Synthetic gene expression regulatory switches

Abstract

Provided herein are modified cells comprising "Synthetic Gene Expression Regulatory Switches" (SynGERS) that are turned on only when needed to help white blood cells grow, persist, and eliminate tumors. SynGERS expression cassettes comprise a 5' sensor that acts on a minimal promoter (minP) followed by the enhancer and repressor units. SynGERS induce potent anti-tumor function by CAR T cells with improved persistence and proliferation in solid tumors.

Description

SYNTHETIC GENE EXPRESSION REGULATORY SWITCHESREFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of United States provisional application number 63 / 575,320, filed April 5, 2024, the entire contents of which are incorporated herein by reference.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on April 3, 2025, is named BACMP0013WO.xml and is 5,009 bytes in size.BACKGROUND1. Field

[0003] The present invention relates generally to the fields of oncology and immunology. More particularly, it concerns synthetic switches that induce bidirectional gene modulation in a cell state-specific manner.2. Description of Related Art

[0004] Cell state-specific bidirectional modulation of gene expression is desired in several disciplines focused on modified properties of engineered cells including regenerative medicine and cancer immunotherapy. For example, chimeric antigen receptor (CAR) T cells can produce up to a 90% complete response rates in patients with acute lymphoblastic leukemia and have the potential to eliminate solid tumors (Maude et al., 2014). However, early -phase studies have shown only modest benefit against solid tumors (Ahmed et al., 2015; Ahmed et al., 2017; Shi et al., 2020; Heczey et al., 2017; Louis et al., 2011). The immunosuppressive solid tumor microenvironment (TME) induces T-cell dysfunction due to the lack of survival factors, presence of inhibitory molecules, and chronic antigen exposure leading to T-cell exhaustion (Majzner et al., 2019). These changes correspond to broad cell state changes associated with bidirectional alterations of key regulatory genes’ expression. Additional genetic manipulation of CAR T cells through overexpression of positive or inhibition of negative regulators of T-cell function can significantly enhance the antitumor properties of these tumor- specific effectors in preclinical solid tumor models (Batra et al., 2020; Belk et al.,2022; Lynn et al., 2019; Legut et al., 2022; Yao et al., 2021; Mai et al., 2023; Wei et al., 2019; Carnevale et al., 2022; Jung et al., 2022). However, mechanisms for ensuring that CAR T-cell proliferation and effector functions are induced only when encountering neoplastic cells are needed.SUMMARY

[0005] Provided herein are a plurality of expression constructs, each independently comprising: (a) a cell state-specific sensor; (b) an enhancer unit that encodes a protein that promotes cell survival, expansion, and / or persistence; and (c) a repressor unit that encodes an inhibitory RNA that downregulates the expression of a protein involved in T-cell dysfunction.

[0006] The cell state-specific sensor may comprise a minimal promoter. The cell statespecific sensor may comprise binding sites for a transcription factor that is activated in response to immune cell activation. The cell state-specific sensor may respond to activation of NFAT, NR4A2, BLIMP1, EGR2, or TOX.

[0007] The enhancer unit may encode a protein that promotes T-cell survival. The enhancer unit may encode interleukin- 15 (IL15), JUN, BACH2, LTBR, or BATF3. The repressor unit may encode a miRNA or shRNA. The repressor unit may downregulate the expression of TOX, ARIDla, BTG1, BLIMP1, RASA2, REGNASE1, or PRDM1. The enhancer unit and the repressor unit may be separated by a cleavable peptide.

[0008] Provided herein are expression constructs comprising an expression construct comprising, from 5’ to 3’, (a) cell state- specific sensor that responds to activation of NR4A2 and comprises a minimal promoter; (b) an enhancer unit that encodes IL15; (c) a repressor unit that encodes an inhibitor RNA that downregulates the expression of TOX, BLIMP 1, BTG1, RASA2, or REGENASE1 ; (d) a promoter; and (e) a chimeric antigen receptor. The expression construct may further comprise an induction reporter positioned between the enhancer unit and the repressor unit, in which case there will be a cleavable peptide positioned between the enhancer unit and the induction reporter.

[0009] A preferred expression construct based on NR4A2 sensing inducing IL15 secretion and TOX repression is provided in SEQ ID NO: 1. The expression construct may comprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to nucleotides 1-1441 of SEQ ID NO: 1. The expression construct maycomprise a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 1. The cell state-specific sensor that responds to activation of NR4A2 corresponds to nucleotides 1-64 of SEQ ID NO: 1. The minimal promoter corresponds to nucleotides 74-145 of SEQ ID NO: 1. The IL15 coding sequence corresponds to nucleotides 156-641 of SEQ ID NO: 1. The T2A sequence corresponds to nucleotides 642-695 of SEQ ID NO: 1. The Q8 coding sequence corresponds to nucleotides 696-1109 of SEQ ID NO: 1. The artificial microRNA targeting TOX corresponds to nucleotides 1121-1441 of SEQ ID NO: 1. The SFFV promoter corresponds to nucleotides 1442-1865 of SEQ ID NO: 1. The GPC3-CAR coding sequence corresponds to 1897-3270 of SEQ ID NO: 1.

[0010] Provided herein are methods for screening the library of expression constructs provided herein. The methods may comprise introducing the plurality of expression constructs into a population of cells and selecting for a desired phenotype. The desired phenotype or enhanced function may be growth, persistence, and / or anti-tumor activity. The screen may be performed in vitro or in vivo.

[0011] The method may comprise introducing the plurality of expression constructs into a plurality of T cells, activating the T cells, selecting a T cell that exhibits an enhanced function, and identifying the expression construct in the selected T cell. The plurality of T cells may further comprise a chimeric antigen receptor. The plurality of T cells may further comprise a truncated EGF-receptor.

[0012] Provided herein are modified T cells comprising an expression cassette comprising an enhancer unit that encodes a protein that promotes cell survival, expansion, and / or persistence and a repressor unit that encodes an inhibitory RNA that downregulates the expression of a protein involved in T-cell dysfunction, wherein the expression of the enhancer unit and repressor unit under the control of a cell state- specific sensor. The modified T cells may further comprise a chimeric antigen receptor. The expression cassette may be under the control of a NR4A2 cell state-specific sensor. The enhancer unit may be IL15. The repressor may knockdown expression of TOX, BTG1, RASA2, BLIMP1, or REGNASE1.

[0013] Provided herein are methods for treating cancer in a subject, the method comprising administering to the subject an effective amount of the modified T cells provided herein. The modified T cells may be autologous or allogeneic to that patient. The cancer maybe a solid tumor. The cancer may be a Wilms tumor, hepatoblastoma, rhabdomyosarcoma, or rhabdoid tumor. The patient may be a child.

[0014] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0016] FIGS. 1A-1B. (FIG. 1A) Schematic of an exemplary SynGERS expression cassette: The cassette consists of the 5’ sensor, acting on a minimal promoter (minP) followed by the enhancer and repressor units. A separate promoter (SFFV) drives the cargo (e.g., GBBz CAR (GPC 3 -specific second-generation CAR incorporating the 41BB endodomain)). Q8 tag for induction detection. (FIG. IB) Schematic of exemplary SynGERS library where the cargo unit is a GBBz CAR (GPC3-specific second-generation CAR incorporating the 41BB endodomain). The cargo unit incorporates the human truncated EGFR (huEGFRt) which is translated into a separate peptide with the use of a T2A sequence. *See Example 1 for calculation specifics.

[0017] FIG. 2. Activation dependent induction and repression of target genes. T cells were transduced with indicated SynGERS and pre- and post- activation gene expression induction (eGFP) and repression (MHC Class I) was measured by FACS. One representative of three independent experiments.

[0018] FIG. 3. Generation of NR4a2 sensors: Transduced cells were stimulated via TCR and induction of GFP expression was measured by FACS. Combined results from three independent experiments.

[0019] FIG. 4A-4D. Functionality of SynGERS-CAR T Cells Against GPC3+ Tumor Cells: A, screening of inducible SynGERS-CAR T cell library in repeat killing assay against Huh7-eGFP cells. Tumor cells were seeded one day before the co-culture in T cell medium. Next day, CAR T cells were added at a 1:1 effector to target ratio. % of killing was calculated as: 100 * (mean eGFP index sample / maximum mean eGFP index). Once killing was complete, CAR T cells were analyzed and added to fresh tumor cells at the same ratio. CAR was detected by flow cytometry using AF647-labeled anti-mouse Fab2antibody (Jackson). eGFP expression was detected with the IncuCyte system (Sortarious). Data is presented as mean of two donors. B,C and D, functionality of top performant inducible SynGERS-CAR T cells with their constitutive SynGERS controls in repeat killing assay against G401-eGFP, Huh7 and HepG2 tumor cells respectively. The assays were performed following the same protocol described in panel A. Data is presented as the mean of 4 donors from two independent experiments.

[0020] FIG. 5. Annexin V expression at round 3 of repeat killing assay against HepG2- eGFP cells. Tumor cells were seeded a day prior to the co-culture. The next day, CAR T cells were added at a 1 : 1 effector to target ratio. Once killing was complete, CAR T cells were analyzed and added to fresh tumor cells at the same ratio. Annexin V was detected via flow cytometry using Annexin V- PerCP-Cy5.5 from BioLegend. Data are shown as mean of two donors.

[0021] FIG. 6. PD-1 and memory markers expression at round 3 of repeat killing assay against HepG2-eGFP cells. Tumor cells were seeded a day prior to the co-culture in T cell medium. The assay was performed following the same protocol described in Figure 6. Data are presented as mean of 2 donors.

[0022] FIG. 7. Expression of memory markers. Data are shown as mean of two donors. TN (CCR7+CD45RO-): Naive, DP (CCR7+CD45RO+): CM (Central Memory); CD45RO+CCR7-: EM (Effector Memory), DN (CCR7-CD45RO-): EFF (Effector). Cons (Constitutive expression vector). Within each marker on the X-axis, the data points represent, from left to right, eQ3-rScrambe, eIL15-rTOX, eIL15-rBLIMPl, eJUN-rBLIMPl, eIL15- rBTGl, eIL15-rRASA2, eJUN-rRASA2, eIL15-rREGNASEl, eIL15-rScrambe, Cons-eIL15- rTOX, Cons-eIL15-rBLIMPl, Cons-eJUN-rBLIMP 1 , Cons-eIL15-rBTGl, Cons-eIL15- rRASA2, Cons-eJUN-rRASA2, Cons-eILI5-rREGNASEI, and Cons-eIL15-rScramble.

[0023] FIGS. 8A-8B. Functionality of SynGERS-CAR T cells against HepG2-eGFP- Luciferase cells in vivo. NSG MHC I / II DK mice received a single i.v. dose of 5xl06SynGERS-CAR T cells on day 7 post i.p. Injection of IxlO6HepG2-eGFP-Luciferase cells, non-transfected cells, eQ8-rScramble, and eIL15-rScramble served as controls (N=4 or 5 per group as indicated in the image). (FIG. 8 A) Quantification of bioluminescence. (FIG. 8B). Kaplan-Meier survival.

[0024] FIGS. 9A-9B. Functionality of SynGERS-CAR T cells against Huh7-eGFP- Luciferase cells in vivo. NSG MHC I / II DK mice received a single i.v. dose of 5xl06SynGERS-CAR T cells on day 7 post i.p. Injection of IxlO6Huh7-eGFP-Lucif erase cells, nontransfected cells (NT), eQ8-rScramble (CAR), and eIL15-rScramble served as controls (N=5 per group). (FIG. 8A) Quantification of bioluminescence. (FIG. 8B). Kaplan-Meier survival.DETAILED DESCRIPTION

[0025] Provided herein are “Synthetic Gene Expression Regulatory Switches” (SynGERS) that are turned on only when needed to help white blood cells grow, persist, and eliminate tumors. SynGERS expression cassettes comprise a 5’ sensor that acts on a minimal promoter (minP) followed by the enhancer and repressor units. A separate promoter drives CAR expression. These results provided herein demonstrate the potential for SynGERS to induce potent anti-tumor function by CAR T cells with improved persistence and proliferation in solid tumors.

[0026] CAR T cells have been shown to mediate up to a 90% complete response rate and approximately 50% durable remission rate in children with acute lymphoblastic leukemia and have the potential to mediate responses in pediatric solid tumor patients as well (Maude et al., 2018). Early-phase studies evaluating CAR T cells have shown only modest benefit in this patient population due to several fundamental differences between solid and hematologic neoplasms (Ahmed et al., 2015; Ahmed et al., 2017; Shi et al., 2020; Heczey et al., 2017; Louis et al., 2011). These include the immunosuppressive solid tumor microenvironment (TME) that induces T-cell dysfunction due to the lack of survival factors, presence of inhibitory molecules, and chronic antigen exposure leading to T-cell exhaustion (Maizner et al., 2019). Additional genetic manipulation of CAR T cells, through overexpression of positive regulators (i.e., interleukin- 15 (IL15), JUN, BACH2, LTBR, BATF3) or inhibition of negative regulators (i.e., ARIDla, BTG1, RASA2, REGNASE1, PRDM1 ) of T cell function, can significantly enhancethe antitumor properties of these tumor-specific effectors in preclinical solid tumor models (Batra et al., 2020; Belk et al., 2022; Lynn et al., 2019; Legut et al., 2022; Yao et al., 2021; Mai et al., 2023; Wei et al., 2019; Carnevale et al., 2022; Jung et al., 2022). It is unclear which of these strategies provides the most potent antitumor effect and whether the combination of positive and negative regulator modulation can safely and synergistically enhance CAR T-cell function. Combining IL15 overexpression and BTG1 repression in CAR-NKT and T cells enhances their antitumor activity (Heczey et al., 2023); thus, systematic testing of additional regulatory combinations was undertaken. As such synthetic regulatory programs are engaged, it is crucial to ensure that CAR T-cell proliferation and effector function are induced only when engaging neoplastic cells. To accomplish these objectives, a library of Synthetic Gene Expression Regulator Switch (SynGERS) was designed, consisting of a cell state-specific sensor (NFAT, NR4A2 - activation; BLIMP1 - terminal differentiation; EGR2 - dysfunction; TOX - exhaustion) (Murphy et al., 2012) acting on a minimal promoter that drives 1) overexpression of specific genes associated with T-cell survival, and 2) RNA interferencebased downregulation of T cell dysfunction-related target genes (FIG. 1). A lend viral, single expression cassette-based vector system was developed that modulates gene expression via SynGERS and constitutively expresses the clinically validated CAR targeting glypican-3 (GPC3). These SynGERS were tested in GPC3-CAR T cells given that GPC3 is an established immunotherapeutic target expressed by several pediatric solid tumor types, it has been safe thus far in clinical testing, and GPC3-CAR T cells co-expressing IL 15 can mediate anti-tumor responses (Steffin et al., 2023).

[0027] Constitutive overexpression of positive T cell regulators such as JUN (Lynn et al., 2019) or IL15 (Batra et al., 2020; Hoyos et al., 2010), as well as knockout of negative regulators such as ARIDla (Belk et al., 2022) or PRDM1 (encoding BLIMP1) (Jung et al., 2022) improves CAR T-cell function. However, these genes have been shown to be overexpressed in neoplastic cells, raising the concern that constitutive overexpression or knockout may lead to unwanted toxicity and negatively affect long-term survivors. SynGERS regulate gene expression in a tumor antigen-dependent manner, which minimizes this risk while maximizing potential synergistic effects of overexpressing / downregulating genes that affect T-cell function. In addition, the SynGERS-CAR cassette may constitutively express the truncated EGF-receptor (FIG. IB), enabling effective elimination of the cells using clinically available cetuximab, if needed.

[0028] Chronic antigen stimulation in the presence of inhibitory factors and without supportive signals causes T cells to undergo robust transcriptional and epigenetic reprogramming, rendering them dysfunctional (unable to proliferate or produce cytokines and cytotoxic molecules) (Pereira et al., 2017; Blank et al., 2019). To prevent this, combined modulation of positive and negative regulators may be necessary as has been demonstrated in tumor-infiltrating T cells responsive to checkpoint inhibition, which have increased TCF7 and repressed TOX expression. Thus, SynGERS that mediate bidirectional gene expression address a key barrier in genetic engineering.

[0029] The SynGERS platform employs several innovative strategies with the potential to introduce significant progress in CAR T-cell engineering to safely maximize antitumor function. First, a new activation-specific inducible promoter is used. During physiologic T-cell activation, interrelated signaling cascades transmit prompts from the extracellular space into the nucleus including the phosphorylation of Nuclear factor of activated T-cells (NF AT) (Murphy et al., 2012). Several NFAT-based T-cell activation reporting systems have been developed with tunable output intensity dependent on the number of DNA-binding sequence (DBS) repeats. Nuclear receptor 4a2 (NR4a2) is downstream of NFAT and is expressed at a high level in exhausted T cells (Pereira et al., 2017; Blank et al., 2019). Four NFAT-based and seven NR4a2 -based DBS sequences were screened, and a novel NR4a2-specific DBS domain that induces the highest peak output signal while maintaining reversible activation specificity was identified. This NR4a2-based sensor unit was validated within a set of SynGERS to maximize tumor-antigen dependent CAR T cell function (FIG. 3). Second, SynGERS provide for repression of genes mediated by artificial micro RNAs (amiRs). CRISPR / Cas9 and shRNA- based methods can be used to downregulate gene expression; however, these strategies cannot be used in an inducible context required for cell state-specific systems using clinically validated vectors due to the large size of Cas9 or the requirement for an additional promoter to drive shRNA expression. Artificial microRNAs (amiRs) can be expressed via inducible promoters given their small size (~300bp) and can even be expressed in frame with other protein-encoding genes in the same mRNA (Zhu et al., 2007). As proof-of-principle, a miR30-based amiR targeting beta-2-microglobulin (B2M) was validated, which generated effective repression of major histocompatibility complex (MHC) class I expression, and replacing the target gene specific sequence of this miR30 backbone confirmed the ability of it to repress other genes (i.e. TOX, not shown). Third, SynGERS provide for the modulation of cell state- specific, bidirectional gene expression. Combining cell state-specific (i.e., activation, terminaldifferentiation, or exhaustion) and tumor-engagement specific regulation of positive / negative mediators of T-cell function has not been previously demonstrated in eukaryotic cells. This strategy is based on Boolean logic and follows “IF-THEN (+X AND -Y)” for the first time to maintain safety while maximizing CAR T-cell antitumor activity. Lastly, the optimized lentiviral expression cassette used for development enable an effective transition to the clinic upon completion of the outlined research.I. Definitions

[0030] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0031] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0032] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0033] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.II. Synthetic Gene Expression Regulatory Switches

[0034] Provided herein are “Synthetic Gene Expression Regulatory Switches” that are turned on in a cell state-specific manner to increase and reduce the expression of target genes of interest resulting in a desired phenotype. For example, the SynGERS may be turned on only when needed to help white blood cells grow, persist, and eliminate tumors. SynGERSexpression cassettes comprise a 5’ sensor that acts on a minimal promoter (minP) followed by the enhancer and repressor units.A. Cell State-Specific Sensors

[0035] Cell state-specific sensors have binding sites for transcription factors that are activated in response to immune cell activation. For example, after engagement of the T-cell receptor, the transcription factor NF AT is dephosphorylated, allowing it to translocate to the nucleus and active key target gene expression. As such, binding of the activated transcription factor triggers gene expression from a minimal promoter that is operably linked to the cell state-specific sensor, thereby causing up-regulated transcription of the downstream enhancer and repressor units. Examples of such transcription factors that are activated in response to immune cell activation include NF AT, NR4A2, BLIMP 1, EGR2, and TOX. TheB. Enhancer Units

[0036] Enhancer units are genes that promote T-cell survival. These gene can be, for example, cytokines or transcription factors. For example, cytokine interleukin- 15 (IL- 15) mediates development and survival of immune cells. Additional examples of genes that promote T-cell survival include CJUN, BACH2, LTBR, BATF3, and TCF7. c-Jun induces CAR T exhaustion resistance. BATF3 promotes CD8+ T-cell survival. TCF7 encodes TCF1, a key transcription factor responsible for T-cell self-renewal. Bach2 is expressed in T cells and maintains the homeostasis of T-cell subsets. LTBR increases T cell effector functions.C. Repressor Units

[0037] Repressor units encode RNA interference-based negative regulators of gene expression to prevent T-cell dysfunction. For example, RASA2 is a RAS GTPase-activating protein (RasGAP). Knockdown of RASA2 in T cells increases activation, cytokine production, metabolic activity, and persistence in cancer cell killing. Additional examples of genes involves in T-cell dysfunction includes ARIDla, BTG1, REGNASE1, and PRDM1. REGNASE-1 knockdown reprograms CD8+ T cells into long-lived effector cells with improved persistence. Inhibition of PRDM1 expression decreases the ratio of T-reg cells. BTG1 actively suppresses T-cell activation and counteracts tonic signaling to maintain T cell quiescence. B TGI -deficient T cells have increased proliferation and spontaneous activation. ARIDla protein is a subunit of the SWI / SNF chromatin remodeling complex. Knockdown of AridlA reverses T-cell exhaustion.

[0038] “RNA interference (RNAi)” is the process of sequence-specific, post- transcriptional gene silencing initiated by siRNA. During RNAi, siRNA induces degradation of target mRNA with consequent sequence-specific inhibition of gene expression. An “inhibitory RNA,” “RNAi,” “small interfering RNA” or “short interfering RNA” or “siRNA” molecule, “short hairpin RNA” or “shRNA” molecule, or “miRNA” is a RNA duplex of nucleotides that is targeted to a nucleic acid sequence of interest. As used herein, the term “siRNA” is a generic term that encompasses the subset of shRNAs and miRNAs. An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of an RNA molecule. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In certain embodiments, the siRNAs are targeted to the sequence encoding huntingtin. In some embodiments, the length of the duplex of siRNAs is less than 30 base pairs. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length. In some embodiments, the length of the duplex is 19 to 25 base pairs in length. In certain embodiment, the length of the duplex is 19 or 21 base pairs in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In certain embodiments, the loop is 18 nucleotides in length. The hairpin structure can also contain 3' and / or 5' overhang portions. In some embodiments, the overhang is a 3' and / or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

[0039] shRNAs are comprised of stem-loop structures which are designed to contain a 5' flanking region, siRNA region segments, a loop region, a 3' siRNA region and a 3' flanking region. Most RNAi expression strategies have utilized short-hairpin RNAs (shRNAs) driven by strong polIII-based promoters. Many shRNAs have demonstrated effective knock down of the target sequences in vitro as well as in vivo, however, some shRNAs which demonstrated effective knock down of the target gene were also found to have toxicity in vivo.

[0040] miRNAs are small cellular RNAs (~22 nt) that are processed from precursor stem loop transcripts. Known miRNA stem loops can be modified to contain RNAi sequences specific for genes of interest. miRNA molecules can be preferable over shRNA molecules because miRNAs are endogenously expressed. Therefore, miRNA molecules are unlikely toinduce dsRNA-responsive interferon pathways, they are processed more efficiently than shRNAs, and they have been shown to silence 80% more effectively.

[0041] A recently discovered alternative approach is the use of artificial miRNAs (pri- miRNA scaffolds shuttling siRNA sequences) as RNAi vectors. Artificial miRNAs more naturally resemble endogenous RNAi substrates and are more amenable to Pol-II transcription (e.g., allowing tissue-specific expression of RNAi) and polycistronic strategies (e.g., allowing delivery of multiple siRNA sequences). See U.S. Pat. No. 10,093,927, which is incorporated by reference.

[0042] The transcriptional unit of a “shRNA” is comprised of sense and antisense sequences connected by a loop of unpaired nucleotides. shRNAs are exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional siRNAs. “miRNAs” stem-loops are comprised of sense and antisense sequences connected by a loop of unpaired nucleotides typically expressed as part of larger primary transcripts (pri- miRNAs), which are excised by the Drosha-DGCR8 complex generating intermediates known as pre-miRNAs, which are subsequently exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional siRNAs. “Artificial miRNA” or an “artificial miRNA shuttle vector”, as used herein interchangably, refers to a primary miRNA transcript that has had a region of the duplex stem loop (at least about 9-20 nucleotides) which is excised via Drosha and Dicer processing replaced with the siRNA sequences for the target gene while retaining the structural elements within the stem loop necessary for effective Drosha processing. The term “artificial” arises from the fact the flanking sequences (~35 nucleotides upstream and ~40 nucleotides downstream) arise from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein the term “miRNA” encompasses both the naturally occurring miRNA sequences as well as artificially generated miRNA shuttle vectors.

[0043] The siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six Ts.

[0044] In designing RNAi there are several factors that need to be considered, such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the siRNA that is introduced into the organism will typicallycontain exonic sequences. Furthermore, the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene-specific sequences. Preferably the siRNA exhibits greater than 80%, 85%, 90%, 95%, 98%, or even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences less than about 80% identical to the target gene are substantially less effective. Thus, the greater homology between the siRNA and the gene to be inhibited, the less likely expression of unrelated genes will be affected.

[0045] In addition, the size of the siRNA is an important consideration. In some embodiments, the present invention relates to siRNA molecules that include at least about 19- 25 nucleotides and are able to modulate gene expression. Tn the context of the present invention, the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, the siRNA is from about 19 nucleotides to about 25 nucleotides in length. For example, the siRNA may be 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length.D. Additional Components

[0046] The SynGERS may further comprise a reporter tag for monitoring induction. The reporter tag may be positioned between the enhancer unit and the repressor unit. The reporter tag may be a polypeptide that is detectable using a specific antibody. The reporter tag may be a polypeptide that produces fluorescence or luminescence.

[0047] The SynGERS may have a cleavable peptide located between the enhancer unit and the downstream reporter tag. In some cases, the cleavable peptide may be a self-cleavable peptide, such as, for example, a 2 A peptide. The 2 A peptide may be a T2A peptide, a P2A peptide, an E2A peptide, or a F2A peptide. The presence of this peptide provides for separation of the enhancer unit and the reporter tag following protein translation. In some cases, the cleavable peptide may be a cleavage site for a widely expressed, endogenous endoprotease, such as, for example, furin, prohormone convertase 7 (PC7), paired basic amino-acid cleaving enzyme 4 (PACE4), or subtilisin kexin isozyme 2 (SKI-1).III. CAR T Cells

[0048] Provided herein are CAR T cells that comprise a SynGERS expression cassette. Chimeric antigen receptor (CAR) molecules are recombinant fusion proteins and are distinguished by their ability to both bind antigen and transduce activation signals via immunoreceptor activation motifs (ITAMs) present in their cytoplasmic tails in order to activate genetically modified immune effector cells for killing, proliferation, and cytokine production. Receptor constructs utilizing an antigen-binding moiety (for example, generated from single chain antibodies (scFv)) afford the additional advantage of being “universal” in that they bind native antigen on the target cell surface in an HLA-independent fashion.

[0049] Embodiments of the CARs described herein include nucleic acids encoding an antigen- specific CAR polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen-binding domain. A CAR may recognize an epitope comprised of the shared space between one or more antigens. Optionally, a CAR can comprise a hinge domain positioned between the transmembrane domain and the antigen binding domain. A CAR may further comprise a signal peptide that directs expression of the CAR to the cell surface. For example, a CAR may comprise a signal peptide from GM-CSF. A CAR may also be co-expressed with a membrane-bound cytokine to improve persistence. For example, a CAR may be co-expressed with membrane-bound IE- 15.

[0050] Depending on the arrangement of the domains of the CAR and the specific sequences used in the domains, immune effector cells expressing the CAR may have different levels activity against target cells. Different CAR sequences may be introduced into immune effector cells to generate engineered cells, the engineered cells selected for elevated SRC, and the selected cells tested for activity to identify the CAR constructs predicted to have the greatest therapeutic efficacy.

[0051] A chimeric antigen receptor can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. A nucleic acid sequence encoding the several regions of the chimeric antigen receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning (genomic library screening, PCR, primer-assisted ligation, scFv libraries from yeast and bacteria, site-directed mutagenesis, etc.). The resulting coding region can be inserted into an expression vector andused to transform a suitable expression host allogeneic or autologous immune effector cells, such as a T cell or an NK cell.

[0052] The chimeric construct may be introduced into immune effector cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression. Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into immune effector cells. Suitable vectors for use in accordance with the method of the present invention are non-replicating in the immune effector cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.A. Antigen binding domains

[0053] An antigen binding domain may comprise complementarity determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen binding fragments thereof. The antigen binding regions or domains may comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular mouse, human, or humanized monoclonal antibody. The fragment can also be any number of different antigen binding domains of an antigen-specific antibody. The fragment may be an antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells. In certain aspects, VH and VL domains of a CAR are separated by a linker sequence, such as a Whitlow linker.

[0054] The prototypical CAR encodes a scFv comprising VH and VL domains derived from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains (e.g. costimulatory domains and signaling domains). Thus, a CAR may comprise the LCDR1-3 sequences and the HCDR1-3 sequences of an antibody that binds to B7-H3. In further aspects, however, two of more antibodies that bind to an antigen of interest are identified and a CAR is constructed that comprises: (1) the HCDR1-3 sequences of a first antibody that binds to the antigen; and (2) the LCDR1-3 sequences of a second antibody that binds to the antigen. Such a CAR that comprises HCDR and LCDR sequences from two different antigen binding antibodies may have the advantage of preferential binding toparticular conformations of an antigen (e.g., conformations preferentially associated with cancer cells versus normal tissue).

[0055] Alternatively, a CAR may be engineered using VH and VL chains derived from different mAbs to generate a panel of CAR+ immune effector cells. The antigen binding domain of a CAR may contain any combination of the LCDR1-3 sequences of a first antibody and the HCDR1-3 sequences of a second antibody.B. Hinge domains

[0056] A CAR polypeptide may include a hinge domain positioned between the antigen binding domain and the transmembrane domain. In some cases, a hinge domain may be included in CAR polypeptides to provide adequate distance between the antigen binding domain and the cell surface or to alleviate possible steric hindrance that could adversely affect antigen binding or effector function of CAR-modified immune effector cells. The hinge domain may comprise a sequence that binds to an Fc receptor, such as FcyR2a or FcyRla. For example, the hinge sequence may comprise an Fc domain from a human immunoglobulin (e.g. , IgG 1 , IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD or IgE) that binds to an Fc receptor.

[0057] A CAR hinge domain may be derived from human immunoglobulin (Ig) constant region or a portion thereof including the Ig hinge, or from human CD8 a transmembrane domain and CD8a-hinge region, or from a CD28-hinge region. A CAR hinge domain may comprise a hinge-CH2-CH3 region of antibody isotype IgG4. The hinge domain (and / or the CAR) may not comprise a wild type human IgG4 CH2 and CH3 sequence. Point mutations may be introduced in antibody heavy chain CH2 domain to reduce glycosylation and non-specific Fc gamma receptor binding of CAR-modified immune effector cells.

[0058] A CAR hinge domain may comprise an Ig Fc domain that comprises at least one mutation relative to wild type Ig Fc domain that reduces Fc-receptor binding. For example, the CAR hinge domain can comprise an IgG4-Fc domain that comprises at least one mutation relative to wild type IgG4-Fc domain that reduces Fc-receptor binding. A CAR hinge domain may comprise an IgG4-Fc domain having a mutation (such as an amino acid deletion or substitution) at a position corresponding to L235 and / or N297 relative to the wild type IgG4- Fc sequence. For example, a CAR hinge domain can comprise an IgG4-Fc domain having a L235E and / or a N297Q mutation relative to the wild type IgG4-Fc sequence. A CAR hinge domain may comprise an IgG4-Fc domain having an amino acid substitution at position L235for an amino acid that is hydrophilic, such as R, H, K, D, E, S, T, N or Q, or that has similar properties to an “E,” such as D. A CAR hinge domain may comprise an IgG4-Fc domain having an amino acid substitution at position N297 for an amino acid that has similar properties to a “Q,” such as S or T.

[0059] The hinge domain may comprise a sequence that is about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an IgG4 hinge domain, a CD8a hinge domain, a CD28 hinge domain, or an engineered hinge domain.C. Transmembrane domains

[0060] The antigen- specific extracellular domain and the intracellular signalingdomain may be linked by a transmembrane domain. Polypeptide sequences that can be used as part of transmembrane domain include, without limitation, the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human CD3^ domain, a cysteine mutated human CD3q domain, or other transmembrane domains from other human transmembrane signaling proteins, such as CD16, CD8, inducible costimulatory (ICOS) and erythropoietin receptor. For example, the transmembrane domain may comprise a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of those provided in U.S. Patent Publication No. 2014 / 0274909 (e.g. a CD8 and / or a CD28 transmembrane domain) or U.S. Patent No. 8,906,682 (e.g. a CD8a transmembrane domain), both incorporated herein by reference. Transmembrane regions may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In certain specific aspects, the transmembrane domain can be 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a CD8a transmembrane domain or a CD28 transmembrane domain.D. Intracellular signaling domains

[0061] The intracellular signaling domain of a CAR is responsible for activation of at least one of the normal effector functions of the immune cell engineered to express the CAR. The term “effector function” refers to a specialized function of a differentiated cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Effector function in a naive, memory, or memory-type T cell includes antigen-dependent proliferation. Thus the term “intracellular signaling domain” refers to theportion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. The intracellular signaling domain may be derived from the intracellular signaling domain of a native receptor. Examples of such native receptors include the zeta chain of the T-cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon), MB1 chain, B29, Fc RIII, Fc RI, and combinations of signaling molecules, such as CD3^ and CD28, CD27, 4-1BB / CD137, ICOS / CD278, IL-2R0 / CD122, IL-2Rot / CD132, DAP10, DAP12, CD40, OX40 / CD134, MYD88 / CD40, KIR2DA2 and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins can be used.

[0062] While the entire intracellular signaling domain may be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term “intracellular signaling domain” is thus meant to include a truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal, upon CAR binding to a target. One or multiple cytoplasmic domains may be employed, as so-called third generation CARs have at least two or three signaling domains fused together for additive or synergistic effect, for example the CD28 and 4- IBB can be combined in a CAR construct. In certain specific aspects, the intracellular signaling domain comprises a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a CD3^ intracellular domain, a CD28 intracellular domain, a CD137 intracellular domain, or a domain comprising a CD28 intracellular domain fused to the 4- IBB intracellular domain.E. T Cells

[0063] The T cells may be derived from the blood, bone marrow, lymph, umbilical cord, or lymphoid organs. The T cells may be human T cells. The T cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. The cells may include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+cells, CD8+cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and / orautologous. For off-the-shelf technologies, the cells may be derived from pluripotent and / or multipotent cells, such as stem cells, such as induced pluripotent stem cells (iPSCs).

[0064] Among the sub-types and subpopulations of T cells (e.g., CD4+and / or CD8+T cells) are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), natural killer T cells (NKT), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and gamma / delta T cells.

[0065] One or more of the T cell populations may be enriched for or depleted of cells that are positive for a specific marker, such as surface markers, or that are negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells).

[0066] T cells may be separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+or CD8+selection step is used to separate CD4+helper and CD8+cytotoxic T cells. Such CD4+and CD8+populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0067] CD8+T cells may be further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. Enrichment for central memory T (TCM) cells may be carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations.

[0068] The T cells may be autologous T cells. In this method, tumor samples are obtained from patients and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g.,collagenase, trypsin or DNase). Single-cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL-2). The cells are cultured until confluence (e.g., about 2xl06lymphocytes), e.g., from about 5 to about 21 days, preferably from about 10 to about 14 days.

[0069] The cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days.

[0070] Expansion can be accomplished by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin- 15 (IL-15), with IL-2 being preferred. The non-specific T-cell receptor stimulus can include around 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from OrthoMcNeil®, Raritan, N.J.), a human monoclonal antibody cocktail containing anti- CD3 / CD28 / CD2 (available from STEMCELL Technologies, Vancouver, Canada). Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growth factor, such as 300 lU / ml IL-2 or IL-15, with IL-2 being preferred. The in vz7ro-induced T-cells are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.

[0071] The autologous T-cells can be modified to express a T-cell growth factor that promotes the growth and activation of the autologous T-cells. Suitable T-cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable methods of modification are known in the art. See, for instance, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and lohn Wiley & Sons, NY, 1994. In particular aspects, modified autologous T- cells express the T-cell growth factor at high levels. T-cell growth factor coding sequences,such as that of IL- 12, are readily available in the art, as are promoters, the operable linkage of which to a T-cell growth factor coding sequence promote high-level expression.F. Engineering of Immune Effector Cells

[0072] The cells may comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. The nucleic acids may be heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. The nucleic acids may not be naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).IV. Methods of Screens

[0073] Provided herein are methods for identifying combinations of cell state-specific sensors, enhancer units, and repressor units that help white blood cells grow, persist, and eliminate tumors. The methods may be performed in vitro or in vivo. In one embodiment, the identification method is performed in vitro. An in vitro method comprises contacting library of CAR T cells that have been modified to contain SynGERS with target tumor cells. In such an embodiment, the CAR that is comprised by the immune cells comprises affinity for the target tumor cell. The step of contacting the T cell library with target tumor cells represents a “challenge,” e.g., a tumor cell challenge. Each challenge results in an enriched CAR T cell library. For example, upon a first challenge, a first enriched CAR T cell library may be isolated. Upon one or more challenges, e.g., two or more challenges, three or more challenges, four or more challenges, five or more challenges, a second or more, e.g., a third or more, a fourth or more, a fifth or more, sixth or more enriched CAR T cell library may be isolated. Upon successive challenges, the final enriched CAR T cell library may comprise T cells a SynGER that confers enhanced T cell functions (e.g., T cell persistence, T cell efficacy).

[0074] In one embodiment, the method is performed in vivo. An in vivo screening method comprises infusing a SynGERS-modified CAR immune cell library into a target tumorbearing model organism. For example, the SynGERS-modified CAR T cell library comprises T cells that express a CAR having affinity for a specific antigen, and the T cell library is infused into a tumor bearing organism, wherein the organism has a tumor that expresses the specific antigen. Tumor infiltrating lymphocytes are then isolated from the infused, tumor-bearingorganism, thereby resulting in an enriched CAR T cell library. The infusion step is akin to the “challenge” step of an in vitro screening method as described herein. As such, in some embodiments, multiple infusions (“challenges”) can be performed successively to further enrich the resulting isolated CAR T cell library.

[0075] A suitable model organism for use in an in vivo screening method of the present invention includes, without limitation, a mouse, a rat, a non-human primate, and a pig. A suitable model organism generally includes an organism that has a natural immune cell repertoire (e.g., T cell repertoire). In some embodiments, where the identification method is performed in vivo, the SynGERS-modified CAR immune cell (e.g., T cell) library is generated from a population of immune cells (e.g., T cells) that are obtained from the same animal as the subject of infusion. In some embodiments, where the identification method is performed in vivo, the SynGERS-modified CAR immune cell (e.g., T cell) library is generated from a population of immune cells (e.g., T cells) that are obtained from a different animal as the subject of infusion. The skilled artisan would readily be able to determine the appropriate source of immune cells and the appropriate infusion subject.

[0076] Once an enriched CAR immune cell library (e.g., an enriched CAR T cell library) is isolated, standard sequencing methods may be used to identify SynGER that regulates immune cell function, immune cell memory, and / or immune cell persistence (e.g., T cell function, T cell memory, and / or T cell persistence). Several methods of DNA extraction and analysis are encompassed in the methods provided herein. The skilled artisan would be able to determine SynGERS that are enriched in the enriched CAR immune cell library.V. Cancer Treatment

[0077] The modified cells (e.g., T cells) described herein may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells may be administered.

[0078] In one aspect, the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a modified T cell of the present invention. In another aspect, the invention includes a method of treating a disease or condition in a subject comprising administering to a subject in need thereof a population of modified T cells.

[0079] Also included is a method of treating a disease or condition in a subject in need thereof comprising administering to the subject a genetically edited modified cell (e.g., genetically edited modified T cell). In one embodiment, the method of treating a disease or condition in a subject in need thereof comprises administering to the subject a genetically edited modified cell comprising a CAR.

[0080] Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. In some embodiments, the cell therapy, e.g., adoptive T cell therapy is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0081] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0082] In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0083] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or conditionover time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0084] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0085] The modified immune cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplary cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancers may be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0086] In one embodiment, the cancer is a solid tumor or a hematological tumor. In one embodiment, the cancer is a carcinoma. In one embodiment, the cancer is a sarcoma. In one embodiment, the cancer is a leukemia. In one embodiment, the cancer is a solid tumor.

[0087] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms’ tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocyto a, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

[0088] Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0089] Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0090] The administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like.

[0091] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0092] In some embodiments, the populations or sub-types of cells, such as CD8+and CD4+T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4+to CD8+ratio), e.g., within a certain tolerated difference or error of such a ratio.

[0093] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of theforegoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges. In some embodiments, the dose of total cells and / or dose of individual sub-populations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 10sand 106cells I kg body weight, for example, at or about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104and at or about 109T cells / kilograms (kg) body weight, such as between 10sand 106T cells I kg body weight, for example, at or about 1 x 105T cells / kg, 1.5 x 105T cells / kg, 2 x 105T cells / kg, or 1 x 106T cells / kg body weight. In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO5cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg about IxlO8cells / kg, from about IxlO8cells / kg about IxlO9cells / kg, from about IxlO9cells / kg about IxlO10cells / kg, from about IxlO10cells / kg about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 1x10scells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about 5xl07total cells. In some embodiments, a suitable dosage is from about IxlO8total cells to about 5xlO8total cells. In some embodiments, a suitable dosage is from about 1.4xl07total cells to about l.lxlO9total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.

[0094] In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

[0095] In certain embodiments, the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

[0096] As such, the modified immune cells comprising a CAR of the present invention when used in a method of treatment as described herein, enhances the ability of the modified immune cells in carrying out their function. Accordingly, the present invention provides a method for enhancing a function of a modified immune cell for use in a method of treatment as described herein.VI. Pharmaceutical Composition and Formulations

[0097] Also provided are populations of immune cells of the invention, compositions containing such cells and / or enriched for such cells, such as in which cells expressing the CAR and the SynGERS make up at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[0098] Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0099] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are avariety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non- ionic surfactants such as polyethylene glycol (PEG).

[0100] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001 % to about 4% by weight of the total composition. Methods for preparing admini strable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21 st ed. (May 1, 2005).

[0101] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective forthe purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0102] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, intratumoral, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0103] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents,and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0104] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0105] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.VII. Examples

[0106] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - SynGERS Design

[0107] Cell state-specific bidirectional modulation of gene expression is desired in several disciplines focused on modified properties of engineered cells including regenerative medicine and cancer immunotherapy. To address this need, the inventors developed Synthetic Gene Expression Regulatory Switches (SynGERS) and tested them in tumor antigen specific, chimeric antigen receptor (CAR) T cells. SynGERS are designed to be turned on only when needed to help white blood cells grow, persist, and eliminate tumors.

[0108] The cell state-specific synthetic gene expression regulatory switches (SynGERS) maximize the expansion, persistence, and antitumor activity of chimeric antigen receptor (CAR) T cells for treatment of solid tumors. To accomplish this goal, a lentiviral,single expression cassette-based vector system that modulates gene expression via SynGERS and constitutively expresses a clinically validated CAR targeting glypican-3 (GPC3) was developed (FIG. 1A). A library of SynGERS was designed (FIG. IB) and preliminary data generated confirming the functionality of each SynGERS module.

[0109] The SynGERS cassette consists of a 5’ sensor, acting on a minimal promoter (minP), that drives expression of enhancer and repressor units, which are separated by a 2A peptide sequence (FIG. 1A). The 5’ sensor is a cell state-specific sensor (e.g., responding to NFAT, NR4A2 (FIG. 3), BLIMP1, EGR2, or TOX) (Seo et al., 2019; Yoshikawa et al., 2022; Jung et al., 2023; Wagle et al., 2021) acting on a minimal promoter. The enhancer unit is a specific gene regulating T-cell survival (e.g., interleukin- 15 (IL15), JUN, BACH2, LTBR, or BATF3). The repressor unit is an RNA interference-based downregulation of a gene to prevent T-cell dysfunction (e.g., a negative regulator of ARIDla, BTG1, RASA2, REGNASE1 , or PRDME). A Q8 tag is also present to allow for induction detection.

[0110] A separate promoter (SFFV) drives the GBBz CAR (GPC3-specific second- generation CAR incorporating the 41BB endodomain) (FIG. IB). The CAR unit incorporates the human truncated EGFR (huEGFRt), which is translated into a separate peptide with the use of a T2A sequence.

[0111] SynGERS sense intracellular signals in the form of DNA-binding proteins recognizing specific DNA sequences in the “sensor unit.” For NFAT, NR4a2 (Blank et al., 2019), and early growth response 2 (EGR2) (Wagle et al., 2021), the respective DNA binding sequences are used for the sensor units. For TOX and BL1MP1, DNA sequences from the promoter region of the corresponding genes are used as these transcription factors act as repressors in the majority of cases, and the SynGERS system aims to initiate transcription of the transgene upon binding of the transcription factors. The sensor unit acts on a synthetic minimal promoter (minP), which drives transcription of enhancer and repressor units in a single mRNA transcript. During translation, the enhancer unit separates and traffics to its destination (endoplasmic reticulum for secreted IL15, membrane bound lymphotoxin- receptor (LTBR) and cytoplasm for CJUN, TCF1 (encoded by the TCF7 gene), and BACH2). Target genespecific amiRs are cleaved from the mRNA tail (O’Brien et al., 2018). Results confirm the functionality of each SynGERS unit and feasibility of the high-throughput in vitro screening strategy to differentiate SynGERS with superior antitumor properties.Example 2 - Effective, bidirectional control of target genes

[0112] To test the functionality of enhancer (e) and repressor (r) units, SynGERS incorporating an NF AT (sNFAT-eGFP-rB2M) sensor (s) were built. For the repression unit, an artificial microRNA (amiR) cassette targeting B2M was placed downstream of the enhancer unit driving eGFP. This amiR is based on the endogenous miR30 microRNA precursor. Activated primary T cells were transduced, activation allowed to subside, and then the cells reactivated. A functional SynGERS was expected to induce expression of GFP and repression of B2M, leading to downregulation of MHC class I. Indeed, T cells expressing the evaluated SynGERS construct mediated combined eGFP upregulation and downregulation of MHC class I molecules following T cell reactivation (FIG. 2) validating the intended functionality of the SynGERS.Example 3 - NR4a2-specific sensor unit induces higher output signal

[0113] To develop additional cell state-specific sensor units, activation- specific input sensing was maximized through the generation of a set of DNA binding sequences (DBS) based on published and computer-predicted binding sites for NF AT and NR4A2. For each sensor, four DBS sequences were placed upstream of a minimal promoter driving eGFP (Uchibori et al., 2019) and cloned into a lentiviral vector. The most sensitive NF AT sensor unit (an established reporter of activation, data not shown) was selected and compared with new NR4A2 sensors in primary T cells. After transduction and activation via the T cell receptor, eGFP output signal was quantified by FACS. The NR4A2-DBS1 sensor module outperformed the NFAT sensor (FIG. 3). The corresponding NFAT and NR4A2 sensor sequences were selected for further development, providing two levels of activation-specific control for downstream enhancer and repressor units.Example 4 - SynGERS -CAR T cells showed enhanced expansion and persistence in vitro

[0114] To identify SynGERS that promote expansion and persistence of GPC3-CAR T cells, a library of SynGERS-CAR was screened for optimal combinations of T cell functionpromoting genes IL-15, TCF7, JUN, BACH2, LTBR) and repression inhibitory genes (ARIDla, BTG1, RASA2, REGNASE1, PRDMl ) providing superior expansion, persistence and killing function in repeat tumor challenge assays against GPC3+ tumor cells. First, the expansion and persistence of SynGERS-GPC3-CAR T cells in vitro was quantified using repeat tumor challenge assays, defined cell phenotype (including markers for activation,exhaustion, and effector / memory differentiation), and the ability of the cells to resist apoptosis co-cultured with fresh tumor cells was determined.

[0115] Expansion and persistence of CAR T cells predicts clinical therapeutic activity. To identify the SynGERS with the most potent effect on these parameters, the expansion and persistence of SynGERS CAR T cells was quantified in vitro using repeat tumor challenge assays. CAR T cells were co-cultured twice weekly with fresh solid tumor cells (hepatoblastoma - HepG2, hepatocellular carcinoma - Huh7, and malignant rhabdoid tumor - G401) (Batra et al., 2020; Lopez-Terrada et al., 2009; Miao et al., 2013; Garvin et al., 1993) at a 1:1 effector to target ratio (E:T) using a multi-plate setup that enables parallel assessment of all constructs and controls. To ensure fair comparisons, cell concentrations and total cell numbers were kept constant among the different groups by normalizing differences at each killing cycle. SynGERS comparison was defined by proliferative capacity and killing function. The first screening using Huh7-eGFP cells showed eight SynGERS with improved killing and / or expansion compared to the CAR-only control (FIG. 4A). These 8 SynGERS were then used to perform three more repeat killing assays with the three eGFP-labeled cell lines. These repeat killing assays demonstrated five SynGERS (eIL15-rTOX, eIL15-rBLIMPl, eIL15- rBTGl, eIL15-rRASA2, and eIL15-rREGNASEl) had superior killing and expansion than the CAR-only control (eQ8-rScramble) (FIGS. 4B, 4C and 4D).

[0116] The exhaustion phenotype of SynGERS-CAR T cells at the end of the repeat killing assay (PD1, TIM3, LAG3) was defined, as well as the memory and effector differentiation (CCR7, CD45RO). Additionally, apoptosis was measured in a separate experiment using Annexin-V.

[0117] The best performing SynGERS-CAR were characterized by their ability to mediate high levels of CAR T cell expansion and persistence, and low levels of Annexin V (FIG. 5) and PD-1 expression (FIG. 6). Top performing SynGERS were also characterized by a higher percentage of effector-like CAR T cells compared to the CAR-only (eQ8-rScramble) control (FIG. 7). Based on these experiments, the top 5 SynGERS-CAR were selected for in vivo testing.

[0118] These results demonstrate the potential for SynGERS to induce potent antitumor function by CAR T cells with improved persistence and proliferation in solid tumors.

[0119] These data also provide evidence that cell state-specific, bidirectional gene expression modulation can be accomplished to alter functional properties of cells and should be applicable to engineered cell therapeutics beyond CAR T cells, including regenerative medicine.Example 5 - SynGERS-CAR T cells showed enhanced anti-tumor effect and survival in vivo

[0120] To identify SynGERS that promote enhanced anti-tumor effects and survival in GPC3-CAR T cells, the top SynGERS-CAR identified in the in vitro screening (eIL15-rTOX, eIL15-rBLIMPl, eIL15-rBTGl, eIL15-rRASA2, and eIL15-rREGNASEl) were tested in vivo against Huh7 and HepG2 cells. NSG MHC Ell DK mice received a single i.v. dose of 5xl06SynGERS-CAR T cells on day 7 post i.p. injection of either 0.5xl06Huh7-eGFP-Luciferase cells or HepG2-eGFP-Luciferase cells. Non-transduced cells, eQ8-rScramble, and eIL15- rScramble served as controls.

[0121] In both experiments theeIL15-rTOX SynGERS-CAR T cells showed prolonged survival and anti-tumor effect in comparison to the CAR-only (eQ8-rScramble) or eIL15- rScramble controls (FIGS. 8 and 9).* * *

[0122] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.Ahmed et al., HER2-Specific Chimeric Antigen Receptor-Modified Virus-Specific T Cells for Progressive Glioblastoma: A Phase 1 Dose-Escalation Trial. JAMA Oncol. 3:1094- 1101, 2017.Ahmed et al., Human Epidermal Growth Factor Receptor 2 (HER2)-Specific Chimeric Antigen Receptor-Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma. Journal of Clinical Oncology 33: 1688- 1696, 2015.Batra et al., Glypican-3-Specific CAR T Cells Coexpressing IL15 and IL21 Have Superior Expansion and Antitumor Activity against Hepatocellular Carcinoma. Cancer Immunol Res 8:309-320, 2020.Belk, J. A. et al. Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence. Cancer Cell 40:768-786 e767 , 2022.Blank et al., Defining “T cell exhaustion”. Nat Rev Immunol. 19:665-674, 2019.Carnevale et al., RASA2 ablation in T cells boosts antigen sensitivity and long-term function.Nature 609: 174-182, 2022.Garvin et al., The G401 cell line, utilized for studies of chromosomal changes in Wilms’ tumor, is derived from a rhabdoid tumor of the kidney. The American Journal of Pathology 142:375-380, 1993.Heczey et al.m CAR T Cells Administered in Combination with Lymphodepletion and PD- 1 Inhibition to Patients with Neuroblastoma. Mol Ther 25:2214-2224, 2017.Heczey et al., Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results. Nature Medicine IN PRESS, 2023.Hoyos et al., Engineering CD19-specific T lymphocytes with interleukin- 15 and a suicide gene to enhance their anti-lymphoma / leukemia effects and safety. Leukemia 24:1160-1170, 2010.Jung et al., BLIMP1 and NR4A3 transcription factors reciprocally regulate antitumor CAR T cell sternness and exhaustion. Sci Transl Med 14:eabn7336, 2022.Jung et al., Type I Interferon Signaling via the EGR2 Transcriptional Regulator Potentiates CAR T cell-intrinsic Dysfunction. Cancer Discovery, 2023.Legut et al., A genome-scale screen for synthetic drivers of T cell proliferation. Nature 603:728-735, 2022.Lopez-Terrada et al., Hep G2 is a hepatoblastoma-derived cell line. Human Pathology 40:1512- 1515, 2009.Louis et al., Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood 118:6050-6056, 2011.Lynn et al., c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 576:293-300, 2019.Mai et al., Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells. Proc Natl Acad Sci USA 120:e2218632120, 2023.Majzner & Mackall, Clinical lessons learned from the first leg of the CAR T cell journey. Nature Medicine 25:1341-1355, 2019.Maude et al., Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 378:439-448, 2018.Maude et al., Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371:1507-1517, 2014.Miao et al., Knockdown of GPC3 inhibits the proliferation of Huh7 hepatocellular carcinoma cells through down-regulation of YAP. Journal of Cellular Biochemistry 114:625-631 , 2013.Murphy et al., Janeway’s Immunobiology. New York: Garland Science; 2012.O'Brien et al., Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Frontiers in Endocrinology 9, 2018.Pereira et al., Transcriptional and epigenetic regulation of T cell hyporesponsiveness. J Leukoc Biol. 102:601-615, 2017.Seo et al., TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8(+) T cell exhaustion. Proc Natl Acad Sci USA 116: 12410-12415, 2019.Shi et al., Chimeric Antigen Receptor-Glypican-3 T-Cell Therapy for Advanced Hepatocellular Carcinoma: Results of Phase I Trials. Clin Cancer Res 26:3979-3989, 2020.Steffin et al., GPC3-CAR T Cells Co-Expressing IL15 Mediate Potent Antitumor Activity in Liver Cancer Patients Associated with Toxicity That Can Be Mitigated Using iC9 Safety Switch. 26th Annual Conference of the American Society of Cell and Gene Therapy, 2023.Uchibori et al., Functional Analysis of an Inducible Promoter Driven by Activation Signals from a Chimeric Antigen Receptor. Molecular Therapy Oncolytics 12: 16-25, 2019.Wagle et al., Antigen-driven EGR2 expression is required for exhausted CD8(+) T cell stability and maintenance. Nat Commun 12:2782, 2021. Wei et al., Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy. Nature 576:471-476, 2019.Yao et al., BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8(+) T cells. Nature Immunology 22:370-380, 2021.Yoshikawa et al., Genetic ablation of PRDM1 in antitumor T cells enhances therapeutic efficacy of adoptive immunotherapy. Blood 139:2156-2172, 2022.Zhu et al., A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs. BMC Mol Biol. 8:98, 2007.

Claims

CLAIMS1. An expression construct comprising, from 5’ to 3’, (a) cell state-specific sensor that responds to activation of NR4A2 and comprises a minimal promoter; (b) an enhancer unit that encodes eIL15; (c) a repressor unit that encodes an inhibitor RNA that downregulates the expression of TOX, BLIMP1, BTG1, RASA2, or REGENASE1 ; (d) a promoter; and (e) a chimeric antigen receptor, wherein the cell state-specific sensor comprises a nucleotide sequence corresponding to nucleotides 1-64 of SEQ ID NO: 1.

2. A plurality of expression constructs, each independently comprising:(a) a cell state-specific sensor;(b) an enhancer unit that encodes a protein that promotes cell survival, expansion, and / or persistence; and(c) a repressor unit that encodes an inhibitory RNA that downregulates the expression of a protein involved in T-cell dysfunction.

3. The constructs of claim 2, wherein the cell state-specific sensor comprises a minimal promoter.

4. The constructs of claim 2, wherein the cell state-specific sensor comprises binding sites for a transcription factor that is activated in response to immune cell activation.

5. The constructs of any one of claims 2-4, wherein the cell state-specific sensor responds to activation of NFAT, NR4A2, BLIMP 1, EGR2, or TOX.

6. The constructs of any one of claims 2-5, wherein the enhancer unit encodes a protein that promotes T-cell survival.

7. The constructs of any one of claims 2-6, wherein the enhancer unit encodes interleukin- 15 (IL15), JUN, BACH2, LTBR, or BATF3.

8. The constructs of any one of claims 2-7, wherein the repressor unit encodes a miRNA or shRNA.

9. The constructs of any one of claims 2-8, wherein the repressor unit downregulates the expression of ARIDla, BLIMP1, BTG1, RASA2, REGNASE1 , TOX, or PRDM1.

10. The constructs of any one of claims 2-9, wherein the enhancer unit and the repressor unit are separated by a cleavable peptide.

11. A method for screening the expression constructs of any one of claims 2-10, the method comprising introducing the plurality of expression constructs into a plurality of T cells, activating the T cells, selecting a T cell that exhibits an enhanced function, and identifying the expression construct in the selected T cell.

12. The method of claim 11, wherein the enhanced function is growth, persistence, and / or anti-tumor activity.

13. The method of claim 11 or 12, wherein the plurality of T cells further comprise a chimeric antigen receptor.

14. The method of any one of claims 11-13, wherein the plurality of T cells further comprise a truncated EGF-receptor.

15. The method of any one of claims 11-14, wherein the screen is performed in vitro or in vivo.

16. A modified T cell comprising an expression cassette comprising an enhancer unit that encodes a protein that promotes cell survival, expansion, and / or persistence and a repressor unit that encodes an inhibitory RNA that downregulates the expression of a protein involved in T-cell dysfunction, wherein the expression of the enhancer unit and repressor unit under the control of a cell state-specific sensor.

17. The modified T cell of claim 16, further comprising a chimeric antigen receptor.

18. A method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the modified T cells of claim 16 or 17.

19. The method of claim 18, wherein the modified T cells are autologous or allogeneic to that patient.

20. The method of claim 18 or 19, wherein the cancer is a solid tumor.

21. The method of claim any one of claims 18-20, wherein the cancer is a Wilms tumor, hepatoblastoma, rhabdomyosarcoma, or rhabdoid tumor.

22. The method of any one of claims 18-21, wherein the patient is a child.

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