US20230104151A1

US20230104151A1 - A method for treating disease using foxp3+cd4+ t cells

Info

Publication number: US20230104151A1
Application number: US17/979,437
Authority: US
Inventors: Ashley Mahne; John Lee; Lih-Yun Hsu; Jeffrey Greve
Original assignee: Kyverna Therapeutics Inc
Current assignee: Kyverna Therapeutics Inc
Priority date: 2020-11-10
Filing date: 2022-11-02
Publication date: 2023-04-06
Also published as: US20220143134A1; US20220169687A1; US11446357B2; WO2022103789A1

Abstract

This document relates to methods and materials for treating a mammal having an autoimmune disease. For example, materials and methods for producing a T cell comprising a FOXP3 polypeptide and one or more transcription factors are provided herein. Methods and materials for treating a mammal having an autoimmune disease comprising administering to a mammal having an autoimmune disease an effective amount of a T cell are also provided herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 17/523,431, filed on Nov. 10, 2021, which claims priority to U.S. Provisional Patent Application No. 63/111,905, filed on Nov. 10, 2020, the content of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named 47902-0016003_SL_ST26.xml. The XML file, created on Nov. 1, 2022, is 60,819 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

BACKGROUND

This document relates to methods and materials for treating a mammal having an autoimmune disease. For example, this document provides materials and methods for producing a T cell comprising a forkhead box P3 (FOXP3) polypeptide and one or more transcription factors. This document also provides methods and materials for treating a mammal having an autoimmune disease, where the methods include administering to a mammal having an autoimmune disease an effective amount of the T cell.
Autoimmunity is a common disease in the United States, with more than 20 million people suffering from one of 81 known autoimmune diseases. Regulatory T cells (Tregs) are a subpopulation of T cells that modulate the immune system and maintain tolerance to self-antigens. Tregs play a role in preventing or treating autoimmune disease (Sakaguchi et al., Int'l Immun., 21(10):1105-1111 (2009)). FOXP3, a transcription factor expressed in Tregs, has been implicated in maintaining Treg immunosuppressive functions (Hort et al., Science, 299:1057-1061 (2003)). FOXP3⁺ Tregs may impair (e.g., eliminate and/or inhibit) responder T cells involved in causing autoimmune disease by a granzyme-dependent or perforin-dependent mechanism (Trzonkowski et al., Clin. Immunol., 112:258-67 (2004)). FOXP3⁺ Tregs also may impair (e.g., eliminate and/or inhibit) responder T cells involved in causing autoimmune disease, by delivering a negative signal to responder T cells via up-regulation of intracellular cyclic AMP, which causes inhibition of responder T cell proliferation (Gondex et al., J. Immunol., 174:1783-6 (2005)).

SUMMARY

This document provides methods and materials that can be used to treat mammals identified as having an autoimmune disease. For example, this document provides materials and methods for a T cell containing a FOXP3 polypeptide and one or more transcription factors. In another example, this document provides materials and methods for producing a T cell containing a FOXP3 polypeptide, one or more transcription factors, and a therapeutic gene product. This document also provides materials and methods for producing a T cell containing a FOXP3 polypeptide, one or more transcription factors, and a therapeutic gene product and/or a binding agent. In addition, this document provides methods and materials for treating a mammal having an autoimmune disease, where the methods include administering to the mammal an effective amount of a T cell (e.g., any of the T cells described herein). The methods and materials provided herein can provide a way to enhance and/or stabilize the immunosuppressive effects of a T cell in order to treat the autoimmune disease.
In general, one aspect of this document features a method for increasing T cell function, where the method includes introducing into a T cell: (i) a first nucleic acid sequence encoding a FOXP3 polypeptide; and (ii) a second nucleic acid sequence encoding one or more transcription factors. In some embodiments, the one or more transcription activators, when present in a mammalian cell, elicit a T reg phenotype in the mammalian cell as compared to when the one or more transcription factors is/are not present in the mammalian cell. In some embodiments, the first nucleic acid sequence can include a mutation that results in nuclear localization of the FOXP3 polypeptide. In some embodiments, the mutation that results in nuclear localization of the FOXP3 polypeptide can be in a sequence encoding a nuclear export sequence. In some embodiments, the nuclear export sequence can include an amino acid substitution selected from the group of L69A, L71A, L74A, L76A, L242A, L246A, and L248A. In some embodiments, the first nucleic acid sequence can include a mutation that results in stabilization of the FOXP3 polypeptide. In some embodiments, the mutation that results in stabilization of the FOXP3 polypeptide can change the level of phosphorylation of the FOXP3 polypeptide compared to FOXP3 polypeptide not having the mutation. In some embodiments, the mutation can result in the expression of a FOXP3 polypeptide having an amino acid substitution selected from the group of S19A, S33A, S57A, S58A, S59A, T115A, S418D, and S422A. In some embodiments, the mutation that results in the stabilization of the FOXP3 polypeptide can change the level of acetylation of the FOXP3 polypeptide compared to FOXP3 polypeptide that not having the mutation. In some embodiments, the mutation can result in the production of a FOXP3 polypeptide having an amino acid substitution mutation selected from the group of K31R, K206R, K216R, K227R, K250R, K252R, K268R, and K277R. In some embodiments, the one or more transcription factors can be selected from the group of: BLIMP1, EOS, ROR-gt, FOXO1, GATA1, HELIOS, ID2, ID3, IRF4, LEF1, SATB1, GATA3, NFATc2, RUNX1, BC111b, Foxp1, Fox4, BACH2, STAT3, and XBP1. In some embodiments, the one or more transcription factors can be selected from selected form the group of: BLIMP1, EOS, GATA1, HELIOS, GATA3, and NFATc2. In some embodiments, the transcription factor can be BLIMP-1.
In some embodiments, the introducing step further includes introducing a nucleic acid construct, where the nucleic acid construct includes the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the nucleic acid construct can further include a promoter operably linked to the first nucleic acid sequence. In some embodiments, the first nucleic acid sequence can be 5′ positioned relative to the second nucleic acid sequence in the nucleic acid construct. In some embodiments, the nucleic acid construct further can include an additional nucleic acid sequence between the first nucleic acid sequence and the second nucleic acid sequence, where the additional nucleic acid sequence operably links the second nucleic acid sequence to the first nucleic acid sequence. In some embodiments, the second nucleic acid sequence is 5′ positioned relative to the first nucleic acid sequence in the nucleic acid construct. In some embodiments, the nucleic acid construct further includes an additional nucleic acid sequence between the second nucleic acid sequence and the first nucleic acid sequence, where the additional nucleic acid sequence operably links the first nucleic acid sequence to the second nucleic acid sequence. In some embodiments, the additional nucleic acid sequence can encode an internal ribosome entry site (IRES) sequence or a self-cleaving amino acid. In some embodiments, the additional nucleic acid sequence can include a promoter or enhancer.
In some embodiments, the introducing step further includes introducing a third nucleic acid sequence encoding a therapeutic gene product into the T cell, where the third nucleic acid sequence is operably linked to a promoter. In some embodiments, the therapeutic gene product can be an antigen-binding antibody fragment or antibody that is capable of binding to an IL-6, an IL-6R, an IFN alpha receptor, or a TGF beta receptor polypeptide. In some embodiments, the therapeutic gene product can be an antigen-binding fragment or antibody that is capable of binding to a IL-6 polypeptide or an IL-6R polypeptide.
In some embodiments, the nucleic acid sequence construct further includes a third nucleic acid sequence encoding the therapeutic gene product. In some embodiments, the introducing step further can include introducing a third nucleic acid sequence encoding a therapeutic gene product into the T cell, where the third nucleic acid sequence is operably linked to a promoter. In some embodiments, the therapeutic gene product can be an antigen-binding antibody fragment or antibody that is capable of binding to an IL-6, an IL-6R, an IFN alpha receptor, or a TGF beta receptor polypeptide. In some embodiments, the therapeutic gene product is an antigen-binding fragment or antibody that is capable of binding to an IL-6 polypeptide or an IL-6R polypeptide. In some embodiments, the third sequence can be 5′ positioned relative to the first sequence and the second sequence, where the third sequence is operably linked a promoter. In some embodiments, the third sequence can be 3′ positioned relative to the first and second sequence, where the third sequence is operably linked to the first sequence and/or the second sequence.
In some embodiments, the introducing step further includes introducing a fourth nucleic acid sequence encoding a binding agent into the T cell, where the fourth nucleic acid sequence is operably linked to a promoter. In some embodiments, the nucleic acid construct further includes a fourth nucleic acid sequence encoding a binding agent. In some embodiments, the binding agent can be an antibody or antigen-binding fragment. In some embodiments, the antigen-binding domain can be an antigen-binding fragment selected from the group of a Fab, a F(ab′)₂fragment, a scFV, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antigen-binding fragment can be a scFv that is capable of binding to an antigen on an autoimmune cell. In some embodiments, the scFv is capable of binding to a cell adhesion molecule. In some embodiments, the cell adhesion molecule can be ICAM-1, VCAM-1, or MADCAM-1. In some embodiments, the binding agent can be a LFA-1 polypeptide. In some embodiments, the binding agent is a chimeric antigen receptor, where the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain, where the extracellular domain includes an antibody or antigen-binding fragment capable of binding to an antigen on an autoimmune cell, and where the intracellular domain includes a cytoplasmic signaling domain and one or more co-stimulatory domains. In some embodiments, the antigen-binding domain is an antigen-binding fragment can be selected from the group of a Fab, a F(ab′)₂fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antigen-binding fragment can be a scFv that is capable of binding to an antigen on an autoimmune cell. In some embodiments, the scFv can be capable of binding to a cell adhesion molecule. In some embodiments, the cell adhesion molecule can be ICAM-1, VCAM-1, or MADCAM-1. In some embodiments, the cytoplasmic signaling domain can be a CD3 zeta domain. In some embodiments, the co-stimulatory domain can include at least one of a CD48, 4-1BB, ICOS, X-40, or CD27 domain. In some embodiments, the fourth sequence can be 5′ positioned relative to the first sequence and the second sequence, where the fourth sequence is operably linked a promoter. In some embodiments, the fourth sequence can be 3′ positioned relative to the first and second sequence, where the fourth sequence is operably linked to the first sequence and/or the second sequence.
In some embodiments, the nucleic acid construct further includes a third sequence encoding any of the therapeutic gene products described herein and a fourth sequence encoding any of the binding agents described herein. In some embodiments, the third sequence can be operably linked to a promoter and/or operably linked the first sequence and/or second sequence, and where the fourth sequence is operably linked to a promoter and/or operably linked the first sequence and/or second sequence.
In some embodiments, the nucleic acid construct can include a viral vector selected from the group of a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. In some embodiments, the viral vector can be a lentiviral vector. In some embodiments, the introducing step includes viral transduction.
In some embodiments, the T cell is a CD4⁺ T cell or a CD4⁺/CD45RA⁺ T cell. In some embodiments, the method further includes: obtaining a T cell from a patient or obtaining T cells allogenic to the patient. In some embodiments, the method further includes: treating the obtained T cells to isolate a population of cells enriched for CD4⁺ T cells or CD4⁺/CD45RA⁺ T cells.
In another aspect, this document features a T cell produced by any of the methods described herein. In another aspect, this document features a composition including any of the T cells described herein.
In another aspect, this document features a T-cell including: a first nucleic acid sequence encoding a FOXP3 polypeptide; and a second nucleic acid sequence encoding one or more transcription factors. In some embodiments, the one or more transcription factors, when present in a mammalian cell, elicit a T reg phenotype in the mammalian cell as compared to when the transcription factor is not present in the mammalian cell. In some embodiments, the nuclear export sequence of the FOX3P polypeptide can include an amino acid substitution selected from the group of L69A, L71A, L74A, L76A, L242A, L246A, and L248A. In some embodiments, the first nucleic acid sequence can include a mutation that results in stabilization of the FOXP3 polypeptide. In some embodiments, the mutation that results in stabilization of the FOXP3 polypeptide can change the level of phosphorylation of the FOXP3 polypeptide compared to FOXP3 polypeptide not having the mutation. In some embodiments, the mutation results in the production of a FOXP3 polypeptide having an amino acid substitution selected from the group of S19A, S33A, S57A, S58A, S59A, T115A, S418D, and S422A. In some embodiments, the mutation that results in the stabilization of the FOXP3 polypeptide can change the level of acetylation of the FOXP3 polypeptide compared to FOXP3 polypeptide that not having the mutation. In some embodiments, the mutation results in the production of a FOXP3 polypeptide having an amino acid substitution mutation selected from the group of K31R, K206R, K216R, K227R, K250R, K252R, K268R, and K277R. In some embodiments, the one or more transcription factors can be selected from the group of: BLIMP1, EOS, ROR-gt, FOXO1, GATA1, HELIOS, ID2, ID3, IRF4, LEF1, SATB1, GATA3, NFATc2, RUNX1, BC111b, Foxp1, Fox4, BACH2, STAT3, and XBP1. In some embodiments, the one or more transcription factors can be selected from selected form the group of: BLIMP1, EOS, GATA1, HELIOS, GATA3, and NFATc2. In some embodiments, the transcription factor can be BLIMP-1. In some embodiments, the first nucleic acid sequence can be operably linked to a promoter. In some embodiments, the second nucleic acid sequence can be operably linked to a promoter.
In some embodiments, the T-cell further includes a third nucleic acid sequence encoding a therapeutic gene product into the T cell, where the third nucleic acid sequence is operably linked to a promoter. In some embodiments, the therapeutic gene product can be an antigen-binding antibody fragment or antibody that is capable of binding to an IL-6, an IL-6R, an IFN alpha receptor, or a TGF beta receptor polypeptide. In some embodiments, the therapeutic gene product can be an antigen-binding fragment or antibody that is capable of binding to a IL-6 polypeptide or an IL-6R polypeptide.
In some embodiments, the T-cell further includes introducing a fourth nucleic acid sequence encoding a binding agent into the T cell, where the fourth nucleic acid sequence is operably linked to a promoter. In some embodiments, the binding agent can be an antibody or antigen-binding fragment. In some embodiments, the antigen-binding domain can be an antigen-binding fragment selected from the group of a Fab, a F(ab′)₂fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antigen-binding fragment can be a scFv that is capable of binding to an antigen on an autoimmune cell. In some embodiments, the scFv is capable of binding to a cell adhesion molecule. In some embodiments, the cell adhesion molecule can be ICAM-1, VCAM-1, or MADCAM-1. In some embodiments, the binding agent can be a LFA-1 polypeptide.
In some embodiments, the binding agent is a chimeric antigen receptor, where the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain, where the extracellular domain includes an antibody or antigen-binding fragment capable of binding to an antigen on an autoimmune cell, and where the intracellular domain includes a cytoplasmic signaling domain and one or more co-stimulatory domains. In some embodiments, the antigen-binding domain can be an antigen-binding fragment selected from the group of a Fab, a F(ab′)₂fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antigen-binding fragment can be a scFv that is capable of binding to an antigen on an autoimmune cell. In some embodiments, the scFv is capable of binding to a cell adhesion molecule. In some embodiments, the cell adhesion molecule can be ICAM-1, VCAM-1, or MADCAM-1. In some embodiments, the cytoplasmic signaling domain can be a CD3 zeta domain. In some embodiments, the co-stimulatory domain can include at least one of a CD48, 4-1BB, ICOS, X-40, or CD27 domain.
In another aspect, this document features a composition including a T cell produced using any of the methods described herein.
In another aspect, this document features a method of producing a T cell population expressing an exogenous FOXP3 polypeptide and one or more transcription factors, where the method includes culturing a T cell (e.g., any of the exemplary T cells described herein) in growth media under conditions sufficient to expand the population of T cells.
In another aspect, this document features a population of T cells produced using any of the methods described herein. In another aspect, this document features a composition including the population of T cells produced using any of the methods described herein.
In another aspect, this document features a vector including a first nucleic acid sequence encoding a FOXP3 polypeptide and a second nucleic acid sequence encoding a one or more transcription factors. In some embodiments, the one or more transcription factors, when present in a mammalian cell, elicit a T reg phenotype in the mammalian cell as compared to when the transcription factor is not present in the mammalian cell. In some embodiments, the nuclear export sequence of the FOX3P polypeptide can include an amino acid substitution selected from the group of L69A, L71A, L74A, L76A, L242A, L246A, and L248A. In some embodiments, the first nucleic acid sequence can include a mutation that results in stabilization of the FOXP3 polypeptide. In some embodiments, the mutation that results in stabilization of the FOXP3 polypeptide can change the level of phosphorylation of the FOXP3 polypeptide compared to FOXP3 polypeptide not having the mutation. In some embodiments, the mutation results in the production of a FOXP3 polypeptide having an amino acid substitution selected from the group of S19A, S33A, S57A, S58A, S59A, T115A, S418D, and S422A. In some embodiments, the mutation that results in the stabilization of the FOXP3 polypeptide can change the level of acetylation of the FOXP3 polypeptide compared to FOXP3 polypeptide that not having the mutation. In some embodiments, the mutation can result in the production of a FOXP3 polypeptide having an amino acid substitution selected from the group of K31R, K206R, K216R, K227R, K250R, K252R, K268R, and K277R. In some embodiments, the one or more transcription factors can be selected from the group of: BLIMP1, EOS, ROR-gt, FOXO1, GATA1, HELIOS, ID2, ID3, IRF4, LEF1, SATB1, GATA3, NFATc2, RUNX1, BC111b, Foxp1, Fox4, BACH2, STAT3, and XBP1. In some embodiments, the one or more transcription factors can be selected from selected form the group of: BLIMP1, EOS, GATA1, HELIOS, GATA3, and NFATc2. In some embodiments, the transcription factor can be BLIMP-1.
In some embodiments, the vector further includes a promoter operably linked to the first nucleic acid sequence. In some embodiments, the first nucleic acid sequence can be 5′ positioned relative to the second nucleic acid in the vector. In some embodiments, the vector further includes an additional nucleic acid sequence between the first nucleic acid sequence and the second nucleic acid sequence, where the additional nucleic acid sequence operably links the second nucleic acid sequence to the first nucleic acid sequence. In some embodiments, the second nucleic acid sequence can be 5′ positioned relative to the first nucleic acid sequence in the vector. In some embodiments, the vector further includes an additional nucleic acid sequence between the second nucleic acid sequence and the first nucleic acid sequence, where the additional nucleic acid sequence operably links the first nucleic acid sequence to the second nucleic acid sequence. In some embodiments, the additional nucleic acid sequence can encode an internal ribosome entry site (IRES) sequence or a self-cleaving amino acid. In some embodiments, the additional nucleic acid sequence can include a promoter or enhancer.
In some embodiments, the vector further includes a third nucleic acid sequence encoding a therapeutic gene product. In some embodiments, the therapeutic gene product can be an antigen-binding antibody fragment or antibody that is capable of binding to an IL-6, an IL-6R, an IFN alpha receptor, or a TGF beta receptor polypeptide. In some embodiments, the therapeutic gene product can be an antigen-binding fragment or antibody that is capable of binding to an IL-6 polypeptide or an IL-6R polypeptide. In some embodiments, the third nucleic acid sequence can be 5′ positioned relative to the first sequence and the second sequence, where the third nucleic acid sequence is operably linked to a promoter. In some embodiments, the third nucleic acid sequence can be 3′ positioned relative to the first and second nucleic acid sequence, where the third nucleic acid sequence is operably linked to the first nucleic acid sequence and/or the second nucleic acid sequence.
In some embodiments, the vector further includes a fourth nucleic acid sequence encoding a binding agent. In some embodiments, the binding agent can be an antibody or antigen-binding fragment. In some embodiments, the antigen-binding domain can be an antigen-binding fragment selected from the group of a Fab, a F(ab′)₂fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antigen-binding fragment can be a scFv that is capable of binding to an antigen on an autoimmune cell. In some embodiments, the scFv is capable of binding to a cell adhesion molecule. In some embodiments, the cell adhesion molecule can be ICAM-1, VCAM-1, or MADCAM-1. In some embodiments, the binding agent can be a LFA-1 polypeptide.
In some embodiments, the binding agent is a chimeric antigen receptor, where the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain, where the extracellular domain includes an antibody or antigen-binding fragment capable of binding to an antigen on an autoimmune cell, and where the intracellular domain includes a cytoplasmic signaling domain and one or more co-stimulatory domains. In some embodiments, the antigen-binding domain can be an antigen-binding fragment selected from the group of a Fab, a F(ab′)₂fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antigen-binding fragment can be a scFv that is capable of binding to an antigen on an autoimmune cell. In some embodiments, the scFv is capable of binding to a cell adhesion molecule. In some embodiments, the cell adhesion molecule can be ICAM-1, VCAM-1, or MADCAM-1. In some embodiments, the cytoplasmic signaling domain can be a CD3 zeta domain. In some embodiments, the co-stimulatory domain includes at least one of a CD48, 4-1BB, ICOS, X-40, or CD27 domain.
In some embodiments, the fourth nucleic acid sequence can be 5′ positioned relative to the first nucleic acid sequence and the second nucleic acid sequence, where the fourth nucleic acid sequence is operably linked a promoter. In some embodiments, the fourth nucleic acid sequence can be 3′ positioned relative to the first and second nucleic acid sequence, where the fourth nucleic acid sequence is operably linked to the first nucleic acid sequence and/or the second nucleic acid sequence. In some embodiments, the third nucleic acid sequence is operably linked to a promoter and/or operably linked the first nucleic acid sequence and/or second nucleic acid sequence, and where the fourth nucleic acid sequence is operably linked to a promoter and/or operably linked the first nucleic acid sequence and/or second nucleic acid sequence.
In some embodiments, the vector includes a viral vector selected from the group of a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. In some embodiments, the viral vector can be a lentiviral vector.
In another aspect, this document features a composition including any of the vectors described herein. In another aspect, this document features a kit including any of the compositions described herein.
In another aspect, this document features a method of treating an autoimmune disease or disorder in a patient including administering any of the T cells described herein, or any of the compositions described herein. In some embodiments, the subject can be previously diagnosed or identified as having an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder can be lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Graves disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, or polyarteritis nodosa. In some embodiments, the administering of the autologous or allogenic T cell population can include intravenous injection or intravenous infusion. In some embodiments, the administering can result in amelioration of one or more symptoms of the autoimmune disease or disorder.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram showing an exemplary targetable cell with enforced expression of a FOXP3 polypeptide. Enforced expression of a FOXP3 polypeptide results in a core Treg suppressive program (e.g., IL-2 consumption and increase in CD25 expression, an increase in adenosine, an increase in CD39 expression, and expression of CTLA-4).

FIG. 2 is a diagram showing an exemplary targetable cell with enforced expression of a FOXP3 polypeptide and a therapeutic gene product. Expression of a therapeutic gene product in addition to a FOXP3 polypeptide can result in enhancement of a core Treg program. Examples of suitable therapeutic gene products include, without limitation, IL6R scFv, IFNαR scFv, IL-10, IL-4, IL-13, and any other anti-fibrotic-related output.

DETAILED DESCRIPTION

This document provides methods and materials that can be used to treat mammals identified as having an autoimmune disease. For example, this document provides materials and methods for producing a T cell containing a FOXP3 polypeptide and one or more transcription factors (miRNA). In another example, this document provides materials and methods for producing a T cell containing a FOXP3 polypeptide, one or more transcription factors, and a therapeutic gene product. In a third example, this document also provides materials and methods for producing a T cell containing a FOXP3 polypeptide, one or more transcription factors, and a binding agent. In a fourth example, this document provides materials and methods for producing a T cell containing a FOXP3 polypeptide, one or more transcription factors, a therapeutic gene product, and a binding agent. In addition, this document provides methods and materials for treating a mammal having an autoimmune disease, where the methods include administering to the mammal an effective amount of a T cell produced using any of the methods described herein.
This document provides methods and materials for introducing into a T cell (e.g., CD4⁺ T cell, CD4⁺CD45RA⁺ T cell, CD4⁺CD62L⁺ T cell, or central memory T cell) a first nucleic acid sequence encoding a FOXP3 polypeptide and a second nucleic acid sequence encoding one or more transcription factors. In some embodiments, the one or more transcription factors, when present in a mammalian cell, elicits a T reg phenotype in the mammalian cell as compared to when the transcription factor(s) is/are not present in the mammalian cell.
In some embodiments, a first nucleic acid sequence encoding a FOXP3 polypeptide having one or more mutations is introduced into a T cell (e.g., CD4⁺ T cell, CD4⁺CD45RA⁺ T cell, CD4⁺CD62L⁺ T cell, or central memory T cell). For example, a mutation in the first nucleic acid sequence encoding a FOXP3 polypeptide can include, without limitation, mutations that result in an amino acid substitution that changes the stability (e.g., level of phosphorylation or acetylation), function (e.g., transcriptional regulation), or sub-cellular localization (e.g., nuclear localization) of the encoded FOXP3 polypeptide.
In some embodiments, a FOXP3 polypeptide can have an amino acid substitution in one or more nuclear export sequences (NES) that can result in nuclear localization of the FOXP3 polypeptide. Transducing cells with a FOXP3 polypeptide having one or more amino acid substitutions, amino acid insertions, and/or amino acid deletions in the nuclear export sequences can result in establishment, maintenance, or enhancement of a FOXP3 polypeptide-dependent expression profile that is indicative of expression profiles seen in native Treg cells (e.g., Treg cells isolated from a healthy human). In some cases, a cell (e.g., a CD4⁺ T cell) with a FOXP3 polypeptide-dependent expression profile can have increased immunosuppressive function. For example, a cell transduced with a FOXP3 polypeptide having one or more amino acid substitutions, amino acid insertions, and/or amino acid deletions as described herein can have increased expression of genes that are transcriptional targets of a FOXP3. Increased expression of these genes (e.g., Il-2, Ctla-4, and Tnfrsf18) can result in increased Treg cell function (e.g., inhibition of responder cell proliferation). In some embodiments, a FOXP3 polypeptide can having one or more amino acid substitutions, amino acid insertions, and/or amino acid deletions within a sequence encoding a NES. In cases where the FOXP3 polypeptide includes one or more amino acid deletions, the one or more deletions can be within a part of a NES (e.g., deletion of a part of a NES, deletion of an entire NES, or deletion of a larger fragment containing a NES sequence (e.g., corresponding to exon 2 or exon 7 of a FOXP3 polypeptide). For example, a FOXP3 polypeptide having the amino acids corresponding to exon 2-deleted (FOXP3d2), amino acids corresponding to exon 7 deleted (FOXP3d7), or amino acids corresponding to exon 2 and 7-deleted (FOXP3d2d7) can result in the nuclear localization of the FOXP3 polypeptide. In some embodiments, point mutations in the first nucleic acid sequence encoding the nuclear export sequences (e.g., NES1, having an amino acid sequence set forth in SEQ ID NO: 4, and NES2, having the amino acid sequence of SEQ ID NO: 5) can be any mutation (e.g., nucleic acid substitution, insertion, and/or deletion) that results in a change within the amino acid sequence of NES1 and/or NES2 and renders the nuclear export signal non-functional. Amino acid substitutions in NES1 and/or NES2 that can result in nuclear localization of a FOXP3 polypeptide include, without limitation: of L69A, L71A, L74A, L76A, L242A, L246A, and L248A. FOXP3 polypeptides harboring any one or more of these amino acid substitutions, amino acid insertions, and/or amino acid deletions can sequestered to the nucleus.
In some embodiments, the first nucleic acid sequence encoding the FOXP3 polypeptide can encode one or more fragments of a full length FOXP3 polypeptide (e.g., a full length FOXP3 polypeptide such as version NP_001107849.1). In some embodiments, a cell can be transduced with a first nucleic acid sequence encoding a FOXP3 polypeptide that includes at least the regions of FOXP3 that have DNA-binding properties (e.g., polypeptide fragments of FOXP3 that can bind to a ATAACA DNA sequence) (Li et al., Acta Biochim. Biophysc. Sin., 49(9):792-99 (2017)).
In some embodiments, an amino acid substitution in a FOXP3 polypeptide that changes the level of phosphorylation can stabilize the FOXP3 polypeptide (e.g., increase the half-life of the FOXP3 polypeptide). For example, a mutation in a first nucleic acid sequence encoding a FOXP3 polypeptide can result in an amino acid substitution that changes the level of phosphorylation of the FOXP3 polypeptide compared to a FOXP3 polypeptide not having the amino acid substitution. Non-limiting examples of amino acid substitutions that can change the level of phosphorylation of the FOXP3 polypeptide include S19A, S33A, S57A, S58A, S59A, T115A, S418D, and S422A.
In some embodiments, an amino acid substitution in a FOXP3 polypeptide is a phosphomimetic amino acid substitution. Phosphomimetics are amino acid substitutions that mimic a phosphorylated polypeptide or can encourage phosphorylation at a particular amino acid position, thereby activating or deactivating the polypeptide. For example, the phosphorylation of Ser418 can be enforced by a phospho-serine mimetic substitution of that residue into an alanine or aspartate. A mutation can be made in the first nucleic acid sequence encoding a FOXP3 polypeptide to produce a FOXP3 polypeptide having the S418D substitution. The S418D residue then serves as phosphomimetic amino acid residue. Additional amino acid residues that can be substituted to produce phosphomimetic amino acid residues include serines at positions 19, 33, 41, 88, and 422, threonines at sites 114 and 175 in FOXP3. See, Morawski, et al., J Biol Chem., 288(34): 24494-24502 (2013). For example, phosphomimetics of these sites can be engineered by substituting the serine or threonine for alanine. These phosphomimetics can enhance the stability and immunosuppressive activity of a FOXP3 polypeptide.
In some embodiments, an amino acid substitution in a FOXP3 polypeptide that changes the level of acetylation can stabilize the FOXP3 polypeptide (e.g., increase the half-life of the FOXP3 polypeptide). For example, a mutation in a first nucleic acid sequence encoding a FOXP3 polypeptide can result in an amino acid substitution that changes the level of acetylation of the FOXP3 polypeptide compared to a FOXP3 polypeptide not having the amino acid substitution. Non-limiting examples of amino acid substitutions that can change the level of acetylation of the FOXP3 polypeptide include K31R, K206R, K216R, K227R, K250R, K252R, K268R, and K277R.
In some embodiments, a second nucleic acid encoding one or more transcription factors is introduced into a T cell (e.g., CD4⁺ T cell, CD4⁺CD45RA⁺ T cell, CD4⁺CD62L⁺ T cell, or central memory T cell) along with the first nucleic acid sequence encoding the FOXP3 polypeptide. In some embodiments, introducing a first nucleic acid sequence encoding a FOXP3 polypeptide and a second nucleic acid sequence encoding one or more transcription factors into a CD4⁺ T cell enhances the suppressive activity of the T cell. In some embodiments, introducing a second nucleic acid sequence encoding one or more transcription factors into a CD4⁺ T cell elicits a T reg phenotype (e.g., immune suppression phenotype) in the T cell as compared to when the one or more transcription factors is/are not present in the mammalian cell. For example, introducing a second nucleic acid sequence encoding an NFATC2 polypeptide into a T cell (e.g., CD4⁺ T cell or any of the other exemplary T cells described herein) can induce a T reg phenotype (e.g., immune suppression phenotype) in the T cell. In another example, introducing a second nucleic acid sequence encoding a GATA3 polypeptide into a T cell (e.g., CD4+ T cell or any of the other exemplary T cells described herein) can induce a T reg phenotype (e.g., immune suppression phenotype) in the T cell. Non-limiting examples of transcription factors that can be used to enhance the T reg phenotype of a T cell include BLIMP1, EOS, ROR-γt, FOXO1, GATA1, HELIOS, ID2, ID3, IRF4, LEF1, SATB1, GATA3, NFATc2, RUNX1, BC111b, Foxp1, Fox4, BACH2, STAT3, and XBP1. For example, a first nucleic acid sequence encoding the FOXP3 polypeptide and a second nucleic acid sequence encoding BLIMP-1 polypeptide can be introduced into a T cell (e.g., CD4⁺ T cell, CD4⁺CD45RA⁺ T cell, CD4⁺CD62L⁺ T cell, or central memory T cell). Dntmt3a is responsible for methylation of genomic DNA encoding FOXP3 causing downregulation of FOXP3 and reducing the immunosuppressive functionality of the T cell. BLIMP1 blocks the upregulation of Dnmt3a. (See Garg, et al., Cell Reports, 26:1854-1868 (2019)). Expression of BLIMP1 prevents methylation (e.g., silencing) of FOXP3 thereby enabling continued expression of FOXP3 and maintenance of the T reg phenotype in the T cell. A T reg phenotype can include, e.g., one or more of IL-2 consumption, an increase in CD25 expression, an increase in adenosine, an increase in CD39 expression, and expression of CTLA-4. Additional markers of a T reg phenotype are known in the art.
This document provides methods and materials for introducing into a T cell (e.g., CD4⁺ T cell, CD4⁺CD45RA⁺ T cell, CD4⁺CD62L⁺ T cell, or central memory T cell) a first nucleic acid sequence encoding a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) and a second nucleic acid sequence encoding one or more transcription factors (e.g., any of the exemplary transcription factors described herein), and a therapeutic gene product. Any appropriate therapeutic gene product that enhances the immunosuppressive effects of a T cell (e.g., a CD4⁺CD45⁺ T cell) can be used. Examples of therapeutic gene products include, without limitation, antigen or antigen-binding fragments directed to interferon alpha receptor 1 (IFNAR1), interleukin 10 (IL-10, interleukin 4 (IL-4), interleukin 13 (IL-13), interleukin 6 (IL-6), IL-6 receptor (IL-6R), and any other anti-fibrotic agent. In some embodiments, the therapeutic gene product can enhance the immunosuppressive effect of the transduced cell. For example, a therapeutic gene product can be any polypeptide or other agent that prohibits an IL-6 polypeptide from binding to an IL-6 receptor (IL-6R). In such cases, a therapeutic gene product can be an antagonist for IL-6R (e.g., an antibody or antigen-binding fragment that binds to IL-6R) and/or blocking antibody or antigen-binding fragment of IL-6 (e.g., a scFv capable of binding to IL-6). Additional examples of therapeutic gene products include, without limitation, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, peptide hormones, metabolic enzymes, receptors, T cell receptors, chimeric antigen receptors (CARs), transcriptional activators, transcriptional repressors, translation activators, translational repressors, immune-receptors, apoptosis inhibitors, apoptosis inducers, immune-activators, and immune-inhibitors.
This document provides methods and materials for introducing into a T cell (e.g., CD4⁺ T cell, CD4⁺CD45RA⁺ T cell, CD4⁺CD62L⁺ T cell, or central memory T cell) a first nucleic acid sequence encoding a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) and a second nucleic acid sequence encoding one or more transcription factors (e.g., any of the exemplary transcription factors described herein), a therapeutic gene product (e.g., any of the exemplary therapeutic gene products as described herein), and a binding agent. Also provided herein are methods and materials for introducing into a T cell (e.g., CD4⁺ T cell, CD4⁺CD45RA⁺ T cell, CD4⁺CD62L⁺ T cell, or central memory T cell) a first nucleic acid sequence encoding a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) and a second nucleic acid sequence encoding one or more transcription factors (e.g., any of the exemplary transcription factors described herein), and a binding agent.
As used herein, “binding agent” refers to any variety of extracellular substance that binds with specificity to its cognate binding partner. In some embodiments, a cell (e.g., a CD4⁺CD45RA⁺ T cell) can be transduced with nucleic acid sequences encoding a mutated FOXP3 polypeptide as described herein, one or more transcription factors, and a binding agent. In some embodiments, a binding agent can be any polypeptide that enhances the immunosuppressive effect of a T cell (e.g., a CD4⁺CD45RA⁺ T cell). In some embodiments, a binding agent can be a polypeptide that binds to molecules found specifically on autoimmune cells or tissues. For example, a binding agent can be a lymphocyte function associated antigen-1 (LFA-1) polypeptide. An LFA-1 can bind to cell adhesion molecules on the surface of cells associated with autoimmune diseases. Examples of binding partners for LFA-1 include, without limitation, ICAM-1, VCAM-1 and MADCAM-1. In another example, a binding agent can be a polypeptide that binds to a VCAM-1 polypeptide (e.g., a scFv capable of binding to a VCAM-1 polypeptide). In yet another example, a binding agent can be a polypeptide that binds to a MADCAM-1 polypeptide (e.g., a scFv capable of binding to a MADCAM-1 polypeptide). In some embodiments, a binding agent can be a chimeric antigen receptor (CAR) as described herein where the CAR has an extracellular domain, a transmembrane domain, and an intracellular domain. In cases where the binding agent is a CAR, the extracellular domain includes a polypeptide capable of binding to a molecule found specifically on autoimmune cells or tissues. For example, the extracellular domain can include an scFV capable of binding to antigen on an autoimmune cell.
As used herein, “FOXP3” refers to the FOXP3 gene or protein that is a transcription factor in the Forkhead box (Fox) family of transcription factors (Sakaguchi et al., Int'l Immun., 21(10):1105-1111 (2009); Pandiyan, et al., Cytokine, 76(1):13-24 (2015)), or a variant thereof (e.g., a FOXP3 protein having one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty amino acid substitutions, amino acid deletions, or amino acid insertions as compared to a wildtype FOXP3 protein). In some embodiments, when preparing a T cell to be used in the treatment of a mammal having an autoimmune disease by administering to the mammal the T cell, FOXP3 refers to human FOXP3 or a variant thereof. An example of a wildtype human FOXP3 polypeptide includes, without limitation, NCBI reference sequence: NP 001107849.1 or a fragment thereof.
As used herein, “nuclear localization” means an increase in the level of FOXP3 (e.g., any of the FOXP3 polypeptides described herein) in the nucleus of a mammalian cell (e.g., any of the T cells described herein) as compared to a control mammalian cell (e.g., a mammalian cell expressing wildtype FOXP3 or a mammalian cell not genetically modified to include any of a first, second, third, and fourth nucleic acid sequences as described herein).
In some embodiments referring to a first nucleic acid sequence encoding a FOXP3 (e.g., full length FOXP3) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 99% and 100%) identical to:

(SEQ ID NO: 1)

AGTTTCCCACAAGCCAGGCTGATCCTTTTCTGTCAGTCCACTTCACCAAG

CCTGCCCTTGGACAAGGACCCGATGCCCAACCCCAGGCCTGGCAAGCCCT

CGGCCCCTTCCTTGGCCCTTGGCCCATCCCCAGGAGCCTCGCCCAGCTGG

AGGGCTGCACCCAAAGCCTCAGACCTGCTGGGGGCCCGGGGCCCAGGGGG

AACCTTCCAGGGCCGAGATCTTCGAGGCGGGGCCCATGCCTCCTCTTCTT

CCTTGAACCCCATGCCACCATCGCAGCTGCAGCTCTCAACGGTGGATGCC

CACGCCCGGACCCCTGTGCTGCAGGTGCACCCCCTGGAGAGCCCAGCCAT

GATCAGCCTCACACCACCCACCACCGCCACTGGGGTCTTCTCCCTCAAGG

CCCGGCCTGGCCTCCCACCTGGGATCAACGTGGCCAGCCTGGAATGGGTG

TCCAGGGAGCCGGCACTGCTCTGCACCTTCCCAAATCCCAGTGCACCCAG

GAAGGACAGCACCCTTTCGGCTGTGCCCCAGAGCTCCTACCCACTGCTGG

CAAATGGTGTCTGCAAGTGGCCCGGATGTGAGAAGGTCTTCGAAGAGCCA

GAGGACTTCCTCAAGCACTGCCAGGCGGACCATCTTCTGGATGAGAAGGG

CAGGGCACAATGTCTCCTCCAGAGAGAGATGGTACAGTCTCTGGAGCAGC

AGCTGGTGCTGGAGAAGGAGAAGCTGAGTGCCATGCAGGCCCACCTGGCT

GGGAAAATGGCACTGACCAAGGCTTCATCTGTGGCATCATCCGACAAGGG

CTCCTGCTGCATCGTAGCTGCTGGCAGCCAAGGCCCTGTCGTCCCAGCCT

GGTCTGGCCCCCGGGAGGCCCCTGACAGCCTGTTTGCTGTCCGGAGGCAC

CTGTGGGGTAGCCATGGAAACAGCACATTCCCAGAGTTCCTCCACAACAT

GGACTACTTCAAGTTCCACAACATGCGACCCCCTTTCACCTACGCCACGC

TCATCCGCTGGGCCATCCTGGAGGCTCCAGAGAAGCAGCGGACACTCAAT

GAGATCTACCACTGGTTCACACGCATGTTTGCCTTCTTCAGAAACCATCC

TGCCACCTGGAAGAACGCCATCCGCCACAACCTGAGTCTGCACAAGTGCT

TTGTGCGGGTGGAGAGCGAGAAGGGGGCTGTGTGGACCGTGGATGAGCTG

GAGTTCCGCAAGAAACGGAGCCAGAGGCCCAGCAGGTGTTCCAACCCTAC

ACCTGGCCCCTGACCTCAAGATCAAGGAAAGGAGGATGGACGAACAGGGG

CCAAACTGGTGGGAGGCAGAGGTGGTGGGGGCAGGGATGATAGGCCCTGG

ATGTGCCCACAGGGACCAAGAAGTGAGGTTTCCACTGTCTTGCCTGCCAG

GGCCCCTGTTCCCCCGCTGGCAGCCACCCCCTCCCCCATCATATCCTTTG

CCCCAAGGCTGCTCAGAGGGGCCCCGGTCCTGGCCCCAGCCCCCACCTCC

GCCCCAGACACACCCCCCAGTCGAGCCCTGCAGCCAAACAGAGCCTTCAC

AACCAGCCACACAGAGCCTGCCTCAGCTGCTCGCACAGATTACTTCAGGG

CTGGAAAAGTCACACAGACACACAAAATGTCACAATCCTGTCCCTCACTC

AACACAAACCCCAAAACACAGAGAGCCTGCCTCAGTACACTCAAACAACC

TCAAAGCTGCATCATCACACAATCACACACAAGCACAGCCCTGACAACCC

ACACACCCCAAGGCACGCACCCACAGCCAGCCTCAGGGCCCACAGGGGCA

CTGTCAACACAGGGGTGTGCCCAGAGGCCTACACAGAAGCAGCGTCAGTA

CCCTCAGGATCTGAGGTCCCAACACGTGCTCGCTCACACACACGGCCTGT

TAGAATTCACCTGTGTATCTCACGCATATGCACACGCACAGCCCCCCAGT

GGGTCTCTTGAGTCCCGTGCAGACACACACAGCCACACACACTGCCTTGC

CAAAAATACCCCGTGTCTCCCCTGCCACTCACCTCACTCCCATTCCCTGA

GCCCTGATCCATGCCTCAGCTTAGACTGCAGAGGAACTACTCATTTATTT

GGGATCCAAGGCCCCCAACCCACAGTACCGTCCCCAATAAACTGCAGCCG

AGCTCCCCA.

In some embodiments referring to a first nucleic acid sequence encoding a FOXP3 polypeptide having a mutation in exon 2, the nucleic acid sequence corresponding to FOXP3 exon 2 is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to: CCTGCCCTTGGACAAGGACCCGATGCCCAACCCCAGGCCTGGCAAGCCCTCGGCCC CTTCCTTGGCCCTTGGCCCATCCCCAGGAGCCTCGCCCAGCTGGAGGGCTGCACCCA AAGCCTCAGACCTGCTGGGGGCCCGGGGCCCAGGGGGAACCTTCCAGGGCCGAGAT CTTCGAGGCGGGGCCCATGCCTCCTCTTCTTCCTTGAACCCCATGCCACCATCGCAG CTGCAG (SEQ ID NO: 2). In some embodiments referring to a first nucleic acid sequence encoding a FOXP3 polypeptide having a deleted exon 2, the nucleic acid sequence that is deleted from full length FOXP3 polypeptide (SEQ ID NO: 1) is SEQ ID NO: 2 or a fragment of SEQ ID NO: 2.
In some embodiments referring to a first nucleic acid sequence encoding a FOXP3 polypeptide having a mutation in exon 7, the nucleic acid sequence corresponding to FOXP3 exon 7 is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to: CTGGTGCTGGAGAAGGAGAAGCTGAGTGCCATGCAGGCCCACCTGGCTGGGAAAAT GGCACTGACCAAGGCTTCATCTGTG (SEQ ID NO: 3). In some embodiments referring to a first nucleic acid sequence encoding a FOXP3 polypeptide having a deleted exon 7, the nucleic acid sequence that is deleted from full length FOXP3 (SEQ ID NO: 1) is SEQ ID NO: 3 or a fragment of SEQ ID NO: 3. In some embodiments referring to a first nucleic acid sequence encoding a FOXP3 polypeptide having a deleted exon 2 and a deleted exon 7, the nucleic acid sequences that are deleted from full length FOXP3 (SEQ ID NO: 1) are SEQ ID NO: 2 or a fragment thereof and SEQ ID NO: 3 or a fragment thereof.
In some embodiments referring to a mutation in a nuclear export sequence of FOXP3, the amino acid sequence corresponding to the NES1 is QLQLPTLPL (SEQ ID NO: 4). In some embodiments referring to a mutation in a nuclear export sequence of FOXP3, the amino acid sequence corresponding to the NES2 is VQSLEQQLVL (SEQ ID NO: 5).
As used herein, the term “chimeric antigen receptor” or “CAR” refers to a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain. In some cases, the extracellular domain can comprise an antigen-binding domain as described herein. In some cases, the transmembrane domain can comprise a transmembrane domain derived from a natural polypeptide obtained from a membrane-binding or transmembrane protein. For example, a transmembrane domain can include, without limitation, a transmembrane domain from a T cell receptor alpha or beta chain, a CD3 zeta chain, a CD28 polypeptide, or a CD8 polypeptide. In some cases, the intracellular domain can comprise a cytoplasmic signaling domain as described herein. In some cases, the intracellular domain can comprise a co-stimulatory domain as described herein.
As used herein, “T-cell function” refers to a T cell's (e.g., any of the exemplary T cells described herein) survival, stability, and/or ability to execute its intended function. For example, a CD4⁺ T cell can have an immunosuppressive function. A CD4⁺ T cell including a first nucleic acid sequence encoding a FOXP3 polypeptide can have a FOXP3-dependent expression profile that increases the immunosuppressive function of the T cell. For example, a cell transduced with a mutated FOXP3 polypeptide as described herein can have increased expression of genes that are transcriptional targets of a FOXP3 that can result in increased T reg cell function. In some embodiments, a T cell is considered to have T reg function if the T cell exhibits or maintains the potential to exhibit an immune suppression function.
As used herein, the term “activation” refers to induction of a signal on an immune cell (e.g., a B cell or T cell) that to results in initiation of the immune response (e.g., T cell activation). In some cases, upon binding of an antigen to a T cell receptor (TCR) or an exogenous chimeric antigen receptor (CAR), the immune cell can undergo changes in protein expression that result in the activation of the immune response. In some cases, a TCR or CAR includes a cytoplasmic signaling sequence that can drive T cell activation. For example, upon binding of the antigen, a chimeric antigen receptor comprising an intracellular domain that includes a cytoplasmic signaling sequence (e.g., an immune-receptor tyrosine-based inhibition motifs (ITAM)) that can be phosphorylated. A phosphorylated ITAM results in the induction of a T cell activation pathway that ultimately results in a T cell immune response. Examples of ITAMs include, without limitation, CD3 gamma, CD3 delta, CD3 epsilon, TCR zeta, FcR gamma, FcR beta, CD5, CD22, CD79a, and CD66d.
As used herein, the term “stimulation” refers to stage of TCR or CAR signaling where a co-stimulatory signal can be used to achieve a robust and sustained TCR or CAR signaling response. As described herein, a co-stimulatory domain can be referred to as a signaling domain. In some cases, a signaling domain (e.g., co-stimulatory domain) can be a CD27, CD28, OX40, CD30, CD40, B7-H3, NKG2C, LIGHT, CD7, CD2, 4-1BB, or PD-1.
In some embodiments where the chimeric antigen receptor polypeptide includes a CD3 zeta cytoplasmic signaling domain, the CD3 zeta cytoplasmic signaling domain has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALF LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP QRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR (NCBI Reference No.: NP_932170) (SEQ ID NO: 13), or a fragment thereof that has activating or stimulatory activity.
In some embodiments where the chimeric antigen receptor polypeptide includes a CD28 co-stimulatory domain, the CD28 co-stimulatory domain is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to:

(SEQ ID NO: 6)

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVL

ACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR

DFAAY.

Transcription Factors

As used herein, the term “transcription factor” refers to a polypeptide possessing one or more domains that bind to a DNA-regulatory sequence (e.g., promoter, enhancer, or silencer) to modulate the rate of gene transcription. This may result in increased or decreased gene transcription, protein synthesis, and subsequent altered cellular function.
As used herein, BLIMP1 also known as PRDM1 refers to PR/SET domain 1 polypeptide. When preparing a T cell or treating a mammal with a T cell, BLIMP1 or PRDM1 refers to human BLIMP1 or PRDM1. An example of a human BLIMP1 or PRDM1 polypeptide includes, without limitation, NCBI reference sequence: NP_001189.2. In some embodiments referring to a second nucleic acid sequence encoding a BLIMP1 (e.g., full length BLIMP1) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to:

(SEQ ID NO: 7)

AACACAGACAAAGTGCTGCCGTGACACTCGGCCCTCCAGTGTTGCGGAGA

GGCAAGAGCAGCGACCGCGGCACCTGTCCGCCCGGAGCTGGGACGCGGGC

GCCCGGGCGGCCGGACGAAGCGAGGAGGGACCGCCGAGGTGCGCGTCTGT

GCGGCTCAGCCTGGCGGGGGACGCGGGGAGAATGTGGACTGGGTAGAGAT

GAACGAGACTTTTCTCAGATGTTGGATATTTGCTTGGAAAAACGTGTGGG

TACGACCTTGGCTGCCCCCAAGTGTAACTCCAGCACTGTGAGGTTTCAGG

GATTGGCAGAGGGGACCAAGGGGACCATGAAAATGGACATGGAGGATGCG

GATATGACTCTGTGGACAGAGGCTGAGTTTGAAGAGAAGTGTACATACAT

TGTGAACGACCACCCCTGGGATTCTGGTGCTGATGGCGGTACTTCGGTTC

AGGCGGAGGCATCCTTACCAAGGAATCTGCTTTTCAAGTATGCCACCAAC

AGTGAAGAGGTTATTGGAGTGATGAGTAAAGAATACATACCAAAGGGCAC

ACGTTTTGGACCCCTAATAGGTGAAATCTACACCAATGACACAGTTCCTA

AGAACGCCAACAGGAAATATTTTTGGAGGATCTATTCCAGAGGGGAGCTT

CACCACTTCATTGACGGCTTTAATGAAGAGAAAAGCAACTGGATGCGCTA

TGTGAATCCAGCACACTCTCCCCGGGAGCAAAACCTGGCTGCGTGTCAGA

ACGGGATGAACATCTACTTCTACACCATTAAGCCCATCCCTGCCAACCAG

GAACTTCTTGTGTGGTATTGTCGGGACTTTGCAGAAAGGCTTCACTACCC

TTATCCCGGAGAGCTGACAATGATGAATCTCACACAAACACAGAGCAGTC

TAAAGCAACCGAGCACTGAGAAAAATGAACTCTGCCCAAAGAATGTCCCA

AAGAGAGAGTACAGCGTGAAAGAAATCCTAAAATTGGACTCCAACCCCTC

CAAAGGAAAGGACCTCTACCGTTCTAACATTTCACCCCTCACATCAGAAA

AGGACCTCGATGACTTTAGAAGACGTGGGAGCCCCGAAATGCCCTTCTAC

CCTCGGGTCGTTTACCCCATCCGGGCCCCTCTGCCAGAAGACTTTTTGAA

AGCTTCCCTGGCCTACGGGATCGAGAGACCCACGTACATCACTCGCTCCC

CCATTCCATCCTCCACCACTCCAAGCCCCTCTGCAAGAAGCAGCCCCGAC

CAAAGCCTCAAGAGCTCCAGCCCTCACAGCAGCCCTGGGAATACGGTGTC

CCCTGTGGGCCCCGGCTCTCAAGAGCACCGGGACTCCTACGCTTACTTGA

ACGCGTCCTACGGCACGGAAGGTTTGGGCTCCTACCCTGGCTACGCACCC

CTGCCCCACCTCCCGCCAGCTTTCATCCCCTCGTACAACGCTCACTACCC

CAAGTTCCTCTTGCCCCCCTACGGCATGAATTGTAATGGCCTGAGCGCTG

TGAGCAGCATGAATGGCATCAACAACTTTGGCCTCTTCCCGAGGCTGTGC

CCTGTCTACAGCAATCTCCTCGGTGGGGGCAGCCTGCCCCACCCCATGCT

CAACCCCACTTCTCTCCCGAGCTCGCTGCCCTCAGATGGAGCCCGGAGGT

TGCTCCAGCCGGAGCATCCCAGGGAGGTGCTTGTCCCGGCGCCCCACAGT

GCCTTCTCCTTTACCGGGGCCGCCGCCAGCATGAAGGACAAGGCCTGTAG

CCCCACAAGCGGGTCTCCCACGGCGGGAACAGCCGCCACGGCAGAACATG

TGGTGCAGCCCAAAGCTACCTCAGCAGCGATGGCAGCCCCCAGCAGCGAC

GAAGCCATGAATCTCATTAAAAACAAAAGAAACATGACCGGCTACAAGAC

CCTTCCCTACCCGCTGAAGAAGCAGAACGGCAAGATCAAGTACGAATGCA

ACGTTTGCGCCAAGACTTTCGGCCAGCTCTCCAATCTGAAGGTCCACCTG

AGAGTGCACAGTGGAGAACGGCCTTTCAAATGTCAGACTTGCAACAAGGG

CTTTACTCAGCTCGCCCACCTGCAGAAACACTACCTGGTACACACGGGAG

AAAAGCCACATGAATGCCAGGTCTGCCACAAGAGATTTAGCAGCACCAGC

AATCTCAAGACCCACCTGCGACTCCATTCTGGAGAGAAACCATACCAATG

CAAGGTGTGCCCTGCCAAGTTCACCCAGTTTGTGCACCTGAAACTGCACA

AGCGTCTGCACACCCGGGAGCGGCCCCACAAGTGCTCCCAGTGCCACAAG

AACTACATCCATCTCTGTAGCCTCAAGGTTCACCTGAAAGGGAACTGCGC

TGCGGCCCCGGCGCCTGGGCTGCCCTTGGAAGATCTGACCCGAATCAATG

AAGAAATCGAGAAGTTTGACATCAGTGACAATGCTGACCGGCTCGAGGAC

GTGGAGGATGACATCAGTGTGATCTCTGTAGTGGAGAAGGAAATTCTGGC

CGTGGTCAGAAAAGAGAAAGAAGAAACTGGCCTGAAAGTGTCTTTGCAAA

GAAACATGGGGAATGGACTCCTCTCCTCAGGGTGCAGCCTTTATGAGTCA

TCAGATCTACCCCTCATGAAGTTGCCTCCCAGCAACCCACTACCTCTGGT

ACCTGTAAAGGTCAAACAAGAAACAGTTGAACCAATGGATCCTTAAGATT

TTCAGAAAACACTTATTTTGTTTCTTAAGTTATGACTTGGTGAGTCAGGG

TGCCTGTAGGAAGTGGCTTGTACATAATCCCAGCTCTGCAAAGCTCTCTC

GACAGCAAATGGTTTCCCCTCACCTCTGGAATTAAAGAAGGAACTCCAAA

GTTACTGAAATCTCAGGGCATGAACAAGGCAAAGGCCATATATATATATA

TATATATATCTGTATACATATTATATATACTTATTTACACCTGTGTCTAT

ATATTTGCCCCTGTGTATTTTGAATATTTGTGTGGACATGTTTGCATAGC

CTTCCCATTACTAAGACTATTACCTAGTCATAATTATTTTTTCAATGATA

ATCCTTCATAATTTATTATACAATTTATCATTCAGAAAGCAATAATTAAA

AAAGTTTACAATGACTGGAAAGATTCCTTGTAATTTGAGTATAAATGTAT

TTTTGTCTTGTGGCCATTCTTTGTAGATAATTTCTGCACATCTGTATAAG

TACCTAAGATTTAGTTAAACAAATATATGACTTCAGTCAACCTCTCTCTC

TAATAATGGTTTGAAAATGAGGTTTGGGTAATTGCCAATGTTGGACAGTT

GATGTGTTCATTCCTGGGATCCTATCATTTGAACAGCATTGTACATAACT

TGGGGGTATGTGTGCAGGATTACCCAAGAATAACTTAAGTAGAAGAAACA

AGAAAGGGAATCTTGTATATTTTTGTTGATAGTTCATGTTTTTCCCCCAG

CCACAATTTTACCGGAAGGGTGACAGGAAGGCTTTACCAACCTGTCTCTC

CCTCCAAAAGAGCAGAATCCTCCCACCGCCCTGCCCTCCCCACCGAGTCC

TGTGGCCATTCAGAGCGGCCACATGACTTTTGCATCCATTGTATTATCAG

AAAATGTGAAGAAGAAAAAAATGCCATGTTTTAAAACCACTGCGAAAATT

TCCCCAAAGCATAGGTGGCTTTGTGTGTGTGCGATTTGGGGGCTTGAGTC

TGGGTGGTGTTTTGTTGTTGGTTTTTGTTGCTTTTTTTTTTTTTTTTTTT

TTAATGTCAAAATTGCACAAACATGGTGCTCTACCAGGAAGGATTCGAGG

TAGATAGGCTCAGGCCACACTTTAAAAACAAACACACAAACAACAAAAAA

CGGGTATTCTAGTCATCTTGGGGTAAAAGCGGGTAATGAACATTCCTATC

CCCAACACATCAATTGTATTTTTTCTGTAAAACTCAGATTTTCCTCAGTA

TTTGTGTTTTTACATTTTATGGTTAATTTAATGGAAGATGAAAGGGCATT

GCAAAGTTGTTCAACAACAGTTACCTCATTGAGTGTGTCCAGTAGTGCAG

GAAATGATGTCTTATCTAATGATTTGCTTCTCTAGAGGAGAAACCGAGTA

AATGTGCTCCAGCAAGATAGACTTTGTGTTATTCTATCTTTTATTCTGCT

AAGCCCAAAGATTACATGTTGGTGTTCAAAGTGTAGCAAAAAATGATGTA

TATTTATAAATCTATTTATACCACTATATCATATGTATATATATTTATAA

CCACTTAAATTGTGAGCCAAGCCATGTAAAAGATCTACTTTTTCTAAGGG

CAAAAAAAAAAAAAAAAAAAAAAGAACACTCCTTTCTGAGACTTTGCTTA

ATACTTGGTGACCTCACAATCACGTCGGTATGATTGGGCACCCTTGCCTA

CTGTAAGAGACCCTAAAACCTTGGTGCAGTGGTGGGGACCACAAAACAAC

CAGGGAGGAAGAGATACATCATTTTTTAGTATTAAGGACCATCTAAGACA

GCTCTATTTTTTTTTTGCCACTTTATGATTATGTGGTCACACCCAAGTCA

CAGAAATAAAAAACTGACTTTACCGCTGCAATTTTTCTGTTTTCCTCCTT

ACTAAATACTGATACATTACTCCAATCTATTTTATAATTATATTTGACAT

TTTGTTCACATCAACTAATGTTCACCTGTAGAAGAGAACAAATTTCGAAT

AATCCAGGGAAACCCAAGAGCCTTACTGGTCTTCTGTAACTTCCAAGACT

GACAGCTTTTTATGTATCAGTGTTTGATAAACACAGTCCTTAACTGAAGG

TAAACCAAAGCATCACGTTGACATTAGACCAAATACTTTTGATTCCCAAC

TACTCGTTTGTTCTTTTTCTCCTTTTGTGCTTTCCCATAGTGAGAATTTT

TATAAAGACTTCTTGCTTCTCTCACCATCCATCCTTCTCTTTTCTGCCTC

TTACATGTGAATGTTGAGCCCACAATCAACAGTGGTTTTATTTTTTCCTC

TACTCAAAGTTAAAACTGACCAAAGTTACTGGCTTTTTACTTTGCTAGAA

CAACAAACTATCTTATGTTTACATACTGGTTTACAATGTTATTTATGTGC

AAATTGTCAAAATGTAAATTAAATATAAATGTTCATGCTTTACCAAAA.

As used herein, EOS also known as Ikzf4 refers to Ikaros family zinc finger 4 polypeptide. When preparing a T cell or treating a mammal with a T cell, EOS or Ikzf4 refers to human EOS or Ikzf4. An example of a human EOS or Ikzf4 polypeptide includes, without limitation, NCBI reference sequence: NP_001338018.1. In some embodiments referring to a second nucleic acid sequence encoding a EOS (e.g., full length EOS) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to:

	(SEQ ID NO: 8)
	CCCTTCTCAGGTGAAGCTGCTGATGGAGATGGAGCC

	GCCGCCACCGCCGCCTCTGAGCGCCCGGGTCCTGGC

	TCCGGCCCGGCGACTGCCGCCGCCTCAGTGACCCCA

	CTCCCCCCGCACTGGGCCGCCCGGGCCAGAGTGGGG

	GACCCCCGCCCCCTCGCCTCCCTCTCCCCCAACACT

	GTCCCCTCTCCCCAACCCCTCACAGCCTGCGCGCGC

	GCGGAGACACCTCAGTCTACATGGGGAGGACAGAGA

	AGCGCAAAGAACAAGAGAAAAGATGCATCCATCTGA

	GATCTAAAAGGAGACAATGAGAATCTCTTTAAAATG

	GACATAGAAGACTGCAATGGCCGCTCCTATGTGTCT

	GTAGGACCAATGAAGGAATTATTGGCATGCACTAAA

	GGAGATAGCAAGATGGGTCAGACACACATATGAGAG

	TCATTGGCAACACCCGGGTAATGTAAGGAATCCACG

	CTTCCTGGAAGGTGAGTGGCTGGGCTCACCCCTGCC

	TGCCACTGAGACGCAGACATGCATACACCACCCGCA

	CTCCCTCGCCGTTTCCAAGGCGGCGGCCGCGTTCGC

	ACCCCAGGGTCTCACCGGCAAGGGAAGGATAATCTG

	GAGAGGGATCCCTCAGGAGGGTGTGTTCCGGATTTC

	TTGCCTCAGGCCCAAGACTCCAACCATTTTATAATG

	GAATCTTTATTTTGTGAAAGTAGCGGGGACTCATCT

	CTGGAGAAGGAGTTCCTCGGGGCCCCAGTGGGGCCC

	TCGGTGAGCACCCCCAACAGCCAGCACTCTTCTCCT

	AGCCGCTCACTCAGTGCCAACTCCATCAAGGTGGAG

	ATGTACAGCGATGAGGAGTCAAGCAGACTGCTGGGG

	CCAGATGAGCGGCTCCTGGAAAAGGACGACAGCGTG

	ATTGTGGAAGATTCATTGTCTGAGCCCCTGGGCTAC

	TGTGATGGGAGTGGGCCAGAGCCTCACTCCCCTGGG

	GGCATCCGGCTGCCCAATGGCAAGCTCAAGTGTGAC

	GTCTGCGGCATGGTCTGTATTGGACCCAACGTGCTC

	ATGGTGCACAAGCGCAGTCACACTGGTGAAAGGCCC

	TTCCATTGCAACCAGTGTGGTGCCTCCTTCACCCAG

	AAGGGGAACCTGCTGCGCCACATCAAGCTGCACTCT

	GGGGAGAAGCCCTTTAAATGTCCCTTCTGCAACTAT

	GCCTGCCGCCGGCGTGATGCACTCACTGGTCACCTC

	CGCACACACTCAGTCTCCTCTCCCACAGTGGGCAAG

	CCCTACAAGTGTAACTACTGTGGCCGGAGCTACAAA

	CAGCAGAGTACCCTGGAGGAGCACAAGGAGCGGTGC

	CATAACTACCTACAGAGTCTCAGCACTGAAGCCCAA

	GCTTTGGCTGGCCAACCAGGTGACGAAATACGTGAC

	CTGGAGATGGTGCCAGACTCCATGCTGCACTCATCC

	TCTGAGCGGCCAACTTTCATCGATCGTCTGGCCAAT

	AGCCTCACCAAACGCAAGCGTTCCACACCCCAGAAG

	TTTGTAGGCGAAAAGCAGATGCGCTTCAGCCTCTCA

	GACCTCCCCTATGATGTGAACTCGGGTGGCTATGAA

	AAGGATGTGGAGTTGGTGGCACACCACAGCCTAGAG

	CCTGGCTTTGGAAGTTCCCTGGCCTTTGTGGGTGCA

	GAGCATCTGCGTCCCCTCCGCCTTCCACCCACCAAT

	TGCATCTCAGAACTCACGCCTGTCATCAGCTCTGTC

	TACACCCAGATGCAGCCCCTCCCTGGTCGACTGGAG

	CTTCCAGGATCCCGAGAAGCAGGTGAGGGACCTGAG

	GACCTGGCTGATGGAGGTCCCCTCCTCTACCGGCCC

	CGAGGCCCCCTGACTGACCCTGGGGCATCCCCCAGC

	AATGGCTGCCAGGACTCCACAGACACAGAAAGCAAC

	CACGAAGATCGGGTTGCGGGGGTGGTATCCCTCCCT

	CAGGGTCCCCCACCCCAGCCACCTCCCACCATTGTG

	GTGGGCCGGCACAGTCCTGCCTACGCCAAAGAGGAC

	CCCAAGCCACAGGAGGGGTTATTGCGGGGCACCCCA

	GGCCCCTCCAAGGAAGTGCTTCGGGTGGTGGGCGAG

	AGTGGTGAGCCTGTGAAGGCCTTCAAGTGTGAGCAC

	TGCCGTATCCTCTTCCTGGACCACGTCATGTTCACT

	ATCCACATGGGCTGCCATGGCTTCAGAGACCCTTTT

	GAGTGCAACATCTGTGGTTATCACAGCCAGGACCGG

	TACGAATTCTCTTCCCACATTGTCCGGGGGGAGCAT

	AAGGTGGGCTAGCAACCTCTCCCTCTCTCCTCAGTC

	CACCACTCCACTGCCCTGACTACAGGCATTGATCCC

	TGTCCCCACCATTTCCCAAGGAGTTTTGCTTTGTAG

	CCCTCACTACTGGCCACCTGACCTCACACCTGACCC

	TGACCCCTCCTCACCTATTCTCTTCCTCTATCCTGA

	CCGATGTAAGCATTGTGATGAAACAGATCTTTTGCT

	TATGTTTTTCCTTTTTATCTTCTCTCATCCCAGCAT

	ACTGAGTTATTTATTAATTAGTTGATTTATTTTTGC

	CTTTTTAAATTTTAACTTATATCAGTCACTTGCCAC

	TCCCCCACCCTCCTGTCCACAACTCCTTTCCACTTT

	AGGCCAATTTTTCTCTCTTAGATCTTCCAGCAGCCC

	CAGGGGTAGGAAGCTCCTCTTAGTACTAAGAGACTT

	CAAGCTTCTTGCTTTAAGTCCTCACCCTTTACATTA

	TCTAATTCTTCAGTTTTGATGCTGATACCTGCCCCC

	GGCCCTACCTTAGCTCTGTGGCATTATATCTCCTCT

	CTGGGACTCTTCAACCTGGTACTCCATACCTCTTGT

	GCCCTCTCACTTTAGGCAGCTTGCACTATTCTTGAA

	TGAATGAAGAATTATTTCCTCATTTGGAAGTAGGAG

	GGACTGAAGAAATTCTCCCCAGGCACTGTGGGACTG

	AGAGTCCTATTCCCCTAGTAATAGGTCATATTCCCC

	TAGTAATATGAGTTCTCAAAGCCTACATTCAGGATC

	TCCCTCTAGGATGTGATAGATCTGGTCCCTCTCCTT

	GAACTACCCCTCCACACGCTCTAGTCCCTTCAACCT

	ACCGGTCTATTAAGTGGTGGCTTTTCTCTCCTTGGA

	GTGCCCCAATTTTATATTCTCAGGGGCCAAGGCTAG

	GTCTGCAACCCTCTGTCTCTGACAGATTGGGAGCCA

	CAGGTGCCTAATTGGGAACCAGGGCATGGGAAAGGA

	GTGGGTCAAAATTCTTCTCTTTCTCCTCCACCTCTC

	AAACTTCTTCACTATAGTGACCTTCCTAGGCTCTCA

	GGGGCTCCTTCAGTCCCCATCCTATGAGAAACTAGT

	GGGTTGCTGCCTGATGACAAGGGGTTGTTTCAGCCC

	CTCAGTCATGCTGCCTTCTGCTGCTCCCTCCCAGCA

	GGATTCACCCTCTCATTCCCGGGCTCCTGGGCCCTG

	TTCTTAGGATCAGTGGCAGGGAGAAACGGGTATCTC

	TTTTCTCTCTTCTAATTTTCAGTATAACCAAAAATT

	ATCCCAGCATGAGCACGGGCACGTGCCCTTCACCCC

	ATTCCACCCTTGTTCCAGCAAGACTGGGATGGGTAC

	AACTGAACTGGGGTCTTCCTTTACTACCCCCTTCTA

	CACTCAGCTCCCAGACACAGGGTAGGAGGGGGGACT

	GCTGGCTACTGCAGAGACCCTTGGCTATTTGAGTAA

	CCTAGGATTAGTGAGAAGGGGCAGAAGGAGATACAA

	CTCCACTGCAAGTGGAGGTTTCTTTCTACAAGAGTT

	TTCTGCCCAAGGCCACAGCCATCCCACTCTCTGCTT

	CCTTGAGATTCAAACCAAAGGCTGTTTTTCTATGTT

	TAAAGAAAAAAAAAAGTAAAAACCAAACACAACACC

	TCACAAGTTGTAACTCTTGGTCCTTCTCTCTCTCCT

	TTTCTCTTCCCTTCCTTCCCCTTCCATCTTTCTTTC

	CACATGTCCTTTCCTTATTGGCTCTTTTACCTCCTA

	CTTTTCTCACTCCCTATCAGGGATATTTTGGGGGGG

	GATGGTAAAGGGTGGGCTAAGGAACAGACCCTGGGA

	TTAGGGCCTTAAGGGCTCTGAGAGGAGTCTACCTTG

	CCTTCTTATGGGAAGGGAGACCCTAAAAAACTTTCT

	CCTCTTTGTCCTCCTTTTTCTCCCCCACTCTGAGGT

	TTCCCCAAGAGAACCAGATTGGCAGGGAGAAGCATT

	GTGGGGCAATTGTTCCTCCTTGACAATGTAGCAATA

	AATAGATGCTGCCAAGGGCAGAAAATGGGGAGGTTA

	GCTCAGAGCAGAGTAGTCTCTAGAGAAAGGAAGAAT

	CCTCAACGGCACCCTGGGGTGCTAGCTCCTTTTTAG

	AATGTCAGCAGAGCTGAGATTAATATCTGGGCTTTT

	CCTGAACTATTCTGGTTATTGAGCCCTTCCTGTTAG

	ACCTACCGCCTCCCACCTCTTCTGTGTCTGCTGTGT

	ATTTGGTGACACTTCATAAGGACTAGTCCCTTCTGG

	GGTATCAGAGCCTTAGGGTGCCCCCATCCCCTTCCC

	CAGTCAACTGTGGCACCTGTAACCTCCCGGAACATG

	AAGGACTATGCTCTGAGGCTATACTCTGTGCCCATG

	AGAGCAGAGACTGGAAGGGCAAGACCAGGTGCTAAG

	GAGGGGAGAGGGGGCATCCTGTCTCTCTCCAGACCA

	TCACTGCACTTTAACCAGGGTCTTAGGTACAAAATC

	CTACTTTTCAGAGCCTTCCAGCTCTGGAACCTCAAA

	CATCCTCATGCTCTCTCCCAGCTCCTTTTGCATAAA

	AAAAAAAGTAAAGAAAAAGAAAAAAAAATACACACA

	CACTGAAACCCACATGGAGAAAAGAGGTGTTTCCTT

	TTATATTGCTATTCAAAATCAATACCACCAACAAAA

	TATTTCTAAGTAGACACTTTTCCAGACCTTTGTTTT

	TTTGTGTCAGTGTCCAAGCTGCAGATAGGATTTTGT

	AATACTTCTGGCAGCTTCTTTCCTTGTGTACATAAT

	ATATATATATACATATATATATATATTTTTAATCAG

	AAGTTATGAAGAACAAAAAGAAAAAATAAACACAGA

	AGCAAGTGCAATACCACCTCTCTTCTCCCTCTCTCC

	TAGGGTTTCCTTTGTAGCCTATGTTTGGTGTCTCTT

	TTGACCTTTACCCCTTCACCTCCTCCTCTCTTCTTC

	TGATTCCCCTCCCCCCCTTTTTTAAAGAGTTTTTCT

	CCTTTCTCAAGGGGAGTTAAACTAGCTTTTGAGACT

	TATTGCAAAGCATTTTGTATATGTAATATATTGTAA

	GTAAATATTTGTGTAACGGAGATATACTACTGTAAG

	TTTTGTACTGTACTGGCTGAAAGTCTGTTATAAATA

	AACATGAGTAATTTAACA.

As used herein, GATA1 refers to a GATA binding protein 1 polypeptide. When preparing a T cell or treating a mammal with a T cell, GATA1 refers to human GATA1. An example of a human GATA1 polypeptide includes, without limitation, NCBI reference sequence: NP_002040.1. In some embodiments referring to a second nucleic acid sequence encoding a GATA1 (e.g., full length GATA1) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to:

	(SEQ ID NO: 9)
	ACACTGAGCTTGCCACATCCCCAAGGCGGCCGAACC

	CTCCGCAACCACCAGCCCAGGTTAATCCCCAGAGGC

	TCCATGGAGTTCCCTGGCCTGGGGTCCCTGGGGACC

	TCAGAGCCCCTCCCCCAGTTTGTGGATCCTGCTCTG

	GTGTCCTCCACACCAGAATCAGGGGTTTTCTTCCCC

	TCTGGGCCTGAGGGCTTGGATGCAGCAGCTTCCTCC

	ACTGCCCCGAGCACAGCCACCGCTGCAGCTGCGGCA

	CTGGCCTACTACAGGGACGCTGAGGCCTACAGACAC

	TCCCCAGTCTTTCAGGTGTACCCATTGCTCAACTGT

	ATGGAGGGGATCCCAGGGGGCTCACCATATGCCGGC

	TGGGCCTACGGCAAGACGGGGCTCTACCCTGCCTCA

	ACTGTGTGTCCCACCCGCGAGGACTCTCCTCCCCAG

	GCCGTGGAAGATCTGGATGGAAAAGGCAGCACCAGC

	TTCCTGGAGACTTTGAAGACAGAGCGGCTGAGCCCA

	GACCTCCTGACCCTGGGACCTGCACTGCCTTCATCA

	CTCCCTGTCCCCAATAGTGCTTATGGGGGCCCTGAC

	TTTTCCAGTACCTTCTTTTCTCCCACCGGGAGCCCC

	CTCAATTCAGCAGCCTATTCCTCTCCCAAGCTTCGT

	GGAACTCTCCCCCTGCCTCCCTGTGAGGCCAGGGAG

	TGTGTGAACTGCGGAGCAACAGCCACTCCACTGTGG

	CGGAGGGACAGGACAGGCCACTACCTATGCAACGCC

	TGCGGCCTCTATCACAAGATGAATGGGCAGAACAGG

	CCCCTCATCCGGCCCAAGAAGCGCCTGATTGTCAGT

	AAACGGGCAGGTACTCAGTGCACCAACTGCCAGACG

	ACCACCACGACACTGTGGCGGAGAAATGCCAGTGGG

	GATCCCGTGTGCAATGCCTGCGGCCTCTACTACAAG

	CTACACCAGGTGAACCGGCCACTGACCATGCGGAAG

	GATGGTATTCAGACTCGAAACCGCAAGGCATCTGGA

	AAAGGGAAAAAGAAACGGGGCTCCAGTCTGGGAGGC

	ACAGGAGCAGCCGAAGGACCAGCTGGTGGCTTTATG

	GTGGTGGCTGGGGGCAGCGGTAGCGGGAATTGTGGG

	GAGGTGGCTTCAGGCCTGACACTGGGCCCCCCAGGT

	ACTGCCCATCTCTACCAAGGCCTGGGCCCTGTGGTG

	CTGTCAGGGCCTGTTAGCCACCTCATGCCTTTCCCT

	GGACCCCTACTGGGCTCACCCACGGGCTCCTTCCCC

	ACAGGCCCCATGCCCCCCACCACCAGCACTACTGTG

	GTGGCTCCGCTCAGCTCATGAGGGCACAGAGCATGG

	CCTCCAGAGGAGGGGTGGTGTCCTTCTCCTCTTGTA

	GCCAGAATTCTGGACAACCCAAGTCTCTGGGCCCCA

	GGCACCCCCTGGCTTGAACCTTCAAAGCTTTTGTAA

	AATAAAACCACCAAAGTCCTGAAA.

As used herein, IKZF2 refers to a IKAROS family zinc finger 2 polypeptide. When preparing a T cell or treating a mammal with a T cell, IKZF2 refers to human IKZF2. An example of a human IKZF2 polypeptide includes, without limitation, NCBI reference sequence: NP_001072994.1. In some embodiments referring to a second nucleic acid sequence encoding a IKZF2 (e.g., full length IKZF2) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to:

	(SEQ ID NO: 10)
	GCTAACCCTGCTCCTCGCTGAAGATGGAGGAAGTAA

	AAACAGGATTACCCTTAGCTACAGATCCACTGCCTT

	AGTTTCCACCACCAACTGCAGTGCACAAACACACGT

	TAGGCACAGGAAAGAAAGAAAGACAGAGGACACATT

	AACAGTAAACACAAACAAAAGGGTGATGGGATTATT

	TTACTGCATGCACTGCTGAGCCCGACATTGTCACCT

	CCTCTTTGAGGGGTTAGAAGAAGCTGAGATCTCCCG

	ACAGAGCTGGAAATGGTGATGAATCTTTTTTAATCA

	AAGGACAATTTCTTTTCATTGCACTTTGACTATGGA

	AACAGAGGCTATTGATGGCTATATAACGTGTGACAA

	TGAGCTTTCACCCGAAAGGGAGCACTCCAATATGGC

	AATTGACCTCACCTCAAGCACACCCAATGGACAGCA

	TGCCTCACCAAGTCACATGACAAGCACAAATTCAGT

	AAAGCTAGAAATGCAGAGTGATGAAGAGTGTGACAG

	GAAACCCCTGAGCCGTGAAGATGAGATCAGGGGCCA

	TGATGAGGGTAGCAGCCTAGAAGAACCCCTAATTGA

	GAGCAGCGAGGTGGCTGACAACAGGAAAGTCCAGGA

	GCTTCAAGGCGAGGGAGGAATCCGGCTTCCGAATGG

	TGAACGCCCCTTCCACTGTAACCAGTGTGGAGCTTC

	TTTTACTCAGAAGGGCAACCTTCTGAGACACATAAA

	GTTACACTCTGGAGAGAAGCCGTTCAAATGTCCTTT

	CTGTAGCTACGCCTGTAGAAGAAGGGACGCCCTCAC

	AGGACACCTCAGGACCCATTCTGTGGGTAAACCTCA

	CAAGTGCAACTACTGTGGACGAAGCTACAAGCAGCG

	CAGTTCACTGGAGGAGCACAAGGAACGCTGCCACAA

	CTATCTCCAGAATGTCAGCATGGAGGCTGCTGGGCA

	GGTCATGAGTCACCATGTACCTCCTATGGAAGATTG

	TAAGGAACAAGAGCCTATTATGGACAACAATATTTC

	TCTGGTGCCTTTTGAGAGACCTGCTGTCATAGAGAA

	GCTCACGGGGAATATGGGAAAACGTAAAAGCTCCAC

	TCCACAAAAGTTTGTGGGGGAAAAGCTCATGCGATT

	CAGCTACCCAGATATTCACTTTGATATGAACTTAAC

	ATATGAGAAGGAGGCTGAGCTGATGCAGTCTCATAT

	GATGGACCAAGCCATCAACAATGCAATCACCTACCT

	TGGAGCTGAGGCCCTTCACCCTCTGATGCAGCACCC

	GCCAAGCACAATCGCTGAAGTGGCCCCAGTTATAAG

	CTCAGCTTATTCTCAGGTCTATCATCCAAATAGGAT

	AGAAAGACCCATTAGCAGGGAAACTGCTGATAGTCA

	TGAAAACAACATGGATGGCCCCATCTCTCTCATCAG

	ACCAAAGAGTCGACCCCAGGAAAGAGAGGCCTCTCC

	CAGCAATAGCTGCCTGGATTCCACTGACTCAGAAAG

	CAGCCATGATGACCACCAGTCCTACCAAGGACACCC

	TGCCTTAAATCCCAAGAGGAAACAAAGCCCAGCTTA

	CATGAAGGAGGATGTCAAAGCTTTGGATACTACCAA

	GGCTCCTAAGGGCTCTCTGAAGGACATCTACAAGGT

	CTTCAATGGAGAAGGAGAACAGATTAGGGCCTTCAA

	GTGTGAGCACTGCCGAGTCCTTTTCCTAGACCATGT

	CATGTACACCATTCACATGGGTTGCCATGGCTACCG

	GGACCCACTGGAATGCAACATCTGTGGCTACAGAAG

	CCAGGACCGTTATGAGTTTTCATCACACATTGTTCG

	AGGGGAGCACACATTCCACTAGGCCTTTTCATTCCA

	AAGGGGACCCCTATGAAGTAAAGAACTGCACATGAA

	GAAATACTGCACTTACAATCCCACCTTTCCTCAAAT

	GTTGACATACCTTTTATTTTTTTTAATATTATTACT

	GTTGATAATTCTTATTTTGTGGAGGCAGTGTCATTT

	GCTCTGCCTAATTACGATAAGGAAGAAACAGAAGAG

	AGAAGGGGCGGGAATATTGTTTCTTTATCACCTGGC

	TTGTTTATTTTGTGGGAATTTAAGAGCAGTCCATTT

	CTACCAAGGCATATCATGCTTTGAAAAATCACTTGA

	TTCATAAAGATTCACCTAAGAGATTCTGATTTGCCA

	CTGATATTCAGAATTATGATGGAAGACAGGAAAGTT

	CAGAGTTTTCTGGGTAGGACTTTGGTGGTTTAAAAA

	TGGTATAAGTAACTTTATTCTTGAAAGAAGAATGTG

	TTTCAAACTGTAAACCAATTTTTTGTTCTTCAGAGA

	TCATGGAACACAAACACATTGTTATTTTCAGTGATA

	ACTCCTAAGAGGAGCTGAGTTGTTGTGGGTTCTATG

	TTTACTTCCCCTATGGAATTTATAATTCAGTATGTT

	TTACACTGTACCATATAGCAAAACTTTTAAACTACA

	GGTAGTTAAGGGCCACCTACAATACATCTGAGGTCC

	TGTGATCTTATTTTTCTAAACGTAAGCACTGTTTTT

	CCATAGTTTTGATGACTGGCATTTTATAGACACCCT

	GGCAGCCTTACTTTTAACACCTTTAAGGAATAGTAT

	TTTTATGTAGTTTTCAGAATAACATATGGTCTAAGA

	GTGGATAAAAGGCAGTCAATAATTTCTGGGAGGGAC

	TTCTACTTTCATAAATTTGTTTGAGAGGTTTTCTTT

	TAAAGTTGTAATGTGATGGCAGCATAGTATATGTAT

	TTGTTTCTAAAAGTATGCTTACGATTGTCACTTTAT

	CAGCATTTAATCAGTGTTAACCAGTCAGCAGAAAAA

	TATAATTATGCTAACAGTAGGGGGAGAAAACCCACT

	TAGAAATCCCTTTTCTGGTATTTCTCTTTTCACTAG

	TTTTTTTCAAGATGTGACCTCCCGGTGTTCTGTCCA

	TAGTTCATTCATCCTTTACTCTTCGAGTAGAAGGTC

	TTAAAAGTCTTCCTGTCGGCTGTTTCTTTCAAAATC

	TCCTCAGAGCAATTGCTAATTTGGCCTGAATCTGGT

	AACTTGAACCCTGTAAGGTTACAGAACTAGGGCTAT

	TTATTTTAGCATTTCTTCAGTAGTATTTACTACTCT

	TGTTGCAAAGAAAAGGGAATGGGACTTCTTTGTAAC

	CTGTACCTTGGACAACAGATAAAAGAAACAAAAAAA

	TAAGAAAGTTTACTTTTACCCTTCTTGGAGTCTAGA

	ATGTGACAGAACCCCCAAAGGAAAGTCCTGCACATT

	TTTCTGTTTCCAAAACATTTAATTGTGTAAGTCCTT

	GTCAGAAATGAATCTCAATCCCTTAGTATAGAATTC

	CCCTTACATGGTATAGGTTGCCATATTTCATGTGCA

	GATTTTAATTTCATTTATGTGGGCGCTCTGTTTTTT

	CTTTGCAGTCCAGCCACATTAGAGGGGAGGAACCGA

	GTGATATTGATTCAAGTCATTTTAGGGGGACATACT

	TGGAAGGCAGAACTTGCTGCTTCTGTTTGGGGAGGA

	CAGACCTGACTGTGACTGGATTATCTGATAACCATT

	TGTGAATACTGAAATTCTGTTAGGCAGTAACTGATA

	ACTGCTCTAAAGGATCATTAAATAGGATGCTGAAAT

	TATGTATCTTAATACAGTGTGGTATGAGAATTACCA

	AGTCAAGAGAATTGTGGACATAAGCAAGTTTGGCCC

	CAATACTGCTCTTAACTCATTTTCCAGCTTACTATT

	TGCTATTTAAATGGTAGGCACCAGCTAAGCACTTCT

	AAGCACTAACACAGCTAGAACTAGGCAAAAATGGTT

	AGAACTCAGCTCTCTTCTACTAGTCCCTGTCATAAT

	TATTTTTGGGAAAATGTCCAAACTGCCCCCTTTAAA

	TCTAAGGGAATGCACCAAAACAGAGATATATAGAAT

	GTCAACCATTTCATTTTTTTTTTTCTGCATGCCTTG

	GTACATAGTGAACATACAACCTATTTAAAGATAAAG

	CATGTTTTTGAGACTCGCTCACCCCCCCCCACCCAA

	CCACTCCCAAATAATAATTGGGATGCCATTTTTTTT

	CCTTTTGGATGAGGTAAATAATTTTAAGGTTCACAA

	TTTTGTCTTTTACTGCAATTTAAGGAAACATTTGGA

	TGTCAGTCAATATGTTCATAATTTTGGCTGTGTGCG

	AATTTCTGCTGGCATTATCTATGAATTTTCTTCCTA

	CTTATTTTTTTTTCAGTATATGAACAATCATGTATC

	TACCTGCCCCAGGATGAAACTAAATTTAGGTGGACC

	CTAAACCTTATGAAGACAGTGCTGAGGCACTTTCCT

	TTTCTGATTTCATCTTTTTGGGAATCTGTTTTATTG

	AAGGTAGTTAGTAGTTGAGAGTGCATTTGCTACAAG

	CATATACTTGTATCTTCCTAGCTTCATGAGGAACAG

	AAAGAGGTGGATATGGCTCAGGGTGTGGCAGGGACA

	ATTGAGGACAAAGTCAATTCAAATTTGTGGGTCAGA

	AAGAATTTTTGTGGACGTAGTGTTTTTGGAGAAACT

	CTGGATGGTTATATGTGCATGCCTTTTCTTCAAAAG

	GAAATACGCAAGGTTGTAGCATCTAAAAATAAACAT

	AAGAGTCAGACACCAAATAAATCAAGTTTTACATAA

	CAGTTGTATGCCCAGTTTGTTTAGGTGAGATTTCAC

	ATTACAGAAAGTATTTGAGGAGCATGAAAATGGGTT

	ATCTTCTGTATTTTCCAGTTTGGCAAAAGTTCAGAA

	TTTCATCACATTGCTTTGCCCTAATTTTGCCCAGAA

	TTTTATCTTAGCCTCTCTCTGACAGTGATGAATCAT

	GCTCAAAAGCCATTCTAATTGGACCTTTTTAAGACA

	GGGAAAGGGATCAGTAGGCGGATTGGAAGAAATTTC

	AAGTCATTGAAATATTCCATTGAGATTTCCTAAAGG

	GACAAAATTGGGAAAATAAGAAACTACGACTTAGAT

	TTGGCTACGTAGTAGAAAGTATCTCCCCTACATACA

	TACAGGCAATTGTATGTATGAATCATAGGGTATATG

	TGTGTGTATACTACACACACATTCTTTTAAAGAGAA

	TTCATGGAAAAAAAAGCAGTTGGAGTGATCAGATGT

	ATTGCAAAAACATACAGAGAATTTAAATGACAGTTA

	ATACCAAGAAATTAGTTGGGTTTACTTTATCAGGTC

	GTAATAGGAATCACTAAAGAAGTTACTAGTGTGTCT

	TTAGGACCAGTGGCAACTCTTAAACTAAAACTTTGG

	GTCCTTATTATCTACTTACAGAACAAAGTGAAACAA

	ACAATGATTAAGCTGATTGGATATACATTCAAAGAT

	ATTTAATGTAAAGTTTTTTGGAATACGAAGAAAATT

	CAGAAAATAAATATTATCAACAGTTACTTATTGGCA

	AATAGAGAAAGACAAGAATAGTTTAGTGAGCCCGGT

	ATTTTGTTTTTATAGTTTTTATCTCAGTTGTACAAC

	TCACAAAACCATGAAGTCTTTGGTATTTTATAAATG

	TTTAACAAAATTTACATCAGATTAAGGCATTTAGAT

	GAAAATTATTATGTTCTCACTATCTTCCAAATTTTA

	TTTCATCCTATCTCCAAAATGATTTCTTAGGGTACA

	AAAAGAGCAGACGGGGCTGTAAAAATACAAGCAAAA

	AACTGTGTGCCCCTAGTTTCAGGCAGAACTTAAACT

	GTCAGAGGTACTAGCTACATGATTTGTTTTTTAACT

	TTGGATTGTTCACGTCCAAAAATGGATAAATTACAT

	TTGTGTTTATCATCAGTTGCATTTTATGTATTATTT

	TAATAAATACTATCTGAATGAAGACTATTCTAAACC

	AGAAAATTCCCCAAATCCAAAAGAAAAAAAAAGTGG

	GAAGAGGTGAAATTGAAGTTTGTGTATATGAAAGTT

	ATCTTAGACATATTTTTAATTCTCCAGTTTCTGCAA

	AATAATTAAAATATACAGTAACTGGTCTCCTAAATC

	CTGAATTTAATGTATTAAATACTTATGTTCTTTATA

	TTGGTGCCTTTTTAAAATGCATTGAGAGTGTTGGTT

	AGCTGTTGCAGCTGTACAACACTTTTAATATGCATT

	TTTAAAAATCACTTAAAATTGAGTACTATATAATTC

	ATCTCTGCATTTTTAGTGCAAATCTTTAGAGCAATT

	TCTAATAGAGAAATTTTCAGCTCAGCTGTTAAAAGG

	AAAAGGAAACTTTGAAACTAGACTTTACTACCTTTT

	TAGTTTCATAGTATTTCTGAATATGATTACAAGATT

	ATGCAGGTAAAATATAGAGTGAAACTTTACCTGTGA

	ATTGAATTATAATTTGTGTTTTTGTTTTGTTTTTAA

	GGAAGAATAAGTTCTGTATCAAACAAGAATTTATTA

	GATAATTTTTTGGTCAATAAAATACAGTATTCATTT

	GGATTTTCATCTCCAGACTAGTATTGTTCTAGTCTT

	GGAATCTGTATTTTCTAATCTGTTAGAAAATAGAGA

	TTGAAAATTGATGGAATAATGTGAAAAAGCAGGTAA

	TTAATTCTCCTTGAACAAAGCAAAACTGAACAGTCA

	TATCACATTGCTATTCTCCAAAGCATAATCTCAAAT

	GGTTTCATATCATGGTTGTGTATTACTTGCAATGGG

	TGTGTTAGGATATGACAGCTTTTTAAAAAAATGAGC

	TGCTGGTTATACAAAGCAAATGGCATATGACCAAGA

	AGCTGTGATATGCTAGTGTTTCTTTTTATCATAGTG

	TATTACTAGGCCAAATAATGACACCTTGAATATTTT

	TACATTTATTGCAGAAACCTTAAACTTTGGAATTTC

	CATAAGGTTTTTATGTAATATTCTATTTCTAGCTTT

	TTAGTTTTATCTTGCTGTACTGTAAGTTTGAGGATA

	TTTTTCACCTGCACTCTTAGGAATAAGTTCATAATT

	CTGTTTATGGGGCTTTCCTCCCATAACACTGCATTT

	GTATATTTTCTGTATAAAATATGTGTTGTGTATTAA

	CCTTTATCCCATACAGAGAGTGGTACATGAATGACT

	AGTTTTCTAAGATGTCCTTTTTATTGTGAATAAAAT

	ATAAAAGTTAAAGGCCCTCTGCTAAGTCACATAAAG

	TACAGCATATAAGTTCATATAGGTACAAATAAATGA

	GTTTGCAGTGAATTGGGCCTTCAAATTACCTCAAGT

	GACAGATAGTAAGAAAAGCTTCTTGAGCAGGTGGAG

	GTCACTGAATCCCCTACTATGCACTTACCAAGATTT

	TACTTACTTTAATTTACTGGAAATTGATTTTTTAAA

	AAATGACTACACTGTAACAAGGGAAGGGATCTGGGT

	TTTTTTGTTGTTTTATTCTTGTTTTTTTTAAGTAGT

	TCAAATTCTGAAACTGTGATTTAAAAATTTTTTACA

	GTCAAGCATTCTGATTTTGAACATAACTCCCTTCCC

	TTTCTGTGTAACAAAGGTCTCTCTGTTATCTCTTAA

	ATTTTGTTACATCTCCCTCAGCCTCTTTCTTTGTCC

	GTCTCCCTTCTGTCATTGTCTATGGATGTTTACCTC

	TCTGTTCTCCTAAAAGTTTGAAGATTAGGTCAACTC

	TTATTTCTAGTTCATTGGTAATTTAATCTTAATTTT

	TTTTTCGTGATTTTTGTTGGTTGTATAATCTGCTGA

	CGTATTTTTATACTCAAGTGTAGTTTTCTATTAAAA

	AGAAAAGTGGTTGGATTAAAAATAGTAAGCTATGTA

	ACCCTCATGTTACTTTCACTTTCAAATATTGGGTAC

	CTAAAACATTACTTCAGAGATTATGTAATCCTATTA

	TAGTATGTTTGCTTTCCTTTATTGTTGGATTTTACA

	TTCTGATTTGGCTTTCCTCCAAAAAATGTATATCAT

	GAAAGACTAGACAGTTATTTGCAAGTGTTTAGAAAG

	GTGTTAAAAATGTAAAGCAAAGAGTCTTAACTTTCT

	CCTAATTGGGAGAAAAATGCTTTAACATTACTATAA

	TAATATTCCAGGTTTGGAGGGGGTCTCCAGGCCCCA

	TATTTGCTGTTAATAGTTGGACCTTTTAGACCATGT

	GTTATTTGCAATCCCAGAATGATTGCTTCTGCTATT

	AGTTAAAAAGATACTATTCTTTTCTTTCTGTACAAG

	TGCAATACTCCCCTTGAAGTCTTAAAAACTATGGTG

	ATTTTTTTTTCTTTTCTGACCTATTCTTCCTTTAGC

	TAATGACAAAAAGAAACTCATAAAAGTCATAGTATG

	TTAAAGGACACAACAAGCAAAGAGAAAAACACTCCA

	CAATCAAAAGATTACAGAATGTGGAAACCACTAGTC

	TGATCTCATGGTATCTTTATTTAAGCTAAATTTCCA

	TGGAAATTAGTAATCTTTTGCTTGAAAAATGTGTCC

	TAAAGTTGAACTTTTTACAGATTGAATCTTCTTAGA

	CCCTCGCCCAATGCTCTAAATTAAGAACCTAATACT

	TAATATTTTTATTTTACTTCTCCCCTTTTAGAAATA

	AACTTTTAAATAAAAGCAAAGCACTTAGCTGAGTTT

	TAAACACTTACATATCACCTATTGGAGAAATTTTTT

	TTAAAAATATTTGGAGCAGTCCTGTTTTCATACAAA

	TTTAAGTAAGAGGTATTTTTCTTATACATATTTATA

	TGTAGTGTGCTAATTTTCTTTTTTTATACCTGTGTC

	CCTGTAGTAAAACTGCTGTAATATAAATACATGTTT

	TGTTAAAAGATAACATTTCTTTGGCATTTCTTTTAA

	AGGCAGTTACTGCATTTCTGCATTTGTACAGTATGT

	GTCTTGGCCATTTTAGATATTCTTTCTTTAACAATA

	CCAAAGGTAATTAGACTATTTTAAAGACTAATTGCT

	TGACAGTTTCTAGGGTATTTTGTGTTTTAGAAGCAA

	AAAAAGAAAAAAAAATAGGTCAAACCAGTAAACCTC

	ATTTTTTTTCAAACTAATAATTTGGGGAAATAAAAA

	CTATTGTTTAAAAAAGAAATATATATATATATATAT

	AAATATATATGTAAAGTTAAAATTCCATACCTTGTA

	TGTCAGGTTTGCTAAGTGTAATGTAGTTTTTTTAAG

	GCTCAAATACCATACCTCAGAAAATGAGGTTTACTA

	TGGAAATACTGAAACAGTCTTTGCAGCTGTGTGACA

	AGTCACTCTACTACATACTGATTTGGAGACCTCCGC

	TAAATAGTTTTATCACTGCAGACTAAAATGTGGGAC

	TTGTATCTTCTTTGTTTTTAATGCACACACATACAT

	GTTCTGTGCATGTATGTGGTTACTGTGTATATGTGT

	ATGAGTGTTGTATATGCATGTGTGAGTGTGTGTCTG

	TATGTGTGTACAACTAAAGAAGCTGCAGAAACTTTG

	TAATACTTTGTGAAAAGGATTATATTATAAAGGTTT

	GTACTGTCTGAGTGCACAGCTACTGGAATAAATTTA

	GGGAATCTCAGGAACAAGCATATAATTTGTCCAAGA

	TTTATTTCTTCTCAGAAGTGTAAGTGCAGTTTTTAA

	TTCTGTATATTATTTAATATTTTACCAATAAAATAA

	ACTTCTGACATAAAAA.

As used herein, GATA3 refers to a GATA binding protein 3 polypeptide. When preparing a T cell or treating a mammal with a T cell, GATA3 refers to human GATA3. An example of a human GATA3 polypeptide includes, without limitation, NCBI reference sequence: NP_001002295.1. In some embodiments referring to a second nucleic acid sequence encoding a GATA3 (e.g., full length GATA3) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to:

	(SEQ ID NO: 11)
	GAACACTGAGCTGCCTGGCGCCGTCTTGATACTTTC

	AGAAAGAATGCATTCCCTGTAAAAAAAAAAAAAAAA

	TACTGAGAGAGGGAGAGAGAGAGAGAAGAAGAGAGA

	GAGACGGAGGGAGAGCGAGACAGAGCGAGCAACGCA

	ATCTGACCGAGCAGGTCGTACGCCGCCGCCTCCTCC

	TCCTCTCTGCTCTTCGCTACCCAGGTGACCCGAGGA

	GGGACTCCGCCTCCGAGCGGCTGAGGACCCCGGTGC

	AGAGGAGCCTGGCTCGCAGAATTGCAGAGTCGTCGC

	CCCTTTTTACAACCTGGTCCCGTTTTATTCTGCCGT

	ACCCAGTTTTTGGATTTTTGTCTTCCCCTTCTTCTC

	TTTGCTAAACGACCCCTCCAAGATAATTTTTAAAAA

	ACCTTCTCCTTTGCTCACCTTTGCTTCCCAGCCTTC

	CCATCCCCCCACCGAAAGCAAATCATTCAACGACCC

	CCGACCCTCCGACGGCAGGAGCCCCCCGACCTCCCA

	GGCGGACCGCCCTCCCTCCCCGCGCGCGGGTTCCGG

	GCCCGGCGAGAGGGCGCGAGCACAGCCGAGGCCATG

	GAGGTGACGGCGGACCAGCCGCGCTGGGTGAGCCAC

	CACCACCCCGCCGTGCTCAACGGGCAGCACCCGGAC

	ACGCACCACCCGGGCCTCAGCCACTCCTACATGGAC

	GCGGCGCAGTACCCGCTGCCGGAGGAGGTGGATGTG

	CTTTTTAACATCGACGGTCAAGGCAACCACGTCCCG

	CCCTACTACGGAAACTCGGTCAGGGCCACGGTGCAG

	AGGTACCCTCCGACCCACCACGGGAGCCAGGTGTGC

	CGCCCGCCTCTGCTTCATGGATCCCTACCCTGGCTG

	GACGGCGGCAAAGCCCTGGGCAGCCACCACACCGCC

	TCCCCCTGGAATCTCAGCCCCTTCTCCAAGACGTCC

	ATCCACCACGGCTCCCCGGGGCCCCTCTCCGTCTAC

	CCCCCGGCCTCGTCCTCCTCCTTGTCGGGGGGCCAC

	GCCAGCCCGCACCTCTTCACCTTCCCGCCCACCCCG

	CCGAAGGACGTCTCCCCGGACCCATCGCTGTCCACC

	CCAGGCTCGGCCGGCTCGGCCCGGCAGGACGAGAAA

	GAGTGCCTCAAGTACCAGGTGCCCCTGCCCGACAGC

	ATGAAGCTGGAGTCGTCCCACTCCCGTGGCAGCATG

	ACCGCCCTGGGTGGAGCCTCCTCGTCGACCCACCAC

	CCCATCACCACCTACCCGCCCTACGTGCCCGAGTAC

	AGCTCCGGACTCTTCCCCCCCAGCAGCCTGCTGGGC

	GGCTCCCCCACCGGCTTCGGATGCAAGTCCAGGCCC

	AAGGCCCGGTCCAGCACAGAAGGCAGGGAGTGTGTG

	AACTGTGGGGCAACCTCGACCCCACTGTGGCGGCGA

	GATGGCACGGGACACTACCTGTGCAACGCCTGCGGG

	CTCTATCACAAAATGAACGGACAGAACCGGCCCCTC

	ATTAAGCCCAAGCGAAGGCTGTCTGCAGCCAGGAGA

	GCAGGGACGTCCTGTGCGAACTGTCAGACCACCACA

	ACCACACTCTGGAGGAGGAATGCCAATGGGGACCCT

	GTCTGCAATGCCTGTGGGCTCTACTACAAGCTTCAC

	AATATTAACAGACCCCTGACTATGAAGAAGGAAGGC

	ATCCAGACCAGAAACCGAAAAATGTCTAGCAAATCC

	AAAAAGTGCAAAAAAGTGCATGACTCACTGGAGGAC

	TTCCCCAAGAACAGCTCGTTTAACCCGGCCGCCCTC

	TCCAGACACATGTCCTCCCTGAGCCACATCTCGCCC

	TTCAGCCACTCCAGCCACATGCTGACCACGCCCACG

	CCGATGCACCCGCCATCCAGCCTGTCCTTTGGACCA

	CACCACCCCTCCAGCATGGTCACCGCCATGGGTTAG

	AGCCCTGCTCGATGCTCACAGGGCCCCCAGCGAGAG

	TCCCTGCAGTCCCTTTCGACTTGCATTTTTGCAGGA

	GCAGTATCATGAAGCCTAAACGCGATGGATATATGT

	TTTTGAAGGCAGAAAGCAAAATTATGTTTGCCACTT

	TGCAAAGGAGCTCACTGTGGTGTCTGTGTTCCAACC

	ACTGAATCTGGACCCCATCTGTGAATAAGCCATTCT

	GACTCATATCCCCTATTTAACAGGGTCTCTAGTGCT

	GTGAAAAAAAAAATGCTGAACATTGCATATAACTTA

	TATTGTAAGAAATACTGTACAATGACTTTATTGCAT

	CTGGGTAGCTGTAAGGCATGAAGGATGCCAAGAAGT

	TTAAGGAATATGGGAGAAATAGTGTGGAAATTAAGA

	AGAAACTAGGTCTGATATTCAAATGGACAAACTGCC

	AGTTTTGTTTCCTTTCACTGGCCACAGTTGTTTGAT

	GCATTAAAAGAAAATAAAAAAAAGAAAAAAGAGAAA

	AGAAAAAAAAAGAAAAAAGTTGTAGGCGAATCATTT

	GTTCAAAGCTGTTGGCCTCTGCAAAGGAAATACCAG

	TTCTGGGCAATCAGTGTTACCGTTCACCAGTTGCCG

	TTGAGGGTTTCAGAGAGCCTTTTTCTAGGCCTACAT

	GCTTTGTGAACAAGTCCCTGTAATTGTTGTTTGTAT

	GTATAATTCAAAGCACCAAAATAAGAAAAGATGTAG

	ATTTATTTCATCATATTATACAGACCGAACTGTTGT

	ATAAATTTATTTACTGCTAGTCTTAAGAACTGCTTT

	CTTTCGTTTGTTTGTTTCAATATTTTCCTTCTCTCT

	CAATTTTTGGTTGAATAAACTAGATTACATTCAGTT

	GGCCTAAGGTGGTTGTGCTCGGAGGGTTTCTTGTTT

	CTTTTCCATTTTGTTTTTGGATGATATTTATTAAAT

	AGCTTCTAAGAGTCCGGCGGCATCTGTCTTGTCCCT

	ATTCCTGCAGCCTGTGCTGAGGGTAGCAGTGTATGA

	GCTACCAGCGTGCATGTCAGCGACCCTGGCCCGACA

	GGCCACGTCCTGCAATCGGCCCGGCTGCCTCTTCGC

	CCTGTCGTGTTCTGTGTTAGTGATCACTGCCTTTAA

	TACAGTCTGTTGGAATAATATTATAAGCATAATAAT

	AAAGTGAAAATATTTTAAAACTA.

As used herein, NFATC2 refers to a nuclear factor of activated T cells 2 polypeptide. When preparing a T cell or treating a mammal with a T cell, NFATC2 refers to human NFATC2. An example of a human NFATC2 polypeptide includes, without limitation, NCBI reference sequence: NP_001129493.1. In some embodiments referring to a second nucleic acid sequence encoding a NFATC2 (e.g., full length NFATC2) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to:

	(SEQ ID NO: 12)
	GCGTTGCCTCTGGAGTAAGCCGGATCGCGGAGCCGC

	GCCGACTCCGCCGAGCCGGGAGCCGGGAGGCGCGCA

	GCTCCCGGGTCGCTCCGAGGCTCCTCGGCCAGGGCA

	GCCCCGCGGGCACGCGGTAGAGAAGACGGCGTCCCC

	TCGGCTGCTGGTCGATACAAACAGATCCCCCTTTCC

	AAACACGCGCCAAGTCCCCGTGCCCTCCAGATGCAG

	AGAGAGGCTGCGTTCAGACTGGGGCACTGCCATCCC

	CTCCGCATCATGGGGTCTGTGGACCAAGAAGAGCCG

	AATGCACATAAGGTCGCCAGCCCACCCTCCGGACCC

	GCATACCCCGATGATGTCCTGGACTATGGCCTCAAG

	CCATACAGCCCCCTTGCTAGTCTCTCTGGCGAGCCC

	CCCGGCCGATTCGGAGAGCCGGATAGGGTAGGGCCG

	CAGAAGTTTCTGAGCGCGGCCAAGCCAGCAGGGGCC

	TCGGGCCTGAGCCCTCGGATCGAGATCACTCCGTCC

	CACGAACTGATCCAGGCAGTGGGGCCCCTCCGCATG

	AGAGACGCGGGCCTCCTGGTGGAGCAGCCGCCCCTG

	GCCGGGGTGGCCGCCAGCCCGAGGTTCACCCTGCCC

	GTGCCCGGCTTCGAGGGCTACCGCGAGCCGCTTTGC

	TTGAGCCCCGCTAGCAGCGGCTCCTCTGCCAGCTTC

	ATTTCTGACACCTTCTCCCCCTACACCTCGCCCTGC

	GTCTCGCCCAATAACGGCGGGCCCGACGACCTGTGT

	CCGCAGTTTCAAAACATCCCTGCTCATTATTCCCCC

	AGAACCTCGCCAATAATGTCACCTCGAACCAGCCTC

	GCCGAGGACAGCTGCCTGGGCCGCCACTCGCCCGTG

	CCCCGTCCGGCCTCCCGCTCCTCATCGCCTGGTGCC

	AAGCGGAGGCATTCGTGCGCCGAGGCCTTGGTTGCC

	CTGCCGCCCGGAGCCTCACCCCAGCGCTCCCGGAGC

	CCCTCGCCGCAGCCCTCATCTCACGTGGCACCCCAG

	GACCACGGCTCCCCGGCTGGGTACCCCCCTGTGGCT

	GGCTCTGCCGTGATCATGGATGCCCTGAACAGCCTC

	GCCACGGACTCGCCTTGTGGGATCCCCCCCAAGATG

	TGGAAGACCAGCCCTGACCCCTCGCCGGTGTCTGCC

	GCCCCATCCAAGGCCGGCCTGCCTCGCCACATCTAC

	CCGGCCGTGGAGTTCCTGGGGCCCTGCGAGCAGGGC

	GAGAGGAGAAACTCGGCTCCAGAATCCATCCTGCTG

	GTTCCGCCCACTTGGCCCAAGCCGCTGGTGCCTGCC

	ATTCCCATCTGCAGCATCCCAGTGACTGCATCCCTC

	CCTCCACTTGAGTGGCCGCTGTCCAGTCAGTCAGGC

	TCTTACGAGCTGCGGATCGAGGTGCAGCCCAAGCCA

	CATCACCGGGCCCACTATGAGACAGAAGGCAGCCGA

	GGGGCTGTCAAAGCTCCAACTGGAGGCCACCCTGTG

	GTTCAGCTCCATGGCTACATGGAAAACAAGCCTCTG

	GGACTTCAGATCTTCATTGGGACAGCTGATGAGCGG

	ATCCTTAAGCCGCACGCCTTCTACCAGGTGCACCGA

	ATCACGGGGAAAACTGTCACCACCACCAGCTATGAG

	AAGATAGTGGGCAACACCAAAGTCCTGGAGATACCC

	TTGGAGCCCAAAAACAACATGAGGGCAACCATCGAC

	TGTGCGGGGATCTTGAAGCTTAGAAACGCCGACATT

	GAGCTGCGGAAAGGCGAGACGGACATTGGAAGAAAG

	AACACGCGGGTGAGACTGGTTTTCCGAGTTCACATC

	CCAGAGTCCAGTGGCAGAATCGTCTCTTTACAGACT

	GCATCTAACCCCATCGAGTGCTCCCAGCGATCTGCT

	CACGAGCTGCCCATGGTTGAAAGACAAGACACAGAC

	AGCTGCCTGGTCTATGGCGGCCAGCAAATGATCCTC

	ACGGGGCAGAACTTTACATCCGAGTCCAAAGTTGTG

	TTTACTGAGAAGACCACAGATGGACAGCAAATTTGG

	GAGATGGAAGCCACGGTGGATAAGGACAAGAGCCAG

	CCCAACATGCTTTTTGTTGAGATCCCTGAATATCGG

	AACAAGCATATCCGCACACCTGTAAAAGTGAACTTC

	TACGTCATCAATGGGAAGAGAAAACGAAGTCAGCCT

	CAGCACTTTACCTACCACCCAGTCCCAGCCATCAAG

	ACGGAGCCCACGGATGAATATGACCCCACTCTGATC

	TGCAGCCCCACCCATGGAGGCCTGGGGAGCCAGCCT

	TACTACCCCCAGCACCCGATGGTGGCCGAGTCCCCC

	TCCTGCCTCGTGGCCACCATGGCTCCCTGCCAGCAG

	TTCCGCACGGGGCTCTCATCCCCTGACGCCCGCTAC

	CAGCAACAGAACCCAGCGGCCGTACTCTACCAGCGG

	AGCAAGAGCCTGAGCCCCAGCCTGCTGGGCTATCAG

	CAGCCGGCCCTCATGGCCGCCCCGCTGTCCCTTGCG

	GACGCTCACCGCTCTGTGCTGGTGCACGCCGGCTCC

	CAGGGCCAGAGCTCAGCCCTGCTCCACCCCTCTCCG

	ACCAACCAGCAGGCCTCGCCTGTGATCCACTACTCA

	CCCACCAACCAGCAGCTGCGCTGCGGAAGCCACCAG

	GAGTTCCAGCACATCATGTACTGCGAGAATTTCGCA

	CCAGGCACCACCAGACCTGGCCCGCCCCCGGTCAGT

	CAAGGTCAGAGGCTGAGCCCGGGTTCCTACCCCACA

	GTCATTCAGCAGCAGAATGCCACGAGCCAAAGAGCC

	GCCAAAAACGGACCCCCGGTCAGTGACCAAAAGGAA

	GTATTACCTGCGGGGGTGACCATTAAACAGGAGCAG

	AACTTGGACCAGACCTACTTGGATGATGAGCTGATA

	GACACACACCTTAGCTGGATACAAAACATATTATGA

	AACAGAATGACTGTGATCTTTGATCCGAGAAATCAA

	AGTTAAAGTTAATGAAATTATCAGGAAGGAGTTTTC

	AGGACCTCCTGCCAGAAATCAGACGTAAAAGAAGCC

	ATTATAGCAAGACACCTTCTGTATCTGACCCCTCGG

	AGCCCTCCACAGCCCCTCACCTTCTGTCTCCTTTCA

	TGTTCATCTCCCAGCCCGGAGTCCACACGCGGATCA

	ATGTATGGGCACTAAGCGGACTCTCACTTAAGGAGC

	TCGCCACCTCCCTCTAAACACCAGAGAGAACTCTTC

	TTTTCGGTTTATGTTTTAAATCCCAGAGAGCATCCT

	GGTTGATCTTAATGGTGTTCCGTCCAAATAGTAAGC

	ACCTGCTGACCAAAAGCACATTCTACATGAGACAGG

	ACACTGGAACTCTCCTGAGAACAGAGTGACTGGAGC

	TTGGGGGGATGGACGGGGGACAGAAGATGTGGGCAC

	TGTGATTAAACCCCAGCCCTTGCGTTCGTTTTTCCA

	GGTCACAGATACAGCTCCTGTACCTTTTGAAGGCAA

	GGAGTTCTCAGAGCAACCAAAGGAACGTGACCCAAG

	AGCCCAGCTTACAGGCTGAAGAAACCCAAAACCCTC

	GATAGAGACAGAAACTGAACTGTCAGTCCTTAGAGC

	TCGCCCAGTCCATGCCACAACTGGGCCACAGCTAAA

	GCTTTATTTTTGAATTCTCATTCCAAAACCAAACTG

	TCTTGCCCAGACAAGATCACCTGTTAAGACTTCTTG

	GCGTTAAGTTATGACATGTATACGCGTTTGTTATTA

	TTATTTTTTCTGCTTTAAAAGGCTGACCAGGGCACC

	TAGCCCTGGAGCTGTCTTGGCGAGCTGTTCTTTAAC

	CCCTGCAGCACGCAGTCCTGCTAACACAATTTCCAT

	AGACTTGGGGGGCTGACCCAGGCTGCAGAGAGCAAG

	CACCTGTCTGCTGCAGCTGTACAACCTGGATGCTTT

	GCAAGGTTCCGGCTTGCTTTCTTCCTAGCAGCCAGA

	GTGCTTTTCCGTAAAGCGGTGGAGAATCTCAAGCAT

	GTGCATTTAATTGAGGAATAGCAGAAGGGCTAAAGC

	AACCAAGAAAAGAAGTGTGGGTATTTTTGTTAAGTA

	AAACAGCCCAAGTGCTTCTGGAGGTGGGTTTCTACC

	AAGATAGAGGAAAAGGGCTGAATTCCCTCTAAGTGG

	GACAGCCGAGCTCAGGATGTGCTTCCCAGCTTCACT

	GGTTAATTTGACCTGAACCTATTTAAAGATCCCTTC

	TGCCCCTGAAGACCTATCCGCACTCAAATTCTAACA

	TGAAGAAATCTACTCGAATGCATCCTTTACTTTGAA

	TGAGCTCTATTCGGTTGCATGTTATATGTGATTTCC

	TTCCTCCCAACTGTTTCCACTGAGCGCACCCAGTCT

	CCCCTAGTCTTCCTCTGTGGGTGTGATTTTTGTGAT

	TTTTACAAACAAAACCCTTGAAGTTCTTGGCAGATG

	TGTTTGTTTCTGTTTGCATGTACTGCAGATACCCCA

	GGACAAGCGGGGGATTCATTTTTCAGCCATTCAGTT

	GTTTCCTCAATAATCCGCAGCAAAGTGAAAATATTC

	TTAGCACTCAGACTGTACTTAGAGTGTTTTCTCAGT

	CCAGTCTGTACAGTCTGTAGGCAGAAGGCCTCAGAA

	GAAAGTCATGGCCACTCAGTGCCCACTGTGGGCTTT

	GTAAGTCCTGGCTCTCCCGTCAAGGTTACCCAGAGG

	TAAAAGCTTCCTGGGAGTGGGGCCAGGTGTGTTTGG

	CACTCCAGATAGAAGGCAAAATGCTCAGATTCGGGC

	CTGTGCACTTGTATGCAACCTGTCGGTCGATACCTA

	GCATTTATTTTTCCCTGACAATGAACGACCTTTCCC

	TCACCCACCCTAAGCTCAAAGAGTTTAGCAAAATTC

	TCTTTTAAATAAACAGAATGCCAGTAAGAGGTTGAC

	CCCTACCATGGAACTTCTGGGATGCTAAATACTTCC

	TCATGAACAAAATAAGTTCCTTATTATAAGTTCCTT

	ATACTAGCAGCTTCACCTAAAGAATTTTCTCTCCAG

	CAATATTGACTTCACTGGGGAAAAGCCAAGAGTGTG

	TGGTGAGTGATTTGTTCTCACTCGACCTGGCTAGGA

	CTGGCTAGGAGCTGTTTTTTGTACATGAGGGAATTT

	GGGCTTTCCTCAGTTATCTGAATGTTTTACCCAAGT

	GCCTTCCTGCTATTGTAGCAAAGTAGCTCAGCTTCC

	TTGTCCACAGGGTGAAAAAGGACTAATGCATTTTCC

	ATCAGTTTTCTAACTATGTTAGCAAAAACGGCCTCC

	TGGTAGCTCAACCTCCTGTACGCGTGTGTGTGTGTA

	ATACACACACAAATAAACCCCTCTGTTTTTCTAAGA

	CATCTTAGCTGGATATTATAGGAAGCACTTTCATAA

	ACAACTGTAACAAATCGCAAAGGAAAGAGAAACAAA

	AGCATTAGATTTGAGACATAAACAGGCAAGAGAAAG

	TGTATTAGGAACTGACAGCTATCAAGGAAGTTTTGT

	CAGTTACAAATGCTAGGAGGAAATTTTGCCAAGAAG

	GATGGCTCATGAAATATTTCCAGTACGGGAAGAGGC

	AATAAGATCCTCTAAGAGAATGAGAAAGTAGGGGTG

	TCTAAATGGTAAAGATGGGTGTGTTGCACGTGTGTT

	AGAAGGATCTCAGTTGAGTGAAGGTTTGCACTGCTA

	CATCTAAGTTAATGTAAATATGTAGCACTCTGACAG

	GTCTACCGTGTTGCTGAATGTAGTATATTTCCAAAG

	TTTGCAAGTCTTCCTGTATTGTACAAAGATGCTGCT

	GCTTGATAATATGTATAGCAATCCAGATTAGTATGT

	TATTAAATTTTATTTTCTTACCTGTATTTTTATGCT

	TTTTACCTGTCCTCAAAATATTACACCCCTGTTGGA

	ATTAGATTTATATTTATAAATGGTCAGAAATCTTTT

	TAAGTGTCTCTTTTTACACATAGGTTGATTTTTTTT

	TCTTAAGAGAAATGATGTATTCTTGAAACATTTGTT

	ACTCATTCCAGGAAACAAAAACCCATATAATAAAAC

	CCCCACTCAGAGCCTGTTAGTCACCTCTCTAGAAGA

	TGGCATCTCAGGAGAAGGAATGGCTTTGTGGAAGAA

	GGAATCACCTTTTTCTTGCTCAAGAATTATGCTGAC

	TTCAGCCCTGAGCCTGGATCTGGTCACTGAGAATCA

	TCAAGTGTCTAGATCCTCCCCCCAAAATAACTAATT

	TAGTAGGTGATTTTGATTTTAAAAAATTGACACCAA

	AACCCTGCCTGCATTGTAATGGAATTCGAAAAGAAT

	TCATGTTCACAGAACTCAACGTTCAGGCTAATATTT

	ACAGAAGGGACCAAATCTAAATCCTGGTAGATAACT

	CCTGTATGCTTTATCCAAAGGACACCCACAGTTTTC

	CAGCATAGATATAACCAAGGATGAATTGATTCCTTC

	AAAGAACTGGGAGGCACGGATATTGCATTTTTTGTT

	TACATCCAGTAGCCAAGACGCCTCAGTGAGCCAGTC

	TTGGGCAGAGGCTGTCACATTTAGGCAGATTGGAAG

	TTGGTATGTTCTAATTCTCACTCTGGACTACAGTGA

	GGCTGAATTTATCATGTCAAAAAAAAAAAAAAAAAA

	AGACCTTTCCAAGTGCTTTCTATTGCTCAGAATTGA

	AAGAATGTTTTCATTTCAAGTTTACAAGAGGCATGG

	ATGGAGTTGTGACGTTCTTGACAAGCTGGGCTAACC

	TTTCCCGAACTTGTTTCCCGGAGGCAAGGTGCTCGG

	TGACCCAGCGCATCTTAACCTTGGGTCTCCTAGGCT

	CGAGGCTAGGGCATTACGTTTCGTGGAACCAAAGCA

	GCCAATTGCATAGCAAGTATTTTCCTGCATTCCAAT

	TAAATGCTTAAGAAAAAGCAGCATCCTATAAAATTG

	TGATCATAAACATCCATTTCCCTCAGCTTTTGTGAG

	TGCCTTGACTTACAGCCAACATCACTGTTTAACTCA

	GTCTGTTTAAAAACAAACTTTTCTGGTGGTTGATAA

	CAGAGAGTTGCTCCCTGAGCCATCAGGGTCCTGGGA

	GCTGGAAGTGAAAGGGTTATTAACATTCTACCTTTA

	TGCAGCTGTTGGCTGACCAGAATAAACTCCCTGCTG

	AGTTCAAGCTTTGAATGGAATGGATGCAAATGATGT

	TGTTTCCATTAGAGCAGGTGCTCACAGCATTCTGAT

	TGGCCTGAGCAGACCGAGGCTATGGCTGTTGGGACA

	AGCTTAGCATCCTGGACATCTTGTCAAAGAACCTCA

	CTCACCCCTCTGGCCTCTACAGCCCTCAGAGGAGAG

	AAAACCAATTCTCCAACAAACAGGTCTCTCCAACAT

	GGTGGTGCTGGCAGGCTTAGGTTTAGAAAATCCTGA

	CTGTTAAAGGCGTTTGAATACATCACATTCCTATGC

	AAATGTTTTTAATCTCCAGTTTAATGTAGTTTATTT

	TTCCTATATGTAAAGTATTTTTATACGGCTTGTATC

	ATGATAGTTTAGCAATAAAACAGTTGGAAGCAA.

As used herein, XBP1 also known as refers to an X-box binding protein 1 polypeptide. When preparing a T cell or treating a mammal with a T cell, XBP1 refers to human XBP1. An example of a human XBP1 polypeptide includes, without limitation, NCBI reference sequence: NP_005071.2. In some embodiments referring to a second nucleic acid sequence encoding a XBP1 (e.g., full length XBP1) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%) identical to:

	(SEQ ID NO: 14)
	GAGCATGCTCCCGCTGCAGTTAACTAGCCCAACCTA

	TTTCTTTAATTCAGCCCATCCCTTCGTTTCCCTTAA

	GGGATACTTTTAGTTAATTTAATATCTATAGAAACA

	ATGCTAATGACTGGTTTGCTGTTAATAAATATGTGG

	GTAAATCTCTGTTCAGGGTTCTCAGCTCTGAAGGTT

	GTAAGATCCCTGATTTCCCACTTCACACCTCTATAT

	TTCCTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTC

	TCACTCTCGCCCAGGCTGGAGTGCAGTGGCACGATC

	TCTGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGC

	CATTCTCCCGCCTCAGCCTTCCGAGTAGCTGGGACT

	ACAGGCGCCCGCCACTACGCCCGGCTAATTTTTTGT

	ATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCA

	GGATGGTCTTGATCTCCTGACTTCGTGATCCGCCTG

	CCTCGGCCTCCGAAAGTGCTGGGATTACAAGCGTGA

	GCCACCGCGCCCGGCCTCACACCTCTATATTTCTGT

	GTGTGTGTCTTTAATTCCTCTAGCACTGCTGGGTTA

	GGGTCTCCCTGACCGAGCTGGTCTCGGCAGATAAGG

	TTTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTG

	ACTTCAGGGGATCCCCGCCCCAGCCTCCCAAAGAGC

	TGGGATTACGGGCATGAGTCACCGTGCCCAGCCAAT

	TTTCTTTTGTTTTTTCTTTTGAGACAGGATCTCACT

	CTGTCACCCAGGCTTGAATGCAGTGGTACCATCTCG

	GCTCACTGCAGCCTCAATCTTCTGGGCTCAAATGAT

	CCTCCCACCTTAGCCTCCCGAGCAGCTGGGGCTACA

	AGTGCACACTACCAAGCCCAGCTAATTTTTTTTTTT

	TTTTTTTTTTTTTGAGACAGAGTCTTGCTGTGTCCC

	TCACCCAGGTTGGAGAGCAGTGGTTCGATCTTGGCT

	CACTACAACCTCTGCCTCCCGTGTTCAAGCAATTCT

	CGTGCCTCAGCCTCCTCAGTAGCTGGGATTACAGGC

	ACGTGCCACCATGCCCAGTTAATTTTTGTATTTTTA

	ATAGAGACGGGGTTTCGCCATGTTGACCAGGCTGGT

	CTTGAACCCCTGACCTCAGCCTCCCAAAGTGCTGAG

	ATTACAGGTGTGAGCCGACATGCTAGGCCTATACAT

	TTCAAAATTATGTTGCTATGTTCATAAAGATGTATA

	TATGGTAACTTGTACCTTCAATCAACATGAAATACC

	CTTCTTTGTCCTTTTAATGCCTTTATGATAAATTCT

	GTCTCATATTAATATTGCTACATATGCTTTCTTTCC

	ATAAACATTTCCATAAACATAAAAATGGCTGGTAAG

	TCATTTTCCTTTTTTTTAAAAAAATTTTTGTTTTTT

	AGAGGCAGGAGCTCATTCTGTCTCCCAGGTTGGAGT

	ACAATGGTTCAATCATAGCTCATAGTTTACTGCAGC

	CTCGAACTCCTGGGTTCAAGGGATCTTACCACCTCC

	GTCTTCCGAGCAGCTGGGACTACAGGTGCAAGTCAC

	CACGCCTGGTTAATTTTTTTAAATTTTTTGTAGAGA

	CAAGGTCACAATATGTTTCCCAGCCTGGTCTTGAAC

	TCCTGGCCTCAAGCAATCCTCCTGCCTTGAGAAATA

	TAGTAAACAAAAAATGTGAAATAACATGGCAGAAAT

	AAGTCCAAATAAATAAATAATCAAAAATAAATACAA

	ATGATTTATATTCTCTTCTTAAAAGAGAGCTCTGAG

	AAACCCCAAAGCCAGCTATATGTTGTTTATAAAGAG

	ACATACATAAAACAAAACAGCATGATTAAGAAGATA

	ATATAACCCATTCACATTTATGTTTTATTATTTATA

	TATTTGGACTTATTCCTGCCATGTTATTTTCTGTTT

	TCTGCTTACCAGTGTACAGTATTTTTCTGTTTTCCC

	TTTTCTGGAATGCCTATTTATTTCTGTTCCTGTTTT

	GTCCACCCTTTCCTGACTGATTCTTTCTGAATAATG

	ACTTTTTTTTTTTTTTTTTTTTTTTTTGAGAAAGTC

	TCACTCTGTTGACCAGGCTGGAGTGCAATGGCACAA

	TCTTGGCTAATTGCAACCTCTGCCTCCCAGGTTCAA

	GACATTATCCTGCCTCAGCCTCCCCAGTAGCTGAGA

	TTACAGGCGCCCCCCACCATGTCCGGCTAATTTTTG

	TATTTTTAGTAGAGACTGGGTTTCACCATGTTGGCC

	AGGCTGGTCTCGAACTCCTGATCTCAGGTGATCTGC

	CCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGGG

	TGAGCCACCGCGTTTGGCCTCAAAGACCGAGAACTT

	TGTAATTTATATATTTTATAGCTCTTATCACAGGTG

	TCTAGTAAATATTTTTAAACACTTATGGCACCTGAT

	GCAAGAATTACCAGGTTCATTTTATAGAGAGGATAT

	GAAACTGTCCAAGGGTTTGGACTCACATGTTCAAGA

	CTGCATGGACAGCAATCTGTAGTGGGTCAAATTATT

	GTTTTTAGTATGATTTAAAGTGTTTGTCAAAAATAT

	AAAAGTTTTGAAAACAAGCTGGGGAAGTGAATTTCA

	ATATCGCATTAACTAAGATCAAAGTGCAATTCATCA

	ACCTTTTTTCCCCATCCCGCACCCTGTGCTTTCTCT

	ACTCAGTTACTCACTACACCCTGCTGGACTAAAAGG

	GTCCTCCAGCATTTTCTTTCTTACACAGTGAAAGAC

	ATTCTCTTGGCATTAATAAATGTTCACTTAATAAAT

	AAAAAGGGCCGGGCTCTGTGGTTCCTGCCTGCAATC

	CCAGCAGTTTGGGAGGCCAAGGCAAGAGGATCGCTT

	GAGCCTAGGAGTTCCAGCCTAGGCAACGTGGCGAAA

	CCCAGTCTCAAAAAAAAAAAAAAGGAAAAAAAAGGC

	ATCAAAAAATAAAACGTAACAGGTGGCATGACATGA

	CATGACTTTTCTAACAGCCTCTTACAGCTTTCCAAG

	GTCTTTTAATATGAAGCTATAGGTCTCGGCTAGAAG

	ACACCTCCAGACTTCTCCCAAAACATTTCAGAGGCC

	CGGAGTAAGTCTCCCCACATCTGAAGGCACATCAGA

	ACCCAGGTGGCCCAAGCTGATGAGAGTTAAACAGGA

	AGTTGGTTTCTTGGTCCGGCAGAGACTCCAATCACC

	CCCACCTCTTTTCCAACCCACAGGACAGCACGTGCT

	CAGGAGGCTCTGGAGTTGGGACAGCCCAGTTAAAAA

	AAAAAAAATCATTGATTTCCCTCCCAACGAAGAGGG

	AGAAAACACGTTAGGAGACTCGTGGCCCAGTCCTGG

	CAAAAACCAAAACTATGTCCCTTTAGAGGGCTTAGA

	TATCAAGAGATGGACTTGCTTTTAGTTCTTTTTCCC

	ATCCTGTTCCCTCCCTACCAAAATAAAATTGACCAG

	CTAATCCGACTTAATAACACTAAAGAATTACTTAGG

	AACCTGCTATCTTAACATTTCACTTTTTGCATATCC

	TCCAAATACCAGGTAGCAGTCTTACTACTGTTTGCA

	CCCCTAGAACCTGGAATAGTGCTGCCCGCAGAGGAG

	GAAGCAATAATTACTTGTTAGAGAAGGTATTGCTGT

	GCATTTCTGGGGAATTTCACATTTTGTAATTTGCTT

	TAAAAAAAGTGGACAGGCATATTTACGGGGGTTTCT

	CGGACTTCTCCATGTTAATATTCGTGTGTATAAATC

	GCTCCCGTGCTGCTCTCTGGGGGCCCCTCTTTCACA

	AACACCTGGCCACCCTCACGCCACAATGGCCAGGCA

	GGAACCTCGACCTCCCCTCGGAGAGGGGGCTCAGGG

	TCAACCCCGGGGTCTCAGTCTCTACATGTGACGTTT

	TCCTGTCCCCTCATTTAAAATAACAAGAGGCTGGGC

	GCAGTGGCTTACGCCTGTAATCCCAGCACTTTGGGA

	GGCCGAGGCGGGCGATCACGAGGTCAGGAGATGGAG

	ACCATCCTGGCCAACACGGTGAAACCCCGCCTCTAC

	TAAACTACAAAAAATTAGCCGGGTGTGGTGGCGGGC

	GCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGG

	AGAATTGCTTGAACCCGGAGGCGAAGGTTGCAGTGA

	GCTGAGATCTCGCCACTGCACTCCAGCCTGGTGACA

	GAGCCTGACTCCGTCTCAAAAAATAAGAAAAAAAAA

	TAAAATAAAAATAATAGAGGCCGAAGCGGGAGGTTC

	ACTTGAGCTCAGAAGTTCGAGATCAGCCTGGGCAAC

	ACAGTGAGACCTCGTTTCTATTTAAAAAATAAAATA

	AAACTAAATTTAAAAAAATGCACGCTCATAGTACAA

	ACTTTAGAAATGGAACGAAAAACTAAAATTGAAGGT

	ATTCCCCTCCAACCCAGAGATAACACCTATCGTTTA

	TTAAGCCCTCACTATTGTTAAACTTAGTTTTAAAGG

	GCACGATCTCATTTCTTAAAGACTTCTATTCCGCAG

	AATTTCTTTCCAGGCTTTTTTCTTTTTCTTTTTTTG

	AGACGGAGTCTCGCTCTGTCGCCCAGGCCGGGGTGC

	AGTGGCGCGATCTCGGCTCACTGAAACCTCTGTCCA

	GTCTTTTCGAACCCAAGGCCCAACTGCGCTCTATCT

	CGACTTTCGGCTCCACTCGGATCCCGAAGTGGCGCA

	CGAGATAAAATGTTGTCAGGCTGAGGTAATTCTCTG

	TTAGTCCCGGTAAAAATTCGTCAGTCTGGAAAGCTC

	TCGGTTTGGAATTAAATTCTGTCACTCCGGATGGAA

	ATAAGTCCGCTTAAGGGGGGAAAATCCGTTTGTGGA

	GGACACGCTCCCGCACGTAACCCCCCGCGGAAAATG

	ACCCCAAGTACCTTTGGCCAGGGATTGCCGCTGCCA

	CGCCGGACTCCATAGCCACGGTCCTGAAACGCCCCG

	CCGGGCAGGCCGGACCAATGGACGCCGAGCTCGGCC

	GTGCGTCACGCGACGCTGGCCAATCGCGGAGGGCCA

	CGACCGTAGAAAGGCCGGGCGCGGCGAGGCTGGGCG

	CTGGGCGGCTGCGGCGCGCGGTGCGCGGTGCGTAGT

	CTGGAGCTATGGTGGTGGTGGCAGCCGCGCCGAACC

	CGGCCGACGGGACCCCTAAAGTTCTGCTTCTGTCGG

	GGCAGCCCGCCTCCGCCGCCGGAGCCCCGGCCGGCC

	AGGCCCTGCCGCTCATGGTGCCAGCCCAGAGAGGGG

	CCAGCCCGGAGGCAGCGAGCGGGGGGCTGCCCCAGG

	CGCGCAAGCGACAGCGCCTCACGCACCTGAGCCCCG

	AGGAGAAGGCGCTGAGGAGGTGGGCGAGGGGCCGGG

	GTCTGGGGCCAGATCTGAAGCCGGGACTAGGGACAG

	GGGCAGGGGCAGGGGCTGGGAGCGGGGACCCAGCAC

	TGGCCGCCCCGCAGGGCTCCGTCGCCTTTGGCCTGG

	CGGGTCGGTGCCAGCGTGGCGCGGGGCGGGGCAGGA

	AGCCCGGACTGACCGGATCCGCCACGCTGGGAACCT

	AGGGCGGCCCAGGGCTCTTTTCTGTACTTTTTAACT

	CTCTCGTTAGAGATGACCAGAGCTGGGGATGCGGGC

	ACCTGTCTTCCAGGCCCTCTTGCTGTGTGGCCGCAG

	ACTGGTGGTTCAGCCTCTTAACTCGGACATGAGGTC

	GAATAATCTGTTTTGGTTTACTGCTATTTCTGGAGA

	GGCGCGGAGCTGAAATAACAGAGCTGTTGAAAGGGC

	TGGGAATTCTGCGAGGCTCACTGGTCTAGCTCAGTA

	TCTGCGTTCTTAAAATGGAACCTACTTCATGAGGTC

	TTTGGGGAGATTGAGACTTGGATATAATGTGCCTAG

	CACTTAGTCCTCCGTAAATGTTCACTCTTTTGTGAT

	CATTGTGCCTTCTGTGATTTATGAAGTGTCTCTTCT

	GAGTTAATTCTTTTAAAAAAAAAAGTGTCTCCTCCA

	ACAGACACGGACCCATCAGCAGGTCACTGCCTAGGA

	TCTCAACACTAGAGATCAGGGAGTGGCATCAGCCTC

	TCCCTTTTCTAAATTGGACTGGGGGACGGAGGGTTG

	ATGTCATAGCAAGATTGCAGCCTTCACTAGATTAAT

	GAGGCCAGGTTGGATCCTGTTTAAGAGAACTGGAGA

	CAGGAAGCAGCGGGGGAATAGATGGGGAAAGAGGAA

	AGTTCCTTATGATGCAAGATGAATAGTGTGTGTGTC

	CAGCCCCAGTGCTGTGACGGGGATGAGTCTGAGGTG

	GACGGATGATGCAATATAGGAGAGAATAAAGCAGGT

	CTTCGAGCTAGATTGACAGAAGACTGTATTTTTTAT

	TTTGTTTTATTGAGGGGAGGAGCCTGAAGTGTATTT

	TATCATTAGTCTGTCTTATACTGTAAATAAAAATGA

	AAGCACCAGCTGGTAAAGTTTTCAAATAAAGACATA

	AATAAGGTTTGATATGACTCAGTGTGGTATGTTCCT

	TCTCTTCCTAGGAAACTGAAAAACAGAGTAGCAGCT

	CAGACTGCCAGAGATCGAAAGAAGGCTCGAATGAGT

	GAGCTGGAACAGCAAGTGGTAGATTTAGAAGAAGAG

	GTAAAACTACTTAAGGTCAAACTCTTTTATCCATTG

	TATACCCTTCCTTGGTGAATGTTCTGATATTTGCTT

	CCCATCCCAAGTTGTTTCAGCCCCTATTAGAATACA

	ATTGAATATATGATTAAAAGTTAAACTAGGCTGGGC

	ATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGA

	GCCTGAGTTGGGCAGATCACTTGAAGCCAGCAGTTT

	GAGACCAGCCTAGCCAACATGGTAAAATCCCGTCTC

	TACCCAAAAATATACCAAAAAAAAAAAAAAAAAAAA

	GGCCAAGCGTGAGTGCCTGTAGTCCCAGCTACTCGG

	GAGGTTGAGGTGGGAGGATTGTTTGAACCTGGGAGA

	GGGAGGTTGCAGTGAGCTGAGATCGCACCACTGCAC

	TCCAGCCTGGGCAACAGAGTGAGACTCTGTCTCAAG

	AAAAAAAAAAAAAGTTTGCTGGGCACCGGGGCTCAC

	ACCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGG

	TAGATAACTTGAGATCAGGAGTTCGAGACCAGCCTG

	ACCAACGTGGTGAAACCCCATCTCTATTAAAAATAC

	AAAAATTAGCCGGGTGTCGTGGCAGGCACCTGTAAT

	CCCAGCTGCTCCGGAGGCTGACGCAGGAGAATCACT

	TGAACCCAGGAGGCGGAGGTTGCAGTGAGCTGAGAT

	CACGAGATCATGCCACTGCACTCCAGTCTGGGCGAC

	AGAGCAAAAACCCTGTCTCAAAAAAAAAAAAAAAGT

	TAATCTAAGTTAGGACAGAGAGTTGGTGAAGTGGTG

	AAGCTTGTTGAGGGCAGAAGTGATTGACTTTGTGGC

	ATTTGGTGCTAGATGTATCTCAAAGTAGATGGATTT

	AACAATGTTTATTGAGTTTGTAGTAAGAAATTAGCA

	AGGGCTAATAGGAAATAATTGCTTAAACTTTACATT

	CTTCCTGGCATGGCCAGAAATTCACTAAAGGTTCCT

	TTCCCCCTCTAGGGTCCACCTGTTAATCAATCTTAA

	ATTGTTGCCAATTACACATCTTGAATACATAGAGAT

	TATTTATATTGTTTTTTTAACCCCTTGGTCAATTTG

	CATATATTGAGCTTTTTAAAGTTTTAATCATTAGTT

	GGTTCTTCTAAGAATCATGAGTCAGGAGCAGGGATT

	TTTTTTAACTTATTTTGGATTTATAGTCACCACTAC

	CACTTTTATTATTACCTGCCAGTTCAAGATAGTTAT

	TTATTTTTATTTTATATTATTATTATTATTATTATC

	ATCATCATTATTTTGAGATGGAGTCTCACTCTGTTG

	CCCAGGCTGGAGTGCAGTGGTGCAATCTCGGCTCAC

	TGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCCC

	TGCTTCAGCCTCCAGATTAGCTGGGATTACAGGCAC

	CCCTCACCACATCCAGCTAATTTTTGGATTTTTTAG

	TAGAGATGGGGGTTTGCCATGTTGGCCAGGCTGGTT

	TTGAACTCTTGACCTCAGGTGATCCACCTGCCTTGG

	CCTCCCAAAGTGTTAGGATTACAAGTGTGAGCCACC

	GAGCCTGGCCAAGATAGTTTAAAAAAAAAATTATAT

	CTACATTAAAGCCACAAGTCACCCTTTGCTGAAGTC

	AGTATTAGTAGTTGGAAGCAGTGTGTTATTCTTGAC

	CCCATGAAGTGGCACTTATTAAGTAGCTTGCTTTTC

	CATAATTATGGCCTAGCTTTTTAAAACCTACTATGA

	ACACCACAAGCATAGAGTTTTCCAAAAGTTCAAGAA

	GGAAAGGAAACCAATTATACTGAATCAGGTAGATTC

	TTAACTGAAATAATTAGATGTTTTAATAGCCTCTTA

	TGAACTTTCTTCCAGAACCAAAAACTTTTGCTAGAA

	AATCAGCTTTTACGAGAGAAAACTCATGGCCTTGTA

	GTTGAGAACCAGGAGTTAAGACAGCGCTTGGGGATG

	GATGCCCTGGTTGCTGAAGAGGAGGCGGAAGCCAAG

	GTAAATCATCTCCTTTATTTGGTGCCTCATGTGAGT

	ACTGGTTCCAAGTGACATGACCCAGCGATTATGTTT

	ACAGTCTGGACTTCTGATCAAGAGCGTTCTTGAAAT

	TTTCCTTCAGTTTTAAGACATTTTCATGCAGGCAGA

	GTGTTCTTCCCCTAAAGGCACTTGACACTCATTTTT

	TAAGTGTGTAGTGAACAGTACTAAGATCTAATAATG

	AAAACAAGTTACATGGCTCCCTAAGAACAAGTACTA

	ACAAATGCAGTAGCCAACAAGATTACCATGCAATCA

	TTAAGGAGAACCAAAGTAAGAGAGCCACTCAAACCA

	GATTTTGAACGCTACTAAAATTAAAGTAGTTCTTTG

	ATGAATATGAATGAGTAGGGAAAGGATTCTTTGTAA

	TAGTGATACCTCTGTGGTAAGAGAAGGGTGGTATGT

	GAGTTTTAGTCTACAGATTATGGCAAATTCAGTGAC

	AACAATCAAATGGTCTAAGATTGACAGTAGCACAGT

	TTTACTCTGTGAAGGTAATGTTCAGGACAAATTTCA

	AGAAAACTAGAAAACCATTCTTTACAGCTGAAATCT

	TTCCCTAACCATTGTTATTTCCACTTTTAAGTCCTC

	AAGAGATGAGAAAAGGGAGGTAAGGCTTCCTTATAC

	ATTTCCTGCACAATGAAACATTTTTCCTCCTCCAGG

	CAAAGATTCAAGCAGAACTGGCAAATATCTTATCTT

	GCTCTTCTCAATAATAATAATGTTGTTAGATAATAA

	AGTTCTATAGCAATTTAACCCTAGAATCTTTTTGAA

	AAGTAATTCTTTAAAGTTGAGAATCACAGCTGTCTA

	GCAAGCATTTCCTTGGGCACTTGAAGCTGTTTATTC

	ACTTTGGTCTTTCCTCCCAGGGGAATGAAGTGAGGC

	CAGTGGCCGGGTCTGCTGAGTCCGCAGCACTCAGAC

	TACGTGCACCTCTGCAGCAGGTGCAGGCCCAGTTGT

	CACCCCTCCAGAACATCTCCCCATGGATTCTGGCGG

	TATTGACTCTTCAGATTCAGAGGTAGGGATCATTCT

	GACTTATTAAAGAGCTATATAACCAGTTAATTCCAT

	CTGTTTGATGCTTGACATCCCTAACTAGACAGATGA

	GGGTTGAAGTTAGTTTTTGGTGGGGTTGGAGGTGAA

	CATCAACTACCTTCCTAGTTCCAGGTAATATAGAAC

	ATGGAGTGAAGTGTAGATAAATGGGTCTGGTGGGTC

	CCGAGGTCATCTTATCACATAATGACTAATTTACAT

	TATGGAACCCAGTACAAAGTGTTCCAGTTAGATTTT

	CCATTGTATTCTGACAGTTGTACTTCATTTAATTTT

	TGCCTCTTACAGTCTGATATCCTGTTGGGCATTCTG

	GACAACTTGGACCCAGTCATGTTCTTCAAATGCCCT

	TCCCCAGAGCCTGCCAGCCTGGAGGAGCTCCCAGAG

	GTCTACCCAGAAGGACCCAGTTCCTTACCAGCCTCC

	CTTTCTCTGTCAGTGGGGACGTCATCAGCCAAGCTG

	GAAGCCATTAATGAACTAATTCGTTTTGACCACATA

	TATACCAAGCCCCTAGTCTTAGAGATACCCTCTGAG

	ACAGAGAGCCAAGCTAATGTGGTAGTGAAAATCGAG

	GAAGCACCTCTCAGCCCCTCAGAGAATGATCACCCT

	GAATTCATTGTCTCAGTGAAGGAAGAACCTGTAGAA

	GATGACCTCGTTCCGGAGCTGGGTATCTCAAATCTG

	CTTTCATCCAGCCACTGCCCAAAGCCATCTTCCTGC

	CTACTGGATGCTTACAGTGACTGTGGATACGGGGGT

	TCCCTTTCCCCATTCAGTGACATGTCCTCTCTGCTT

	GGTGTAAACCATTCTTGGGAGGACACTTTTGCCAAT

	GAACTCTTTCCCCAGCTGATTAGTGTCTAAGGAATG

	ATCCAATACTGTTGCCCTTTTCCTTGACTATTACAC

	TGCCTGGAGGATAGCAGAGAAGCCTGTCTGTACTTC

	ATTCAAAAAGCCAAAATAGAGAGTATACAGTCCTAG

	AGAATTCCTCTATTTGTTCAGATCTCATAGATGACC

	CCCAGGTATTGTCTTTTGACATCCAGCAGTCCAAGG

	TATTGAGACATATTACTGGAAGTAAGAAATATTACT

	ATAATTGAGAACTACAGCTTTTAAGATTGTACTTTT

	ATCTTAAAAGGGTGGTAGTTTTCCCTAAAATACTTA

	TTATGTAAGGGTCATTAGACAAATGTCTTGAAGTAG

	ACATGGAATTTATGAATGGTTCTTTATCATTTCTCT

	TCCCCCTTTTTGGCATCCTGGCTTGCCTCCAGTTTT

	AGGTCCTTTAGTTTGCTTCTGTAAGCAACGGGAACA

	CCTGCTGAGGGGGCTCTTTCCCTCATGTATACTTCA

	AGTAAGATCAAGAATCTTTTGTGAAATTATAGAAAT

	TTACTATGTAAATGCTTGATGGAATTTTTTCCTGCT

	AGTGTAGCTTCTGAAAGGTGCTTTCTCCATTTATTT

	AAAACTACCCATGCAATTAAAAGGTACAATGCAGCA

	TCCTTGTTTGATTTCTTCTAGGGCCGTAAGTCTTGT

	TTTCTCTCCAGATGTTTATCTGTGTGCTGTGGTAGG

	AATTAATCCAACTGAAGTGAGCCTAACGCTTTTTAA

	AGTGACTGAAGGCTTTTCCACCTTAATTACTGCCTG

	CTTTAATTCTGGACTGCCATAAGTGATATAAGCTAT

	AATTTGAGCAGTTACTGTCTTTCTGAGACAGATTCT

	TGAGCCTAACTGACCAATATCACAGCTAGTAAGTGG

	AAGAGCTAGAACCCTAACCACTATTTGCTACACCAT

	CTTATAAATGTTAAACAAGGACACACCATCACATAT

	CGAGATTCTCTTGCCCTTATTATGGGAATTAAGAGC

	ATTTTCTAGACTGAAACTCCCTATTTTCAACTCTGC

	CACTGGTAAGCTGGGTAACCCAGGGGTTATATATAA

	TCACTTATTTCCTCATCTGTAAAGTTGGATAATGGT

	ATCTCTAAAGGTTAAGATTCAAAGAGACGATGCATT

	ATAAGCATTTAGTATATGCTAGGCACCATCCTAAAC

	ACTGGAAAGTTAGTTAGTTATTATCTCCTAATCCAC

	TTTGGAAGGGTTTTAATCTCTTCCAGAATTATATTT

	ACTCAAGAATTTGTTTCATCAAAGAATAAACCTCGG

	CCAGGCGCGGTGGCTCATGCCTGTAATCCCAGCACT

	TTGGGAGGCTGAGGCGGGTGGATCACGAGGTCAGGA

	GATCGAGACCATCCTGCCTAACATGGGGAAACCCTG

	TCTCTACTAAAATTACAAAAAATTAGCCAGGCGTGG

	TGGTGGGCGCCTGTAATCCCAGCTACTTGGGAGGCT

	GAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGC

	TTGCGGTGAGGGGAGATCGCGCCACTGCACTCCAGC

	CTGGGCAACAGAGCGAGACTCTGTCTCAAAAAATAA

	ATAAATAAATAAATAAATAAATAAATAAACCTCTTC

	AAGAAAAAATCCTAGTGATATTAATACAACTCCCAA

	AGACTTGATAACCTCCTCATCCTTCATAGCATCTTT

	TCCTTGGAAATCTTACAAGGTTTTACAGGACTTTAC

	TTATTTATAAAAATTTCACCTATGCCAGTAGATGAA

	ATCATTCTATGCCAATTTAGCATTTAAATGCTATGT

	TCCCAACTTACAAAGACTAACTCTGGGGAGGTCAAA

	GTGAATGAGTAGAAAAAAGGCAGGATTCAGAGAATC

	CCAAGCAGCAAGGCAAAGTGGATTATAGAATACCTT

	TGGTGTAGGCCAGGTGTAGTGGCTCACGCTTGTAAT

	CCCAACACTTTGGGAGGCTGAGGTGGGCGGATCACC

	TGAGGTCAGGAGTTCATGGCCAGCCTGACCAACATA

	GTGAAACCCCATCTCTAGTAAAAATACAAAATTAGC

	TGGGTGTGGTGGCGCATATGCCTGTAATCCCAGCTA

	CTCAGGAGGCTGAGGCGGCAGAATCACTTGAACCCG

	GGAGGCAGAGGATGCAGCGAGCCGAGATCGTGCCAT

	TGCACTCCAGCCTGGGCAACAAGAGCGAAACTCCAT

	TTAAAAAAGAAAAAAAAAAATAGAATGCCTTTCATG

	TAGTGACTGGAGGCAAGTCAGCTAGCTGCCTTCAAG

	ATCCGGTCGTTGAAGCCAGGGCCCAATCCTGGTGCT

	CAGCAATACAAACTTGCTTAGGCTCTTAAGTTTCTT

	CAGAAACAGGCCAGGCATGGTGGCTCACACCTATAA

	TCCCAGCACTTTGGGAGGCCGAGGCCAGCAGATTGC

	TTGGTTCAAGACTAGCCTGGACAACATGGCAAACCC

	GTCTCTCCATGAAAAGTAAAAAAAAATAGCCAGGCA

	TGGTGGTGTGCACTGGTGGTCACAGCCACTCAGGAA

	GCTGAGGTGGGAGGATCGCTTGAGGCCAGGGGGCAG

	AGGTTGCAGTCAGCCAAGATCGCAGCACTGCACTCC

	AGACTGGGTGAAAAAGCAAGACTGCCTAAAAAAAAA

	AAGGTTCTGTATATAAG.

As used herein, FOXO1 refers to a forkhead box 1 polypeptide. When preparing a T cell or treating a mammal with a T cell, FOXO1 refers to human FOXO1. An example of a human FOXO1 polypeptide includes, without limitation, NCBI reference sequence: NP_002006.2.
As used herein, ID2 refers to an inhibitor or DNA binding 2 polypeptide. When preparing a T cell or treating a mammal with a T cell, ID2 refers to human ID2. An example of a human ID2 polypeptide includes, without limitation, NCBI reference sequence: NP_002157.2.
As used herein, ID3 refers to an inhibitor or DNA binding 3 polypeptide. When preparing a T cell or treating a mammal with a T cell, ID3 refers to human ID3. An example of a human ID3 polypeptide includes, without limitation, NCBI reference sequence: NP_002158.3.
As used herein, IRF4 refers to a interferon regulatory factor 4 polypeptide. When preparing a T cell or treating a mammal with a T cell, IRF4 refers to human IRF4. An example of a human IRF4 polypeptide includes, without limitation, NCBI reference sequence: NP_001182215.1.
As used herein, LEF1 refers to a lymphoid enhancer binding factor 1 polypeptide. When preparing a T cell or treating a mammal with a T cell, LEF1 refers to human LEF1. An example of a human LEF1 polypeptide includes, without limitation, NCBI reference sequence: NP_001124185.1.
As used herein, SATB1 refers to a SATB1 homeobox 1 polypeptide. When preparing a T cell or treating a mammal with a T cell, SATB1 refers to human SATB1. An example of a human SATB1 polypeptide includes, without limitation, NCBI reference sequence: NP_001124482.1.
As used herein, RUNX1 refers to a RUNX family transcription factor 1 polypeptide. When preparing a T cell or treating a mammal with a T cell, RUNX1 refers to human RUNX1. An example of a human RUNX1 polypeptide includes, without limitation, NCBI reference sequence: NP_001001890.1.
As used herein, BCL11b refers to a BAF chromatin remodeling complex subunit BCL11b polypeptide. When preparing a T cell or treating a mammal with a T cell, BCL11b refers to human BCL11b. An example of a human BCL11b polypeptide includes, without limitation, NCBI reference sequence: NP_001269166.1.
As used herein, FOXP1 refers to a forkhead box P1 polypeptide. When preparing a T cell or treating a mammal with a T cell, FOXP1 refers to human v. An example of a human FOXP1 polypeptide includes, without limitation, NCBI reference sequence: NP_001012523.1.
As used herein, FOXP4 refers to a forkhead box P4 polypeptide. When preparing a T cell or treating a mammal with a T cell, FOXP4 refers to human v. An example of a human FOXP4 polypeptide includes, without limitation, NCBI reference sequence: NP_001012426.1.
As used herein, BACH2 refers to a BTB domain and CNC homolog 2 polypeptide. When preparing a T cell or treating a mammal with a T cell, BACH2 refers to human BACH2. An example of a human BACH2 polypeptide includes, without limitation, NCBI reference sequence: NP_001164265.1.
As used herein, STAT3 refers to a signal transducer and activator of transcription 3 polypeptide. When preparing a T cell or treating a mammal with a T cell, STAT3 refers to human STAT3. An example of a human STAT3 polypeptide includes, without limitation, NCBI reference sequence: NP_001356441.1.
As used herein XBP1 refers to an X-box binding protein 1 polypeptide. When preparing a T cell or treating a mammal with a T cell, XBP1 refers to human XBP1. An example of a human XBP1 polypeptide includes, without limitation, NCBI reference sequence: NP_005071.2.

Antigen-Binding Domains

As used herein, the term “antibody,” “antigen-binding domain,” or “antigen-binding fragment” refers to an intact immunoglobulin or to an antigen-binding portion thereof. In some embodiments, a binding agent refers to an intact immunoglobulin or to an antigen-binding portion thereof. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Examples of antigen-binding portions include Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen-binding to the polypeptide. As used herein, the term “scFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Included in the definition are single domain antibody, including camelids. In some cases, the antibody is human or humanized.
In some embodiments, any of the “antigen-binding domains,” “antibodies,” “ligand binding domains,” or “binding agents” described herein can bind specifically to a target selected from the group of: CD16a, CD28, CD3 (e.g., one or more of CD3α, CD3β, CD3δ, CD3ε, and CD3γ), CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, LAG3, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein (e.g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6), HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGFDD, a ligand of TGF-β receptor II (TGF-βRII), a ligand of TGF-βRIII, a ligand of DNAM1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NK30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-D, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.
In some embodiments, any of the “antigen-binding domains,” “antibodies,” “ligand binding domains,” or “binding agents” further include a secretion signal peptide. For example, a nucleic acid sequence encoding a binding agent further includes a nucleic acid sequence encoding a secretion signal peptide.
As used herein, ICAM-1 refers to intercellular adhesion molecule 1 polypeptide. When preparing the T cell or treating a mammal with the T cell, ICAM-1 refers to human ICAM-1. An example of a human ICAM-1 polypeptide includes, without limitation, NCBI reference sequence: NP_000192.2 or a fragment thereof.
As used herein, VCAM-1 refers to vascular cell adhesion molecule 1 polypeptide. When preparing the T cell or treating a mammal with the T cell, VCAM-1 refers to human VCAM-1. An example of a human VCAM-1 polypeptide includes, without limitation, NCBI reference sequence: NP_001069.1 or a fragment thereof.
As used herein, LFA-1 also known as ITGB2 refers to lymphocyte function associated antigen-1 (LFA-1) polypeptide or integrin subunit beta 2 (ITGB2) polypeptide. When preparing the T cell or treating a mammal with the T cell, LFA-1 or ITGB2 refers to human LFA-1 or ITGB2. An example of a human LFA-1 or ITGB2 polypeptide includes, without limitation, NCBI reference sequence: NP_000620.2 or a fragment thereof
As used herein, TGFBR2 refers to transforming growth factor beta receptor 2. When preparing the T cell or treating a mammal with the T cell, TGFBR2 refers to human TGFBR2. An example of a human TGFBR2 polypeptide includes, without limitation, NCBI reference sequence: NP_001020018.1 or a fragment thereof.
As used herein, IFNAR1 refers to interferon (alpha and beta) receptor 1. When preparing the T cell or treating a mammal with the T cell, IFNAR1 refers to human IFNAR1. An example of a human IFNAR1 polypeptide includes, without limitation, NCBI reference sequence: NP_000620.2 or a fragment thereof.

Methods of Producing T Cells

As described herein, any appropriate method of producing cells (e.g., T cells) comprising a FOXP3 polypeptide and one or more transcription factors can be used to generate the T cells as described herein. In some embodiments, a cell (e.g., a T cell) that is transduced with the nucleic acid sequences described herein is isolated from a mammal (e.g., a human) using any appropriate method (e.g., magnetic activated sorting or flow cytometry-mediated sorting). In some cases, nucleic acid sequences encoding a FOXP3 polypeptide and one or more transcription factors can be transformed into a cell (e.g., a T cell) along with nucleic acid sequences encoding a therapeutic gene product and/or a binding agent. For example, a T cell can be made by transducing nucleic acid sequences encoding a FOXP3 polypeptide and one or more transcription factors into a cell (e.g., a T cell) using a lentivirus. In another example, a T cell can be made by transducing nucleic acid sequences encoding a FOXP3 polypeptide, one or more transcription factors, and a therapeutic gene product into a cell (e.g., a T cell) using a lentivirus. In yet another example, a T cell can be made by co-transducing nucleic acid sequences encoding a FOXP3 polypeptide, one or more transcription factors, a therapeutic gene product, and a binding agent into an immune cell (e.g., a T cell) using a lentivirus. In all cases described herein, the nucleic acid sequences are operably linked to a promoter or are operably linked to other nucleic acid sequences using a self-cleaving 2A polypeptide or IRES sequence.
Methods of introducing nucleic acids and expression vectors into a cell (e.g., a eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection. As used herein, “transformed” and “transduced” are used interchangeably.
In some embodiments, the transformed cell can be an immune cell, an epithelial cell, an endothelial cell, or a stem cell. In some embodiments, the transformed cell is an immune cell selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell and a cytotoxic T cell. In some examples, the immune cell is a NK cell, and the detection of a memory NK cell can include, for example, the detection of the level of one or more of IL-12, IL-18, IL-33, STAT4, Zbtb32, DNAM-1, BIM, Noxa, SOCS1, BNIP3, BNIP3L, interferon-γ, CXCL16, CXCR6, NKG2D, TRAIL, CD49, Ly49D, CD49b, and Ly79H. A description of NK memory cells and methods of detecting the same is described in O'Sullivan et al., Immunity 43:634-645, 2015. In some examples, the immune cell is a T cell, and the detection of memory T cells can include, e.g., the detection of the level of expression of one or more of CD45RO, CCR7, L-selectin (CD62L), CD44, CD45RA, integrin αeβ7, CD43, CD4, CD8, CD27, CD28, IL-7Rα, CD95, IL-2Rβ, CXCR3, and LFA-1. Additional examples of T-cells that can be transduced are described herein.

Nucleic Acids/Vectors

Also provided herein are nucleic acids sequences that encode any of the polypeptides described herein. For example, nucleic acid sequences are included that encode for a FOXP3 polypeptide, one or more transcription factors, a therapeutic agent comprising a polypeptide, and a binding agent comprising a polypeptide. Also provided herein are vectors that include any of the nucleic acid sequences encoding any of the polypeptides described herein. For example, the polypeptides include, without limitation, a FOXP3 polypeptide, one or more transcription factors, a therapeutic agent comprising a polypeptide, and a binding agent comprising a polypeptide.
Any of the vectors described herein can be an expression vector. For example, an expression vector can include a promoter sequence operably linked to the sequence encoding any of the polypeptides as described herein. Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. In some cases, a vector can include sufficient cis-acting elements that supplement expression where the remaining elements needed for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the T cells as described herein. Any appropriate promoter (e.g., EF1 alpha) can be operably linked to any of the nucleic acid sequences described herein. Non-limiting examples of promoters to be used in any of the vectors or constructs described herein include EF1a, SFFV, PGK, CMV, CAG, UbC, MSCV, MND, EF1a hybrid, and/or CAG hybrid. As used herein, the term “operably linked” is well known in the art and refers to genetic components that are combined such that they carry out their normal functions. For example, a nucleic acid sequence is operably linked to a promoter when its transcription is under the control of the promoter. In another example, a nucleic acid sequence can be operably linked to other nucleic acid sequence by a self-cleaving 2A polypeptide or an internal ribosome entry site (IRES). In such cases, the self-cleaving 2A polypeptide allows the second nucleic acid sequence to be under the control of the promoter operably linked to the first nucleic acid sequence. The nucleic acid sequences described herein can be operably linked to a promoter. In some cases, the nucleic acid sequences described herein can be operably linked to any other nucleic acid sequence described herein using a self-cleaving 2A polypeptide or IRES. In some cases, the nucleic acid sequences are all included on one vector and operably linked either to a promoter upstream of the nucleic acid sequences or operably linked to the other nucleic acid sequences through a self-cleaving 2A polypeptide or an IRES.

Compositions

Also provided herein are compositions (e.g., pharmaceutical compositions) that include at least one of any of the polypeptides (e.g., FOXP3 polypeptides, one or more transcription factors, therapeutic polypeptides, and binding agent polypeptides), any of the cells, or any of the nucleic acids or vectors described herein. In some embodiments, the compositions include at least one of the any of polypeptides (e.g., FOXP3 polypeptides, one or more transcription factors, therapeutic polypeptides, and binding agent polypeptides) described herein. In some embodiments, the compositions include any of the cells (e.g., any of the cells described herein including any of the cells produced using any of the methods described herein). In some embodiments, the pharmaceutical compositions are formulated for different routes of administration (e.g., intravenous, subcutaneous). In some embodiments, the pharmaceutical compositions can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline).

Cells

Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acid sequences described herein that encode any of the polypeptides (e.g., FOXP3 polypeptides, one or more transcription factors, therapeutic polypeptides, and/or binding agent polypeptides) described herein. Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include any of the vectors described herein. In some embodiments, the cells are any of the exemplary types of T cells described herein or known in the art.
In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human) cells. Non-limiting examples of mammalian cells include Chinese hamster ovary cells and human embryonic kidney cells (e.g., HEK293 cells).

Methods of Treatment

Also provided herein are methods of treating a mammal (e.g., a human) having an autoimmune disease that includes administering to the mammal (e.g., human) a therapeutically effective amount of a cell (e.g., any of the exemplary T cells described herein) or any of the compositions (e.g., pharmaceutical compositions) described herein.
In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the autoimmune diseases in the mammal (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the autoimmune disease in the mammal prior to treatment). For example, a mammal having an autoimmune disease having been administered a T cell as described here can experience a reduction in inflammation or autoantibody production.
Any appropriate method of administration can be used to administer the T cells to a mammal (e.g. a human) having an autoimmune disease. Examples of methods of administration include, without limitation, parenteral administration and intravenous injection.
A pharmaceutical composition containing the T cells and a pharmaceutically acceptable carrier or buffer can be administered to a mammal (e.g., a human) having an autoimmune disease. For example, a pharmaceutical composition (e.g., a T cell along with a pharmaceutically acceptable carrier) to be administered to a mammal having an autoimmune disease can be formulated in an injectable form (e.g., emulsion, solution and/or suspension). In some embodiments, a pharmaceutical composition containing the T cells can include phosphate buffered saline.
Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in a pharmaceutical composition described herein can include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
Effective dosage can vary depending on the severity of the autoimmune disease, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments, and the judgment of the treating physician. An effective amount of a T cell can be any amount that reduces inflammation and autoantibody production within a mammal having an autoimmune disease without producing significant toxicity to the mammal. For example, an effective amount of T cells administered to a mammal having an autoimmune disease can be from about 1×10⁶cells to about 1×10¹⁰(e.g., from about 1×10⁶to about 1×10⁹, from about 1×10⁶to about 1×10⁸, from about 1×10⁶to about 1×10⁷, from about 1×10⁷to about 1×10¹⁰, from about 1×10⁷to about 1×10⁹, from about 1×10⁷to about 1×10⁸, from about 1×10⁸to about 1×10¹⁰, from about 1×10⁸to about 1×10⁹, or form about 1×10⁹to about 1×10¹⁰) cells. In some cases, the T cells can be a purified population of immune cells generated as described herein. In some cases, the purity of the population of T cells can be assessed using any appropriate method, including, without limitation, flow cytometry. In some cases, the population of T cells to be administered can include a range of purities from about 70% to about 100%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 90%, from about 90% to about 100%, from about 80% to about 100%, from about 80% to about 90%, or from about 90% to 100%. In some cases, the dosage (e.g., number of T cells to be administered) can adjusted based on the level of purity of the T cells.
The frequency of administration of a T cell can be any frequency that reduces inflammation or autoantibody production within a mammal having an autoimmune disease without producing toxicity to the mammal. In some cases, the actual frequency of administration can vary depending on various factors including, without limitation, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in frequency of administration.
An effective duration for administering a composition containing a T cell can be any duration that reduces inflammation or autoantibody production within a mammal having an autoimmune disease without producing toxicity to the mammal. In some cases, the effective duration can vary from several days to several months. In general, the effective treatment duration for administering a composition containing a T cell to treat an autoimmune disease can range in duration from about one month to about five years (e.g., from about two months to about five years, from about three months to about five years, from about six months to about five years, from about eight months to about five years, from about one year to about five years, from about one month to about four years, from about one month to about three years, from about one month to about two years, from about six months to about four years, from about six months to about three years, or from about six months to about two years). In some cases, the effective treatment duration for administering a composition containing a T cell can be for the remainder of the life of the mammal.
In some cases, a course of treatment and/or the severity of one or more symptoms related to autoimmune disease can be monitored. Any appropriate method can be used to determine whether the autoimmune disease is being treated. For example, immunological techniques (e.g., ELISA) can be performed to determine if the level of autoantibodies present within a mammal being treated as described herein is reduced following the administration of the T cells. Remission and relapse of the disease can be monitored by testing for one or more markers of autoimmune disease.
Any appropriate autoimmune disease can be treated with a T cell as described herein. In some cases, an autoimmune disease caused by the accumulation of autoantibodies can be treated with a T cell as described herein. Examples of autoimmune diseases include, without limitation, lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, and polyarteritis nodosa.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1. T Cell Transduced with Nucleic Acid Sequences Encoding FOXP3 and BLIMP1

A set of experiments is performed to assess the effect of co-expression of a BLIMP1 polypeptide and a FOXP3 polypeptide. In these experiments, CD4⁺ T cells are transduced with a lentivirus where the lentiviral vector includes a first nucleic acid sequence encoding a FOXP3 polypeptide harboring mutations in NES1 and NES2 that result in nuclear localization of FOXP3 and a second nucleic acid sequence encoding BLIMP1 polypeptide. The vector includes an EF1α promoter. Lentivirus is produced in HEK293 cells according to standard protocols.
CD4⁺ T cells are counted and checked for viability. Next cells are re-suspended in fresh serum free ImmunoCult T cell expansion media at a concentration of 10⁶cells/mL. Then 500 μL (˜500,000 cells) of the cell suspension is aliquoted to each well. The cells are then cultured in the presence of CD3/CD28 for 1-2 days prior to addition of virus. Different concentrations of lentiviral particles are added to each well for the desired target MOI. The plates are then sealed with parafilm, and the cells are spun in a table top centrifuge at 300×g for 5 minutes. After spinoculation, the cells are incubated at 37° C. The cells are then assessed for FOXP3 expression and cellular localization, BLIMP1 expression, and expression of a T reg phenotype.

Example 2

Table 1 (below) shows the percentage of Mean Fluorescence Intensity (MFI) as compared to donor-matched expanded Tregs.
Each column represents values for synReg transduced with FOXP3 alone or co-transduced with FOXP3 and the indicated modifier. Each row displays data for the specified marker. Values are displayed as mean of 3 donors±SD, * p<0.05, ** p<0.01 by paired t-test of co-transduced modifier versus FOXP3 alone.

TABLE 1

Percentage of Mean Fluorescence Intensity (MFI) as compared to donor-matched expanded Tregs.

FOXP3	FOXP3	FOXP3	FOXP and	FOXP3 and	FOXP3	FOXP3 and
only	and ID2	and ID3	GATA1	GATA3	and XBP1	SATB1

CTLA4	119.6 ± 24.5	248.3 ± 100.8	226.1 ± 58.5, *	207.5 ± 48.0	167.5 ± 19.5, *	121.5 ± 22.4	129.2 ± 31.7
CD25	206.9 ± 130.8	322.0 ± 202.6	274.7 ± 166.8	293.8 ± 142.8	230.2 ± 115.1	281.3 ± 147.9, *	257.1 ± 168.0
ICOS	239.9 ± 123.0	564.1 ± 355.1	467.8 ± 284.5	205.3 ± 115.2	242.7 ± 119.8	249.7 ± 138.2	239.3 ± 134.4
LAG3	168.28 ± 78.8	318.6 ± 146.7	256.0 ± 102.5, *	254.7 ± 38.5	207.9 ± 62.5	200.0 ± 84.8, *	191.4 ± 77.7, **

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. A vector comprising:

(i) a first nucleic acid sequence encoding a FOXP3 polypeptide; and

(ii) a second nucleic acid sequence encoding one or more transcription factor(s) selected from the group consisting of: ID2, ID3, GATA1, GATA3, XBP1, and SATB1.

2. The vector of claim 1, wherein the one or more transcription factor(s), when present in a human cell, elicit(s) a T reg phenotype in the human cell as compared to when the one or more transcription factor(s) is/are not present in the human cell.

3. The vector of claim 1, wherein the one or more transcription factor(s) is ID2.

4. The vector of claim 1, wherein the one or more transcription factor(s) is ID3.

5. The vector of claim 1, wherein the one or more transcription factor(s) is GATA1.

6. The vector of claim 1, wherein the one or more transcription factor(s) is GATA3.

7. The vector of claim 1, wherein the one or more transcription factor(s) is XBP1.

8. The vector of claim 1, wherein the one or more transcription factor(s) is SATB1.

9. The vector of claim 1, wherein the vector further comprises a promoter operably linked to the first nucleic acid sequence.

10. The vector of claim 1, wherein the first nucleic acid sequence is positioned 5′ relative to the second nucleic acid sequence in the vector.

11. The vector of claim 10, wherein the vector further comprises an additional nucleic acid sequence between the first nucleic acid sequence and the second nucleic acid sequence, wherein the additional nucleic acid sequence operably links the second nucleic acid sequence to the first nucleic acid sequence, and the additional nucleic acid sequence (i) encodes an internal ribosome entry site (IRES) sequence or a self-cleaving amino acid, or (ii) comprises a promoter or an enhancer.

12. The vector of claim 1, wherein the second nucleic acid sequence is positioned 5′ relative to the first nucleic acid sequence in the vector, and the second nucleic acid sequence is operably linked to a promoter.

13. The vector of claim 12, wherein the vector further comprises an additional nucleic acid sequence between the second nucleic acid sequence and the first nucleic acid sequence, wherein the additional nucleic acid sequence operably links the first nucleic acid sequence to the second nucleic acid sequence, and the additional nucleic acid sequence (i) encodes an internal ribosome entry site (IRES) sequence or a self-cleaving amino acid, or (ii) comprises a promoter or an enhancer.

14. The vector of claim 1, wherein the vector further comprises a third nucleic acid sequence encoding a therapeutic gene product.

15. The vector of claim 14, wherein the therapeutic gene product is an antibody or antigen-binding fragment that is capable of specifically binding to an IL-6, an IL-16R, an IFN alpha receptor, or a TGF beta receptor polypeptide.

16. The vector of claim 14, wherein the third nucleic acid sequence is operably linked to a promoter.

17. The vector of claim 14, wherein the vector further comprises a fourth nucleic acid sequence encoding a binding agent, wherein the binding agent is an antibody, an antigen-binding fragment, or a chimeric antigen receptor.

18. The vector of claim 17, wherein the fourth nucleic acid sequence is operably linked to a promoter.

19. The vector of claim 17, wherein the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antibody or antigen-binding fragment that is capable of specifically binding to an antigen on an autoimmune cell, and the intracellular domain comprises a cytoplasmic signaling domain and one or more co-stimulatory domain(s).

20. The vector of claim 19, wherein the extracellular domain specifically binds to a cell adhesion molecule selected from the group consisting of: ICAM-1, VCAM-1, and MAdCAM-1.

21. The vector of claim 19, wherein the cytoplasmic signaling domain is a CD3 zeta domain and the one or more co-stimulatory domain(s) comprise(s) at least one of a CD48 domain, a 4-1BB domain, an ICOS domain, an OX-40 domain, and a CD27 domain.

22. The vector of claim 1, wherein the vector comprises a viral vector selected from the group consisting of: a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector.

23. A composition comprising:

(i) a first vector comprising a first nucleic acid sequence encoding a FOXP3 polypeptide and a promoter operably linked to the first nucleic acid sequence; and

(ii) a second vector comprising a second nucleic acid sequence encoding one or more transcription factor(s) selected from the group consisting of: ID2, ID3, GATA1, GATA3, XBP1, and SATB1, and a promoter operably linked to the second nucleic acid sequence.

24. The composition of claim 23, wherein the one or more transcription factor(s), when present in a human cell, elicit(s) a T reg phenotype in the human cell as compared to when the one or more transcription factor(s) is/are not present in the human cell.

25. The composition of claim 1, wherein the one or more transcription factor(s) is ID2.

26. The composition of claim 1, wherein the one or more transcription factor(s) is ID3.

27. The composition of claim 1, wherein the one or more transcription factor(s) is GATA1.

28. The composition of claim 1, wherein the one or more transcription factor(s) is GATA3.

29. The composition of claim 1, wherein the one or more transcription factor(s) is XBP1.

30. The composition of claim 1, wherein the one or more transcription factor(s) is SATB1.