Compositions and Methods for Preventing T Cell Exhaustion

Abstract

Compositions comprising T cells modified to overexpress FOXO1 and methods of using the same are provided herein. Methods for treating a disease or disorder in a subject are provided, including administration of the modified T cells. Methods for preventing exhaustion of engineered T cells are also provided, including introducing a nucleic acid that overexpresses FOXO1 into the T cells.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 334,462, filed on April 25, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to compositions comprising T cells modified to overexpress FOXO1 and methods of using the same.

[0003] Sequence Listing Submission The content of the electronic sequence listing entitled "STDU2 - 39531 - 601_SQL.xml" (size: 4,128 bytes; and creation date April 24, 2023) is hereby incorporated by reference in its entirety.

Background Art

[0004] The efficacy of chimeric antigen receptor (CAR) T cells is limited by T cell exhaustion, in which chronic CAR signaling induces progressive loss of function. Exhaustion is often targeted by checkpoint inhibitors. However, the majority of patients do not respond to these agents, and their effectiveness in combination with CAR T cells has not been shown in clinical trials. Memory T cells, which are long - lived, highly proliferative progenitor cells that give rise to effector T cells upon re - encounter with an antigen, are essential for anti - tumor immune responses and are associated with responses to checkpoint blockade and adoptive T cell therapy. CAR T cells infused into patients / animals lack a large number of memory T cells. Poor CAR - T persistence and recurrence in patients / animals are associated with this deficit in memory CAR - T cell formation.

Summary of the Invention

Means for Solving the Problems

[0005] Compositions are provided herein that include isolated T cells containing exogenous nucleic acids encoding Forkhead box protein O1 (FOXO1). In some embodiments, FOXO1 is wild-type FOXO1. In some embodiments, FOXO1 is FOXO1 that is fully or partially nuclear-localized. In some embodiments, FOXO1 is constitutively expressed from the exogenous nucleic acid. In some embodiments, FOXO1 is fused to a motif (e.g., degron, destabilizing domain, etc.) that regulates expression levels or enhances intracellular degradation.

[0006] In some embodiments, the isolated T cells maintain functionality under conditions where unmodified T cells exhibit exhaustion.

[0007] In some embodiments, the isolated T cells further comprise a nucleic acid encoding a recombinant receptor. In some embodiments, the recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is specific for a tumor antigen.

[0008] In some embodiments, FOXO1 and the engineered receptor are encoded by separate nucleic acids. In some embodiments, FOXO1 and the engineered receptor are encoded by a single nucleic acid. In some embodiments, FOXO1 and the engineered receptor are expressed under different promoters.

[0009] In some embodiments, the isolated T cells are derived from a biological sample from a subject. In some embodiments, the T cells are isolated from a tumor sample. In some embodiments, the T cells are expanded ex vivo.

[0010] In some embodiments, the composition further comprises at least one therapeutic agent.

[0011] A method of treating a disease or disorder in a subject, the method comprising administering to a subject having the disease or disorder an effective amount of the disclosed composition, is also provided herein. In some embodiments, the T cells are autologous to the subject. In some embodiments, FOXO1 is overexpressed in the T cells prior to exposure to the antigen. In some embodiments, the method further comprises administering to the patient / animal one or more chemotherapeutic agents.

[0012] In some embodiments, the disease or disorder includes an infectious disease or cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the recombinant receptor is specific for the cancer.

[0013] In some embodiments, administering decreases the number of cancerous cells in the patient / animal. In some embodiments, administering decreases and / or eliminates the tumor burden in the patient / animal. In some embodiments, administering exhibits enhanced cancer treatment as compared to administration of unmodified T cells. In some embodiments, administering results in lower expression of inhibitory receptors in the subject as compared to administration of unmodified T cells.

[0014] A method for preventing exhaustion of engineered T cells, the method comprising introducing into the engineered T cells a nucleic acid that overexpresses FOXO1, is further provided herein. In some embodiments, the nucleic acid promotes constitutive expression of FOXO1 from the nucleic acid.

[0015] In some embodiments, FOXO1 is wild-type FOXO1. In some embodiments, FOXO1 is fully or partially nuclear-localized FOXO1. In some embodiments, FOXO1 is fused to a motif (e.g., degron, destabilizing domain, etc.) that regulates the expression level or enhances intracellular degradation.

[0016] In some embodiments, the engineered T cells comprise a nucleic acid encoding a recombinant receptor. In some embodiments, the recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is specific for a tumor antigen. In some embodiments, FOXO1 is overexpressed prior to exposure to the antigen.

[0017] In some embodiments, FOXO1 and the engineered receptor are encoded by separate nucleic acids. In some embodiments, FOXO1 and the engineered receptor are encoded by a single nucleic acid. In some embodiments, FOXO1 and the engineered receptor are expressed under different promoters.

[0018] In some embodiments, the method further comprises administering the T cells to a subject in need thereof. In some embodiments, the subject has cancer.

[0019] Other aspects and embodiments of the disclosure will become apparent in light of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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Mode for Carrying Out the Invention

[0021] In this specification, to enhance CAR-T cell therapy, the expression and function of the master transcription factor FOXO1, wild-type (FOXO1-WT) or nuclear-localized mutant FOXO1 (FOXO1-AAA), were utilized. Ectopic expression of FOXO1 in human CAR-T cells conferred a more memory-like phenotype in vitro (based on cell surface markers) and enhanced tumor-responsive killing in CAR-T cells targeting CD19 or Her2. These effects were independent of the co-stimulatory domain used in the CAR (e.g., CD28 vs. 4-1BB). Furthermore, using an in vitro T cell exhaustion model (HA-28z) in which a high-affinity GD2-targeting CAR spontaneously aggregates and continuously signals in the absence of antigen, ectopic expression of FOXO1 resulted in suppression of exhaustion, lower expression of inhibitory receptors, a more memory-like surface phenotype, and enhanced functionality in response to tumors.

[0022] Constitutive expression of FOXO1-WT in CAR-T cells resulted in enhanced tumor suppression in vivo using the following CAR-T / xenograft models: HA-28z vs. GD2-expressing Nalm6 leukemia (liquid tumor); CD19-BBz vs. Nalm6 leukemia (liquid tumor); Her2-BBz vs. 143B osteosarcoma (solid tumor).

[0023] The headings of the sections used in this section and throughout the disclosure of this specification are for merely organizational purposes and are not intended to be limiting.

[0024] 1. Definitions As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)" and variations thereof are intended to be non-limiting transitional phrases, terms or words that do not exclude the possibility of additional acts or structures. When used herein, including a particular sequence or a particular SEQ ID NO. generally means that at least one copy of said sequence is present in the peptide or polynucleotide described. However, two or more copies are also contemplated. The singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments "comprising", "consisting of" and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly described.

[0025] Regarding the description of numerical ranges herein, each number intervening therebetween is explicitly contemplated with the same degree of precision. For example, for the range of 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are contemplated, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.

[0026] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear, but in the event of potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. Further, unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include singulars.

[0027] As used herein, "nucleic acid" or "nucleic acid sequence" refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine and uracil, and adenine and guanine, respectively (see Albert L. Lehninger, Principles of Biochemistry, 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variant thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The composition of the polymer or oligomer can be heterogeneous or homogeneous, and the polymer or oligomer can be isolated from a naturally occurring source or produced artificially or synthetically. Further, the nucleic acid can be DNA or RNA, or a mixture thereof, and can exist permanently or transiently in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, the nucleic acid or nucleic acid sequence includes other types of nucleic acid structures, such as, for example, DNA / RNA helices, peptide nucleic acids (PNA), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 41(14):4503-4510 (2002)) and U.S. Patent No. 5,034,506), locked nucleic acids (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122:8595-8602 (2000)) and / or ribozymes. Thus, the term "nucleic acid" or "nucleic acid sequence" can include chains containing unnatural nucleotides, modified nucleotides, and / or non-nucleotide components (e.g., "nucleotide analogs") that can exhibit the same function as natural nucleotides, and further, as used herein, the term "nucleic acid sequence" refers to DNA or RNA of genomic or synthetic origin that can be oligonucleotides, nucleotides or polynucleotides and fragments or portions thereof, and can be single-stranded or double-stranded and can represent a sense strand or an antisense strand.The terms "nucleic acid", "polynucleotide", "nucleotide sequence" and "oligonucleotide" are used interchangeably. These terms refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0028] As used herein, the term "percent sequence identity" refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence or amino acids in an amino acid sequence that are identical to the corresponding nucleotides or amino acids in a reference sequence after aligning two sequences to achieve the maximum percent identity and introducing gaps as necessary. Numerous mathematical algorithms are known for obtaining optimal alignments and calculating identity between two or more sequences and are incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof), and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also disclosed, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7):951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0029] As used herein, the terms "provide," "administer," and "introduce" are used interchangeably herein and refer to the placement of a composition of the present disclosure into a subject by a method or route that results in at least partial localization of the composition to a desired site. The composition can be administered by any suitable route that results in delivery to a desired location within the subject.

[0030] "Subject" or "patient / animal patient" can be human or non-human and can include, for example, an animal strain or species used as a "model system" for research purposes, such as the mouse models described herein. Similarly, a patient / animal patient can include either an adult / adult or a juvenile / young individual (e.g., a child). Further, a patient / animal patient can mean any organism, preferably a mammal (e.g., human and non-human), to which administration of a composition contemplated herein can be beneficial. Examples of mammals include any member of the class Mammalia: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs, but are not limited thereto. Examples of non-mammals include, but are not limited to, birds, fish, etc. In one embodiment, the mammal is a human.

[0031] As used herein, "treat," "treatment," etc. mean the slowing, halting, or reversal of the progression of a disease or disorder when a compound or composition described herein is provided to a suitable control subject. This term also means reversing the progression of such a disease or disorder to the point of removing or significantly reducing the symptoms. Thus, "treat" means applying or administering a composition described herein to a subject when the subject has a disease or symptoms of a disease, and the goal is to cure, heal, alleviate, reduce, change, treat, remit, improve, or affect the disease or symptoms of the disease.

[0032] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment, e.g., an "insert fragment", can be ligated or incorporated so as to bring about replication of the ligated segment in a cell.

[0033] 2. Compositions Disclosed herein are compositions comprising isolated T cells comprising an exogenous nucleic acid encoding Forkhead box protein O1 (FOXO1).

[0034] In some embodiments, FOXO1 is wild-type FOXO1. In some embodiments, FOXO1 comprises an amino acid sequence having at least about 70% (about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or 100%) similarity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, FOXO1 comprises the amino acid sequence of SEQ ID NO: 1.

[0035] In some embodiments, FOXO1 is FOXO1 that is fully or partially nuclear-localized (e.g., FOXO1-AAA or FOXO1-3A as disclosed herein). In some embodiments, FOXO1 comprises an amino acid sequence having at least about 70% (about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or 100%) similarity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, FOXO1 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, FOXO1 is not nuclear-localized FOXO1. In some embodiments, FOXO1 is a mutant form of FOXO1 that is partially nuclear-localized, e.g., having an increased likelihood of being present in the nucleus compared to wild-type but not being fully nuclear-localized. In some embodiments, FOXO1 is not partially nuclear-localized FOXO1.

[0036] SEQ ID NO: 1

[0037]

Chemical formula

[0038] Array number 2

[0039] [Chem.]

[0040] FOXO1 suitable for the disclosed compositions and methods may contain one or more amino acid substitutions or cleavages compared to the corresponding wild-type protein or SEQ ID NO: 1. In some embodiments, FOXO1 is a functional fragment of wild-type FOXO1 or SEQ ID NO: 1. In some embodiments, FOXO1 may contain one or more substitutions in its primary amino acid sequence, but the resulting polypeptide retains its expression level, cellular localization, and / or activity (e.g., promoting and maintaining upregulation of transcription factors known to drive the memory phenotype and / or stemness and effector transcription programs of CAR-T cells) such that it is a functional variant of wild-type FOXO1 or SEQ ID NO: 1. The functional variant preferably retains more than 50% of the activity of the original polypeptide (e.g., prevention of T cell exhaustion).

[0041] An amino acid "substitution" or "replacement" refers to the replacement of one amino acid at a given position or residue with another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as "aromatic" or "aliphatic". Aromatic amino acids contain an aromatic ring. Examples of "aromatic" amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly grouped as "aliphatic". Examples of "aliphatic" amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R or Arg).

[0042] An amino acid substitution or replacement can be conservative, semi-conservative, or non-conservative. The phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid having common properties. A functional way to define common properties among individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, groups of amino acids can be defined when the amino acids within a group are preferentially exchanged with each other and thus are most similar to each other in their effect on the overall protein structure (Schulz and Schirmer, supra).

[0043] Examples of conservative amino acid substitutions include substitutions of amino acids within the above subgroups, e.g., lysine in place of arginine and vice versa so that a positive charge can be maintained, glutamate in place of aspartic acid and vice versa so that a negative charge can be maintained, serine in place of threonine so that a free - OH can be maintained, and glutamine in place of asparagine so that a free - NH2 can be maintained. "Semi - conservative mutations" include amino acid substitutions of amino acids that are within the same group as listed above but not within the same subgroup. For example, the substitution of asparagine with aspartic acid, or the substitution of lysine with asparagine, involves amino acids within the same group but different subgroups. "Non - conservative mutations" include amino acid substitutions between different groups, e.g., lysine in place of tryptophan, phenylalanine in place of serine, etc.

[0044] Exogenous nucleic acids promote overexpression of FOXO1 in T cells. The expression of FOXO1 can be constitutive, regulatable or inducible, cell - type specific, tissue - specific or species - specific. In selected embodiments, isolated T cells constitutively express FOXO1 from exogenous nucleic acids.

[0045] In some embodiments, T cells maintain functionality (e.g., maintain the functionality of T cells exposed to excess antigen) under conditions where unmodified T cells, i.e., T cells that do not express FOXO1 from exogenous nucleic acids, show exhaustion. "T - cell exhaustion" refers to the loss of T - cell function that can occur as a result of an infection (e.g., chronic infection) or disease. T - cell exhaustion is associated with increased expression of exhaustion markers and inhibitory receptors (e.g., PD - 1, TIM - 3 and LAG - 3), apoptosis and decreased cytokine secretion.

[0046] In some embodiments, FOXO1 is fused to a motif (e.g., degron, destabilizing domain, etc.) that regulates the expression level or enhances intracellular degradation. The motif can be fused to the N - terminus or C - terminus of FOXO1. The motif can be linked to FOXO1 via a linker.

[0047] In some embodiments, FOXO1 is fused to a destabilizing domain (DD). The destabilizing domain is a protein domain that regulates the stabilization of a payload (e.g., a protein of interest (e.g., FOXO1)) fused to the DD as a result of the absence or presence of a binding ligand (e.g., a small molecule or drug). For example, some destabilizing domains in the absence of their binding ligand result in the recognition and degradation of the payload fused to the DD by the ubiquitin-proteasome system. While in the presence of their binding ligand, the fused DD and payload are stabilized. In some examples, the stability is dose-dependent. Thus, the presence, absence, or amount of a small molecule ligand that binds to or interacts with the DD can regulate the stability of FOXO1 upon such binding or interaction, and as a result, regulate the function of FOXO1. Thus, the presence of an adjustable destabilizing domain enables the concentration of FOXO1 to be regulated over time using a cognate binding ligand.

[0048] In some embodiments, FOXO1 is fused to one degron or one or more degrons. As used herein, a "degron" is a single amino acid or peptide that can target FOXO1 for degradation. Based on the disclosure herein, any suitable degron that is considered appropriate for the intended use may be used. The degron signals and / or targets a portion of the protein to which it is bound or otherwise associated (e.g., grafted thereon) for its degradation (or otherwise to increase the rate of degradation). Non-limiting examples of degrons include short amino acid sequences, structural motifs, exposed amino acids, etc. The degron can be of prokaryotic or eukaryotic origin and can be used in its naturally occurring form or a non-naturally occurring (i.e., recombinant) form. The degron can be post-translationally modified to target the protein for degradation, and such post-translational modifications include, but are not limited to, ubiquitination, proteolytic cleavage, phosphorylation, methylation, ADP-ribosylation, AMPylation, lipidation, alkylation, nitrosylation, succinylation, SUMOylation, NEDDylation, ISGylation, etc. Useful degrons include ubiquitin-dependent degrons and ubiquitin-independent degrons. For example, in some instances, a protein can be targeted for ubiquitin-independent proteasomal degradation by attachment of an ornithine decarboxylase (ODC) degron, including, but not limited to, mammalian ODCs such as murine ODC including, for example, the C-terminal murine ODC (cODC). In some instances, useful degrons include those described in Takeuchi et al., Biochem. J (2008) 410:401-407 and / or Matsuzawa et al., PNAS (2005) 102(42):14982-7, the disclosures of which are incorporated herein by reference in their entirety.In some examples, proteins can be targeted for ubiquitin-independent proteasomal degradation by post-translational modification of degrons (including, but not limited to, proteolytic cleavage, phosphorylation, methylation, ADP-ribosylation, AMPylation, lipidation, alkylation, nitrosylation, succinylation, SUMOylation, NEDDylation, ISGylation), and such modifications can directly or indirectly result in partial or complete unfolding of the protein or other mechanisms that lead to degradation of the protein.

[0049] The present invention is not limited by the type of T cell modified to overexpress and / or contain an exogenous nucleic acid molecule encoding FOXO1. The T cell can be selected from CD3+ T cells, CD8+ T cells, CD4+ T cells, natural killer (NK) T cells, alpha-beta T cells, gamma-delta T cells, or any combination thereof (e.g., a combination of CD4+ T cells and CD8+ T cells). In some embodiments, the T cell is a memory T cell (e.g., a central memory T cell or an effector memory T cell). In some embodiments, the T cell is a tumor-infiltrating lymphocyte. In some embodiments, the T cell is a cytokine-induced killer cell.

[0050] In some embodiments, the T cell is a naturally occurring T cell. For example, the T cell can be isolated from a subject sample. In some embodiments, the T cell is an anti-tumor T cell (e.g., a T cell having activity against a tumor (e.g., an autologous tumor) that is activated and expands in response to an antigen). Anti-tumor T cells include, but are not limited to, T cells obtained from an excised tumor or tumor biopsy (e.g., tumor-infiltrating lymphocytes (TIL)) and polyclonal or monoclonal tumor-reactive T cells (e.g., expanded ex vivo against tumor antigens presented by autologous or artificial antigen-presenting cells obtained by apheresis). In some embodiments, the T cell is expanded ex vivo.

[0051] In some embodiments, the isolated T cells further comprise a nucleic acid encoding a recombinant receptor. In some embodiments, the recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0052] In certain embodiments, the T cells are genetically modified with a recombinant receptor that recognizes a tumor antigen and responds to the tumor antigen. Such receptors generally consist of an extracellular domain comprising a single-chain antibody (scFv) specific for the tumor antigen linked to an intracellular T cell signaling motif (see, for example, Westwood, J.A. et al, 2005, Proc. Natl. Acad. Sci., USA, 102(52):19051-19056).

[0053] The present invention is not limited by the type of tumor antigen recognized. As used herein, the term "tumor antigen" refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) that is expressed or overexpressed exclusively or predominantly by tumor cells or cancer cells such that the antigen is associated with a tumor or cancer. Cancer antigens may be further expressed by normal cells, non-tumor cells or non-cancerous cells. However, in such cases, the expression of cancer antigens by normal cells, non-tumor cells or non-cancerous cells is not as robust as that by tumor or cancer cells. In this regard, tumor or cancer cells can overexpress the antigen or express the antigen at a significantly higher level compared to the expression of the antigen by normal cells, non-tumor cells or non-cancerous cells. Also, cancer antigens may be further expressed by cells in different states of development or maturity. For example, cancer antigens may be further expressed by cells at the embryonic or fetal stage that are not normally found in adults / adults. Alternatively, cancer antigens may be further expressed by stem cells or progenitor cells that are not normally found in adults / adults.

[0054] A cancer antigen can be an antigen expressed by any cancer or any cell of a tumor. A cancer antigen can be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor. Or, a cancer antigen can be a cancer antigen of more than one type of cancer or tumor (e.g., can be characteristic of more than one type of cancer or tumor). For example, a cancer antigen can be expressed by both breast cancer cells and prostate cancer cells and not be expressed at all by normal, non-tumor or non-cancer cells. Exemplary cancer antigens include, but are not limited to, glycoprotein 100 (gp100), melanoma antigen recognized by T cells 1 (MART-1), melanoma antigen gene (MAGE) family members (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12), New York esophageal squamous cell carcinoma 1 (NY-ESO-1), vascular endothelial growth factor receptor-2 (VEGFR-2), glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, human telomerase reverse transcriptase, prostate specific antigen (PSA), prostate carcinoma tumor antigen-1 (PCTA-1), insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, intestinal carboxylesterase, human epidermal growth factor receptor 2 (HER-2), mesothelin, and epidermal growth factor receptor variant III (EGFR III).

[0055] Any T cell containing a receptor that recognizes a tumor antigen is used in the compositions and methods of the present invention. Examples include CD19, CD20, CD22, receptor tyrosine kinase-like orphan receptor 1 (ROR1), disialoganglioside 2 (GD2), Epstein-Barr virus (EBV) protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen (CEA), prostate acid phosphatase (PAP), CD33 / IL3R, tyrosine protein kinase Met (c-Met) or hepatocyte growth factor receptor (HGFR), prostate-specific membrane antigen (PSMA), glycolipid F77, epidermal growth factor receptor variant III (EGFRvIII), NY-ESO-1, melanoma antigen gene (MAGE) family member A3 (MAGE-A3), melanoma antigen recognized by T cells 1 (MART-1), GP1000, p53, or a receptor that recognizes an antigen selected from other tumor antigens described herein (e.g., a natural or naturally occurring receptor, or a receptor engineered to express a synthetic receptor such as an engineered TCR or CAR), including but not limited to T cells expressing such receptors.

[0056] In some embodiments, the T cells are engineered to express a chimeric antigen receptor (CAR). Any CAR that specifically binds to a desired antigen (e.g., a tumor antigen) can be utilized with the present invention. In certain embodiments, the CAR includes an antigen-binding domain. In certain embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) containing heavy and light chain variable regions that specifically bind to the desired antigen. In some embodiments, the CAR further includes a transmembrane domain (e.g., a T cell transmembrane domain (e.g., the CD28 transmembrane domain)) and a signaling domain that includes one or more immunoreceptor tyrosine-based activation motifs (ITAMs) (e.g., a T cell receptor signaling domain (e.g., the TCR zeta chain)). In some embodiments, the CAR includes one or more costimulatory domains (e.g., a domain that provides a second signal to stimulate T cell activation). The present invention is not limited by the type of costimulatory domain. Indeed, any costimulatory domain known in the art can be used, including but not limited to CD28, OX40 / CD134, 4-1BB / CD137 / TNFRSF9, high-affinity immunoglobulin E receptor-gamma subunit (FcERIγ, ICOS / CD278, interleukin 2 subunit beta (ILRβ) or CD122, cytokine receptor common subunit gamma (IL-2Rγ) or CD132, and CD40. In some embodiments, the costimulatory domain is 4-1BB. In some embodiments, the costimulatory domain is CD28.

[0057] The CAR can include a target-specific binding element, also referred to as an antigen-binding portion. The selection of the portion depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Examples of cell surface markers that can act as ligands for the antigen portion domain in the CAR of the present invention include those associated with viral infections, bacterial infections, and parasitic infections, autoimmune diseases, and cancer cells as described above.

[0058] Depending on the desired antigen to be targeted, the CAR can be engineered to include an appropriate antigen-binding portion that is specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as the antigen-binding portion to be incorporated into the CAR of the present invention.

[0059] FOXO1 and the engineered receptor can be encoded by the same or different nucleic acids. In some embodiments, FOXO1 and the engineered receptor are encoded by a single nucleic acid. In some embodiments, FOXO1 and the engineered receptor are encoded by separate nucleic acids. The (one or more) nucleic acids can include DNA or RNA (e.g., mRNA). In some embodiments, the (one or more) nucleic acids include a vector.

[0060] FOXO1 and the engineered receptor can be expressed using the same, similar (e.g., both weak or both strong), or different promoters. For example, the promoter for FOXO1 can confer a high transcription rate (strong promoter), whereas the promoter for the engineered receptor can confer a low transcription rate (weak promoter) or vice versa. In some embodiments, the promoters for FOXO1 and the engineered receptor can confer a high transcription rate (strong promoter). In some embodiments, the promoters for FOXO1 and the engineered receptor can confer a low transcription rate (weak promoter). Many promoter libraries have been experimentally established, and the selection of promoters and promoter strength is well known to those skilled in the art.

[0061] Furthermore, the nucleic acids of the present disclosure may include a promoter that is constitutive, regulatable or inducible, cell-type specific, tissue-specific or species-specific. In addition to the sequences sufficient to direct transcription, the promoter may also include sequences of other regulatory elements involved in the regulation of transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression are available in the art, including, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1α promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human β-actin promoter, murine β-actin promoter, CBh (chicken β-actin promoter), CAG (hybrid promoter containing CMV enhancer, chicken β-actin promoter and rabbit β-globin splice acceptor), TRE (tetracycline response element promoter), H1 (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), etc., but not limited thereto. Additional promoters that can be used for expression include viral LTRs such as cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Moloney murine leukemia virus (MMLV) LTR, myeloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1-α (EF1-α) promoter with or without EF1-α intron, etc., but not limited thereto. Further promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used such that its expression can be regulated intracellularly.

[0062] Furthermore, inducible expression can be achieved by placing the nucleic acid encoding such a molecule under the control of an inducible promoter / regulatory sequence. Promoters well known in the art can be induced in response to inducers such as metals, glucocorticoids, tetracycline, hormones, etc., and are also contemplated for use with the present invention. Thus, it will be understood that the present disclosure includes the use of any promoter / regulatory sequence known in the art that can operably drive the expression of the desired protein.

[0063] The present disclosure also provides vectors containing nucleic acids and cells containing the nucleic acid or its vector. Vectors can be used to increase the nucleic acid in a suitable cell and / or to enable expression from the nucleic acid (e.g., expression vector). Those skilled in the art will recognize the various vectors available for the propagation and expression of nucleic acid sequences.

[0064] Expression vectors for stable or transient expression can be constructed by conventional methods and introduced into cells. For example, the nucleic acid can be cloned into a suitable expression vector such as a plasmid or viral vector operably linked to an appropriate promoter. The choice of expression vector / plasmid / viral vector is preferably suitable for integration and replication in eukaryotic cells.

[0065] In certain embodiments, the vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the control functions of the expression vector are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40 and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which are incorporated herein by reference.

[0066] Furthermore, the vector may contain, for example, some or all of the following: a selectable marker gene for selecting stable or transient transformants in a host cell; transcription termination and RNA processing signals; 5' and 3' untranslated regions; an internal ribosome entry site (IRES) within the sequence, a multiple cloning site; and a reporter gene for assessing the expression of the chimeric receptor. Methods for making vectors containing appropriate vectors and transgenes are well known and available in the art. Selectable markers include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, neomycin, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamicin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of Saccharomyces cerevisiae (S. cerevisiae).

[0067] When introduced into a cell, the vector can be maintained as a self-replicating sequence or an episomal element, or can be integrated into the host DNA. The nucleic acid can be delivered to the cell by any suitable means.

[0068] To introduce nucleic acids into cells, conventional virus-based and non-virus-based gene delivery methods can be used. Such methods can be used to administer nucleic acids to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., transcripts of the vectors described herein), nucleic acids, and nucleic acids complexed with delivery vehicles.

[0069] Viral vector delivery systems include DNA and RNA viruses that have either an episome or an integrated genome after delivery to a cell. To deliver this nucleic acid to a cell, various viral constructs can be used. Viral vectors include, for example, retroviruses, lentiviruses, adenoviruses, adeno-associated, and herpes simplex virus vectors. Non-limiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenovirus, recombinant lentivirus, recombinant retrovirus, recombinant herpes simplex virus, recombinant poxvirus, phage, and the like. The present disclosure provides vectors, such as retroviruses or lentiviruses, that can integrate into the host genome. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M.A., et al., 2001 Nat. Med. 7(1):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2):249-71, which are incorporated herein by reference.

[0070] The vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a cell. Transfection refers to the uptake of a vector by a cell, regardless of whether the coding sequence is actually being expressed. Numerous methods of transfection, such as lipofectamine, calcium phosphate coprecipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art, are known to those of skill in the art. Transduction refers to the entry of a virus into a cell and the expression (e.g., transcription and / or translation) of the sequences delivered by the viral vector genome. In the case of a recombinant vector, "transduction" generally refers to the entry of a recombinant viral vector into a cell and the expression of the nucleic acid of interest delivered by the vector genome.

[0071] Methods for delivering vectors to cells are well known in the art and can include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles for delivering DNA or RNA; mechanical deformation of DNA, RNA or (e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6):2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vector is delivered to the cell by viral transduction. Nucleic acids can be delivered directly as part of a larger construct such as a plasmid or viral vector, or by, for example, electroporation, lipid vesicles, viral transporters, microinjection and particle guns (biolistic bombardment).

[0072] Furthermore, delivery vehicles such as nanoparticle-based and lipid-based delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery systems, gene guns, hydrodynamic delivery, electroporation or nucleofection microinjection and particle guns. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012;1:27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1;459(1-2):70-83), which are incorporated herein by reference.

[0073] The composition can optionally include at least one additional therapeutic agent such as other drugs for treating T cell exhaustion (e.g., anti-PD-1 checkpoint inhibitors such as nivolumab), or other pharmaceuticals used to treat a subject with an infection or disease associated with T cell exhaustion (e.g., antiviral drugs, antibiotics, antibacterial drugs, or anticancer drugs).

[0074] In some embodiments, at least one additional therapeutic agent comprises at least one chemotherapeutic agent. As used herein, the terms "chemotherapeutic agent" or "anticancer agent" include any small molecule or other drug used in the treatment or prevention of cancer. Chemotherapeutic agents include, but are not limited to, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemaciclib, afinitol (everolimus), alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, and tucatinib. In selected embodiments, the chemotherapeutic agent comprises paclitaxel.

[0075] The composition can include, for example, cytokines, chemokines, and other biological signaling molecules, tumor-specific vaccines, cellular cancer vaccines (e.g., GM-CSF transduced cancer cells), tumor-specific monoclonal antibodies, (e.g., autologous and allogeneic stem cell rescue to enhance the graft-versus-tumor effect, other therapeutic antibodies, molecular targeted therapies, anti-angiogenic therapies, infectious agents for therapeutic purposes (such as tumor localizing bacteria), and gene therapy.

[0076] The composition may contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means any kind of non-toxic inert solid, semi-solid or liquid filler, diluent, encapsulating material, surfactant, cyclodextrin or formulation aid. The carrier may include a single component or a combination of two or more components. Some examples of materials that can function as a pharmaceutically acceptable carrier include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; surfactants such as Cremophor EL, Cremophor RH 60, Solutol HS 15 and polysorbate 80; cyclodextrins such as alpha-CD, beta-CD, gamma-CD, HP-beta-CD, SBE-beta-CD; glycols; propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer, and other non-toxic compatible lubricants, preservatives and antioxidants such as sodium lauryl sulfate and magnesium stearate, can also be present in the composition according to the judgment of the formulator.

[0077] The route of administration and form of the composition determine the type of carrier used. The composition can be in various forms suitable for, for example, systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implant, or parenteral injection) or topical administration (e.g., skin, lung, nose, ear, eye, liposome delivery system, or iontophoresis).

[0078] 3. Method The present disclosure also provides methods for treating a disease or disorder. In some embodiments, the method comprises administering to a subject an effective amount of T cells modified to express and / or contain elevated levels of FOXO1. In some embodiments, the method comprises administering to a subject an effective amount of a composition described herein. The invention is not limited by the type of disease or condition being treated. (e.g., containing and / or using T cells modified to express and / or contain elevated levels of FOXO1) Using the compositions and methods of the present invention, any disease or condition treatable via administration of T cells can be treated in an improved and more effective manner.

[0079] In some embodiments, the administration inhibits or reduces T cell exhaustion (e.g., as compared to a subject receiving the same amount of engineered T cells not modified to express and / or contain elevated levels of FOXO1, such as CAR T cells or T cells comprising a recombinant TCR). In some embodiments, the administration results in lower expression of inhibitory receptors (e.g., programmed cell death 1 (also known as PDCD1 or PD1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)) as compared to administration of unmodified T cells.

[0080] In some embodiments, exogenous FOXO1 is expressed in the T cells prior to introduction of the recombinant receptor into the T cells. In some embodiments, the exogenous FOXO1 and the recombinant receptor are introduced into the T cells simultaneously and the expression is simultaneous or substantially simultaneous. In some embodiments, the recombinant receptor is expressed in the T cells prior to introduction of exogenous FOXO1 into the T cells.

[0081] In some embodiments, FOXO1 is overexpressed in the T cells prior to exposure to the antigen.

[0082] T cells can be isolated from a subject. In some embodiments, the T cells are allogeneic to the subject. In some embodiments, the T cells are autologous to the subject. Thus, T cells can be isolated from a sample from the subject, modified and expanded ex vivo, and returned to the subject.

[0083] In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is an infectious disease. The present invention is not limited by the type of cancer or the type of infectious disease. Indeed, any cancer known in the art for which T cell therapy is used for treatment can be treated with the compositions and methods of the present invention. Similarly, any infectious disease known in the art for which T cell therapy is used for treatment can be treated with the compositions and methods of the present invention.

[0084] In certain embodiments, the present invention provides a method for treating cancer in an individual or for delaying the progression of cancer, or for treating an infectious disease or for delaying the progression of an infectious disease, comprising administering to the individual an effective amount of the modified T cells or a composition thereof, as described herein. In some embodiments, the treatment results in a sustained response in the individual after cessation of treatment.

[0085] The method can be used in a subject having any cancer cell or any type of cancer, such as those described by the National Cancer Institute of the United States. In some embodiments, the cancer can be carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma or seminoma. In some embodiments, the cancer includes solid tumors. In some embodiments, the cancer is metastatic cancer.

[0086] The cancer can be cancer of the bladder, blood, bone, brain, breast, cervix, colon / rectum, endometrium, head and neck, kidney, liver, lung, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testis, thyroid or uterus.

[0087] The methods described herein can be used in the treatment of conditions where enhanced immunogenicity is desired, such as increasing tumor immunogenicity for the treatment of cancer. In some embodiments, the recombinant receptor (e.g., CAR and / or TCR) is specific for the cancer being treated. In some embodiments, the recombinant receptor (e.g., CAR and / or TCR) is common to all cancers.

[0088] In certain embodiments, the present invention demonstrates that treatment of a subject having cancer with a therapeutically effective amount of the disclosed composition is superior to treatment of a subject having cancer with unmodified T cells. In some embodiments, treatment with a therapeutically effective amount of the disclosed composition inhibits the development or proliferation of cancer cells and / or, as a population, makes cancer cells more susceptible to other treatments (e.g., the cell death-inducing activity of cancer therapeutics or radiation therapy). Thus, the compositions and methods of the present invention can be used as a monotherapy (e.g., to induce apoptosis and / or cell cycle arrest in cancer cells in order to kill cancer cells and / or reduce or inhibit the proliferation of cancer cells) or in combination with one or more additional agents such as other anti-cancer agents (e.g., cell death-inducing or cell cycle-disrupting cancer therapeutics or radiation therapy) to render a greater proportion of cancer cells susceptible to death, inhibited cancer cell proliferation, induced apoptosis, and / or cell cycle arrest compared to the corresponding proportion of cells in animals treated with the cancer therapeutic alone or radiation therapy alone.

[0089] In some embodiments, the individual has cancer that is resistant (e.g., has been demonstrated to be resistant) to one or more other forms of anti-cancer treatment (e.g., chemotherapy, immunotherapy, etc.). In some embodiments, resistance includes cancer recurrence or refractory cancer. Recurrence may refer to the re-appearance of cancer at the original site or a new site after treatment. In some embodiments, resistance includes progression of cancer during treatment with chemotherapy. In some embodiments, resistance includes cancers that do not respond to traditional or conventional treatment with chemotherapeutic agents. The cancer may be resistant at the start of treatment or may become resistant during treatment. In some embodiments, the cancer is in an early or late stage.

[0090] In some embodiments, the modified T cells and compositions thereof are used to treat, ameliorate, or prevent cancers (e.g., cancer cells such as chemotherapy-resistant, radiation-resistant, hormone-resistant, etc.) that are characterized by resistance to one or more conventional cancer therapies. In some embodiments, the treatment can completely inhibit the growth of resistant cancer cells and / or, as a population, make such cells more susceptible to cancer therapeutics or radiation therapy (e.g., with respect to their apoptosis-inducing activity).

[0091] In certain embodiments, a therapeutically effective amount of the modified T cell composition reduces the number of cancer cells in a patient / animal subject after such treatment. In certain embodiments, a therapeutically effective amount of the modified T cell composition reduces and / or eliminates the tumor burden in a patient / animal subject after such treatment.

[0092] In conjunction with the methods of the present disclosure, a wide variety of second treatments may be used. The second treatment may be the administration of additional therapeutic agents or may be a second treatment not related to the administration of another agent. Such second treatments include, but are not limited to, surgery, immunotherapy, radiation therapy, or additional chemotherapeutic or anti-cancer agents.

[0093] The second treatment can be administered concurrently with the first treatment, either in the same composition or in a separate composition administered substantially simultaneously with the first composition. In some embodiments, the second treatment can precede or follow the treatment of the first treatment by a time interval ranging from several hours to several months.

[0094] In certain embodiments, the method further comprises administering radiation therapy to the patient / animal subject. In certain embodiments, the radiation therapy is administered before, concurrently with, and / or after the patient / animal subject receives a therapeutically effective amount of the modified T cell composition.

[0095] In certain embodiments, the method further comprises administering to the patient / animal subject one or more anti-cancer agents and / or one or more chemotherapeutic agents. In certain embodiments, the one or more anti-cancer agents and / or one or more chemotherapeutic agents are administered before, concurrently with, and / or after the patient / animal subject receives a therapeutically effective amount of the modified T cell composition. In certain embodiments, the combined treatment of the patient / animal subject with a therapeutically effective amount of the modified T cells and a series of anti-cancer agents results in a greater tumor response and clinical benefit in such patient / animal subjects compared to patient / animal subjects treated with the modified T cells or the anti-cancer drug / radiation alone. Since the dosages for all approved anti-cancer drugs and radiation treatments are known, the present invention contemplates various combinations of all approved anti-cancer drugs and radiation treatments with the modified T cells.

[0096] In some embodiments, the second treatment comprises administration of an antibody. The antibody can target either an antigen specifically expressed by tumor cells or an antigen shared with normal cells. In some embodiments, the antibody can target, for example, CD20, CD33, CD52, CD30, HER (also called erbB or EGFR), VEGF, CTLA-4 (also called CD152), epithelial cell adhesion molecule (EpCAM, also called CD326), and PD-1 / PD-L1. Suitable antibodies include, but are not limited to, rituximab, blinatumomab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, ipilimumab, brentuximab, pertuzumab, etc.). In some embodiments, the additional therapeutic agent can include anti-PD-1 / PD-L1 antibodies including, but not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab. The antibody can also be conjugated to a chemotherapeutic agent. Thus, in some embodiments, the antibody is an antibody-drug conjugate.

[0097] The administration of the second treatment can be administered to the subject by various methods. In any of the uses or methods described herein, the administration can be by various routes known to those of skill in the art, including, but not limited to, oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and / or intraperitoneal administration to a subject in need thereof.

[0098] The present disclosure also provides a method of preventing exhaustion of engineered T cells (e.g., maintaining the functionality of T cells exposed to excessive antigen) that includes introducing a nucleic acid that overexpresses FOXO1 into T cells. In some embodiments, the method further includes administering the engineered T cells to a subject in need thereof.

[0099] "Preventing T cell exhaustion" refers to one or more of the following: decreased expression and / or levels of PD-1, TIM-3, and LAG-3; increased memory cell formation and / or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; increased production and / or secretion of antigen-induced cytokines (e.g., IL-2); enhanced killing ability; increased recognition of tumor targets with low surface antigens; enhanced antigen-responsive proliferation; and a restored functional state of T cells characterized by one or more of lower expression of inhibitory receptors (e.g., programmed cell death 1 (also called PDCD1 or PD1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)).

[0100] Thus, the modified T cells can exhibit increased functionality and / or activity (e.g., increased antigen-induced cytokine production, enhanced killing ability (e.g., increased recognition of tumor targets with low surface antigens), increased memory cell formation, and / or enhanced antigen-responsive proliferation) and / or characteristics of reduced exhaustion (e.g., lower levels of markers indicating exhaustion or inhibitory receptors (e.g., PD-1, TIM-3, LAG-3), and / or lower levels of programmed cell death) compared to unmodified T cells. In the context of therapeutic use, the modified T cells can enhance the clinical efficacy of therapeutic agents (e.g., CAR T cells).

[0101] In some embodiments, the isolated T cells further comprise a nucleic acid encoding a recombinant receptor. In some embodiments, the recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is specific for a tumor antigen.

[0102] The description of FOXO1, recombinant receptor, nucleic acid and its target antigen, subject, and diseases and disorders described above in connection with the compositions of the present invention is also applicable to methods of preventing exhaustion of engineered T cells.

[0103] The effective amount of the modified T cells or compositions disclosed herein can be determined based on the type of disease to be treated, the type of modified T cells, the severity and course of the disease, the clinical status of the individual, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0104] The effectiveness of any method described herein (e.g., treatment of a disease or disorder) can be tested in various models known in the art, such as clinical or preclinical models. The effectiveness of a treatment can refer to any one or more of: extending survival (including overall survival and progression-free survival); resulting in an objective response (including complete or partial response); or improving the signs or symptoms of a disease or disorder (e.g., cancer or infectious disease).

[0105] In some embodiments, the sample is obtained as a baseline for measuring the response to treatment prior to treatment with T cells (e.g., alone or in combination with another treatment described herein). In some embodiments, the sample is a tissue sample (e.g., formalin-fixed and paraffin-embedded (FFPE), archival, fresh, or frozen). In some embodiments, the sample is whole blood. In some embodiments, the whole blood contains immune cells, circulating tumor cells, and any combination thereof.

[0106] For any exemplary cancer model, after developing a tumor, the mice can be placed in a treatment group that receives the treatment or a control treatment. During the course of the treatment, the tumor size (e.g., tumor volume) is measured and the overall survival is also monitored.

[0107] In some embodiments, efficacy can refer to an improvement in one or more factors according to a published set of RECIST guidelines for determining the state of a tumor in a cancer patient / animal, i.e., responsive, stable, or progressive. A responsive subject can refer to, for example, a subject in which the cancer shows improvement according to one or more factors based on the RECIST criteria. A non-responsive subject can refer to, for example, a subject in which the cancer does not show improvement according to one or more factors based on the RECIST criteria.

[0108] Efficacy can also refer to an improvement in one or more immune-related response criteria (irRC). In some embodiments, new lesions are added to the defined tumor burden and, for example, are followed for radiographic progression in subsequent evaluations. In some embodiments, the presence of non-target lesions is included in the assessment of complete response but not in the assessment of radiographic progression. In some embodiments, radiographic progression can be determined only based on measurable disease and / or can be confirmed by consecutive evaluations exceeding 4 weeks from the date first documented.

[0109] The present disclosure further provides a kit containing one or more reagents or other components useful, necessary, or sufficient for performing any of the methods described herein. For example, the kit can include FOXO1 reagents (nucleic acids, vectors, compositions, etc.), recombinant vector reagents (nucleic acids, vectors, compositions, etc.), transfection or administration reagents, negative and positive control samples (e.g., T cells or empty vector DNA), T cells, containers (e.g., microcentrifuge tubes), detection and analysis instruments, software, instructions, and the like.

Example

[0110] 4. Example [Example 1] CAR T cell exhaustion CARs are, most commonly, synthetic proteins that incorporate the T cell receptor (TCR) machinery, combining an intracellular domain that enables T cells to recognize and destroy tumor cells in a major histocompatibility complex-independent manner with an extracellular tumor recognition domain. CAR T cells have shown unprecedented efficacy rates in hematologic malignancies. However, T cell exhaustion, a process by which CD8+ T cells that have experienced chronic antigen stimulation via TCR / CAR gradually lose effector function, has hampered the effectiveness of CAR T cells in liquid and solid tumors. Targeting exhaustion via checkpoint blockade is effective in some patients, but there is a clear need for alternative approaches because it does not reset the epigenetic imprint of exhaustion, convert exhausted cells into memory cells, or improve the effectiveness of CAR T cells.

[0111] A novel model was created in which T cells expressed a high-affinity GD2-targeted CAR (HA-28z) that continuously signaled in the absence of antigen for spontaneous CAR aggregation, thereby simulating continuous antigen exposure. HA-28z CAR T cells rapidly acquired exhausted phenotypic, functional, transcriptional, and epigenetic features. To test whether transient disruption of CAR signaling could functionally reactivate exhausted CAR T cells and promote memory, the HA-28z CAR was modified with a C-terminal destabilizing domain (DD), enabling drug-dependent control of the CAR protein, anti-tumor function, and persistent signaling (Figures 1A - 1B). Transient downregulation of the HA-28z CAR and cessation of persistent CAR signaling, or "quiescence" (Figure 1C), reversed dysfunction, attenuated inhibitory receptor expression, and promoted a memory-like phenotype. Quiescence also induced global transcriptional and epigenetic reprogramming of exhausted CAR T cells, leading to a return to a state more closely resembling healthy memory-like CAR T cells. Epigenetic changes included increased accessibility of motifs bound by memory-related transcription factors (e.g., FOXO family transcription factors, TCF7) in quiescent cells (Figure 17), suggesting their activity in factors involved in the recovery or alleviation of exhaustion. The recovery of dysfunction mediated by quiescence was dependent on EZH2 activity, suggesting a causal relationship between epigenetic reprogramming and enhanced CAR T cell functionality.

[0112] [Example 2] Endogenous FOXO1 for CAR-T Cell Survival and Memory Formation To investigate the function of FOXO1 in human CAR-T cells, CAR-T cells were expanded in the presence of a selective FOXO1 small molecule inhibitor (FOXO1i). FOXO1i inhibited the expansion, persistence, and viability of CAR-T cells in a dose-dependent manner (Figure 2A). Furthermore, FOXO1i dramatically and dose-dependently decreased the expression of memory-related FOXO1 target genes (SEL, IL-7R, TCF7), while simultaneously promoting an effector-like phenotype (Figures 2B–2C), indicating that endogenous FOXO1 promotes and maintains the memory phenotype in human CAR-T cells. These studies demonstrated that FOXO1 biology enhances the persistence and overall efficacy of engineered T cell therapies.

[0113] [Example 3] Ectopic expression of TF via retroviral co-transduction To ectopically express a transcription factor (TF) in primary human T cells, a co-transduction approach was used in which one retrovirus was used to express a CAR and a separate retrovirus was used to express a bicistronic vector with a truncated nerve growth factor receptor (NGFR) in addition to the TF (Figure 3A). Control CAR-T cells were co-transduced with a retrovirus expressing NGFR only. This approach enabled normalization of CAR expression, maximization of TF expression, and identification and / or purification of TF-expressing T cells using NGFR as an alternative surface marker. Expression 3- to 5-fold above endogenous levels was routinely achieved (Figure 3B). Furthermore, a bicistronic vector expressing both TF and CAR under the same constitutive promoter showed similar results (Figures 18A–18C).

[0114] [Example 4] Ectopic expression of FOXO1-WT and FOXO1-AAA does not affect CAR-T cell function during short-term activation Single-cell phenotyping and functional assays were performed on CD19-targeted CAR-T cells with either the CD28 (CD19.28z) or 4-1BB (CD19.BBz) costimulatory domain, with or without ectopic TF expression. Ectopic expression of FOXO1-WT, FOXO1-3A, and TCF1 did not dramatically affect the pre-stimulatory surface phenotype or the extent to which CAR-T cells stimulated with tumor upregulated activation and degranulation markers (CD69 and CD107a, respectively) (Figure 4), induced cell death, or secreted inflammatory cytokines (Figure 5). These results indicated that ectopic FOXO1 overexpression neither improved nor impaired the effector functions of transiently activated CAR-T cells.

[0115] [Example 5] Ectopic FOXO1 upregulates endogenous LEF1 and TCF1 in resting CAR-T cells and sustains their expression during activation Despite similar surface marker expression of resting FOXO1-overexpressing CD19 CAR-T cells compared to controls (Figure 4), it was hypothesized that FOXO1-overexpressing CAR-T cells could drive the expression of memory and stemness-related TFs and the endogenous TCF1 (gene name TCF7), a FOXO1 target gene, that correlate with checkpoint blockade responses and CAR-T cell responses in patients / animals. Indeed, intracellular flow cytometry revealed that ectopic expression of FOXO1-WT and FOXO1-3A enhanced the expression of TCF1 and its associated HMG-box family TF, lymphoid enhancer-binding factor 1 (LEF1), in resting CD19 CAR-T cells (Figure 6). Notably, the expression of these stemness-related TFs was maintained during CAR-T cell activation, in addition to the enhanced expression of the effector-related TFs T-box transcription factor TBX21 (Tbet) and B lymphocyte-induced maturation protein-1 (Blimp-1) (Figure 7). Collectively, these data demonstrated that FOXO1 directly and / or indirectly upregulates TFs known to drive stemness and effector transcriptional programs and confer enhanced functionality during chronic stimulation.

[0116] [Example 6] FOXO1-engineered CD19-targeted CAR-T cells exhibit enhanced function in a model of chronic antigen simulation To examine the effect of ectopic FOXO1 expression on CAR-T cell function during chronic stimulation, CD19-targeted CAR-T cells co-transduced with either NGFR, FOXO1-WT or FOXO1-3A were challenged with repeated tumor stimulation or at a low effector-to-tumor (E:T) ratio. After three tumor challenges, CD19.BBz CAR-T cells were rested for 7 days prior to phenotyping to determine the extent to which FOXO1-engineered CAR-T cells could form memory, an important feature for sustaining the anti-tumor response. CAR-T cells expressing FOXO1-WT and FOXO1-3A maintained significantly higher expression levels of the memory markers CD62L and IL-7R compared to TCF1 or NGFR cells, indicating an enhanced ability for memory formation and / or persistence (Figure 8A). For both CD19.28z and CD19.BBz CAR-T cells, ectopic FOXO1-WT enhanced FOXO1 killing compared to the NGFR control, but paradoxically, FOXO1-3A did not (Figures 8B-8C). Further investigation revealed that FOXO1-WT CAR-T cells exhibited enhanced CAR-T cell expansion at a low E:T ratio compared to NGFR and FOXO1-3A (Figure 8D). These data indicate that FOXO1-WT cells are more potent than FOXO1-3A and NGFR cells in the context of chronic antigen stimulation, and that the constitutive nuclear localization of FOXO1-3A suppresses CAR-T cell expansion or effector function during chronic stimulation.

[0117] [Example 7] Ectopic FOXO1 reduces T cell exhaustion Ectopic TF expression for enhancing CAR-T cell function was also tested in a validated in vitro model of CAR-T cell exhaustion, in which the high-affinity GD2-targeted CAR (HA.28z) promotes antigen-independent aggregation and signaling, as well as the rapid onset of T cell dysfunction. HA.28z CAR-T cells expressing FOXO1-WT and FOXO1-3A both showed altered surface phenotypes with decreased exhaustion markers (e.g., CD39 and PD-1), while memory markers and FOXO1 target genes (e.g., CD62L) were increased (Figs. 9A - 9B). FOXO1-WT cells showed enhanced cytokine secretion and killing compared to controls, while FOXO1-3A cells were not functionally enhanced despite showing a surface phenotype consistent with non-exhausted cells (Figs. 9C - 9E), which was consistent with observations from chronically stimulated CD19-targeted CAR (Fig. 8). Collectively, these data showed that ectopic FOXO1-WT abrogates CAR-T cell exhaustion and confers enhanced function.

[0118] [Example 8] Constitutive expression of FOXO1 confers exhaustion resistance To test whether constitutive FOXO1-WT reduced exhaustion in the HA.28z CAR model or whether transient expression at the time of tumor exposure was sufficient to enhance function, a destabilizing domain was fused to the N-terminus of FOXO1-WT (DD-FOXO1-WT), which enabled precise drug-dependent control of expression (Fig. 10A). HA.28z CAR-T cells expressing DD-FOXO1-WT from day 4 to day 15 showed increased IL-2 secretion in response to tumors, whereas HA.28z CAR-T cells expressing DD-FOXO1-WT starting 24 hours before tumor challenge did not show increased IL-2 secretion (Fig. 10B).

[0119] [Example 9] Ectopic FOXO1 reprograms CAR-T cells metabolically Since FOXO1 promotes memory and stemness in CAR-T cells and these phenotypes are known to correlate with distinct metabolic features compared to effector T cells, we hypothesized that ectopic FOXO1 could drive metabolic reprogramming in CAR-T cells. Using the Seahorse assay, we found that both FOXO1-WT and FOXO1-3A decreased the extracellular acidification rate (basal ECAR) and increased mitochondrial fitness (e.g., spare respiratory capacity, SRC) in both resting CD19-targeted HA.28z CAR-T cells and exhausted HA.28z CAR-T cells (Figure 11A). FOXO1-WT and FOXO1-3A CAR-T cells also showed higher mitochondrial mass than the NGFR control in the unstimulated state (Figure 11B). Antigen stimulation further increased mitochondrial mass in FOXO1-engineered CAR-T cells, while NGFR control cells remained at baseline levels (Figure 11C). Collectively, these data demonstrate that ectopic FOXO1-WT and FOXO1-3A metabolically reprogram CAR-T cells to have features characteristic of memory T cells.

[0120] [Example 10] FOXO1-engineered CAR-T cells mediate enhanced tumor suppression and mouse survival in liquid tumor models Non-therapeutic doses of TF-engineered CAR-T cells or control CAR-T cells (CD19.28z, CD19.BBz, or HA.28z) were injected into mice engrafted with Nalm6 leukemia. FOXO1-WT and CAR-T cells showed significantly enhanced tumor suppression and survival compared to NGFR control and TCF1 cells (Figs. 12A - 12C, 21). FOXO1-3A CAR-T cells also showed enhanced tumor suppression and survival compared to NGFR control and TCF1 cells, but to a lesser extent than FOXO1-WT and CAR-T cells. These data further supported the idea that ectopic FOXO1 enhances CAR-T cell function, particularly in the context of chronic antigen exposure, by alleviating T cell exhaustion and enforcing a stem cell program. Finally, FOXO1-WT HA.28z CAR-T cells, which were more functionally superior to NGFR control cells in vitro, showed enhanced tumor suppression in a leukemia xenograft model in which Nalm6 leukemia cells were engineered to express GD2 (Fig. 12D).

[0121] [Example 11] CAR-T cells expressing FOXO1-WT are superior to FOXO1-3A in an in vivo model of leukemia through enhanced expansion, persistence, and recall capabilities The therapeutic doses of NGFR-purified CD19.28z CAR T cells co-expressing ectopic FOXO1-WT, FOXO1-3A, TCF1, or control NGFR were injected into NSG mice engrafted with Nalm6 leukemia. The circulating levels of CAR-T cells were measured 7 days after CAR-T injection and then every 7 days thereafter (Figure 13A). One week after tumor clearance in all groups (Figure 13B), mice were re-challenged with a high dose of 10e6 Nalm6 to evaluate whether persistent CAR-T cells were responsive to the second challenge. The circulating CAR-T cells detected at each time point remained CAR+ / NGFR+ (Figure 13C), and the CAR surface density was consistent among experimental groups (Figure 13D). Notably, on day 7 after injection, FOXO1-WT cells expanded approximately 28-fold more than the NGFR control and 20-fold more than FOXO1-3A cells (Figures 14A - 14B). FOXO1-3A cells showed robust but delayed expansion, with their circulating levels peaking on day 14 after injection and far exceeding the NGFR control levels, yet still being 1 / 4 to 1 / 5 of the FOXO1-WT levels observed on day 7 (Figures 14A - 14B). FOXO1-WT and FOXO1-3A CAR-T cells showed comparable persistence in mice until day 21, when control NGFR and TCF1 cells were almost undetectable (Figures 14A - 14B). Seven days after tumor re-challenge (day 28), only FOXO1-WT cells showed memory recall ability, which was characterized by a 2 - 3-fold expansion of circulating CAR-T cells in response to the re-challenge and long-term mouse survival (Figure 14). In contrast, the circulating FOXO1-3A CAR-T cell levels continued to decline after re-challenge, and ultimately, these mice as well as those treated with control NGFR and TCF1 cells succumbed to the disease (Figure 14). Importantly, at all time points, the relative frequency of CD8 CAR-T cells was significantly higher in both the FOXO1-WT and FOXO1-3A conditions compared to the control, suggesting a role for FOXO1 in enhancing the expansion and persistence of CD8+ CAR-T cells (Figure 15). In summary, FOXO1-WT and FOXO1-3A CAR-T cells showed enhanced expansion and persistence compared to control NGFR and TCF1 cells.However, FOXO1-WT cells expanded more robustly compared to FOXO1-3A cells, retained their antitumor functionality, and further supported the idea that ectopic FOXO1-WT enhanced CAR-T cell function to a greater extent than ectopic FOXO1-3A.

[0122] [Example 12] FOXO1-engineered CAR-T cells enhanced CAR-T cell targeting to solid tumors In contrast to CD19-targeted CAR-T cells, in which FOXO1-engineered cells were phenotypically and functionally similar to control CAR-T cells at rest and during transient activation, ectopic FOXO1-WT in HER2-targeted CAR-T cells (HER2.28z and HER2.BBz) showed a higher frequency of memory-like T cells at rest and enhanced function in short-term assays (Figures 16A - 16C). Mice engrafted with the osteosarcoma cell line 143B were injected with a non-purified dose of 10e6 CAR-T cells, of which only 50% were NGFR / FOXO1-WT+. FOXO1-WT cells enhanced tumor suppression in this highly aggressive solid tumor model (Figures 16D - 16E), indicating that this approach is broadly applicable to solid tumors in addition to hematological malignancies.

[0123] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in full herein.

[0124] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the present invention. Variations of these preferred embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend for the present invention to be practiced in other embodiments than those specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. Further, unless otherwise indicated herein or clearly contradicted by context, any combination of the above-described elements in all possible variations thereof is included by the present invention.

Claims

1. A composition comprising isolated T cells containing an exogenous nucleic acid encoding forkheadbox protein O1 (FOXO1).

2. The composition according to claim 1, wherein FOXO1 is constitutively expressed from an exogenous nucleic acid.

3. The composition according to claim 1, wherein FOXO1 is fused to a motif that modulates its expression level or enhances its intracellular degradation.

4. The composition according to claim 1, wherein isolated T cells maintain functionality under conditions in which unmodified T cells exhibit exhaustion.

5. The composition according to claim 1, wherein the isolated T cells further comprise nucleic acids encoding recombinant receptors.

6. The composition according to claim 5, wherein the recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

7. The composition according to claim 5, wherein the recombinant receptor is specific to the tumor antigen.

8. The composition according to claim 5, wherein FOXO1 and the manipulated receptor are encoded by separate nucleic acids.

9. The composition according to claim 5, wherein FOXO1 and the engineered receptor are encoded by a single nucleic acid.

10. The composition according to claim 5, wherein FOXO1 and the engineered receptor are expressed under different promoters.

11. The composition according to any one of claims 1 to 5, wherein the isolated T cells are derived from a biological sample from a subject.

12. The composition according to claim 11, wherein isolated T cells are isolated from a tumor sample.

13. The composition according to claim 11, wherein isolated T cells are expanded ex vivo.

14. The composition according to any one of claims 1 to 5, further comprising at least one therapeutic agent.

15. A pharmaceutical composition comprising isolated T cells containing an exogenous nucleic acid encoding forkhead box protein O1 (FOXO1) for use in a method of treating a disease or disorder in a subject, wherein the method comprises administering an effective amount of the composition according to any one of claims 1 to 5 to a subject having the disease or disorder.

16. The pharmaceutical composition according to claim 15, wherein the isolated T cells are autologous to the subject.

17. The pharmaceutical composition according to claim 15, wherein FOXO1 is overexpressed in isolated T cells before exposure to the antigen.

18. The pharmaceutical composition according to claim 15, wherein the disease or disorder includes infectious diseases or cancer.

19. The pharmaceutical composition according to claim 18, wherein the cancer is a solid tumor.

20. The pharmaceutical composition according to claim 18, wherein the recombinant receptor is specific to cancer.

21. The pharmaceutical composition according to claim 18, wherein administration reduces the number of cancer cells in a patient / animal.

22. The pharmaceutical composition according to claim 18, wherein administration reduces and / or eliminates tumor burden in a patient / animal.

23. The pharmaceutical composition according to claim 18, wherein administration exhibits enhanced cancer treatment compared to administration of unmodified T cells.

24. The pharmaceutical composition according to claim 18, further comprising administering one or more chemotherapeutic agents to a patient / animal.

25. The pharmaceutical composition according to claim 15, wherein administration results in lower expression of inhibitory receptors in the subject compared to administration of unmodified T cells.

26. A method for preventing exhaustion of engineered T cells, comprising introducing a nucleic acid that overexpresses FOXO1 into engineered T cells in vitro.

27. The method according to claim 26, wherein FOXO1 is wild-type FOXO1 or a functional fragment or mutant thereof.

28. The method according to claim 26, wherein the nucleic acid promotes the constitutive expression of FOXO1 from the nucleic acid.

29. The method according to claim 26, wherein the manipulated T cells further comprise nucleic acids encoding recombinant receptors.

30. The method according to claim 29, wherein the recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

31. The method according to claim 29, wherein the recombinant receptor is specific to the tumor antigen.

32. The method according to claim 31, wherein FOXO1 is overexpressed before exposure to the antigen.

33. The method according to any one of claims 29 to 32, wherein FOXO1 and the manipulated receptor are encoded by separate nucleic acids.

34. The method according to any one of claims 29 to 32, wherein FOXO1 and the manipulated receptor are encoded by a single nucleic acid.

35. The method according to any one of claims 29 to 32, wherein FOXO1 and the manipulated receptor are expressed under different promoters.

36. A pharmaceutical composition for use in a method according to any one of claims 26 to 32 for preventing exhaustion of engineered T cells, the method comprising introducing a nucleic acid overexpressing FOXO1 into engineered T cells, and the method further comprising administering T cells to a subject requiring administration of T cells.

37. The pharmaceutical composition according to claim 36, wherein the subject has cancer.

38. A composition according to any one of claims 1 to 5, for use in treating a disease or disorder in a subject.

39. A composition comprising an exogenous nucleic acid configured to overexpress FOXO1 for use in preventing exhaustion of manipulated T cells.

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