFFRNHPATW KNAIRHNLSL HKCFVRVESE KGAVWTVDEL

361 EF (SEQ ID NO: 10). In some embodiments, the N- and C-terminally truncated Foxp3 comprises or consists essentially of SEQ ID NO: 10.

In some embodiments, the N- and C-terminally truncated Foxp3 is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of l ggcggggccc atgcctcctc ttcttccttg aaccccatgc caccatcgca gctgcagctg

61 cccacactgc ccctagtcat ggtggcaccc tccggggcac ggctgggccc cttgccccac

121 ttacaggcac tcctccagga caggccacat ttcatgcacc agctctcaac ggtggatgcc

181 cacgcccgga cccctgtgct gcaggtgcac cccctggaga gcccagccat gatcagcctc

241 acaccaccca ccaccgccac tggggtcttc tccctcaagg cccggcctgg cctcccacct

301 gggatcaacg tggccagcct ggaatgggtg tccagggagc cggcactgct ctgcaccttc

361 ccaaatccca gtgcacccag gaaggacagc accctttcgg ctgtgcccca gagctcctac

421 ccactgctgg caaatggtgt ctgcaagtgg cccggatgtg agaaggtctt cgaagagcca

4 81 gaggacttcc tcaagcactg ccaggcggac catcttctgg atgagaaggg cagggcacaa

541 tgtctcctcc agagagagat ggtacagtct ctggagcagc agctggtgct ggagaaggag

601 aagctgagtg ccatgcaggc ccacctggct gggaaaatgg cactgaccaa ggcttcatct

661 gtggcatcat ccgacaaggg ctcctgctgc atcgtagctg ctggcagcca aggccctgtc

721 gtcccagcct ggtctggccc ccgggaggcc cctgacagcc tgtttgctgt ccggaggcac

7 81 ctgtggggta gccatggaaa cagcacattc ccagagttcc tccacaacat ggactacttc

841 aagttccaca acatgcgacc ccctttcacc tacgccacgc tcatccgctg ggccatcctg

901 gaggctccag agaagcagcg gacactcaat gagatctacc actggttcac acgcatgttt

961 gccttcttca gaaaccatcc tgccacctgg aagaacgcca tccgccacaa cctgagtctg

1021 cacaagtgct ttgtgcgggt ggagagcgag aagggggctg tgtggaccgt ggatgagctg

1081 gagttc (SEQ ID NO: 35). In some embodiments, the N- and C-terminally truncated

Foxp3 is encoded by SEQ ID NO: 35.

[118] In some embodiments, the Foxp3 is constitutively active. In some embodiments, the Foxp3 is constitutively active due to a substitution of residue serine 418 to glutamic acid (S418E) relative to SEQ ID NO: 9. In some embodiments, the Foxp3 is constitutively active due to a substitution of glutamic acid for serine at amino acid residue 418 (S418E) relative to SEQ ID NO: 9. In some embodiments, the Foxp3 is constitutively active due to a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3). In some embodiments, the Foxp3 is constitutively active due to a substitution of glutamic acid for serine at amino acid residue 418 relative to SEQ ID NO: 9 and a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S418E, S422A FOXP3).

[119] In some embodiments, the polynucleotide encoding the CAR and the polynucleotide encoding the Foxp3 are configured for translation as a fusion protein comprising the CAR and the Foxp3. The CAR polynucleotide may be 5’ to the Foxp3 polynucleotide, in which case the CAR is expressed as a N-terminal fusion to the Foxp3, or vice-versa. In an embodiment, the CAR is N-terminal to the Foxp3 in the fusion protein. In an embodiment, the fusion protein comprises a cleavage site between the CAR and the Foxp3. In an embodiment, the cleavage site is a 2 A peptide.

[120] Foxp3 plays a crucial role in development and function of Treg cells. (Y agi et al. 2004. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol. 2004 Nov; l6(l 1): 1643-56. Epub 2004 Oct 4; Sadlon et al. (2018) Unravelling the molecular basis for regulatory T-cell plasticity and loss of function in disease. Clinical & Translational Immunology 2018; elOl l .) Constructs and methods from expressing Foxp3 in T cells are described in WO 2007/065957, which is incorporated herein in its entirety.

Chimeric Antigen Receptor Regulatory T cells (CAR-Tregs)

[121] The disclosure provides chimeric-antigen-receptor regulatory T cells (CAR-Tregs). In some embodiments, the CAR-Tregs comprise (a) a first trangene encoding a chimeric antigen receptor (CAR) polypeptide targeting a human leukocyte antigen (HLA), operatively linked to a first promoter; (b) a second transgene encoding Fox3p operably linked a second promoter; and (c) the CAR polypeptide is expressed on the surface of the CAR-Treg; wherein the CAR- Treg is not a natural regulatory T cell (nTreg). In some embodiments, the first and second promoter are the same promoter, and the CAR and the first and second transgenes are encoded by the same polynucleotide. For example, the first and second transgenes are encoded as a fusion protein under control of the same promoter, and separated by a cleavage site.

[122] “Regulatory T lymphocyte” or “Treg cell” or “Treg,” as used in the present specification and claims are synonymous and are intended to have its standard definition as used in the art. Treg cells are a specialized subpopulation of T cells that act in a“regulatory” way to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have sometimes been referred to suppressor T-cells. Treg cells are characterized by expression of the forkhead family transcription factor Foxp3 (forkhead box p3). They may also express CD4 or CD8 surface proteins. They usually also express CD25 (also known as Interleukin 2 receptor subunit alpha, or IL2R).

[123] Regulatory T (Treg) cells are involved in the maintenance of immunological self tolerance and in mitigating deleterious immune responses to both self and non-self (alio) antigens. Tregs comprise both natural and induced subtypes, both of which are considered within the scope of the CAR-Tregs of the instant disclosure. Natural Tregs (nTregs) are cells which originate as a separate cell lineage during development. Peripheral or induced Tregs (iTregs) differentiate from conventional T cell. In some embodiments, CD4+ T cells that are not nTregs and not iTregs can be engineered into Tregs through the expression of constitutively active Foxp3 using the methods and compositions of the disclosure. In some embodiments, the CD4+ T cells are used to make CAR-Tregs using the methods and compositions of the disclosure.

[124] In some embodiments, the Treg expressing the CAR of the disclosure is not naturally occurring (not an nTreg and/or not a iTreg). In some embodiments, the CAR-Treg expresses one or markers characteristic of a Treg. Treg markers include high levels of CD5 (CD25+), low levels of CD 127 (CD127-), or both high CD25 and low CD 127. The levels of CD25 and CD 127 are compared, for example to CD4+ T cell that is not a Treg. In some embodiments, the CAR-Treg cell has a high CD25, high CD4 and low CD27 phenotype.

[125] Tregs also exhibit lower levels of certain cytokines compared to other types of T cells, such as CD4+ T cells that are not Tregs. In some embodiments, the CAR-Treg exhibits lower IFN-gamma expression than a CD4+ T cell. In some embodiments, the CAR-Treg exhibits lower expression and/or lower secretion of IL-2 than a CD4+ T cell. Tregs also exhibit higher levels of expression and/or higher secretion of IL-10 than a CD4+ T cell.

[126] Methods of measuring markers used to characterize Treg cells will be readily to apparent to the person ordinary skill in the art. For example, CAR-Tregs or populations of T cells comprising CAR-Tregs can be cultured using the methods described herein. Following culturing, CAR-Tregs can be collected and stained using antibodies against Treg markers such as FOXP3 (PE), CD25 (APC), and CD 127 (BV421) and expression analyzed using fluorescence activated flow cytometry (FACS) or fluorescence microscopy. Intracellular cytokines can be assayed by activating cells with CD3/28 beads for 48 hours prior to addition of 10 pg/mL of Brefeldin A for 18 hours. CAR-Treg cells can then be harvested, fixed in a suitable immunohistochemistry fixation buffer, and stained in a permeabilization buffer with antibodies such as an anti-IFNy antibody before being analyzed by FACS or fluorescence microscopy.

[127] CAR-Tregs of the disclosure also exhibit suppression when assayed in a Mixed Lymphocyte Reaction (MLR). MLR comprises mixing two populations of lymphocytes together, and measuring the reaction that occurs. After several days, mismatched allogeneic lymphocytes will undergo responses such as blast transformation, DNA synthesis, and proliferation in response to mismatched MHC antigen.

[128] Exemplary MLR assays of the comprise staining one population of lymphocytes with an internal dye such as CFSE, for example CFSE at 2uM for 15 minutes at room temperature. Two populations of lymphocytes are mixed, and CAR-Tregs of the disclosure added to the co culture at varying ratios. After 4 days, supernatant can be collected and stored at -20°C for cytokine analysis. Lymphocytes from the co-culture can be assayed using antibody staining and FACS to see if CD3+ T cell proliferation has occurred. Cytokines in the supernatant can be analyzed by methods available in the art, for example the BC CBA Thl/Th2/Thl7 kit, according to manufacturer’s instructions. Briefly, mixed capture beads and PE detection reagent can be added to all assay samples (including standards), incubated for 3 hours at room temperature (RT), and read on the flow cytometer.

[129] In some embodiments, CAR-Tregs of the disclosure have a persistent phenotype. As used herein,“a persistent phenotype” refers to persistence of CAR-Tregs in a subject following transplantation. In some embodiments, CAR-Tregs persist at least 2 weeks, at least 3, weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 1 1 weeks, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months or at least 2 years after the CAR-Tregs are administered to the subject.

Vectors

[130] In some embodiments, the disclosure provides one or more vectors for use in modifying a chimeric-antigen-receptor regulatory T cell (CAR-Treg cell) to have a persistent Treg phenotype. In some embodiments, the CAR-Treg is for therapeutic use in a transplant patient.

[131] In some embodiments, the one or more vectors comprise (a) a sequence encoding a CAR; and (b) sequence encoding a constitutively active Foxp3.

[132] In some embodiments, the or more vectors and comprise a polynucleotide encoding a chimeric antigen receptor having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2R-beta); and a polynucleotide encoding a constitutively active Foxp3; wherein the two polynucleotides are either the same polynucleotide or different polynucleotides.

[133] The present disclosure encompasses DNA constructs comprising sequences of a CAR and Foxp3, as described herein. In some embodiments, the CAR targets an allele of HLA-A2 and the Foxp3 is constitutively active. In some embodiments, sequence encoding the CAR and the sequence encoding the constitutively active Foxp3 are encoded by the same polynucleotide. In some embodiments, the vector comprises a promoter. In some embodiments, for example those embodiments where the encoding the CAR and the sequence encoding the constitutively active Foxp3 are encoded by the same polynucleotide, the vector comprises a sequence encoding, from 5' to 3', the promoter, the CAR, a post-translational cleavage site and Foxp3. Exemplary post translational cleavage sites include a T2A polypeptide, an E2A polypeptide, an F2A polypeptide and a P2A polypeptide as described herein.

[134] In some embodiments, the vector of the disclosure comprises a sequence encoding a

Foxp3 polypeptide. In some embodiments, the vector comprises a sequence encoding a wild type Foxp3 sequence is operably linked to CAR of the disclosure, for example as a fusion protein comprising a 2A post-translational cleavage site between an 5’ CAR and a 3’ Foxp3 polypeptide.

[135] In some embodiments, the Foxp3 polypeptide is a wild type Foxp3. In embodiments, the vector comprises polynucleotide sequence that shares at least at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of (SEQ ID NO: 34).

[136] In some embodiments, the Foxp3 is a minimal Foxp3, for example an N- and C- terminally truncated Foxp3. In some embodiments, the vector comprises a polynucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 35.

[137] In some embodiments, the Foxp3 is constitutively active. In some embodiments, the vector comprises a polynucleotide sequence encoding a polypeptide comprising a substitution of residue serine 418 to glutamic acid (S418E) relative to SEQ ID NO: 9, a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3) or a combination thereof.

[138] In some embodiments of the vectors of the disclosure, the polynucleotides encoding the CAR and/or the Foxp3 polypeptide are codon-optimized. In some embodiments, the polynucleotides are codon optimized for expression in a mammalian cell. In some embodiments, the polynucleotides are codon optimized for expression in a human cell.

[139] In some embodiments, the disclosure provides a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID: 1. In embodiments, the disclosure provides a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID: 2. In embodiments, the disclosure provides a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID: 3. In embodiments, the disclosure provides a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID: 4. In embodiments, the disclosure provides a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID: 5. In embodiments, the disclosure provides a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID: 6. In embodiments, the disclosure provides a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID: 7. In embodiments, the disclosure provides a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID: 8.

[140] The disclosure also provides vectors in which polynucleotides encoding the CARs and Foxp3 polypeptides of the present disclosure are inserted. In some embodiments, the Foxp3 polypeptide is wild type Foxp3. In some embodiments, the vector comprises a recombinant polynucleotide sequence that shares at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44.

[141] The disclosure also provides vectors in which polynucleotides encoding the CARs and Foxp3 polypeptides of the present disclosure are inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[142] The expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[143] The polynucleotides encoding the CARs, and optionally Foxp3, of the instant disclosure can be cloned into a number of types of vectors. For example, the polynucleotides can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[144] Further, the expression vector may be provided to a CD4+ T cell or Treg cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326, 193).

[145] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[146] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 basepairs (bp) upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[147] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[148] In order to assess the expression of a CAR polypeptide, optionally a Foxp3 polypeptide of the disclosure, or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[149] Reporter genes are used for identifying potentially transfected or transduced cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al, 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[150] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[151] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[152] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[153] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[154] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example,“molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

Methods of Making CAR-Tregs [155] The disclosure provides methods for generating chimeric-antigen-receptor regulatory T cells (CAR-Tregs). The CAR-Tregs have a persistent Treg phenotype. In some embodiments, the methods comprise: (a) providing a population of T cells; and (b) contacting the population of T cells with one or more vectors under conditions sufficient for transduction of the vector; wherein the one or more vectors comprise a first trangene encoding a chimeric antigen receptor (CAR) polypeptide operatively linked to a first promoter and a second transgene encoding a constitutively active Fox3p operably linked a second promoter. In some embodiments, the methods comprise providing a regulatory T cell (Treg cell); and contacting the Treg cell with one or more vectors; wherein the one or more vectors comprise a polynucleotide encoding a constitutively active Foxp3.

[156] In some embodiments, the disclosure provides a method for generating chimeric- antigen-receptor regulatory T cells (CAR-Tregs) capable of exhibiting a persistent Treg phenotype, comprising providing a regulatory T cell (Treg cell); and contacting the Treg cell with one or more vectors; wherein the one or more vectors comprise a polynucleotide encoding a chimeric antigen receptor (CAR) having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2R-beta cytoplasmic domain). Methods for constructing CARs, CAR-T cells, and CAR Tregs are provided by US 2010/0135974 Al, US 7,741,465, US 20030147865, and US 20020137697, which are incorporated herein in their entirety.

[157] In some embodiments, the disclosure provides a method for generating chimeric- antigen-receptor regulatory T cells (CAR-Tregs) having a persistent Treg phenotype, comprising providing a regulatory T cell (Treg cell); and contacting the Treg cell with one or more vectors; wherein the one or more vectors comprise a polynucleotide encoding a constitutively active Foxp3. In an embodiment, the Foxp3 is a minimal Foxp3. In an embodiment, the Foxp3 is constitutively active.

[158] In some embodiment, the disclosure provides a chimeric-antigen-receptor regulatory T cell (CAR-Treg cell) designed to have a persistent Treg phenotype after transplantation into a patient, comprising a polynucleotide encoding a chimeric antigen receptor having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IU-2R-beta).

[159] In some embodiments, the CAR-Treg cell expresses one or more markers of a Treg as described herein. For example, the CAR-Treg has a CD25hi / CD4hi / CD 1271o phenotype. In some embodiments, the CAR-Treg further comprises a polynucleotide encoding a constitutively active Foxp3. In some embodiments, the constitutively active Foxp3 is a minimal Foxp3.

[160] In some embodiments, the CAR-Treg cells are not naturally occurring Tregs. In some embodiments, the CAR-Treg cells are non-Treg CD4+ cells that are induced to transform into Tregs using the compositions and methods of the instant disclosure. For example, expression of constitutively active Foxp3 can induce CAR-Tregs. Induced CAR-Tregs express one or more markers as described herein, for example high CD25 and/or low CD 127 and have cytokine profiles representative of naturally occurring Tregs.

[161] Methods transforming populations of T cells comprising CD4+ cells and Tregs with the vectors of the instant disclosure will be readily apparent to the person of ordinary skill in the art. For example, T cells such as CD4+ T cells and Treg cells can be isolated from PBMCs using a CD4+ T cell negative isolation kit (Miltenyi), according to manufacturer’s instructions. CD4+ T cells can be cultured at a density of 1 x 10^L6 cells/mL in X-Vivo 15 media supplemented with 5% human A/B serum and 1% Pen/strep in the presence of CD3/28 Dynabeads (1: 1 cell to bead ratio) and 300 Units/mL of IL-2 (Miltenyi). After 2 days, CD4+ cells can be transduced with viral vectors, such as lentiviral vectors using methods known in the art. In some embodiments, the viral vector is transduced at a multiplicity of infection (MOI) of 5. Cells can then be cultured in IL-2 for an additional 5 days prior to enrichment.

[162] Methods of activating and culturing populations of T cells, for example population comprising CAR-Tregs of the instant disclosure, will be readily apparent to the person of ordinary skill in the art.

[163] Whether prior to or after genetic modification of CD4+ T or Treg cells to express a CAR and optionally Foxp3, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7, 172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10040846; and U.S. Pat. Appl. Pub. No.

2006/0121005.

[164] Generally, the T cells of the instant disclosure are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Bcsancon. France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1- 2):53-63, 1999).

[165] In some embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in“cis” formation) or to separate surfaces (i.e., in“trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In some embodiments, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

[166] In some embodiments, the two agents are immobilized on beads, either on the same bead, i.e.,“cis,” or to separate beads, i.e.,“trans.” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti- CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1.

[167] Ratios of particles to cells from 1 :500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. In some embodiments, a ratio of 1 : 1 cells to beads is used. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size and on cell size and type.

[168] In further embodiments of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[169] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached to contact the T cells. In one embodiment the cells (for example, CD4+ T cells and Tregs) and beads (for example, DYNABEADS CD3/CD28 T paramagnetic beads at a ratio of 1 : 1) are combined in a buffer. Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. In some embodiments, cells that are cultured at a density of lxlO⁶ cells/mL are used.

[170] In some embodiments, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the beads and T cells are cultured together for 2-3 days.

[171] Conditions appropriate for CD4+ or TReg cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp. and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. In some embodiments, the media comprises X-VIVO-15 media supplemented with 5% human A/B serum, 1% penicillin/streptomycin (pen/strep) and 300 Units/ml of IL-2 (Miltenyi).

[172] The T cells, such as CD4+ T cells and Tregs, are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% C02).

[173] In some embodiments, The CAR-Tregs of the disclosure are autologous. Prior to expansion and genetic modification of the CD4+ or TReg cells of the invention, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation.

[174] In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi- automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[175] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulation of T cells, such as CD4+ T cells or CD4+ Foxp3+ T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD4 -conjugated beads, for a time period sufficient for positive selection of the desired T cells.

[176] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immune -adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD l lb, CD 16, HFA-DR, and CD8. In some embodiments, it may be desirable to enrich for or positively select for regulatory T cells which, in some embodiments, express CD4+ CD25+, and FoxP3+.

[177] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. [178] In some embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10° C. or at room temperature.

[179] T cells for stimulation, or PBMCs from which T cells are isolated can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C. or in liquid nitrogen.

Pharmaceutical Compositions

[180] The disclosure provides pharmaceutical composition comprising the CAR-Tregs of the disclosure and a pharmaceutically acceptable diluent, carrier or excipient.

[181] Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; and preservatives.

Treatment Diseases and Disorders

[182] The disclosure provides a method of treating or inhibiting autoimmune disease, allergic disease, or inflammatory disease in a patient in need thereof, comprising providing to the subject a regulatory T cell (Treg cell) comprising a CAR of the disclosure, and optionally Foxp3. In some embodiments, the method comprises contacting a CD4+ cell or Treg cell with a vector of the present disclosure (e.g. a vector encoding either or both of a CAR and Foxp3, where optionally the CAR comprises an IF-2Rbeta cytoplasmic domain); and administering the Treg cell to the patient. In some embodiments, the method comprises (a) providing a population of the CAR regulatory T cells (CAR-Treg cells) of the disclosure; and (b) administering the population of Treg cells to the patient.

[183] In some embodiments the CAR-Treg cells are autologous. As used herein“autologous CAR-Tregs” refers to CAR-Tregs that were made using T cells isolated from the subject.

[184] In some embodiments, the CAR-Treg cells are allogeneic. As used herein“allogeneic CAR-Tregs” refers to CAR-Tregs that were made using T cells isolated from a different subject.

[185] In some embodiments, the autoimmune disease, allergic disease, or inflammatory disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, and allergic rhinitis.

[186] In some embodiments, the autoimmune disease, allergic disease, or inflammatory disease is an organ-specific inflammatory disease.

[187] In some embodiments, the organ-specific inflammatory disease is selected from the group consisting of kidney disease and lung disease. In some embodiments, the organ-specific inflammatory disease is a disease of any other organ or group of organs in the body (for example, liver, brain, heart intestines, lymph nodes, circulatory system, stomach, spleen etc.).

[188] In some embodiments, the autoimmune disease, allergic disease, or inflammatory disease is graft-versus-host disease (GvHD). In some embodiments, the GvHD is caused by an organ or stem cell transplant. In some embodiments, the stem cells comprise hematopoietic stem cells and/or peripheral blood mononuclear cells (PBMCs). GvHD can also be caused by bone marrow transplants. In some embodiments, the organ or stem cell transplant is allogeneic.

[189] In some embodiments, the autoimmune disease, allergic disease, or inflammatory disease is transplant rejection. In some embodiments, the transplant rejection occurs in response to transplanted blood, bone marrow, bone, skin, heart, kidney, lung, muscle, heart or liver. In some embodiments, the transplant rejection is hyperacute rejection. In some embodiments, the transplant rejection is acute rejection. In some embodiments, the transplant rejection is chronic rejection.

[190] In some embodiments, the CAR-Tregs of the disclosure comprise a CAR that targets a human leukocyte antigen. In some embodiments, the HLA targeted by the CAR is the same HLA allele as a donor. For example, the donor is HLA-A*2:0l and the CAR-Treg targets HLA- A* 2:0.1 In some embodiments, the human leukocyte antigen targeted by the CAR is the same HLA allele as a recipient. All alleles of HLA-A, HLA-B and HLA-C are envisaged as within the scope of the disclosure. [191] In some embodiments, the method increases CD45+ cell engraftment, increases overall survival, decreases serum inflammatory cytokines or a combination thereof. In some embodiments, the method decreases spleen inflammation, liver inflammation, lung inflammation, inflammation of the central nervous system (CNS), inflammation of the skin, inflammation of the pancreas, inflammation of the kidney, inflammation of the pleural cavity, inflammation of the gastrointestinal tract, inflammation of the genitourinary tract, or inflammation of the pelvis. In some embodiments, the disclosure provides a method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ, comprising providing a regulatory T cell (Treg cell); contacting the Treg cell with a vector of the present disclosure (e.g. a vector encoding either or both of a CAR and Foxp3, where optionally the CAR comprises an IL-2Rbeta cytoplasmic domain); and administering the Treg cell to the patient.

[192] In some cases, the method prevents allograft rejection, improving upon concepts described in Noyan et al. (2017) Prevention of Allograft Rejection by Use of Regulatory T Cells With an MHC-Specific Chimeric Antigen Receptor. Am. J. Transplantation 2017; 17: 917-930.

[193] In an embodiment, the patient is transplanted before administering the Treg cell to the patient. In an embodiment, the patient is transplanted after administering the Treg cell to the patient.

[194] In some embodiments of the methods of preventing allograft rejection, the CAR-Treg cells are autologous.

[195] Pharmaceutical compositions of the present disclosure comprising the CAR-Tregs described herein may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[196] When“therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of disease, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the CAR- Treg cells described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, for example between 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. CAR-Treg cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[197] The administration of the subject compositions comprising CAR-Tregs of the disclosure may be carried out in any convenient manner, including by injection, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the CAR-Treg cell compositions of the present disclosure are administered by i.v. injection.

[198] In some embodiments of the present invention, cells activated and expanded using the CARs and methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, such as organ or stem cell transplantation. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art- accepted practices. The dose for CAMPATH, for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S. Pat. No. 6, 120,766).

[199] Exemplary methods for Treg therapy are provide by Romano et al. (2016) Treg therapy in transplantation: a general overview. Transplant International 2017; 30: 745-753. Treg cells can be expanded ex vivo, by methods such as those described in Xia et al. (2009) Prevention of Allograft Rejection by Amplification of Foxp3+CD4+CD25+ Regulatory T Cells. Transl Res. 2009 Feb; 153(2): 60-70.

ENUMERATED EMBODIMENTS

[200] The invention may be defined by reference to the following enumerated, illustrative embodiments: Illustrative Embodiments Set 1 :

1. A method for generating chimeric-antigen-receptor regulatory T cells (CAR-Tregs) capable of exhibiting a persistent Treg phenotype, comprising:

a) providing a regulatory T cell (Treg cell); and

b) contacting the Treg cell with one or more vectors;

wherein the one or more vectors comprise a polynucleotide encoding a chimeric antigen receptor (CAR) having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IF-2R- beta cytoplasmic domain).

2. The method of embodiment 1, wherein the IL-2R-beta cytoplasmic domain comprises one or more STAT5 -recruitment motifs.

3. The method of embodiment 1, wherein the STAT5-recruitment motif(s) consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 46).

4. The method of embodiment 1, wherein the chimeric antigen receptor comprises one or more STAT5 -recruitment motifs outside the IL-2R-beta cytoplasmic domain.

5. The method of embodiment 1, wherein the one or more vectors further comprises a polynucleotide encoding a Foxp3.

6. The method of embodiment 5, wherein the Foxp3 is a minimal Foxp3.

7. The method of embodiment 5, wherein the Foxp3 is constitutively active.

8. The method of embodiment 6, wherein the polynucleotide encoding the CAR and the polynucleotide encoding the Foxp3 are configured for translation as a fusion protein comprising the CAR and the Foxp3.

9. The method of embodiment 7, wherein the CAR is N-terminal to the Foxp3 in the fusion protein.

10. The method of embodiment 7 or 8, wherein the fusion protein comprises a post-translational cleavage site between the CAR and the Foxp3.

11. The method of embodiment 9, wherein the CAR is cleaved from the Foxp3 following translation.

12. A method for generating chimeric-antigen-receptor regulatory T cells (CAR-Tregs) having a persistent Treg phenotype, comprising:

a) providing a regulatory T cell (Treg cell); and

b) contacting the Treg cell with one or more vectors; wherein the one or more vectors comprise a polynucleotide encoding a constitutively active Foxp3.

13. The method of embodiment 12, wherein the Foxp3 is a minimal Foxp3.

14. The method of embodiment 12, wherein the Foxp3 is constitutively active.

15. A chimeric-antigen-receptor regulatory T cell (CAR-Treg cell) designed to have a persistent Treg phenotype after transplantation into a patient, comprising a polynucleotide encoding a chimeric antigen receptor having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2R-beta).

16. The CAR-Treg cell of embodiment 15, wherein the CAR-Treg cell has a CD25hi / CD4^~hi / CD271o phenotype.

17. The CAR-Treg cell of embodiment 15, further comprising a polynucleotide encoding a constitutively active Foxp3.

18. The CAR-Treg cell of embodiment 17, wherein the constitutively active Foxp3 is a minimal Foxp3.

19. A vector for use in modifying a chimeric -antigen-receptor regulatory T cell (CAR-Treg cell) to have a persistent Treg phenotype for therapeutic use in a transplant patient, comprising: a) a polynucleotide encoding a chimeric antigen receptor having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2R-beta); and

b) a polynucleotide encoding a constitutively active Foxp3;

wherein the two polynucleotides are either the same polynucleotide or different polynucleotides.

20. A method of treating or inhibiting autoimmune disease, allergic disease, or inflammatory disease in a patient in need thereof, comprising:

a) providing a regulatory T cell (Treg cell);

b) contacting the Treg cell with the vector of embodiment 19; and

c) administering the Treg cell to the patient.

21. The method of embodiment 20, wherein the autoimmune disease, allergic disease, or inflammatory disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, and allergic rhinitis. 22. The method of embodiment 20, wherein the autoimmune disease, allergic disease, or inflammatory disease is an organ-specific inflammatory disease.

23. The method of embodiment 22, wherein the organ-specific inflammatory disease is selected from the group consisting of kidney disease and lung disease.

24. A method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ, comprising:

a) providing a regulatory T cell (Treg cell);

b) contacting the Treg cell with the vector of embodiment 19; and

c) administering the Treg cell to the patient.

25. The method of embodiment 24, wherein the patient is transplanted before administering the Treg cell to the patient.

26. The method of embodiment 24, wherein the patient is transplanted after administering the Treg cell to the patient.

Illustrative embodiments set 2:

1. A chimeric-antigen-receptor regulatory T cell (CAR-Treg) comprising:

a) a first trangene encoding a chimeric antigen receptor (CAR) polypeptide targeting a human leukocyte antigen (HLA), operatively linked to a first promoter;

b) a second transgene encoding Fox3p operably linked a second promoter; and c) the CAR polypeptide is expressed on the surface of the CAR-Treg;

wherein the CAR-Treg is not a natural regulatory T cell (nTreg).

2. The CAR-Treg of embodiment 1, wherein the first promoter and the second promoter are the same promoter.

3. The CAR-Treg of embodiment 2, wherein the first transgene and the second transgene are both encoded by a polynucleotide that further comprises a sequence encoding a cleavage site between the first and second transgenes.

4. The CAR-Treg of embodiment 3, wherein the post-translational cleavage site is selected from the group consisting of a T2A peptide, an E2A peptide, an F2A peptide and a P2A peptide.

5. The CAR-Treg of embodiment 4, wherein the T2A peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 30).

6. The CAR-Treg of any one of embodiments 1-5, wherein the CAR comprises at least one cytoplasmic activation domain.

7. The CAR-Treg of embodiment 6, wherein the at least one cytoplasmic activation domain is a CD247 molecule ^ϋ3z) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, a DNAX -activating protein of 12 kDa (DAP 12) activation domain, or an IL-2Rbeta cytoplasmic domain.

8. The CAR-Treg of embodiment 7, wherein the 6Ό3z activation domain comprises 3 immunoreceptor tyrosine-based activation motifs (ITAMs).

9. The CAR-Treg of embodiment 8, wherein the Oϋ3z activation domain comprises a sequence of

RVKF SRS AD APAYKQGQN QLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 21).

10. The CAR-Treg of embodiment 7, wherein the CD3z activation domain comprises one ITAM.

11. The CAR-Treg of embodiment 10, wherein the CD3z activation domain comprises a sequence of RVKF SRS AD AP AY QQGQN QLYNELNLGRREEYD VLHMQALPPR (SEQ ID NO: 23).

12. The CAR-Treg of embodiment 7, wherein the IL-2Rbeta cytoplasmic domain comprises one or more STAT5-recruitment motifs.

13. The CAR-Treg of embodiment 12, wherein the STAT5-recruitment motif(s) consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 46).

14. The CAR-Treg of embodiment 1, wherein the chimeric antigen receptor comprises one or more STAT5 -recruitment motifs outside the IL-2Rbeta cytoplasmic domain.

15. The CAR-Treg of any one of embodiments 1-14, comprising at least one co-stimulatory domain.

16. The CAR-Treg of embodiment 15 wherein the co-stimulatory domain is selected from the group consisting of IL-2Rbeta, Fc Receptor gamma (FcRy), Fc Receptor beta (FcR ), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-l), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-l (LFA-l), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional fragments thereof. 17. The CAR-Treg of embodiment 16, wherein the CD28 co-stimulatory domain comprises a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 25).

18. The CAR-Treg of embodiment 16, wherein the IL-2Rbeta co-stimulatory domain comprises a sequence of

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSP GGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 27).

19. The CAR-Treg of any one of embodiments 1-18, comprising a hinge region.

20. The CAR-Treg of embodiment 19, wherein the hinge comprises a CD8a or CD28 hinge.

21. The CAR-Treg of embodiment 20, wherein the CD8a hinge comprises a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 13).

22. The CAR-Treg of embodiment 20, wherein the CD28 hinge comprises a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 15).

23. The CAR-Treg of any one of embodiments 1-22, comprising a transmembrane domain.

24. The CAR-Treg of embodiment 23, wherein the transmembrane domain comprises a CD28 transmembrane domain or an IL-2Rbeta transmembrane domain.

25. The CAR-Treg of embodiment 24, wherein the CD28 transmembrane domain comprises a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 27).

26. The CAR-Treg of embodiment 24, wherein the IL-2Rbeta transmembrane domain comprises a sequence of PWLGHLLV GLSGAFGFIILVYLLI (SEQ ID NO: 19).

27. The CAR-Treg of any one of embodiments 1-26, wherein the Foxp3 is wild-type (WT) Foxp3.

28. The CAR-Treg of embodiment 27, wherein the wild type Foxp3 comprises an amino acid sequence of

MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHA SSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHA RTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFP NPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKG RAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIV AAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPP FTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFV RVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP (SEQ ID NO: 9).

29. The CAR-Treg of any one of embodiments 1-28, wherein the Foxp3 is a minimal Foxp3. 30. The CAR-Treg of embodiment 29, wherein the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C- terminally truncated.

31. The CAR-Treg of embodiment 30, wherein the N- and C-terminally truncated Foxp3 comprises a sequence of

GGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLST

VDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPA

LLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHL

LDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKG

SCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFH

NMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSL

HKCFVRVESEKGAVWTVDELEF (SEQ ID NO: 10).

32. The CAR-Treg of any one of embodiments 1-26, wherein the Foxp3 comprises a substitution of serine for glutamic acid at amino acid 418 relative to SEQ ID NO: 9 (S418E FOXP3).

33. The CAR-Treg of embodiment 27 or 32, wherein the Foxp3 comprises a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3).

34. The CAR-Treg of any one of embodiments 1-33, wherein the Foxp3 is constitutively active.

35. The CAR-Treg of any one of embodiments 1-34, wherein the CAR-Treg cell exhibits a persistent Treg phenotype.

36. The CAR-Treg of embodiment 35, wherein the persistent Treg phenotype persists in a patient after the CAR-Treg is administered to the patient.

37. The CAR-Treg of embodiment 36, wherein the Treg phenotype persists at least 2 weeks, at least 3, weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months or at least 2 years after the CAR-Treg is administered to the subject.

38. The CAR-Treg of any one of embodiments 1-37, wherein the CAR-Treg is CD25+, CD 127- , or both CD25+ and CD127-.

39. The CAR-Treg cell of any one of embodiments 1-37, wherein the CAR-Treg cell has a high CD25, high CD4 and low CD27 phenotype.

40. The CAR-Treg of any one of embodiments 1-39, wherein the CAR-Treg exhibits higher

CD25 expression, lower CD 127 expression, or both compared to a CD4+ T cell. 41. The CAR-Treg of any one of embodiments 1-40, wherein the CAR-Treg exhibits lower IFN -gamma expression than a CD4+ T cell.

42. The CAR-Treg of any one of embodiments 1-41, wherein the CAR-Treg exhibits lower expression and/or lower secretion of IL-2 than a CD4+ T cell.

43. The CAR-Treg of any one of embodiments 1-42, wherein the CAR-Treg exhibits higher expression and/or higher secretion of IL-10 than a CD4+ T cell.

44. The CAR-Treg of any one of embodiments 1-43, wherein the CAR-Treg exhibits suppression in a Mixed Lymphocyte Reaction (MLR) assay.

45. The CAR-Treg of any one of embodiments 1-44, wherein the human leukocyte antigen is an HLA-A2 antigen.

46. The CAR-Treg of embodiment 45, wherein the HLA-A2 antigen is an HLA-A*2:0l, an HLA-A*2:02, HLA-A*2:03, HLA-A*2:05, HLA-A*2:06, HLA-A*2:07 or HLA-A*2:0l l.

47. The CAR-Treg of embodiment 45, wherein the HLA-A2 antigen is HLA-A*02:0l.

48. The CAR-Treg of any one of embodiments 1-44, wherein the human leukocyte antigen is an allele listed in Table 1.

49. A method for generating chimeric-antigen-receptor regulatory T cells (CAR-Tregs) having a persistent Treg phenotype, comprising:

a) providing a population of T cells; and

b) contacting the population of T cells with one or more vectors under conditions sufficient for transduction of the vector;

wherein the one or more vectors comprise a first trangene encoding a chimeric antigen receptor (CAR) polypeptide operatively linked to a first promoter and

a second transgene encoding a constitutively active Fox3p operably linked a second promoter.

50. The method of embodiment 49, wherein a substantial fraction of the population of T cells are not naturally occurring regulatory T cells (nTregs).

51. The method of embodiment 49, wherein the population of T cells comprises CD4+ T cells.

52. The method of embodiment 50, wherein less than 10%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the CD4+ T cells are nTregs.

53. The method of any one of embodiments 49-52, wherein the population of T cells are autologous to a subject.

54. The method of any one of embodiments 49-53, wherein expression of constitutively active

Foxp3 converts CD4+ T cells into regulatory T cells. 55. The method of any one of embodiments 49-54, comprising contacting the T-cell population with the vector in the presence of T-cell stimulatory beads.

56. The method of any one of embodiments 49-55, wherein the one or more vectors comprises a lentiviral vector.

57. The method of embodiment 56, wherein the lentiviral vector comprises a sequence encoding the first transgene and a sequence encoding the second transgene.

58. The method of embodiment 57, first promoter and the second promoter are the same promoter.

59. The method of embodiment 58, wherein the first transgene and the second transgene are both encoded by a polynucleotide that further comprises a sequence encoding a cleavage site between the first and second transgenes.

60. The method of embodiment 59, wherein the lentiviral vector comprises a sequence encoding, in order from 5’ to 3’, the promoter, the first transgene, the cleavage site and the second transgene.

61. The method of embodiment 59 or 60, wherein the cleavage site is selected from the group consisting of a T2A peptide, an E2A peptide, an F2A peptide and a P2A peptide.

62. The method of embodiment 61, wherein the T2A peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 30).

63. The method of any one of embodiments 49-62, wherein the CAR polypeptide comprises at least one cytoplasmic activation domain.

64. The method of embodiment 63, wherein the at least one cytoplasmic activation domain is a CD247 molecule (Oϋ3z) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain or a DNAX-activating protein of 12 kDa (DAP 12) activation domain, or an IL-2Rbeta cytoplasmic domain.

65. The method of embodiment 64, wherein the 6Ό3z activation domain comprises three immunoreceptor tyrosine-based activation motifs (ITAMs).

66. The method of embodiment 65, wherein the 0O3z activation domain comprises a sequence of

67. The method of embodiment 64, wherein the 0O3z activation domain comprises one ITAM. 68. The method of embodiment 67, wherein the Oϋ3z activation domain comprises a sequence of RVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 23).

69. The method of embodiment 64, wherein the IL-2Rbeta cytoplasmic domain comprises one or more STAT5 -recruitment motifs.

70. The method of embodiment 69, wherein the STAT5-recruitment motif(s) consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 46).

71. The method of embodiment 64, wherein the chimeric antigen receptor comprises one or more STAT5 -recruitment motifs outside the IL-2Rbeta cytoplasmic domain.

72. The method of any one of embodiments 49-71, wherein the CAR polypeptide further comprises at least one co-stimulatory domain.

73. The method of embodiment 72, wherein the co-stimulatory domain is selected from the group consisting of IL-2Rbeta, Fc Receptor gamma (FcRy), Fc Receptor beta (FcR ), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-l), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-l (FFA-l), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (FIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional fragments thereof.

74. The method of embodiment 73, wherein the CD28 co-stimulatory domain comprises a sequence of RSKRSRFFHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 25).

75. The method of embodiment 73, wherein the IF-2Rbeta co-stimulatory domain comprises a sequence of

NCRNTGPWFKKVFKCNTPDPSKFFSQFSSEHGGDVQKWFSSPFPSSSFSPGGFAPEIS PFEVFERDKVTQFFPFNTDAYFSFQEFQGQDPTHFV (SEQ ID NO: 27).

76. The method of any one of embodiments 49-75, wherein the CAR polypeptide comprises a hinge region.

77. The method of embodiment 76, wherein the hinge comprises a CD8a or CD28 hinge.

78. The method of embodiment 77, wherein the CD8a hinge comprises a sequence of

TTTPAPRPPTPAPTIASQPFSFRPEACRPAAGGAVHTRGFDFACD (SEQ ID NO: 13). 79. The method of embodiment 77, where, wherein the CD28 hinge comprises a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 15).

80. The method of any one of embodiments 49-79, wherein the CAR polypeptide comprises a transmembrane domain.

81. The method of embodiment 80, wherein the transmembrane domain is a CD28 transmembrane domain or an IL-2Rbeta transmembrane domain.

82. The method of embodiment 81, wherein the CD28 transmembrane domain comprises a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 17).

83. The method of embodiment 81, wherein the IL-2Rbeta transmembrane domain comprises a sequence of PWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 19).

84. The method of any one of embodiments 49-83, wherein the Foxp3 is wild-type (WT) Foxp3.

85. The method of embodiment 84, wherein the wild type Foxp3 comprises an amino acid sequence of

MPNPRPGKPSAPSFAFGPSPGASPSWRAAPKASDFFGARGPGGTFQGRDFRGGAHA SSSSFNPMPPSQFQFPTFPFVMVAPSGARFGPFPHFQAFFQDRPHFMHQFSTVDAHA RTPVFQVHPFESPAMISFTPPTTATGVFSFKARPGFPPGINVASFEWVSREPAFFCTFP NPSAPRKDSTFSAVPQSSYPFFANGVCKWPGCEKVFEEPEDFFKHCQADHFFDEKG RAQCFFQREMVQSFEQQFVFEKEKFSAMQAHFAGKMAFTKASSVASSDKGSCCIV AAGSQGPVVPAWSGPREAPDSFFAVRRHFWGSHGNSTFPEFFHNMDYFKFHNMRPP FTYATFIRWAIFEAPEKQRTFNEIYHWFTRMFAFFRNHPATWKNAIRHNFSFHKCFV RVESEKGAVWTVDEFEFRKKRSQRPSRCSNPTPGP (SEQ ID NO: 9).

86. The method of any one of embodiments 49-83, wherein the Foxp3 is a minimal Foxp3.

87. The method of embodiment 86, wherein the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C-terminally truncated.

88. The method of embodiment 87, wherein the N- and C-terminally truncated Foxp3 comprises a sequence of

GGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLST

VDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPA

LLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHL

LDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKG

SCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFH NMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSL HKCFVRVESEKGAVWTVDELEF (SEQ ID NO: 10).

89. The method of any one of embodiments 49-83, wherein the Foxp3 comprises a substitution of serine for glutamic acid at amino acid 418 relative to SEQ ID NO: 9 (S418E FOXP3).

90. The method of embodiment 85 or 89, wherein the Foxp3 comprises a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3).

91. The method of any one of embodiments 49-90, wherein the Foxp3 is constitutive ly active.

92. The method of any one of embodiments 49-91, wherein the persistent Treg phenotype persists in a patient after the CAR-Treg is administered to the patient.

93. The method of embodiment 92, wherein the Treg phenotype persists at least 2 weeks, at least 3, weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months or at least 2 years after the CAR-Treg is administered to the subject.

94. The method of any one of embodiments 49-93, wherein the CAR-Treg is CD25+, CD 127- , or both CD25+ and CD127-.

95. The method of any one of embodiments 49-93, wherein the CAR-Treg cell has a high CD25, high CD4 and low CD27 phenotype.

96. The method of any one of embodiments 49-95, wherein the CAR-Treg exhibits higher CD25 expression, lower CD 127 expression, or both compared to a CD4+ T cell.

97. The method of any one of embodiments 49-96, wherein the CAR-Treg exhibits lower IFN- gamma expression than a CD4+ T cell.

98. The method of any one of embodiments 49-97, wherein the CAR-Treg exhibits lower expression and/or lower secretion of IL-2 than a CD4+ T cell.

99. The method of any one of embodiments 49-98, wherein the CAR-Treg exhibits higher expression and/or higher secretion of IL-10 than a CD4+ T cell.

100. The method of any one of embodiments 49-99, wherein the CAR-Treg exhibits suppression in a Mixed Lymphocyte Reaction (MLR) assay.

101. The method of any one of embodiments 49-100, wherein the CAR polypeptide comprises an extracellular domain targeting a human leukocyte antigen (HLA).

102. The method of embodiment 101, wherein the human leukocyte antigen is an HLA-A2 antigen. 103. The method of embodiment 102, wherein the HLA-A2 antigen is an HLA-A*2:0l, an HLA-A*2:02, HLA-A*2:03, HLA-A*2:05, HLA-A*2:06, HLA-A*2:07 or HLA-A*2:0l l .

104. The method of embodiment 103, wherein the HLA-A2 antigen is HLA-A* 02:01.

105. The method of embodiment 101, wherein the human leukocyte antigen is an allele listed in Table 1.

106. A vector for use in modifying a chimeric-antigen-receptor T cell (CAR-Treg cell) to have a persistent Treg phenotype for therapeutic use in a transplant patient, comprising:

a) a sequence encoding a CAR; and

b) a sequence encoding a constitutively active Foxp3.

107. The vector of embodiment 106, wherein the sequence encoding the CAR and the sequence encoding the constitutively active Foxp3 are encoded by the same polynucleotide.

108. The vector of embodiment 107, wherein the vector comprises a promoter.

109. The vector of embodiment 108, wherein the promoter comprises an Elongation Growth Factor- la (EF-la).

110. The vector of embodiment of any one of embodiments 106-109, wherein the vector comprises a sequence encoding, from 5’ to 3’, the promoter, the CAR, a cleavage site and Foxp3.

111. The vector of embodiment 110, wherein the cleavage site is selected from the group consisting of a T2A peptide, an E2A peptide, an F2A peptide and a P2A peptide.

112. The vector of embodiment 111, wherein the T2A peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 30).

113. The vector of any one of embodiments 106-112, wherein the Foxp3 is wild-type (WT) Foxp3.

114. The vector of embodiment 113, wherein the wild type Foxp3 comprises an amino acid sequence of

MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHA SSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHA RTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFP NPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKG RAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIV AAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPP FTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFV

RVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP (SEQ ID NO: 9). 115. The vector of any one of embodiments 106-112, wherein the Foxp3 is a minimal Foxp3.

116. The vector of embodiment 115, wherein the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C- terminally truncated.

117. The vector of embodiment 116, wherein the N- and C-terminally truncated Foxp3 comprises a sequence of

GGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLST

VDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPA

LLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHL

LDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKG

SCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFH

NMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSL

HKCFVRVESEKGAVWTVDELEF (SEQ ID NO: 10).

118. The vector of embodiment 106 or 117, wherein the Foxp3 comprises a substitution of serine for glutamic acid at amino acid 418 relative to SEQ ID NO: 9 (S418E FOXP3).

119. The vector of embodiment 118, wherein the Foxp3 comprises a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3).

120. The vector of any one of embodiments 106-119, wherein expression of Foxp3 by CD4+ cells transduced with the vector induces a persistent Treg phenotype.

121. The vector of embodiment 120, wherein the persistent Treg phenotype persists in a patient after the CAR-Treg is administered to the patient.

122. The vector of embodiment 121, wherein the Treg phenotype persists at least 2 weeks, at least 3, weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months or at least 2 years after the CAR-Treg is administered to the subject.

123. A method of treating or inhibiting autoimmune disease, allergic disease, or inflammatory disease in a patient in need thereof, comprising:

a) providing a population of the CAR regulatory T cells (CAR-Treg cells) of any one of embodiments 1-48; and

b) administering the population of Treg cells to the patient.

124. The method of embodiment 123, wherein the Treg cells are autologous. 125. The method of embodiment 123 or 124, wherein the autoimmune disease, allergic disease, or inflammatory disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, and allergic rhinitis.

126. The method of embodiment 123 or 124, wherein the autoimmune disease, allergic disease, or inflammatory disease is an organ-specific inflammatory disease.

127. The method of embodiment 126, wherein the organ-specific inflammatory disease is selected from the group consisting of kidney disease and lung disease.

128. The method of embodiment 123 or 124, wherein the autoimmune disease, allergic disease, or inflammatory disease is graft versus host disease (GvHD).

129. The method of embodiment 128, wherein the GvHD is caused by a stem cell transplant.

130. The method of embodiment 129, wherein the stem cells comprise hematopoietic stem cells, bone marrow cells and/or peripheral blood mononuclear cells (PBMCs).

131. The method of embodiment of any one of embodiments 128-130, wherein the transplant is allogeneic.

132. The method of embodiment 131, wherein the human leukocyte antigen targeted by the CAR is the same HLA allele as a donor.

133. The method of embodiment 131, wherein the human leukocyte antigen targeted by the CAR is the same HLA allele as a recipient.

134. The method of any one of embodiments 128-133, wherein the method increases CD45+ cell engraftment.

135. The method of 108 or 109, wherein the autoimmune disease, allergic disease, or inflammatory disease is transplant rejection.

136. The method of embodiment 135, wherein the transplant rejection is due to transplanted, kidney, liver, heart, lung or skin.

137. The method of embodiment 135 or 136, wherein the human leukocyte antigen targeted by the CAR is the same HLA allele as a donor.

138. The method of embodiment 135 or 136, wherein the human leukocyte antigen targeted by the CAR is the same HLA allele as a recipient.

139. The method of any one of embodiments 123-138, wherein the method increases overall survival.

140. The method of any one of embodiments 123-139, wherein the method decreases serum inflammatory cytokines. 141. The method of any one of embodiments 123-140, wherein the method decreases spleen inflammation, liver inflammation, lung inflammation, inflammation of the central nervous system (CNS), inflammation of the skin, inflammation of the pancreas, inflammation of the kidney, inflammation of the pleural cavity, inflammation of the gastrointestinal tract, inflammation of the genitourinary tract, or inflammation of the pelvis.

142. A method for generating chimeric-antigen-receptor regulatory T cells (CAR-Tregs) capable of exhibiting a persistent Treg phenotype, comprising:

a) providing a regulatory T cell (Treg cell); and

b) contacting the Treg cell with one or more vectors;

wherein the one or more vectors comprise a polynucleotide encoding a chimeric antigen receptor (CAR) having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL- 2Rbeta cytoplasmic domain).

143. The method of embodiment 142, wherein the IL-2Rbeta cytoplasmic domain comprises one or more STAT5-recruitment motifs.

144. The method of embodiment 143, wherein the STAT5-recruitment motif(s) consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 46).

145. The method of embodiment 142, wherein the chimeric antigen receptor comprises one or more STAT5 -recruitment motifs outside the IL-2Rbeta cytoplasmic domain.

146. The method of embodiment 142, wherein the one or more vectors comprises a polynucleotide encoding a Foxp3.

147. The method of embodiment 146, wherein the Foxp3 is wild-type (WT) Foxp3.

148. The method of embodiment 147, wherein the wild type Foxp3 comprises an amino acid sequence of

MPNPRPGKPSAPSFAFGPSPGASPSWRAAPKASDFFGARGPGGTFQGRDFRGGAHA SSSSFNPMPPSQFQFPTFPFVMVAPSGARFGPFPHFQAFFQDRPHFMHQFSTVDAHA RTPVFQVHPFESPAMISFTPPTTATGVFSFKARPGFPPGINVASFEWVSREPAFFCTFP NPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKG RAQCFFQREMVQSFEQQFVFEKEKFSAMQAHFAGKMAFTKASSVASSDKGSCCIV AAGSQGPVVPAWSGPREAPDSFFAVRRHFWGSHGNSTFPEFFHNMDYFKFHNMRPP FTYATFIRWAIFEAPEKQRTFNEIYHWFTRMFAFFRNHPATWKNAIRHNFSFHKCFV RVESEKGAVWTVDEFEFRKKRSQRPSRCSNPTPGP (SEQ ID NO: 9).

149. The method of embodiment 146, wherein the Foxp3 is a minimal Foxp3. 150. The method of embodiment 149, wherein the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C- terminally truncated.

151. The method of embodiment 150, wherein the N- and C-terminally truncated Foxp3 comprises a sequence of

GGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLST

VDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPA

LLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHL

LDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKG

SCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFH

NMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSL

HKCFVRVESEKGAVWTVDELEF (SEQ ID NO: 10).

152. The method of embodiment 146, wherein the Foxp3 comprises a substitution of serine for glutamic acid at amino acid 418 relative to SEQ ID NO: 9 (S418E FOXP3).

153. The method of embodiment 146 or 142, wherein the Foxp3 comprises a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3).

154. The method of any one of embodiments 142-153, wherein the polynucleotide encoding the CAR and the polynucleotide encoding the Foxp3 are configured for translation as a fusion protein comprising the CAR and the Foxp3.

155. The method of embodiment 154, wherein the CAR is N-terminal to the Foxp3 in the fusion protein.

156. The method of embodiment 154 or 155, wherein the fusion protein comprises a post- translational cleavage site between the CAR and the Foxp3.

157. The method of embodiment 156, wherein the post-translational cleavage site is selected from the group consisting of a T2A polypeptide, an E2A polypeptide, an F2A polypeptide and a P2A polypeptide.

158. The method of embodiment 156 or 157, wherein the CAR is cleaved from the Foxp3 following translation.

159. A chimeric-antigen-receptor regulatory T cell (CAR-Treg cell) generated by the methods of any one of embodiments 142-158.

160. The CAR-Treg of embodiment 159, wherein the CAR-Treg phenotype persists after transplantation into a patient. 161. A vector for use in modifying a chimeric-antigen-receptor regulatory T cell (CAR-Treg cell) to have a persistent Treg phenotype for therapeutic use in a transplant patient, comprising: a) a polynucleotide encoding a chimeric antigen receptor having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2Rbeta); and

b) a polynucleotide encoding a constitutively active Foxp3;

162. A method of treating or inhibiting autoimmune disease, allergic disease, or inflammatory disease in a patient in need thereof, comprising administering the CAR-Treg of any one of embodiments 142-160 to the patient.

163. The method of embodiment 162, wherein the autoimmune disease, allergic disease, or inflammatory disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, and allergic rhinitis.

164. The method of embodiment 163, wherein the autoimmune disease, allergic disease, or inflammatory disease is an organ-specific inflammatory disease.

165. The method of embodiment 164, wherein the organ-specific inflammatory disease is selected from the group consisting of kidney disease and lung disease.

166. A method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ, comprising administering to the subject the CAR-Treg cells of any one of embodiments 142-160.

167. The method of embodiment 166, wherein the CAR-Treg cells are autologous.

168. The method of embodiment 167, wherein the patient is transplanted before administering the Treg cell to the patient.

169. The method of embodiment 168, wherein the patient is transplanted after administering the Treg cell to the patient.The invention is further described in the following Examples, which do not limit the scope of the invention described in the embodiments. EXAMPLES

Example 1: Exemplary Gene Constructs

[201] Exemplary polynucleotide sequences useful in the methods of the present disclosure include the following artificial sequences, which are also depicted in FIGs. 1-8 and summarized in Table 2.

Table 2: Exemplary Constructs

Example 2: Transduction of CD25^hl / CD4^hl / CD27¹⁰ phenotype natural regulatory T cells

[202] Splenocytes are isolated from mice and cultured ex vivo in the presence of IL-2, resulting in expansion of a CD25^hl / CD4¹" / CD27¹⁰ population of natural regulatory T cells (nTregs). The nTreg population is enriched using magnetic separating with anti-CD25 conjugated beads and counterselection from anti-CD27 beads. Each of the constructs in Table 2 are transformed into a packaging cell line. The resulting lentiviral particles are harvested and used to transduce the nTregs. After two days of culture, flow cytometric analysis confirms cell- surface expression of a chimerical antigen receptor (CAR) specific to HLA A2 and intracellular expression of Foxp3.

[203] The resulting nTreg population is transplanted into a host animal. Lymph nodes and spleens are harvested after 2, 4, and 7 days. Flow cytometry analysis confirms increased persistence of the Treg phenotype compared to control.

Example 3: Chimeric Antigen Receptors with FOXP3: Converting CD4+ T cells to Regulatory Cells to Suppress Inflammation in Allotransplantation

[204] After organ transplantations such lung transplantation, patients are managed with immunosuppressive agents including calcineurin inhibitor, prednisone, and mycophenolate mofetil to reduce the risk of organ rejection. However, these immunosuppressive medications act to reduce patients’ T cell count, making it difficult to isolate sufficient quantities of regulatory T cells (5-10% of CD4+ T cells) from blood for manufacturing of CAR-Tregs. To overcome this limitation, a method to convert CD4+ T cells into regulatory CAR-T cells with the addition of FOXP3 attached to the CAR construct via a cleavable T2A linker was developed. This method of converting CD4+ T cells into regulatory CAR-T cells dramatically reduces the volume of blood collected from transplant patients and provides higher numbers of starting cells to enable a faster transduction and expansion process. Both in vitro and in vivo studies demonstrate that CD4+ T cells engineered to produce FOXP3 and CAR can be used to suppress autologous CD3 proliferation in mixed lymphocyte reaction (MLR) and inflammation and GvHD progression in a mouse model.

[205] To limit rejection and ensure the best chances for stable engraftment, (organ) allotransplantation requires suppression of endogenous host inflammation. Immune suppressing agents (such as calcineurin inhibitors like Tacrolimus, steroids like prednisone, and CpG/cell-cycle inhibitors like Cellcept, see Table 3) are commonly applied to control rejection by limiting immune responses to non-self (i.e. donor) antigens present on the transferred graft. Although such treatments show varying degrees of efficacy in controlling acute rejection (depending on the context), they often fail to prevent chronic rejection while simultaneously disposing patients to other life-threatening complications, such as infection and cancer (by eliminating immune cell compartments that normally control these threats). Thus, therapies are needed that (a) limit acute and chronic allotransplant rejection (b) without compromising key immune cell compartments.

Table 3. Maintenance of Immunosuppression after Alemtuzumab (Campath) Induction

Table adapted from Lung transplantation management guidelines, University of Pittsburgh Medical Center (2016).

[206] Adoptive transfer of regulatory T cells (i.e., Tregs, which are CD3+, CD4+, FOXP3+ and CD25+) represents an attractive therapeutic intervention strategy to address the complications of allotransplantation by providing immune-suppression that has the potential for well-tolerated, stable (persistent) effects without eliminating key immune cell compartments. [207] However, several factors complicate the use of Tregs for Adoptive Cell Therapy (ACT) to treat allotransplantation. First, because transplant patients are typically on the immune suppressing agents mentioned above, the amount of Tregs available for extraction is insufficient to meet needs for efficient ex vivo expansion to achieve cell numbers required for meaningful therapeutic dosing. Additionally, regulatory T cells proliferate at a significantly reduced rate and are less-stable compared to non-regulatory CD3+ cells, furthering the limitations associated with expanding these cells to obtain an effective dose for patients. In the context of manufacturing treatment products for patients, regardless of treatment status, Tregs may therefore be inappropriate candidates for scalable anti-inflammatory therapy.

[208] To overcome the limitations associated with the ex vivo expansion of Tregs for adoptive cell transfer (ACT), CD4+ T cells can be used in ACT to meet demands for effective dosing. CD4+ T cells are appropriate candidates for this purpose for several reasons: (1) they are present at significantly higher numbers than Tregs and therefore a sufficient number can be extracted for ex vivo expansion, (2) they can be converted to a regulatory phenotype via adoptive transfer and stable expression of the FOXP3 gene and (3) they can be expanded efficiently due to their higher rate of proliferation and increased stability. Moreover, these factors also distinguish CD4+ T cells as superior candidates (compared to Tregs) for adoptive cell therapies for auto-immunity and inflammation indications.

[209] Further control of undesired off-target effects (e.g. compromised immunity leading to infection or cancer) can be achieved via the application of a Chimeric Antigen Receptor (CAR) designed to“home” to and become selectively activated by a target antigen. Generally, donor- specific antigens (e.g. non-self/mismatched pMHCs) present only on the graft are targeted in this system, allowing converted CD4+ cells to exert their effects in a local, context-specific, and controlled manner.

[210] FOXP3 plays an essential role in the regulatory functions of CD4+CD25+ suppressive T cells (Tregs), serving as the identifying marker of the Treg phenotype (Fontenot, J.D. et al. (2003); Lu, L. et al. (2017); Ono, M., et al., (2008) Regulatory T cells and immune tolerance. Cell, 133(5), 775-787). Mutations in FOXP3 yield severe functional deficiencies in Tregs in humans and mice (e.g. in IPEX and scurfy syndromes, respectively), while forced expression of FOXP3 can drive the acquisition of regulatory/suppressive functions in CD4+ cells. Moreover, adoptive gene transfer of FOXP3 into CD4+ cells extracted from patients with IPEX syndrome (marked by deficiencies in Treg function driven by specific FOXP3 mutations) can convert these cells into functional, stable immune-regulatory cells, as mentioned above (Bacchetta, R. et al. (2013) CD4 T Cells from IPEX Patients Convert into Functional and Stable Regulatory T Cells by FOXP3 Gene Transfer. Science Translational Medicine, 5(215); Zheng, S. G. et al. (2018) Targeting IF-2: An Unexpected Effect in Treating Immunological Diseases. Signal Transduction and Targeted Therapy (Nature)).

[211] A diagram of constructs for converting CD4+ T cells to T regulatory cells to suppress inflammation in allotransplantation is shown in FIG. 9. Including FOXP3 in chimeric antigen receptor (CAR) designs offers the opportunity to convert CD4+ cells to a regulatory phenotype with several advantages for application in allotransplantation therapy. First, the ability to convert CD4+ T cells to suppressive cells circumnavigates the key limiting factors associated with the application of Tregs in allotransplantation rejection therapy: the lack of available Tregs due to immune cell-depletion and their poor growth characteristics and fragility. Second, FOXP3 expression in natural Tregs is highly regulated (and prone to silencing) by the methylation status of the upstream non-coding, so-called“CNS2”, region. When CNS2 is methylated, pSTAT5 cannot bind the FOXP3 promoter efficiently, resulting in reduced FOXP3 expression, decreased suppressive function, and an“instable” Treg phenotype (Fotenont, J.D. et al. (2003); Fu, F. et al. (2017); Greene, M. I. et al. (2012) Structural and Biological Features of FOXP3 Dimerization Relevant to Regulatory T Cell Function. Cell Reports, 1(6)). By linking FOXP3 expression to a promoter independent of CNS2 regulation, converted CD4+ cells express FOXP3 more consistently, are more suppressive, and maintain a stable regulatory phenotype. Third, CAR+ cells“home” to target engrafted tissue and exert their effects in a localized, restricted manner. This effect limits off-target suppression events that may lead to other complications, such as infection or cancer, offering an additional level of control for these therapies through higher on-target specificity. Fourth, modifications of FOXP3 itself offer yet another level of control in CAR designs for regulatory conversion of traditional CD4+ cells. For example, mutants that are resistant to inflammatory cytokine -driven turnover of FOXP3 may induce a more stable regulatory phenotype compared to wild-type forms of FOXP3 (Fi, B. (2014) PIM1 Kinase Phosphorylates the Human Transcription Factor FOXP3 at Serine 422 to Negatively Regulate Its Activity under Inflammation. Biological Chemistry, 289(39), 26872-26881; Zhang, J. Z. (2013). Phosphorylation of FOXP3 Controls Regulatory T Cell Function and Is Inhibited by TNF-a in Rheumatoid Arthritis. Nature Medicine, 19(3), 322- 328). Finally, because FOXP3+ cells upregulate CD25 and efficiently bind IF-2, an inherent “kill switch” treatment utilizing anti-CD25 treatments can be applied to quickly control any undesired responses by selectively delivering a toxic payload to CD25+ cells. Transduction and phenotvping

[212] CD4+ T cells were transduced with lentivirus at a multiplicity of infection (MOI) of 5. Additional transduction methods are described in Example 4. After transduction, FOXP3/CAR+ cells were enriched using an optimized method to achieve greater 80% CAR expression (FIG. 10A) . Cells were cultured in XVIVO- 15 media supplemented with 5 % human A/B serum and proliferation was measured using Vi-CEFF over 7 days. CD4+ FOXP3/CAR- T cells demonstrated 3-fold higher proliferation rate compared to Tregs and comparable proliferation rate compared to untransduced CD4+ T cells (FIG. 10B). This result demonstrates that CD4+ T cells can be converted to regulatory T cells while maintaining original proliferative potential. Enriched cells were expanded and rested for 8 days before phenotype analysis was carried out using flow cytometry and fluorescence activated cell sorting (FACS). Cells were labeled with antibodies against CD 127, CD25, and FOXP3. A significant increase in FOXP3, CD25 and decrease in CD 127 in FOXP3/CAR+ cells was observed consistent with the Treg phenotype (FIG. 11B). In addition, cells were activated with CD3/28 beads for 2 days and treated with brefeldin A for 24 hours prior to intracellular staining for IFN-g. Compared to untransduced CD4+ T cells (which were 11.5% IFN-g positive), FOXP3/CAR-T cells had reduced IFN-g production (1.8%) with CD3/28 stimulation (FIG. 11C). This data indicates that FOXP3 transduced CD4+ T cells have a regulatory phenotype (high CD25 and low CD 127), FOXP3 expression, and reduced IF-2 secretion upon activation.

Mixed lymphocyte reaction and cytokine secretion

[213] FOXP3/CAR-T cells were evaluated to see if they were capable of suppression in a mixed lymphocyte reaction (MFR) assay. Further MFR methods are described in Example 4. To perform this assay, CFSE staining was performed on autologous CD3+ T cells from A*02:0l- donor and mismatched, growth arrested FCF721 cells (A*02:0l+) with mitomycin C. Carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) is a cell permeant, non- fluorescent pro-dye which is cleaved by esterases in live cells to produce a green fluorescent molecule that is membrane impermeant. CFSE is an intracellular fluorescent label of living cells that is retained after fixation. CD3+ T cells and FCF721 cells were added at a ratio of 2: 1. CD4+ FOXP3/CAR-T cells were then added to the co-culture at varying ratios of 0.5: 1, 0.2: 1, 0.1 : 1, and 0.05: 1 of CAR T to CD3. After 4 days, CD3 T cell proliferation was quantified using flow cytometry. A drastic increase in CD3 T cell suppression in cells co-cultured with FOXP3/CAR-T cells was observed (FIG. 12). In comparison, CD4+ T cells transduced with

CAR alone and control CD4+ T cells both showed minimal suppression even at high cell concentrations. Cells transduced with WT FOXP3 and S418E FOXP3 showed marginally improved suppressive activity compared to NC truncated FOXP3. This result demonstrates the ability of FOXP3+/CAR+ cells to inhibit A*02 mismatch-induced activation of CD3+ T cells in vitro.

[214] Furthermore, supernatant from the MLR experiment was collected and cytokine levels quantified using cytometric bead array (CBA). A substantial increase in IL-10 secretion by WT FOXP3 and S418E FOXP3/CAR-T cells compared CAR+ cells without FOXP3 was observed (FIG. 13). NC truncated FOXP3+ cells showed a slight increase in IL-10 secretion. Additionally, secretion of proinflammatory cytokines (IL-2 and TNFa) was suppressed in all FOXP3/CAR cell conditions, with the most significant suppression in the WT and S418E FOXP3/CAR-T cells co-cultures (FIG. 13). In summary, both WT FOXP3 and S418E FOXP3/CAR-T cells effectively suppressed CD3+ T cell proliferation in an MLR assay by increased secretion of suppressive cytokines and reduced secretion of proinflammatory cytokines.

Graft-versus-Host Disease Study ( GvHD )

[215] A common model used to study transplantation is GvHD, (graft-versus-host disease). GvHD occurs when the body’s immune-system attacks recipient tissue driven mainly by donor CD8+ T cells contained in the transplant. Murine models of GvHD use human peripheral blood mononuclear cells (PBMCs) that engraft in the mouse usually within 3-4 weeks, although the timeframe may vary. To mimic transplantation, allele-level human leukocyte antigen (HLA- A*02:0l) PBMCs were used to represent the donor and CD4+ cells were used as the recipient.

[216] In order to explore the functional capacity of A2-CAR Tregs and A2-CAR-CD4s to be superior to polyclonal Tregs at preventing xenogeneic GvHD mediated by HLA-A2+ PBMC a mouse model in which human PBMCs engrafted into immunodeficient NOD SCID IL-2Ry -/- (NSG) mice was used to generate xenogeneic GvHD.

[217] In these experiments, 10E7 PBMCs from an HLA-A2+ donor were injected into NSG mice by tail vein injection. The mice were then divided into 4 groups (n=6 per group). Group 1, a control, was not further treated. CD4 T cells or enriched CD4 Treg cells were transduced with lentivirus at an MOI of 5 virus particles/cell. At day 7, CAR+ cells were examined by flow cytometric analysis of kappa light chain positive cells. These cells were then restimulated as previously described and expanded for 6 to 7 days. To test effects of A2 -mediated stimulation, HLA-A2 CAR+ Tregs unfortunately only reached 10 - 13% whereas CAR+ CD4s reach 96% positivity. However, these cells were used for injection. At both 2 and 4 weeks the other groups (2, 3 and 4) were injected with the saline or the cell numbers indicated below (see FIG. 14 Model and Timeline).

Group 1 ; saline only

Group 2; 4xlOE6 polyclonal Treg

Group 3; 4x10E6 CAR- Treg

Group 4; 4xlOE6 CAR-CD4

[218] Body weight and clinical scores were monitored on a daily basis for 9 weeks and blood samples were taken at week 2, 3, 4, 5, 6, 8, 9 and used for flow cytometric analysis of engraftment of PBMCs in peripheral blood and for analysis of peripheral blood cytokine levels as markers of inflammation.

[219] The clinical scores showed little deterioration until about 4 weeks and then a number of mice in each group began to lose weight and show signs of GvHD including bald patches and scratching. These symptoms and clinical score are summarized in the survival graphs shown in FIG. 15 (survival curves). The mice that received HLA-A2+ CAR-CD4 cells had improved survival and delayed onset or reduced of symptoms and, with one exception, those mice that had exhibited signs of GvHD at 4 weeks had improvements following the injection of HLA-A2+ CAR-CD4 cells.

[220] The experiment was stopped at week 9 and the animals were euthanized and blood samples were taken as before for analysis of PBMCs, serum cytokines and lung, liver and spleen tissue taken for pathological analysis.

[221] Engraftment was assessed using engraftment was assessed using flow cytometric analyses of cells in peripheral blood. For flow cytometry analysis, PBMCs were stained with fixable viability dye (FVD) and for surface markers expression and separately staining for intracellular proteins. Before fixation and permeabilization with FOXP3 staining, surface staining was performed for HLA-A2 (BB7.2, Biolegend), CD3, CD4, CD8, hCD45, mCD45 and CD25. Intracellular staining was performed for FOXP3 (Biolegend). As is clearly evident from FIG. 16, the engraftment of was detectable in the peripheral blood of most animals by 3- 4 weeks and reached a plateau by 5- 6 weeks after the initial xenogeneic PBMC injections.

[222] For serum cytokine analysis samples were incubated with CBA beads and were read on an FACS CANTO (BD Biosciences) and results analyzed using FlowJo Software (Tree Star) (FIG. 17, serum cytokines). The data clearly shows that the proinflammatory cytokines IFN gamma and TNF alpha were not elevated in animals from group 4 that had been dosed with

HLA-A2+ CAR-CD4 T cells, suggesting that these cells markedly suppressed inflammation in these animals. This was not the case in any other group as neither the polyclonal Treg nor the HLA-A2+ CAR-Treg markedly reduced serum cytokines.

[223] Pathological analysis of sections from 6 animal lungs in each group 1 - 4 was undertaken for spleen, liver and lung. The observations showed no significant differences were observed in the spleen tissue between the groups as the groups could not be segregated reliably using splenic architecture, with the possible exception of a clear lack of any fibrosis in sections from untreated controls (FIG. 18, spleen pathology). Analysis of liver haemotoxylin and eosin (H&E) stained sections showed that groups 2 and 4 appear to have relatively reduced liver changes relative to other non-control groups, with generally less than 5% degeneration or necrosis (consider minimal). Conversely groups 1 and 3 showed mild (Inflammation and/or degeneration / necrosis affects 10% to 20% of the section) to moderate (Inflammation and/or degeneration / necrosis affects 25% to 40% of the section) (FIG. 19, liver pathology). The most evidence of amelioration of pathology and inflammatory infiltrate changes due to CAR-CD4 cells was evident in the lung pathology of group 4. When compared to animals in groups 1, 2 and 3, the analysis showed that most animals in group 4 the lungs showed either no inflammatory findings and no consolidation of alveolar parenchyma or occasional inflammatory foci (<2% of section) with no consolidation of alveolar parenchyma, whereas lung sections from mice in groups 1, 2 and 3 showed obvious inflammation with consolidation of alveolar parenchyma impacts about 30% to 90%, or all, of the sections (FIG. 20 and 21 , lung pathology). These observations of lung and liver pathology showed that the mice that received CAR-CD4 cells had markedly less infiltrate and pathology than other mice strongly suggesting that these cells could block inflammatory pathology in the GvHD model.

Example 4: Methods

Isolation and transduction ofCD4+ T cells

[224] A*02:0l- PBMCs were isolated from fresh Leukopak (All Cells) using Ficoll density gradient media and frozen in CS-10 and stored in liquid nitrogen. Frozen PBMCs were thawed and CD4+ T cells were isolated using CD4+ T cell negative isolation kit (Miltenyi), according to manufacturer’s instructions. Post isolation, CD4+ T cells were cultured at a density of 1 x 10^L6 cells/mL in X-Vivo 15 media supplemented with 5% human A/B serum and 1% Pen/strep in the presence of CD3/28 Dynabeads (1 : 1 cell to bead ratio) and 300 Units/mL of IL-2 (Miltenyi) . After 2 days, cells were transduced with lentivirus (outsourced) at a MOI of 5. Cells were cultured in IL-2 for an additional 5 days prior to enrichment. Phenotvpins of CD4+ CAR-T cells

[225] Extracellular staining: enriched F0XP3/CAR-T cells were rested for 8 days in media containing 100 Units/mL of IL-2. Cells were collected and stained using antibodies against FOXP3 (PE), CD25 (APC), and CD 127 (BV421) for 30 minutes on ice, and ran on the flow cytometer (BC FACS Canto). Data was analyzed and graphed using FlowJo.

Intracellular cytokine staining: cells were activated with CD3/28 beads for 48 hours prior to addition of 10 pg/mF of Brefeldin A for 18 hours. Cells were harvested, fixed in IC fixation buffer (eBioscience) for 60 minutes at RT, and stained in lx permeabilization buffer (eBioscience) and anti-IFNy antibody for another 60 minutes. Cells were washed and analyzed on the flow cytometer.

Mixed lymphocyte reaction

[226] A*02:0l- CD3+ T cells (same donor as CAR-T cells) were isolated from PBMCs using CD3+ negative selection kit from Miltenyi. Post isolation, CD3+ T cells were stained with CFSE (2uM) for 15 minutes at RT. FCF721 cells (A*02:0l+) were treated with Mitomycin C at lOpg/mF for 30 minutes at 37°C. CD3+ cell and FCF721 cells were co-cultured at a ratio of 1 :0.5 CD3:FCF72l cells. FOXP3/CAR-T cells were then added do the co-culture at varying ratios. After 4 days, supernatant was collected and stored at -20°C for cytokine analysis. Cells were read on the flow cytometer and CD3+ T cell proliferation was analyzed using FlowJo.

[227] Cytokine analysis: supernatant was analyzed using BC CBA Thl/Th2/Thl7 kit, according to manufacturer’s instructions. Briefly, mixed capture beads and PE detection reagent were added to all assay samples (including standards) and incubated for 3 hours at RT, protected from light. Wash samples and read on the flow cytometer.

IL-2 transfection ofHEK293T cells

[228] HEK293T cells were seeded in 12 well plates at a density of 0.3 x l0⁶/mF and cultured overnight. The next day, HEK293T cells were transfected with CAR/FOXP3 IF-2 constructs using Fipofectamine 3000. Cells were incubated for an additional 24 hours and supernatant was harvested and stored at -20°C.

PhosphoSTAT5 staining of primary T cells

[229] CD4+ T cells and FOXP3/CAR-T cells were IF-2 starved and in cultured in X-Vivo 15 media for 36 hours prior to pSTAT5 experiments. IF-2 starved T cells were then cultured in HEK293T secreted media containing IF-2 at various titrations for 20 minutes. Cells were fixed with addition of equal volume of lx IC Fixation buffer (eBioscience) for 15 minutes at RT. Post fixation, cells were permeabilized using ice cold methanol at 4°C for 30 minutes, washed, and stained using anti-pSTAT5 antibody (PE) and analyzed on the flow cytometer.

Example 5: FOXP3 stability

[230] Mutated FOXP3 constructs were made by single- or double-amino acid substitutions at the following locations: none (WT, #75) S418 (S318E, #76), S418/S422 (S418E, S422A, #59). Phosphorylation of S418 in FOXP3 represents a stabilizing event, reducing FOXP3 turnover by preventing binding events with ubiquitinases (negative regulators) and extending FOXP3 half-life. A phosphomimetic (S418E) mutant was designed to recreate this phosphorylation event in a stable, constitutive form no longer dependent upon upstream kinase activity for the extension of FOXP3 half-life (Ono, M. et al. (2008).

[231] For the S422A mutation, upon inflammatory cytokine-signaling (e.g. IF-6), PIM1 is activated via phosphorylation. Activated PIM1 can bind FOXP3 and phosphorylate serine 422, which disrupts FOXP3 chromatin binding activities required for its functionality as a transcription factor. Knockdown of PIM1 promotes FOXP3-induced target gene expression/repression in human Tregs and enhances their immunosuppressive function (Fi et al., 2017)). A mutant (S422A) that alters this serine to an alanine was designed to eliminate the possibility of PIM1 -driven phosphorylation of this region, freeing FOXP3 from PIM1- mediated negative regulation, and stabilizing the anti-inflammatory transcriptional program conveyed by FOXP3 activity.

[232] CD4+ cells were transduced using the methods described in Example 4.

[233] After stable transduction with the indicated CAR constructs (75, 76, or 77), CD4⁺/FOXP3-CAR⁺ cells from were treated with or without cycloheximide (CHX, 50ng/uF) for 18 hours. During this period, CAR⁺/FOXP3⁺ cells were co-treated with either IFNy, TNFa, or IF-6 (at 50ng/uF) to induce inflammatory-cytokine driven turnover of FOXP3. I.e., transduced CD4+ cells were treated with or without cycloheximide (CHX, 50 ng/mF) and proinflammatory cytokines (IFNy, TNFa, or IF-6, 50 ng/uF). After 18 hours, cells were fixed, permeabilized (according to the eBiosciences FOXP3 staining Kit protocol), and analyzed by flow cytometry for their FOXP3 levels (1 hour incubation at room temperature with anti- FOXP3 antibody at a 1 :50 dilution) by flow cytometry.

[234] At 18 hours, cells were fixed and analyzed for FOXP3 levels by flow cytometry. Modified FOXP3 constructs (76 and 59) resisted turnover of FOXP3 compared to their wild- type counterpart (75). The results are shown in FIG. 22. Example 6: ITAM Multiplicity in CAR designs that contain FOXP3

[235] CARs designed with CD3zeta motifs modified to contain single functional ITAM were tested, in combination with various hinge and transmembrane (TM) domains, as shown in Table 4 and FIG. 23. All constructs contain the same a-A*02:0l antigen recognition domain. Table 4: Construct Designs

[236] Exemplary vectors with different configurations hinge, transmembrane domain, intracellular domains and CD3z domains with 1 or 3 ITAMs are provided as SEQ ID NOs: 37- 44, and also included a T2A peptide separating the CAR from wild type FoxP3.

[237] To generate effector cells, Jurkat NFAT-luciferase reporter cells were transfected with the each of the CAR-Foxp3 constructs and allowed to recover overnight. As target cells, MRC- 5 (lung fibroblasts, A* 02: 01+) cells were titrated, seeded and allowed to adhere to the plate surface overnight. On the following day, 10³ per well of CAR-Foxp3 effector cells were added to the target cells. Following incubation at 6 hours, luciferase reagent was added and NFAT- driven luciferase signaling in the effector cells was measured. Signaling activity is plotted as a function of effector-to-target (E:T) cell ratio in FIG. 24A and FIG. 24B.

Claims

1. A chimeric-antigen-receptor regulatory T cell (CAR-Treg) comprising:

wherein the CAR-Treg is not a natural regulatory T cell (nTreg).

2. The CAR-Treg of claim 1, wherein the first promoter and the second promoter are the same promoter.

3. The CAR-Treg of claim 2, wherein the first transgene and the second transgene are both encoded by a polynucleotide that further comprises a sequence encoding a cleavage site between the first and second transgenes.

4. The CAR-Treg of claim 3, wherein the post-translational cleavage site is selected from the group consisting of a T2A peptide, an E2A peptide, an F2A peptide and a P2A peptide.

5. The CAR-Treg of claim 4, wherein the T2A peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 30).

6. The CAR-Treg of any one of claims 1-5, wherein the CAR comprises at least one cytoplasmic activation domain.

7. The CAR-Treg of claim 6, wherein the at least one cytoplasmic activation domain is a CD247 molecule ^ϋ3z) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, a DNAX-activating protein of 12 kDa (DAP 12) activation domain, or an IL-2Rbeta cytoplasmic domain.

8. The CAR-Treg of claim 7, wherein the CD3z activation domain comprises 3 immunoreceptor tyrosine-based activation motifs (ITAMs).

9. The CAR-Treg of claim 8, wherein the 6Ό3z activation domain comprises a sequence of

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDT YDALHMQALPPR (SEQ ID NO: 21).

10. The CAR-Treg of claim 7, wherein the Oϋ3z activation domain comprises one ITAM.

11. The CAR-Treg of claim 10, wherein the CD3z activation domain comprises a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 23) .

12. The CAR-Treg of claim 7, wherein the IL-2Rbeta cytoplasmic domain comprises one or more STAT5 -recruitment motifs.

13. The CAR-Treg of claim 12, wherein the STAT5 -recruitment motif(s) consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 46).

14. The CAR-Treg of claim 1, wherein the chimeric antigen receptor comprises one or more STAT5-recruitment motifs outside the IL-2Rbeta cytoplasmic domain.

15. The CAR-Treg of any one of claims 1-14, comprising at least one co-stimulatory domain.

16. The CAR-Treg of claim 15, wherein the co-stimulatory domain is selected from the group consisting of IL-2Rbeta, Fc Receptor gamma (FcRy), Fc Receptor beta (FcR ), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-l), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-l (LFA-l), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional fragments thereof.

17. The CAR-Treg of claim 16, wherein the CD28 co-stimulatory domain comprises a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRS (SEQ ID NO: 25).

18. The CAR-Treg of claim 16, wherein the IL-2Rbeta co-stimulatory domain comprises a sequence of

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGL APEISPFEVFERDKVTQFFPFNTDAYFSFQEFQGQDPTHFV (SEQ ID NO: 27).

19. The CAR-Treg of any one of claims 1-18, comprising a hinge region.

20. The CAR-Treg of claim 19, wherein the hinge comprises a CD8a or CD28 hinge.

21. The CAR-Treg of claim 20, wherein the CD8a hinge comprises a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 13).

22. The CAR-Treg of claim 20, wherein the CD28 hinge comprises a sequence of CTIEVMYPPPYLDNEKSNGTHHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 15) .

23. The CAR-Treg of any one of claims 1-22, comprising a transmembrane domain.

24. The CAR-Treg of claim 23, wherein the transmembrane domain comprises a CD28 transmembrane domain or an IL-2Rbeta transmembrane domain.

25. The CAR-Treg of claim 24, wherein the CD28 transmembrane domain comprises a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 27).

26. The CAR-Treg of claim 24, wherein the IL-2Rbeta transmembrane domain comprises a sequence of PWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 19).

27. The CAR-Treg of any one of claims 1-26, wherein the Foxp3 is wild-type (WT) Foxp3.

28. The CAR-Treg of claim 27, wherein the wild type Foxp3 comprises an amino acid sequence of

MPNPRPGKPSAPSFAFGPSPGASPSWRAAPKASDFFGARGPGGTFQGRDFRG

GAHASSSSFNPMPPSQFQFPTFPFVMVAPSGARFGPFPHFQAFFQDRPHFMH QLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASL

EWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEP

EDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLA

GKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL

WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYH

WFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKK

RSQRPSRCSNPTPGP (SEQ ID NO: 9).

29. The CAR-Treg of any one of claims 1-28, wherein the Foxp3 is a minimal Foxp3.

30. The CAR-Treg of claim 29, wherein the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C-terminally truncated.

31. The CAR-Treg of claim 30, wherein the N- and C-terminally truncated Foxp3

comprises a sequence of

GGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFM HQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVAS LEWV SREPALLCTFPNP S APRKD STLS A VPQ S S YPLLANGV CKWPGCEKVFEE PEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLA GKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYH WFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEF

(SEQ ID NO: 10).

32. The CAR-Treg of any one of claims 1-26, wherein the Foxp3 comprises a substitution of serine for glutamic acid at amino acid 418 relative to SEQ ID NO: 9 (S418E FOXP3).

33. The CAR-Treg of claim 27 or 32, wherein the Foxp3 comprises a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3).

34. The CAR-Treg of any one of claims 1-33, wherein the Foxp3 is constitutively active.

35. The CAR-Treg of any one of claims 1-34, wherein the CAR-Treg cell exhibits a persistent Treg phenotype.

36. The CAR-Treg of claim 35, wherein the persistent Treg phenotype persists in a patient after the CAR-Treg is administered to the patient.

37. The CAR-Treg of claim 36, wherein the Treg phenotype persists at least 2 weeks, at least 3, weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months or at least 2 years after the CAR-Treg is administered to the subject.

38. The CAR-Treg of any one of claims 1-37, wherein the CAR-Treg is CD25+, CD127-, or both CD25+ and CD127-.

39. The CAR-Treg cell of any one of claims 1-37, wherein the CAR-Treg cell has a high CD25, high CD4 and low CD27 phenotype.

40. The CAR-Treg of any one of claims 1-39, wherein the CAR-Treg exhibits higher CD25 expression, lower CD 127 expression, or both compared to a CD4+ T cell.

41. The CAR-Treg of any one of claims 1-40, wherein the CAR-Treg exhibits lower IFN- gamma expression than a CD4+ T cell.

42. The CAR-Treg of any one of claims 1-41, wherein the CAR-Treg exhibits lower expression and/or lower secretion of IL-2 than a CD4+ T cell.

43. The CAR-Treg of any one of claims 1-42, wherein the CAR-Treg exhibits higher expression and/or higher secretion of IL-10 than a CD4+ T cell.

44. The CAR-Treg of any one of claims 1-43, wherein the CAR-Treg exhibits suppression in a Mixed Lymphocyte Reaction (MLR) assay.

45. The CAR-Treg of any one of claims 1-44, wherein the human leukocyte antigen is an HLA-A2 antigen.

46. The CAR-Treg of claim 45, wherein the HLA-A2 antigen is an HLA-A*2:0l, an HLA- A*2:02, HLA-A*2:03, HLA-A*2:05, HLA-A*2:06, HLA-A*2:07 or HLA-A*2:0l l .

47. The CAR-Treg of claim 45, wherein the HLA-A2 antigen is HLA-A*02:0l .

48. The CAR-Treg of any one of claims 1-44, wherein the human leukocyte antigen is an allele listed in Table 1.

a) providing a population of T cells; and

50. The method of claim 49, wherein a substantial fraction of the population of T cells are not naturally occurring regulatory T cells (nTregs).

51. The method of claim 49, wherein the population of T cells comprises CD4+ T cells.

52. The method of claim 50, wherein less than 10%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the CD4+ T cells are nTregs.

53. The method of any one of claims 49-52, wherein the population of T cells are autologous to a subject.

54. The method of any one of claims 49-53, wherein expression of constitutively active Foxp3 converts CD4+ T cells into regulatory T cells.

55. The method of any one of claims 49-54, comprising contacting the T-cell population with the vector in the presence of T-cell stimulatory beads.

56. The method of any one of claims 49-55, wherein the one or more vectors comprises a lentiviral vector.

57. The method of claim 56, wherein the lentiviral vector comprises a sequence encoding the first transgene and a sequence encoding the second transgene.

58. The method of claim 57, first promoter and the second promoter are the same promoter.

59. The method of claim 58, wherein the first transgene and the second transgene are both encoded by a polynucleotide that further comprises a sequence encoding a cleavage site between the first and second transgenes.

60. The method of claim 59, wherein the lentiviral vector comprises a sequence encoding, in order from 5’ to 3’ , the promoter, the first transgene, the cleavage site and the second transgene.

61. The method of claim 59 or 60, wherein the cleavage site is selected from the group consisting of a T2A peptide, an E2A peptide, an F2A peptide and a P2A peptide.

62. The method of claim 61, wherein the T2A peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 30).

63. The method of any one of claims 49-62, wherein the CAR polypeptide comprises at least one cytoplasmic activation domain.

64. The method of claim 63, wherein the at least one cytoplasmic activation domain is a CD247 molecule (Oϋ3z) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain or a DNAX-activating protein of 12 kDa (DAP 12) activation domain, or an IL-2Rbeta cytoplasmic domain.

65. The method of claim 64, wherein the 6Ό3z activation domain comprises three immunoreceptor tyrosine-based activation motifs (ITAMs).

66. The method of claim 65, wherein the 0O3z activation domain comprises a sequence of

67. The method of claim 64, wherein the 0O3z activation domain comprises one ITAM.

68. The method of claim 67, wherein the Oϋ3z activation domain comprises a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 23) .

69. The method of claim 64, wherein the IL-2Rbeta cytoplasmic domain comprises one or more STAT5-recruitment motifs.

70. The method of claim 69, wherein the STAT5 -recruitment motif(s) consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 46).

71. The method of claim 64, wherein the chimeric antigen receptor comprises one or more STAT5 -recruitment motifs outside the IL-2Rbeta cytoplasmic domain.

72. The method of any one of claims 49-71, wherein the CAR polypeptide further comprises at least one co-stimulatory domain.

73. The method of claim 72, wherein the co-stimulatory domain is selected from the group consisting of IL-2Rbeta, Fc Receptor gamma (FcRy), Fc Receptor beta (FcR ), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-l), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-l (LFA-l), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional fragments thereof.

74. The method of claim 73, wherein the CD28 co-stimulatory domain comprises a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRS (SEQ ID NO: 25).

75. The method of claim 73, wherein the IL-2Rbeta co-stimulatory domain comprises a sequence of NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGL APEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 27).

76. The method of any one of claims 49-75, wherein the CAR polypeptide comprises a hinge region.

77. The method of claim 76, wherein the hinge comprises a CD8a or CD28 hinge.

78. The method of claim 77, wherein the CD8a hinge comprises a sequence of

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 13).

79. The method of claim 77, where, wherein the CD28 hinge comprises a sequence of CTIEVMYPPPYLDNEKSNGTHHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 15) .

80. The method of any one of claims 49-79, wherein the CAR polypeptide comprises a transmembrane domain.

81. The method of claim 80, wherein the transmembrane domain is a CD28 transmembrane domain or an IL-2Rbeta transmembrane domain.

82. The method of claim 81, wherein the CD28 transmembrane domain comprises a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 17).

83. The method of claim 81, wherein the IL-2Rbeta transmembrane domain comprises a sequence of PWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 19).

84. The method of any one of claims 49-83, wherein the Foxp3 is wild-type (WT) Foxp3.

85. The method of claim 84, wherein the wild type Foxp3 comprises an amino acid sequence of

MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRG GAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMH QLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASL EWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEP EDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLA GKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYH WFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKK RSQRPSRCSNPTPGP (SEQ ID NO: 9).

86. The method of any one of claims 49-83, wherein the Foxp3 is a minimal Foxp3.

87. The method of claim 86, wherein the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C-terminally truncated.

88. The method of claim 87, wherein the N- and C-terminally truncated Foxp3 comprises a sequence of

(SEQ ID NO: 10).

89. The method of any one of claims 49-83, wherein the Foxp3 comprises a substitution of serine for glutamic acid at amino acid 418 relative to SEQ ID NO: 9 (S418E FOXP3).

90. The method of claim 85 or 89, wherein the Foxp3 comprises a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3).

91. The method of any one of claims 49-90, wherein the Foxp3 is constitutively active.

92. The method of any one of claims 49-91, wherein the persistent Treg phenotype persists in a patient after the CAR-Treg is administered to the patient.

93. The method of claim 92, wherein the Treg phenotype persists at least 2 weeks, at least 3, weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months or at least 2 years after the CAR-Treg is administered to the subject.

94. The method of any one of claims 49-93, wherein the CAR-Treg is CD25+, CD 127-, or both CD25+ and CD127-.

95. The method of any one of claims 49-93, wherein the CAR-Treg cell has a high CD25, high CD4 and low CD27 phenotype.

96. The method of any one of claims 49-95, wherein the CAR-Treg exhibits higher CD25 expression, lower CD 127 expression, or both compared to a CD4+ T cell.

97. The method of any one of claims 49-96, wherein the CAR-Treg exhibits lower IFN- gamma expression than a CD4+ T cell.

98. The method of any one of claims 49-97, wherein the CAR-Treg exhibits lower expression and/or lower secretion of IL-2 than a CD4+ T cell.

99. The method of any one of claims 49-98, wherein the CAR-Treg exhibits higher expression and/or higher secretion of IL-10 than a CD4+ T cell.

100. The method of any one of claims 49-99, wherein the CAR-Treg exhibits suppression in a Mixed Lymphocyte Reaction (MLR) assay.

101. The method of any one of claims 49-100, wherein the CAR polypeptide comprises an extracellular domain targeting a human leukocyte antigen (HLA).

102. The method of claim 101, wherein the human leukocyte antigen is an HLA-A2 antigen.

103. The method of claim 102, wherein the HLA-A2 antigen is an HLA-A*2:0l, an HLA-A*2:02, HLA-A*2:03, HLA-A*2:05, HLA-A*2:06, HLA-A*2:07 or HLA- A*2:0l l .

104. The method of claim 103, wherein the HLA-A2 antigen is HLA-A*02:0l.

105. The method of claim 101, wherein the human leukocyte antigen is an allele listed in Table 1.

a) a sequence encoding a CAR; and

b) a sequence encoding a constitutively active Foxp3.

107. The vector of claim 106, wherein the sequence encoding the CAR and the sequence encoding the constitutively active Foxp3 are encoded by the same polynucleotide.

108. The vector of claim 107, wherein the vector comprises a promoter.

109. The vector of claim 108, wherein the promoter comprises an Elongation Growth Factor- la (EF-la).

110. The vector of claim of any one of claims 106-109, wherein the vector comprises a sequence encoding, from 5’ to 3’, the promoter, the CAR, a cleavage site and Foxp3.

111. The vector of claim 110, wherein the cleavage site is selected from the group consisting of a T2A peptide, an E2A peptide, an F2A peptide and a P2A peptide.

112. The vector of claim 111, wherein the T2A peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 13).

113. The vector of any one of claims 106-112, wherein the Foxp3 is wild-type (WT) Foxp3.

114. The vector of claim 113, wherein the wild type Foxp3 comprises an amino acid sequence of

MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRG

GAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMH

QLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASL

EWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEP

EDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLA

GKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYH WFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKK RSQRPSRCSNPTPGP (SEQ ID NO: 9).

115. The vector of any one of claims 106-112, wherein the Foxp3 is a minimal Foxp3.

116. The vector of claim 115, wherein the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C- terminally truncated.

117. The vector of claim 116, wherein the N- and C-terminally truncated Foxp3 comprises a sequence of

GGAHAS S S SLNPMPPS QLQLPTLPLVMVAP SGARLGPLPHLQ ALLQDRPHFM HQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVAS LEWV SREPALLCTFPNP S APRKD STLS A VPQ S S YPLLANGV CKWPGCEKVFEE PEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLA GKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYH WFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEF

(SEQ ID NO: 10).

118. The vector of claim 106 or 117, wherein the Foxp3 comprises a substitution of serine for glutamic acid at amino acid 418 relative to SEQ ID NO: 9 (S418E FOXP3).

119. The vector of claim 118, wherein the Foxp3 comprises a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3).

120. The vector of any one of claims 106-119, wherein expression of Foxp3 by CD4+ cells transduced with the vector induces a persistent Treg phenotype.

121. The vector of claim 120, wherein the persistent Treg phenotype persists in a patient after the CAR-Treg is administered to the patient.

122. The vector of claim 121, wherein the Treg phenotype persists at least 2 weeks, at least 3, weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months or at least 2 years after the CAR-Treg is administered to the subject.

a) providing a population of the CAR regulatory T cells (CAR-Treg cells) of any one of claims 1-48; and

b) administering the population of Treg cells to the patient.

124. The method of claim 123, wherein the Treg cells are autologous.

125. The method of claim 123 or 124, wherein the autoimmune disease, allergic disease, or inflammatory disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, and allergic rhinitis.

126. The method of claim 123 or 124, wherein the autoimmune disease, allergic disease, or inflammatory disease is an organ-specific inflammatory disease.

127. The method of claim 126, wherein the organ-specific inflammatory disease is selected from the group consisting of kidney disease and lung disease.

128. The method of claim 123 or 124, wherein the autoimmune disease, allergic disease, or inflammatory disease is graft versus host disease (GvHD).

129. The method of claim 128, wherein the GvHD is caused by a stem cell transplant.

130. The method of claim 129, wherein the stem cells comprise hematopoietic stem cells, bone marrow cells and/or peripheral blood mononuclear cells (PBMCs).

131. The method of claim of any one of claims 128-130, wherein the transplant is allogeneic.

132. The method of claim 131, wherein the human leukocyte antigen targeted by the CAR is the same HLA allele as a donor.

133. The method of claim 131, wherein the human leukocyte antigen targeted by the CAR is the same HLA allele as a recipient.

134. The method of any one of claims 128-133, wherein the method increases CD45+ cell engraftment.

136. The method of claim 135, wherein the transplant rejection is due to transplanted, kidney, liver, heart, lung or skin.

137. The method of claim 135 or 136, wherein the human leukocyte antigen targeted by the CAR is the same HLA allele as a donor.

138. The method of claim 135 or 136, wherein the human leukocyte antigen targeted by the CAR is the same HLA allele as a recipient.

139. The method of any one of claims 123-138, wherein the method increases overall survival.

140. The method of any one of claims 123-139, wherein the method decreases serum inflammatory cytokines.

141. The method of any one of claims 123-140, wherein the method decreases spleen inflammation, liver inflammation, lung inflammation, inflammation of the central nervous system (CNS), inflammation of the skin, inflammation of the pancreas, inflammation of the kidney, inflammation of the pleural cavity, inflammation of the gastrointestinal tract, inflammation of the genitourinary tract, or inflammation of the pelvis.

142. A method for generating chimeric-antigen-receptor regulatory T cells (CAR- Tregs) capable of exhibiting a persistent Treg phenotype, comprising:

a) providing a regulatory T cell (Treg cell); and

b) contacting the Treg cell with one or more vectors; wherein the one or more vectors comprise a polynucleotide encoding a chimeric antigen receptor (CAR) having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL-2Rbeta cytoplasmic domain).

143. The method of claim 142, wherein the IL-2Rbeta cytoplasmic domain comprises one or more STAT5-recruitment motifs.

144. The method of claim 143, wherein the STAT5 -recruitment motif(s) consists of the sequence Tyr-Leu-Ser-Leu (SEQ ID NO: 46).

145. The method of claim 142, wherein the chimeric antigen receptor comprises one or more STAT5 -recruitment motifs outside the IL-2Rbeta cytoplasmic domain.

146. The method of claim 142, wherein the one or more vectors comprises a polynucleotide encoding a Foxp3.

147. The method of claim 146, wherein the Foxp3 is wild-type (WT) Foxp3.

148. The method of claim 147, wherein the wild type Foxp3 comprises an amino acid sequence of

MPNPRPGKPSAPSFAFGPSPGASPSWRAAPKASDFFGARGPGGTFQGRDFRG

GAHASSSSFNPMPPSQFQFPTFPFVMVAPSGARFGPFPHFQAFFQDRPHFMH

QFSTVDAHARTPVFQVHPFESPAMISFTPPTTATGVFSFKARPGFPPGINVASF

EWVSREPAFFCTFPNPSAPRKDSTFSAVPQSSYPFFANGVCKWPGCEKVFEEP

EDFFKHCQADHFFDEKGRAQCFFQREMVQSFEQQFVFEKEKFSAMQAHFA

GKMAFTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSFFAVRRHF

WGSHGNSTFPEFFHNMDYFKFHNMRPPFTYATFIRWAIFEAPEKQRTFNEIYH

WFTRMFAFFRNHPATWKNAIRHNFSFHKCFVRVESEKGAVWTVDEFEFRKK

RSQRPSRCSNPTPGP (SEQ ID NO: 9).

149. The method of claim 146, wherein the Foxp3 is a minimal Foxp3.

150. The method of claim 149, wherein the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C- terminally truncated.

151. The method of claim 150, wherein the N- and C-terminally truncated Foxp3 comprises a sequence of

(SEQ ID NO: 10).

152. The method of claim 146, wherein the Foxp3 comprises a substitution of serine for glutamic acid at amino acid 418 relative to SEQ ID NO: 9 (S418E FOXP3).

153. The method of claim 146 or 142, wherein the Foxp3 comprises a substitution of alanine for serine at amino acid 422 relative to SEQ ID NO: 9 (S422A FOXP3).

154. The method of any one of claims 142-153, wherein the polynucleotide encoding the CAR and the polynucleotide encoding the Foxp3 are configured for translation as a fusion protein comprising the CAR and the Foxp3.

155. The method of claim 154, wherein the CAR is N-terminal to the Foxp3 in the fusion protein.

156. The method of claim 154 or 155, wherein the fusion protein comprises a post- translational cleavage site between the CAR and the Foxp3.

157. The method of claim 156, wherein the post-translational cleavage site is selected from the group consisting of a T2A polypeptide, an E2A polypeptide, an F2A polypeptide and a P2A polypeptide.

158. The method of claim 156 or 157, wherein the CAR is cleaved from the Foxp3 following translation.

159. A chimeric-antigen-receptor regulatory T cell (CAR-Treg cell) generated by the methods of any one of claims 142-158.

160. The CAR-Treg of claim 159, wherein the CAR-Treg phenotype persists after transplantation into a patient.

161. A vector for use in modifying a chimeric-antigen-receptor regulatory T cell (CAR-Treg cell) to have a persistent Treg phenotype for therapeutic use in a transplant patient, comprising:

a) a polynucleotide encoding a chimeric antigen receptor having a cytoplasmic activation domain, said cytoplasmic activation domain comprising a truncated cytoplasmic domain from an interleukin-2 receptor beta-chain (IL- 2Rbeta); and

b) a polynucleotide encoding a constitutively active Foxp3;

162. A method of treating or inhibiting autoimmune disease, allergic disease, or inflammatory disease in a patient in need thereof, comprising administering the CAR- Treg of any one of claims 142-160 to the patient.

163. The method of claim 162, wherein the autoimmune disease, allergic disease, or inflammatory disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, and allergic rhinitis.

164. The method of claim 163, wherein the autoimmune disease, allergic disease, or inflammatory disease is an organ-specific inflammatory disease.

165. The method of claim 164, wherein the organ-specific inflammatory disease is selected from the group consisting of kidney disease and lung disease.

166. A method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ, comprising administering to the subject the CAR-Treg cells of any one of claims 142-160.

167. The method of claim 166, wherein the CAR-Treg cells are autologous.

168. The method of claim 167, wherein the patient is transplanted before administering the Treg cell to the patient.

169. The method of claim 168, wherein the patient is transplanted after administering the Treg cell to the patient.